Nanoligands and a method for promoting cell adhesion and differentiation of stem cells by using the same

Abstract

To provide a nano ligand for accelerating cell adhesion and differentiation of a stem cell, and a method for accelerating cell adhesion and differentiation of the stem cell using the nano ligand.SOLUTION: A nano ligand for accelerating cell adhesion and differentiation of a stem cell comprises: a core which includes a magnetic nanoparticle; a coating layer which is arranged to envelope the core and includes an integrin binding ligand peptide that is negatively charged; and a linker which is arranged between the core and the coating layer. Applying a magnetic field to a basal plate including the nano ligand enables reversible control of a nano ligand slide in addition to temporal and spatial control thereof, and adhesion and differentiation of the stem cell in vivo and in vitro can be efficiently adjusted by the magnetic field based spatiotemporal control.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a nanoligand for promoting cell adhesion and differentiation of stem cells, and a method for promoting cell adhesion and differentiation of stem cells using the nanoligand, specifically, to a method for remotely controlling stem cell adhesion and differentiation using the nanoligand. [Background technology]

[0002] Stem cells are capable of self-renewal and proliferation, and have the ability to differentiate into various cells, such as bone, fat, muscle, cardiac muscle, blood vessels, and cartilage. In recent years, techniques for transplanting stem cells or cells differentiated from stem cells to regenerate damaged tissues and organs by utilizing these characteristics have been widely studied. In addition, active research is being conducted on biomaterials that help stem cells differentiate into specific cells.

[0003] Thus, in vivo ligand presentation technology has been disclosed as a method for efficiently controlling the regenerative effect of stem cells. However, conventional nano-ligand presentation in the body is almost static, and even if it is dynamic, it is not controlled remotely in real time. It has the disadvantage of being unable to induce reversible changes in macroscopic ligand density. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Korean Patent Publication No. 2018-0017704 Summary of the Invention [Problem to be solved by the invention]

[0005] The problem to be solved by the present invention is to provide a nano-ligand that can electrostatically bind to a substrate and move therewith, and to provide a method for using the nano-ligand to induce reversible changes in macroscopic nano-ligand density through real-time remote control, thereby promoting stem cell attachment and differentiation.

Means for Solving the Problem

[0006] The present invention includes a core containing magnetic nanoparticles, a coating layer provided so as to wrap the core and containing an integrin-binding ligand peptide, and a linker provided between the core and the coating layer, and provides a nanoligand for promoting cell adhesion and differentiation of stem cells, wherein the integrin-binding ligand peptide is negatively charged.

[0007] Furthermore, the present invention provides a method for producing the above-mentioned nanoligand for promoting cell adhesion and differentiation of stem cells, including the steps of preparing a core containing magnetic nanoparticles, mixing the core with a first suspension containing a linker to produce a core to which the linker is bound, and mixing the core to which the linker is bound with a second suspension containing an integrin-binding ligand peptide (RGD).

[0008] Furthermore, the present invention provides a method for promoting cell adhesion and differentiation of stem cells, including the steps of producing a nanoligand-presenting substrate by supporting a substrate with an activated surface on a solution containing the above-mentioned nanoligand for promoting cell adhesion and differentiation of stem cells, and applying an external magnetic field after treating the stem cells on the nanoligand-presenting substrate to adjust the adhesion and differentiation of the stem cells.

Advantages of the Invention

[0009] The nanoligand for promoting cell adhesion and differentiation of stem cells according to the present invention is obtained by coating magnetic nanoparticles with a negatively charged ligand and can be easily moved on a substrate by electrostatic binding to the substrate.

[0010] ​Furthermore, the method for promoting cell adhesion and differentiation of stem cells according to the present invention not only controls the sliding of the nanoligand in a temporal and spatial manner by applying a magnetic field to the substrate containing the nanoligand, but also enables reversible control, and can efficiently regulate the adhesion and differentiation of stem cells in vitro and in vivo by magnetic field-based spatio-temporal control.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0012] Hereinafter, in order to describe the present invention more specifically, preferred embodiments according to the present invention will be described in more detail with reference to the accompanying drawings. However, the present invention is not limited to the embodiments described in the present application and may be embodied in other forms.

[0013] The nanoligands for stem cell adhesion and differentiation according to the present invention are substances with remote, spatiotemporal, and reversible controllability, and can mimic extracellular matrix (ECM) remodeling to regulate cell adhesion in the body. Here, the nanoligand is a slidable nanoligand with an amine-functionalized, polyethylene glycol linker, and a superparamagnetic nanomaterial bound to a negatively charged RGD ligand. The method for promoting stem cell adhesion and differentiation according to the present invention binds a slidable nanoligand to a substrate charged with an amount by optimizing electrostatic interactions to exhibit reversible slidability. Therefore, the present invention magnetically attracts slidable nanoligands, adjusts the macroscale nanoligand density, and enables imaging of the slide of macroscale and in situ nanoscale nanoligands. In addition, the present invention can easily achieve spatiotemporal and reversible control by in situ magnetic control of the attraction of slidable nanoligands and stem cell adhesion under in vitro and in vivo conditions. Furthermore, the present invention stimulates mechanosensing-mediated differentiation of stem cells by the in situ magnetic attraction of slidable nanoligands. By remotely controlling spatiotemporal and reversible nanoligand mutations that mimic these extracellular matrices, a method for promoting stem cell adhesion and differentiation is provided that is excellent in regulating the recovery processes of various cells in the body.

[0014] The present invention provides a nanoligand for promoting stem cell adhesion and differentiation, including a core containing magnetic nanoparticles, a coating layer provided to wrap the core and containing an integrin-binding ligand peptide, and a linker provided between the core and the coating layer, wherein the integrin-binding ligand peptide is negatively charged.

[0015] Figure 1 is a schematic diagram showing the nanoligand for promoting stem cell adhesion and differentiation according to the present invention, and the method for stem cell adhesion and differentiation using the same.

[0016] As shown in FIG. 1, the nanoligand of the present invention includes a core containing magnetic nanoparticles and a coating layer containing an integrin-binding ligand peptide bound on the core, and it can be seen that the integrin-binding ligand peptide is a negatively charged integrin peptide. Specifically, the integrin-binding ligand peptide bound on the core may surround the core like a micelle structure. Thereby, the surface charge of the nanoligand can be negatively charged.

[0017] FIG. 3 is a transmission electron microscope (TEM) image of the nanoligand for promoting cell adhesion and differentiation of stem cells according to the present invention, from which the size of the nanoligand can be grasped. Specifically, the nanoligand may have a diameter of 30 to 60 nm. When the diameter of the nanoligand is less than 30 nm, it is difficult to control the movement of the nanoligand, and when it exceeds 60 nm, the adhesion efficiency of stem cells decreases. More specifically, the nanoligand may have a diameter of 30 to 50 nm, or 35 to 45 nm.

[0018] The magnetic nanoparticles are not particularly limited as long as they are magnetic nanoparticles. For example, the nanoligand may have a diameter of 5 to 30 nm. When the diameter of the magnetic nanoparticles is less than 5 nm, the particle size is too small and there are many losses, resulting in a decrease in efficiency. When it exceeds 30 nm, since the diameter of the nanoligand is large, there may be a problem that the adhesion efficiency of stem cells decreases. More specifically, the magnetic nanoparticles may have a diameter of 5 to 15 nm, or 10 to 20 nm. By including the magnetic nanoparticles as described above, the nanoligand of the present invention can promote the adhesion and differentiation of stem cells using a magnetic field.

[0019] Also, the magnetic nanoparticles may be bound to the silica surface. Specifically, the magnetic nanoparticles may have amino-silica bound to the surface. The type of silica may be any one or more of tetraethyl orthosilicate (TEOS) and (3-aminopropyl) triethoxysilane (APTES).

[0020] For example, the nanoligand of the present invention has a structure in which a core and a coating layer are linked by a linker, and the linker may be a polyethylene glycol (PEG)-based linker. Specifically, the polyethylene glycol-based linker may be maleimide-polyethylene glycol-NHS ester (Mal-PEG-NHS ester). By including the linker as described above, the binding force between the core and the coating layer can be increased, and the durability of the nanoligand can be improved.

[0021] The coating layer may be bonded to the core or a linker bonded to the core, or may surround the core. Specifically, the coating layer contains an integrin-binding ligand peptide (RGD), and the integrin-binding ligand peptide may be negatively charged and may contain a negatively charged thiolated integrin-binding ligand peptide. By including the integrin-binding ligand peptide negatively charged as described above, the surface of the nanoligand of the present invention is negatively charged, and thereby, movement on the substrate becomes free due to electrostatic binding to the substrate. Due to these characteristics, the nanoligand is also called a "slidable nanoligand", and can promote the adhesion and differentiation of stem cells through the sliding of the nanoligand on the substrate.

[0022] Furthermore, the present invention provides a method for producing a nanoligand for promoting cell adhesion and differentiation of the above-described stem cells, including the steps of preparing a core containing magnetic nanoparticles, mixing the core with a first suspension containing a linker to produce a core to which the linker is bonded, and mixing the core to which the linker is bonded with a second suspension containing an integrin-binding ligand peptide (RGD).

[0023] In the step of preparing the core, magnetic nanoparticles may be stirred with a silane solution to form a core coated with silane. Specifically, in the step of preparing the core, magnetic nanoparticles may be stirred with an amino-silane solution to form a core coated with amino-silane. The type of silane contained in the silane solution and the type of silica may be any one or more of tetraethyl orthosilicate (TEOS) and (3-aminopropyl) triethoxysilane (APTES).

[0024] Specifically, in the step of manufacturing a core to which a linker is bound, the core and a suspension containing the linker may be stirred for 10 to 20 hours, or 10 to 15 hours, under dark conditions. Thereby, a core to which a linker is bound can be obtained. At this time, while using a permanent magnet, it may be washed two or more times with a solvent to obtain a core to which a linker is bound. The solvent may contain any one or more of dimethylformaldehyde (DMF) and dimethyl sulfoxide (DMSO).

[0025] The linker may be a polyethylene glycol (PEG)-based linker. Specifically, the polyethylene glycol-based linker may be maleimide-polyethylene glycol-NHS ester (Mal-PEG-NHS ester). By binding a linker as described above to the core, the binding force between the core and the coating layer can be increased, and the durability of the nanoligand can be improved.

[0026] Furthermore, the step of mixing with the second suspension may be performed by stirring the core bound to the linker with a suspension containing an integrin-binding ligand peptide (RGD) for 10 to 20 hours, or 10 to 15 hours, under dark conditions. At this time, while using a permanent magnet, it may be washed with a solvent to obtain magnetic nanoparticles (nanoligands) to which a negatively charged integrin-binding ligand peptide is bound. The solvent may contain any one or more of dimethylformaldehyde (DMF) and dimethyl sulfoxide (DMSO).

[0027] Here, a coating layer may be formed on the core by a process of stirring with an integrin-binding ligand peptide. Specifically, the integrin-binding ligand peptide may be negatively charged, or may be a negatively charged thiolated integrin-binding ligand peptide. By forming a coating layer on the core with the integrin-binding ligand peptide negatively charged as described above, the surface of the nanoligand of the present invention is negatively charged, and thereby, movement on the substrate becomes free due to electrostatic coupling with the substrate. Due to these characteristics, the nanoligand is also called a "slidable nanoligand", and can promote the adhesion and differentiation of stem cells through the sliding of the nanoligand on the substrate.

[0028] Furthermore, the present invention provides a method for promoting the adhesion and differentiation of stem cells, including the steps of producing a nanoligand-presenting substrate by supporting a substrate with an activated surface on a solution containing the above-described nanoligand for promoting the adhesion and differentiation of stem cells, and after treating the nanoligand-presenting substrate with a culture solution, applying an external magnetic field to adjust the adhesion and differentiation of stem cells.

[0029] FIG. 1 and FIG. 2 are diagrams schematically showing a method for promoting the adhesion and differentiation of stem cells according to an embodiment of the present invention. As shown in FIGS. 1 and 2, by electrostatically coupling a nanoligand with a negatively charged surface to a positively charged substrate and applying a magnetic field, the adhesion and differentiation of stem cells can be promoted or activated at the portion where the magnetic field is applied. Specifically, since the substrate and the nanoligand are coupled by electrostatic coupling, the nanoligand moves (slides) according to the position where the magnetic field is applied, and thereby, there is an advantage that the density of the nanoligand can be adjusted at the portion where the magnetic field is applied to promote the adhesion and differentiation of stem cells at a desired site.

[0030] Specifically, the steps of manufacturing the nano-ligand presenting substrate may include the step of immersing the surface of the substrate in an acidic solution, the step of supporting the immersed substrate in an aminosilane solution to activate the surface of the substrate, and the step of subjecting the activated substrate to ultrasonic treatment at room temperature. In the step of immersing the surface of the substrate in the acidic solution, it may be immersed in an acidic solution containing one or more of hydrochloric acid and sulfuric acid for 30 minutes to 2 hours, or 30 minutes to 1 hour. Thereby, hydroxyl groups can be bonded to the surface of the substrate, and the surface activation of the substrate can be effectively performed so that the bonding with amino groups becomes easy.

[0031] In the step of activating the surface of the substrate, the substrate may be supported in an aminosilane solution under dark conditions to activate the surface of the substrate. The aminosilane solution may contain (3-aminopropyl)triethoxysilane (APTES). At this time, activating the surface of the substrate means positively charging the surface of the substrate. Specifically, the surface of the substrate can be activated by bonding amine groups onto the substrate. When immersed in the amino-silane solution as described above to activate the surface of the substrate and positively charge the surface of the substrate, the substrate can be bonded to the nano-ligand by electrostatic attraction.

[0032] Also, in the step of subjecting to ultrasonic treatment, a substrate with an activated surface may be supported in a solution containing nano-ligands to manufacture a nano-ligand presenting substrate. Specifically, a substrate with an activated surface was placed in purified water in a solution containing nano-ligands and subjected to ultrasonic treatment, and supported for 30 minutes to 2 hours, or 30 minutes to 1 hour at room temperature.

[0033] The step of regulating the adhesion and differentiation of stem cells may be carried out by applying a magnetic field of 100 to 700 mT for 12 to 48 hours after positioning the nano-ligand presenting substrate in vivo and in vitro. Specifically, the step of regulating the adhesion and differentiation of stem cells may be carried out by applying a magnetic field of 100 to 600 mT, 200 to 600 mT, or 300 to 550 mT for 12 to 36 hours, 24 to 26 hours, or 12 to 24 hours after positioning the nano-ligand presenting substrate in vivo and in vitro. As described above, by applying a magnetic field to the nano-ligand presenting substrate, the adhesion of stem cells to the nano-ligands located on the substrate may be promoted, and the differentiation of the adhered stem cells may be promoted.

[0034] In addition, the step of regulating the adhesion and differentiation of stem cells may be carried out by changing the position of the magnetic field applied to the substrate. Specifically, while applying a magnetic field of 100 to 600 mT, 200 to 600 mT, or 300 to 550 mT, the position of the magnetic field applied to the substrate may be changed to spatially control the adhesion and differentiation of stem cells. For example, a magnetic field may be applied to a part of the substrate to adjust the density of the nano-ligands on the substrate, and the adhesion and differentiation of stem cells may be promoted only at a desired part on the substrate.

[0035] In addition, the step of regulating the adhesion and differentiation of stem cells may be carried out by changing the position of the magnetic field applied to the lower end of the substrate. Specifically, while applying a magnetic field of 100 to 600 mT, 200 to 600 mT, or 300 to 550 mT, the position of the magnetic field applied to the substrate may be changed according to time to temporally and spatially control the adhesion and differentiation of stem cells. More specifically, a magnetic field may be individually applied to each part of the substrate to adjust the density of the nano-ligands located on the substrate according to time, and the degree of promotion of the adhesion and differentiation of stem cells in each part of the substrate may be adjusted. For example, when a magnetic field is applied to the left side of the substrate for 12 to 24 hours and a magnetic field is applied to the right side of the substrate for 24 to 36 hours, the amount of stem cells adhered or differentiated on the left side and the right side of the substrate may be different.

[0036] Hereinafter, embodiments of the present invention will be described. However, the following embodiments are merely preferred embodiments of the present invention, and the scope of the claims of the present invention is not limited to the following embodiments. [Manufacturing Example]

[0037] Manufacturing Example 1 Production of Slidable Nano-Ligand 1) Production of Magnetic Core (MNP) For in-situ reversible control of the slidable nano-ligand, the magnetic core of the slidable nano-ligand was produced as follows. First, about 80 mL of ethanol, 60 mL of deionized water (DI), and 140 mL of heptane were mixed. To this mixture, 36.5 g (120 mmol) of sodium oleate and 10.8 g (40 mmol) of iron(III) chloride hexahydrate were added at 70 °C over 4 hours under an inert environment. After complete mixing, the heptane layer containing iron-oleate was separately collected. After washing with deionized water, the heptane was evaporated. About 5.7 g (20 mmol) of oleic acid and 200 g of 1-octadecene were mixed, and 36 g (40 mmol) of dried iron oleate was added. This mixed solution was maintained at about 100 °C for about 5 minutes and then at about 320 °C for about 30 minutes. After the reaction, the mixture solution was cooled to room temperature, and the collected product using a permanent magnet was washed three times with ethanol and then dispersed in heptane to store the magnetic core.

[0038] 2) Functionalization of Magnetic Core with Amino-Silica (Amino-Silica Coated MNP) About 30 mg of magnetic core nanoparticles were dispersed from heptane into 25 mL of cyclohexane, 5 mL of Triton-X, 5 mL of 1-hexanol, 0.5 mL of NH 4OH and 1 mL of deionized water were sequentially added. The mixed solution was stirred for 30 minutes to stabilize the emulsion. To this emulsion, 12.5 μL of tetraethyl orthosilicate (TEOS) was slowly added and stirred for 10 minutes. Then, 6.25 mL of (3-aminopropyl)triethoxysilane (APTES) was added to the emulsion and stirred for 16 hours. After the reaction, 25 mL of acetone was quickly added to the emulsion, and it was washed with acetone and dimethylformamide (DMF) using a permanent magnet to collect the nanoparticles. The amino-silica-coated MNPs were dispersed in 1 mL of DMF.

[0039] 3) Production of Slidable Nanoligands To complete the nanoassembly of slidable nanoligands, the amino-silica-coated MNPs were sequentially grafted with a polyethylene glycol (PEG) linker to improve the slidability of the nanoligands and grafted with a negatively charged RGD ligand. The PEG linker also serves to prevent cell uptake. Approximately 20 mg of amino-silica-coated MNPs in 1 mL of DMF was added to 5 mg of maleimide-poly(ethylene glycol)-NHS ester (Mal-PEG-NHS ester; Mn = 5000 Da, Biochempeg), and an additional 2 μL of N,N-diisopropylethylamine (DIPEA) was added. The suspension was stirred for 16 hours under dark conditions and then washed with DMF and dimethyl sulfoxide (DMSO) (three times each) using a permanent magnet. The amino-silica-coated PEGylated MNPs in 1 mL of DMSO were added to 0.5 mg of negatively charged thiolated RGD peptide (CDDRGD, GL Biochem), and 0.2% DIPEA and 10 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were subsequently added. The mixed solution was stirred for 16 hours under dark conditions, collected using a permanent magnet, washed with water and DMSO three times, and then maintained in DMSO before electrostatic binding to the substrate.

[0040] Comparative Production Example 1 "No RGD" nanoligands were produced in the same manner as in Production Example 1, except that negatively charged thiolated RGD peptide (CDDRGD, GL Biochem) was not added.

[0041] [Examples] Example 1 Slidable nanoligands, and combinations of such nanoligands and substrates To reversibly bind the slidable nanoligands produced in the above Production Example to a substrate, culture-grade glass coverslips (22 mm × 22 mm) were used. Before binding the negatively charged slidable nanoligands, the glass substrate was aminated to be positively charged. The substrate was immersed in a 1:1 mixture of hydrochloric acid and methanol for 30 minutes to remove any organic contaminants, and then washed three times with deionized water. The substrate was immersed in sulfuric acid for 1 hour to activate the surface with hydroxyl groups, and then washed three times with deionized water. The activated substrate was treated with a 1:1 mixture of APTES and ethanol for 1 hour under dark conditions to functionalize the substrate and provide amine groups. The substrate subjected to amino functionalization was washed three times with ethanol and dried at 100 °C for 1 hour. After diluting a suspension of the slidable nanoligands in DMSO 1:20 with DMSO, this was added to the positively charged amino-functionalized substrate. The slidable nanoligands were sonicated and electrostatically bound to the substrate at room temperature for 1 hour, and then washed three times with DMSO and three times with deionized water to obtain a substrate having slidable nanoligands.

[0042] [Experimental Examples] Experimental Example 1 To confirm the form of the slidable nanoligands according to the present invention, transmission electron microscopy (TEM) and dynamic light scattering / high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) analysis were performed on the slidable nanoligands, and the results are shown in FIGS. 3 and 4.

[0043] In addition, in order to confirm the properties and chemical bonding characteristics of the slidable nanoligands, vibrating sample magnetometry and Fourier transform infrared spectroscopy (FTIR) were performed on the slidable nanoligands, and the results are shown in FIGS. 5 and 6.

[0044] Specifically, for the transmission electron microscope (TEM) experiment to confirm the size and shape characteristics of the slidable nanoligands, TEM imaging was performed using a Tecnai 20 (FEI, USA).

[0045] In addition, the high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) is for confirming the size and shape characteristics of typical slidable nanoligands. HAADF-STEM imaging was performed using a JEOL2100F with a probe size of 1 nm, a condenser aperture of 20 μm, and a collection angle of 80 - 150 mrad for the Z ratio.

[0046] Furthermore, for dynamic light scattering (DLS) analysis to quantify the size distribution profile (hydrodynamic diameter) during the assembly process of the slide nanoligands, DLS measurements (Zetasizer Nano ZS90 Malvern Panalytical, Malvern, UK) were performed.

[0047] In addition, Fourier transform infrared spectroscopy (FTIR) was performed using a GX1 (Perkin Elmer Spectrum, USA) to confirm the characteristics of continuous chemical changes from the deformation of the slidable nanoligands. Before analyzing the sample that had undergone analysis of the changes in chemical bonding characteristics, it was freeze-dried and densely packed with a KBr pellet.

[0048] Vibrating sample magnetometry (VSM) was used to confirm the reversible slidability (superparamagnetism) property of the nanoligands. The magnetic cores of the slidable nanoligands were applied to VSM measurements (EV9; Microsense) at room temperature under an applied magnetic field. The corresponding magnetic moment was displayed in a hysteresis loop after normalizing the magnetic cores with the dry weight in the slidable nanoligands.

[0049] Figure 3 is a nanoscale image of the slidable nanoligands and also a transmission electron microscope image, with a scale bar of 20 nm. (a) in Figure 4 is the result of dynamic light scattering of magnetic nanoparticles (MNPs) with size distribution and amino-silica-coated MNPs, and (b) is a high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) image of the amino-silica-coated MNPs, with a scale bar of 20 nm.

[0050] From Figures 3 and 4, a uniform spherical shape of the slidable nanoligands with a size of about 40 ± 5 nm can be confirmed.

[0051] Figure 5 is the vibrating sample magnetometer hysteresis of the slidable nanoligands, and Figure 6 is the Fourier transform infrared spectral image of the slidable nanoligands of an embodiment. Specifically, it is the Fourier transform infrared spectral image of MNPs, silica-coated MNPs, and RGD ligand-presenting PEG-grafted silica-coated MNPs (RGD-PEG-silica-coated MNPs, corresponding to the slidable nanoligands).

[0052] In Figure 5, superparamagnetism was confirmed with an Ms of 20 emu / g. Thus, the slidable nanoligands according to the present invention can be reversibly slid with superparamagnetic properties. This property is very important for the magnetic manipulation of the sliding of nanoligands both temporally and reversibly.

[0053] As shown in Fig. 6, the Fe-O bond was detected at the absorption peak value of 699 cm -1 in the superparamagnetic iron oxide core nanoparticles. The Si-O bond was detected at the absorption peak value of 1168 cm -1 from the silica shell. In the slidable nanoligand, the PEG linker (M n = 5000 Da) not only improves the sliding property as proven in the prior art, but also suppresses the absorption by cells. CDDRGD represents the C=O bond at the absorption peak of 1152 cm -1 and the amide bond at the absorption peak of 1635 cm -1 . From such TIR analysis, it was confirmed that the assembly of the slidable nanoligand was successful.

[0054] Experimental Example 2 To demonstrate the in-situ reversible and spatio-temporal control of the slidable nanoligand according to the present invention, the slidable nanoligand was photographed with a scanning electron microscope (SEM) and atomic force microscope (AFM) imaging was performed, and the results are shown in Figs. 8 and 9.

[0055] Specifically, as shown in Fig. 7, the present invention binds a slidable ligand to a positively charged substrate for in-situ spatio-temporal control of the sliding of the nanoligand in order to electrostatically control the presentation of the macroscale nanoligand. The electrostatic coupling between the nanoligand and the substrate enables the nanoligand to slide reversibly.

[0056] Here, a scanning electron microscope (SEM) (FE-SEM, FEI, Quanta250FEG) was used to confirm the electrostatic coupling with the substrate of the slidable nanoligands and the characteristics of in-situ reversible and spatio-temporal control of macroscale nanoligand presentation. The substrate was dried using a sputter coater and platinum-coated. The density of the slidable nanoligands bound to the substrate was calculated by Image J software from ten images and illustrated as mean ± standard error. For the in-situ reversible and spatio-temporal control of the macroscale ligand density, a permanent magnet (270 mT) was positioned at the lower left of the substrate for 12 hours, at the lower right for 12 hours, and then at the lower left. The spatio-temporal changes in the macroscale ligand density were measured, and the results are shown in Fig. 8.

[0057] In addition, in-situ magnetic atomic force microscopy (AFM) (Asylum Research, XE-100 System) was performed to confirm the characteristics of in-situ 2D and 3D images of the slidable nanoligands on the substrate. The imaging was performed in air mode AC at 25 °C using an AFM cantilever (Nanosensors, SSS-SEIHR-20) with a spring constant of 5 - 3 N / m and a resonance frequency of 96 - 175 kHz. The AFM imaging was continuously performed with the same scanning area on the opposite side of the magnet in the presence or absence of the magnet to identify the in-situ motion of the slidable nanoligands at the nanoscale. As an experiment for the comparative example, continuous AFM imaging in the same scanning area without a magnet continuously was performed to identify the negligible nanoscale motion of the slidable nanoligands by continuous AFM scanning, and the results are shown in Figs. 8 and 9.

[0058] Figure 8 is an image of the in situ reversible spatiotemporal manipulation of the sliding of macroscale and nanoscale nanoligands. According to Figures 8a - b, the positively charged amino-functionalized substrate is optimally and uniformly bound to the negatively charged slidable nanoligands, as demonstrated by scanning electron microscopy (SEM) and 3D atomic force microscopy (AFM). Also, macroscopically, the density of the nanoligands was calculated to be approximately 17 ± 3 nanoligand particles per μm 2 . The present invention applies a macroscale ligand density and thus enables reversible movement of the nanoligands, which is optimal for in situ spatiotemporal control of the sliding of the nanoligands without aggregation of the slidable nanoligands, and has significantly changed focal adhesion and mechanical transfection of stem cells.

[0059] Figures 8c - d show the results of SEM imaging of the spatiotemporal control experiment. After positioning a permanent magnet at the lower left of the substrate to attract the slidable nanoligands to the left and then switching to the right and returning to the left every 12 hours. Time-lapse SEM imaging shows a fairly high nanoligand particle density on the left, right, and left at 12-hour, 24-hour, and 36-hour time points respectively, thereby fully confirming the spatiotemporal reversibility of the sliding of the nanoligands.

[0060] Figure 8e illustrates, as a comparative example of the spatiotemporal control experiment, the nanoscale displacement of the sliding of the nanoligands by continuous in situ magnetic AFM scanning in a similar region where a magnet is present or absent on the opposite side of the scan area. According to Figure 8e, the slidable nanoligands are clearly confirmed in the absence of a magnet and are moving along the magnet.

[0061] Figure 9 shows an experiment of a comparative example, which is an in-situ atomic force microscope (AFM) image of a slide of nanoligands taken in the absence of a magnet in the same scan. According to Figure 9, black dotted lines are drawn along the slidable nanoligands in two different images. The scale bar was 50 μm. In the absence of a magnet, the nanoscale movement of the slidable nanoligands was confirmed by continuous AFM imaging in the same scan area, but it was negligible.

[0062] Experimental Example 3 The remote control method using slidable nanoligands according to the present invention is to confirm the effect on the regulation of stem cell adhesion by the spatio-temporal reversible regulation of macroscale nanoligand presentation. The in-situ control of the slide of nanoligands is integrin JPEG0007683149000001.jpg45127 It was investigated whether it is possible to adjust integrin ligation and focal adhesion of human mesenchymal stem cells (hMSCs).

[0063] The binding experiment of the slidable nanoligand to integrin β1 was conducted as follows. To evaluate the binding efficiency of the in-situ slidable nanoligand to integrin β1, the slidable nanoligand presentation substrate was cultured with 50 μg / mL of integrin β1 in phosphate buffered saline (PBS) at 4 °C for 12 hours with a permanent magnet positioned at the "lower left" of the substrate. The cultured substrate was fixed with 4% (w / v) paraformaldehyde at room temperature for 10 minutes and immunofluorescently stained for integrin β1 (Santa Cruz Biotechnology) to examine the integrin β1 bound to the slidable nanoligand, and the results are shown in Figure 10.

[0064] In addition, under the in situ control of the nano-ligand slides, in vitro regulation experiments of stem cell adhesion and differentiation were conducted as follows. To investigate the in situ effect of the nano-ligand slides on stem cell adhesion, the substrate containing the slidable nano-ligand of the present invention was sterilized with UV light and blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich) at 37°C for 1 hour to minimize non-specific cell adhesion. Human mesenchymal stem cells (hMSCs, passage 5, Lonza) were placed on the substrate treated at a density of 5000 cells / cm 2 and cultured in a basal medium containing high-concentration glucose DMEM, 10% (v / v) fetal bovine serum, 4 mM L-glutamine, and 50 U / mL penicillin / streptomycin at 37°C and 5% CO2. The adhesion of stem cells was investigated with a permanent magnet (270 mT) located at the "lower left" of the substrate, which promoted the slide of the nano-ligand towards the left. The attached stem cells were imaged from the centers of various positions (left, center, right) of the substrate, and the results are shown in FIGS. 11 and 12.

[0065] FIG. 10 relates to the regulation of integrin β1 binding in the in situ control of slide nano-ligands. (a) is a schematic diagram of the slide nano-ligand with a permanent magnet at the "lower left" of the substrate, and (b) is a confocal immunofluorescence image of integrin β1 clusters bound to the slide nano-ligand at the "left", "middle", and "right" of the substrate. The scale bar is 50 μm, and the confocal immunofluorescence image is indicated by the red arrow. (c) is a graph showing the staining intensity of integrin β1 clusters at the "left", "center", and "right" of the substrate. The data are represented as mean ± standard error (n = 30). Comparison groups with statistically significant differences are described with other alphabetical characters.

[0066] Figure 11 is an image showing the results of an in situ control experiment of the slide of nanoligands according to an embodiment of the present invention. It can be seen that the spatiotemporally reversible attraction of the slidable nanoligands promotes the adhesion of stem cells. These are immunofluorescence images of vinculin, F-actin, and nuclei of stem cells from the static position (a) and the switching position (b) of the magnet. The scale bar is 50 μm.

[0067] Figure 12 is a graph showing the calculated density, area, number of focal adhesions, and aspect ratio of adherent cells after culturing hMSCs together with a permanent magnet located at the "lower left" of the substrate for 48 hours. It can be seen that the in situ maneuvering of macroscale ligand presentation by the attraction of the slide nanoligands promotes the focal adhesion of stem cells. The calculation was performed based on the confocal immunofluorescence images shown in Figure 3a. The data are represented as mean ± standard error (n = 30). Other alphabetic characters were assigned to the comparison groups with statistically significant differences.

[0068] According to Figures 11a - c, 11a, and 12, due to the magnetic attraction of the slidable nanoligands, immunofluorescence staining showed that vinculin was clearly expressed in the FA complex on the left side (magnet), and there were also very high ligation efficacy of integrin β1, high-density stem cell adhesion, and wider focal adhesions.

[0069] Also, as a comparative experiment, the nanoligands of Comparative Production Example 1 were experimented on a substrate with a magnetic field ("No RGD" group), and the nanoligands of Production Example 1 were experimented on a substrate without a magnetic field ("No magnet" group). The results are shown in Figure 13.

[0070] Figure 13 shows the results of an experiment on the adhesion of stem cells through a slidable nanoligand without an RGD ligand or a permanent magnet as a comparative example. (a) is a confocal image of immunofluorescence against vinculin, F-actin, and nuclei after culturing stem cells without RGD or a permanent magnet for 48 hours. The stem cells were imaged from the centers of the "left side", "center", and "right side" of the substrate. The scale bar was 50 μm. (b) is a graph showing the calculated adherent cell density and cell area. The data are represented as mean ± standard error (n = 30). The same alphabet indicates a non-statistical difference from the compared group.

[0071] According to Figure 13, the presentation of the regulation through the nanoligand slide for stem cell adhesion is not effective for the "No RGD" group and the "No magnet" group, but the "No RGD" group shows the minimum non-RGD specific adhesiveness of stem cells by efficient blocking. From this, it can be seen that an RGD ligand and a magnetic field (permanent magnet) are required for the in situ control of slidable nanoligand-mediated stem cell adhesion.

[0072] In addition, it was investigated whether the slidable nanoligand according to the present invention can regulate the adhesion of stem cells by time-regulated switching of the slide of the nanoligand.

[0073] The effect of time-regulated switching of the presentation of the macroscale nanoligand on the adhesion of stem cells was confirmed by positioning the permanent magnet under the left side of the substrate ("ON") or removing the permanent magnet from the substrate ("OFF") by switching between "ON" and "OFF". The effect of spatiotemporally reversible regulation of the slide of the nanoligand on the adhesion of stem cells was confirmed by switching the position of the permanent magnet to two opposite sides from the "lower left" to the "right side" and then to the "left side" of the substrate.

[0074] Figure 14 is a confocal image of immunofluorescence of stem cells for time-regulated switching of macroscale nanoligand presentation according to an embodiment. Specifically, in the case of the left side (magnet side) and the right side (non-magnet side), confocal images of immunofluorescence of vinculin, F-actin, and nuclei of stem cells cultured with a permanent magnet positioned at the lower left of the substrate ("ON") or removed from the substrate ("OFF") after 2 hours or 48 hours are shown. The "ON" and "OFF" conditions were applied continuously for 48 hours after culturing or switched after 12 hours of culturing. The above images were obtained from the center of the left side of the substrate. The scale bar was 50 μm. Thus, it can be seen that the slidable nanoligand of the present invention adjusts the adhesion force of stem cells by macroscale time-regulated switching.

[0075] Figure 15 is a graph calculating the adherent cell density, cell area, and number of focal adhesions for time switching of macroscale nanoligand presentation according to an embodiment. Specifically, a permanent magnet was placed at the lower left of the substrate ("ON") or removed from the substrate ("OFF"), and after culturing stem cells for 12 hours or 48 hours, the density of adherent cells, cell area, and number of focal adhesions on the left side of the substrate were calculated. The "ON" and "OFF" conditions were applied continuously for 48 hours after culturing or switched after 12 hours of culturing. Calculations were based on confocal images of immunofluorescence for the left side (magnet side) and the right side (non-magnet side) shown in Figure 14, and the data are represented as mean ± standard error (n = 30). Other alphabetic characters were assigned to the comparison groups with statistically significant differences.

[0076] As shown in FIGS. 14 and 15, the permanent magnet was placed at the lower left of the substrate ("ON"), or not placed near the substrate for 48 hours ("OFF"). Also, the magnet was placed first and then removed after 12 hours ("ON-OFF"), or the magnet was not placed initially and then placed after 12 hours ("OFF-ON"). As a result, on the left side (magnet side) of the substrate, the adhesion of stem cells was significantly manifested under the "ON" condition. Surprisingly, when the magnet was placed after 12 hours, the attached cell density increased significantly by 66%, the cell area by 51%, and the number of focal adhesions by 59%, and the adhesion of stem cells improved rapidly at 48 hours ("OFF-ON"). This suggests that the nanoligand can be attracted to the magnet at the selected time point. Also, when the magnet was removed after 12 hours, the adhesion of stem cells remained maintained without further increase at 48 hours ("ON-OFF"). This suggests that the nanoligand attracted to the magnet side can remain on the same side as the substrate.

[0077] Furthermore, regarding the present invention, in order to adjust the adhesion of stem cells, the spatiotemporal and reversible adjustment of the sliding of the nanoligand was investigated. As can be seen from FIGS. 8c - d, the position of the magnet was changed every 12 hours, and higher cell adhesion was observed on the left side, right side, and left side at 12 hours, 24 hours, and 36 hours, respectively.

[0078] FIG. 16 is a graph calculating the adherent cell density, cell area, and aspect ratio for the spatiotemporal reversible transformation of the sliding of the nanoligand according to an embodiment. After culturing stem cells for 12 hours, 24 hours, and 36 hours on the "left side and right side" of the substrate shown in FIG. 11b, the density, area, and aspect ratio of adherent cells on the "left side" and "right side" of the substrate were calculated using time-lapse confocal images of vinculin, F-actin, and nuclear immunofluorescence. The position of the permanent magnet was at the "lower left", "lower right", "lower left" of the substrate, and was switched between two opposite sides 3 times for 12 hours each, and the data are represented as mean ± standard error (n = 30). Therefore, it can be seen that the adhesion of stem cells can be controlled by the spatiotemporal reversible switching of the sliding of the nanoligand.

[0079] According to FIGS. 11b and 16, the spatiotemporal reversible switching of the sliding of the nanoligands controls the adhesion of stem cells. As shown in FIG. 11b, after culturing stem cells on the "left side" and "right side" of the substrate for 12 hours, 24 hours, and 36 hours, the density, area, and aspect ratio of adherent cells on the "left side" and "right side" of the substrate are calculated using time-lapse confocal images of vinculin, F-actin, and nuclear immunofluorescence. The position of the permanent magnet was switched between two opposite sides over 3 hours to 12 hours at the "lower left", "lower right", and "upper left" of the substrate. Thereby, by sliding the slidable nanoligands according to the present invention, the spatiotemporal reversibility of cell adhesion is demonstrated, and the tissue penetration and magnetic field-mediated spatiotemporal reversible control of cell adhesion can present a very promising strategy for in vivo application using light.

[0080] Experimental Example 4 Regarding the slidable nanoligands according to the present invention, the following experiments were conducted to confirm the changes in the differentiation of mechanosensitivity-mediated stem cells by in situ time control.

[0081] The proliferation of stem cells having integrin ligation-mediated adhesion and the formation of mature FAs activates mechanosensitive signals that can promote the differentiation of stem cells. Therefore, the present invention examined the osteogenic differentiation of stem cells as a model of mechanosensitivity-mediated differentiation. The remote control of stem cell differentiation provides advantages in the in vivo application of tissue regeneration methods.

[0082] The mechanical transduction-mediated differentiation of stem cells was investigated by placing a permanent magnet at the "lower left" of the substrate under ROCK inhibition (50 μM Y27632) or myosin II inhibition (10 μM blebbistatin). Substrates with RGD ligands or nanoparticles without nanoligands without applying a magnet were used to additionally confirm the effect of the slide of nanoligands that were magnetically controlled for stem cell adhesion. The differentiation of adherent stem cells under the slide of nanoligands was performed in osteogenic induction medium culture (basic growth medium supplemented with 10 mM β-glycerophosphate, 50 μM ascorbic acid-2-phosphate, and 100 nM dexamethasone).

[0083] Also, alkaline phosphatase (ALP) staining-based analysis aimed at analyzing stem cell differentiation under in situ control of the slide of nanoligands was performed as follows. After culturing in osteogenic differentiation medium, the stem cells were washed with PBS, treated with BCIP / NBT liquid (Sigma-Aldrich) at room temperature for 30 minutes under cancer conditions, and then washed with PBS. The stem cells treated as described above were fixed with 4% (w / v) paraformaldehyde for 10 minutes and visualized using an optical microscope. The above ALP-positive cells were calculated from the total cell number by nuclear (DAPI) staining.

[0084] Figure 17 is an immunofluorescence image of the in situ magnetic attraction of slidable nanoligands according to an embodiment with respect to the mechanosensing-mediated differentiation of stem cells. (a) is the immunofluorescence for F-actin, nucleus, RUNX2 and YAP with ALP expression in stem cells in the presence of a magnet. (b) is the immunofluorescence for vinculin under myosin II inhibition (blebbistatin) from stem cells under magnetic conditions for TAZ, integrin β1, FAK, RhoA, YAP (Y27632) under ROCK inhibition, having F-actin and nucleus. The scale bar is 50 μm. According to Figure 17, it can be seen that the in situ magnetic attraction of the slidable nanoligands promotes the mechanosensing-mediated differentiation of stem cells.

[0085] Figure 18 is a confocal image of immunofluorescence in the case of no RGD ligand and no permanent magnet as a comparative example. Specifically, it is a confocal image of immunofluorescence for RUNX2, F-actin, nucleus, and alkaline phosphatase (ALP) after 7 days of stem cell culture, or immunofluorescence for YAP, F-actin, nucleus 2 days after culture. Adherent stem cells are imaged from the left center of the substrate as indicated by the red dotted frame in the schematic drawing. The "No RGD" group and the "No Magnet" group were used as comparison groups. The scale bar indicates 50 μm. According to Figure 18, in the absence of the RGD ligand (No RGD) or in the absence of magnetic control (No Magnet), the differentiation of stem cells by the sliding of the nanoligands is not effectively regulated.

[0086] According to FIGS. 17a and 18, a very high nuclear localization of RUNX2 in immunofluorescent and alkaline phosphatase-positive cells was observed on the left side (magnet side) of the substrate. At the same time, the nuclear localization of YAP, a mechanosensitive transcriptional regulator, was significantly improved from the left side. This is consistent with the temporal switching of stem cell adhesion in FIGS. 14 and 15 on the left side (magnet side) of the substrate.

[0087] FIG. 19 shows the results of a mechanical transfection and differentiation experiment of stem cells with respect to the magnetic attraction of slidable nanoligands according to an embodiment. After culturing the stem cells, it is a graph showing the calculated fluorescence ratio of RUNX2 in the nucleus and cytoplasm, alkaline phosphatase-positive cells, and the fluorescence ratio of YAP in the nucleus and cytoplasm. Specifically, after culturing the stem cells with a permanent magnet positioned at the "lower left" of the substrate, the fluorescence ratio of RUNX2 in the nucleus and cytoplasm, the fluorescence ratio of alkaline phosphatase-positive cells, and the fluorescence ratio of YAP in the nucleus and cytoplasm were calculated. The above calculations were performed using the confocal images of immunofluorescence shown in FIGS. 17a and 18. The data are represented as mean ± standard error (n = 30). Statistically significant differences are indicated by different alphabets. According to FIG. 19, the magnetic attraction of the slidable nanoligands promotes mechanotransduction and differentiation of stem cells.

[0088] Figure 20 is a confocal immunofluorescence image of the in situ time-controlled experiment of the slide of the nanoligand according to an embodiment, and a graph calculating the RUNX2 fluorescence ratio of the nucleus and cytoplasm. (a) is a confocal immunofluorescence image of RUNX2, F-actin, and nucleus 7 days after the culture of stem cells with a permanent magnet positioned at the lower left of the substrate (“ON”) or removed from the substrate (“OFF”). The conditions of “ON” and “OFF” were continuously applied for 7 days of culture (for RUNX2), or switched after 12 hours of culture. The stem cells were imaged from the centers of the “left side” and “right side” of the substrate. The scale bar indicates 50 μm. (b) is a graph calculating the RUNX2 fluorescence ratio of the nucleus and cytoplasm. The data are represented as mean ± standard error (n = 30). Statistically significant differences are indicated by different alphabets. According to Figure 20, the slidable nanoligand of the present invention can temporally regulate the differentiation of stem cells using a magnetic field.

[0089] Figure 21 shows the results of an experiment on mechanotransduction by time-regulated control of a slidable nanoligand in an embodiment. (a) is a confocal immunofluorescence image of RUNX2, F-actin, and nucleus 2 days after culture with a permanent magnet positioned at the lower left of the substrate (“ON”) or removed from the substrate (“OFF”). (b) is a graph calculating the YAP fluorescence ratio of the nucleus and cytoplasm. According to Figure 21, the slidable nanoligand of the present invention can regulate the mechanotransduction of stem cells by time control.

[0090] From Figures 20a - b and 21a - b, the differentiation and mechanotransduction of stem cells were stimulated by more significant nuclear localization of RUNX2 and YAP under the “ON” condition.

[0091] Figure 22 shows the gene expression profiles of RUNX2 and ALP 7 days after culturing stem cells with a permanent magnet positioned at the “lower left” of the substrate for a slidable nanoligand of an embodiment, and the data are presented as mean ± standard error (n = 30). Statistically significant differences are indicated by different alphabets.

[0092] According to FIG. 22, the analysis of gene expression showed that the expressions of RUNX2 and ALP were very high from the left side (magnet side) under the "ON" condition. Interestingly, on the left side (magnet side), the "OFF-ON" and "ON-OFF" conditions showed significant nuclear localization of RUNX2 and YAP comparable to the "ON" state. This indicates that the nanoligand can be attracted towards the magnet and activate cellular signaling events at a predetermined time point, so that the magnet can be placed initially or subsequently to promote mechanotransduction and differentiation of stem cells. Thus, it can be seen that the differentiation of stem cells can be stimulated through in situ control that magnetically attracts the slidable nanoligand.

[0093] In addition, the present invention is investigating a method for promoting the differentiation of stem cells under the slide of in situ nanoligands by integrin ligation-mediated activation of mechanotransduction signals. In the present invention, a magnet was placed at the lower left of the substrate to search for various intracellular mechanosensitive pathways.

[0094] FIG. 23 shows the results calculated from immunofluorescence images of the magnetic attraction of slidable nanoligands in one embodiment. (a) is the quantification of the nuclear and cytoplasmic TAZ fluorescence ratio obtained from the confocal image of immunofluorescence against TAZ shown in FIG. 4b. (b) is a confocal image of immunofluorescence against p-FAK having F-actin and nuclei. (c) is a graph of the YAP fluorescence ratio of the nucleus and cytoplasm under ROCK inhibition (having Y27632) illustrated in FIG. 17b, and (d) is a graph of the cell area of myosin II inhibition (having blebbistatin). The stem cells were cultured for 2 days with a permanent magnet located at the "lower left" of the substrate. The adherent stem cells were imaged from the centers of the "left side" and the "right side" of the substrate. The scale bar indicates 50 μm. The data are represented as mean ± standard error (n = 30). Statistically significant differences are indicated by different alphabets. According to FIG. 23, the slidable nanoligand of the present invention promotes focal adhesion and mechanosensitivity of stem cells by controlling magnetic attraction.

[0095] According to FIGS. 17b and 23a - d, the positions of nuclei with higher TAZ, transcriptional co - activation, and integrin β1 activation by immunofluorescence were on the left side (magnet side) of the substrate. The stable formation of the FA complex involves focal adhesion kinase (FAK) which is phosphorylated to activate mechanosensitive RhoA. On the left side (magnet side), FAK was highly expressed and phosphorylated and activated to stimulate pFAK. The focal adhesion - mediated activation of mechanotransduction stimulated RhoA from the left, which activated rho - associated protein kinase (ROCK) to induce the nuclear localization of YAP, as revealed by ROCK inhibition with Y27632. Also, myosin II inhibition by blebbistatin significantly reduced the cell spreading area on the left side. From these results, it can be seen that the in - situ control of slidable nanoligand materials promotes integrin ligation to form FA assemblies that activate the mechanosensitivity - mediated differentiation of line cells.

[0096] Experimental Example 5 In order to confirm that the slidable nanoligand according to the present invention can spatially regulate the adhesion of stem cells in the body by in - situ control, experiments were conducted as follows.

[0097] The in - situ remote control of the sliding of nanoligands using the slidable nanoligand of the present invention can also be applied in the body. Recently, UV light has been used for the spatial regulation of cell adhesion in the body. However, in this study, it was revealed that UV light can induce serious cytotoxicity because it is highly absorbed by living tissues in the body. In contrast, applying external magnetic - field - based control for the spatial regulation of cell adhesion in the body guarantees desirable tissue permeability and cytocompatibility.

[0098] To investigate the slide In situ effect of nanoligands on the adhesion of stem cells in vivo, a substrate containing slidable nanoligands was subcutaneously implanted into 14 nude mice at about 8 weeks of age. Before implantation, a mixture of 5 μL of zoletil, 2 μL of rompun, and 3 μL of physiological saline was administered to the nude mice by intraperitoneal injection. The back of the mouse was incised with a length of 2 cm. After implantation, hMSCs were injected into the substrate at 100 k / mL, and a permanent magnet was attached under the left side (abdominal side) of the substrate to promote the slide of the nanoligands toward the left. Anesthesia was maintained until the substrate was collected for confocal imaging of immunofluorescence.

[0099] Figure 24 shows the results of a magnetic control experiment on the slide nanoligand attraction of slidable nanoligands according to an embodiment. (a) is an image schematizing the experiment of magnetic control of nanoligands, and (b) is the result of immunofluorescence for human-specific HuNu with actin and nuclei of stem cells injected into a slidable ligand presentation substrate after subcutaneous implantation with (\"ON\") or without (\"OFF\") a magnet. (c) shows the quantification of the cell density and area adhered in vivo. The scale bar is 50 μm. According to Figure 24, the slidable nanoligands promote stem cell adhesion in vivo by magnetic control.

[0100] In the present invention, as shown in Fig. 24a, a substrate representing a slidable nanoligand was subcutaneously implanted on the back of a nude mouse injected with hMSC. Then, a magnet was placed under the left side of the substrate (mouse abdomen) ("ON"), and as a comparative experiment, no magnet was placed ("OFF"). As a result, according to Figs. 24b - c, the confocal images of immunofluorescence against human-specific nuclear antigen (HuNu) having actin and nuclei show that all adherent cells are hMSC, which adhered at a significantly high density of 75% on the left side of the substrate (magnet side) under the "ON" condition, and the cell area was diffused by about 44% more than the right side. In contrast, the stem cells adhered left and right at a compatible cell density and area under the "OFF" condition. From these results, it can be seen that the in situ remote control of the sliding of the nanoligand can be applied to a complex in vivo environment to spatially regulate the adhesion of stem cells. Furthermore, it was confirmed that these in situ remote controls are effective for the spatial regulation of various host cells in the body.

[0101] Therefore, the magnetic field-based spatiotemporal control of the slidable nanoligand according to the present invention can effectively control the results of differentiation by regulating stem cell adhesion and mechanotransduction in vitro and in vivo.

[0102] In the above experimental example, under the in situ control of the sliding of the nanoligand, the immunofluorescence staining-based analysis of the adhesion and differentiation of stem cells was performed as follows. The cultured stem cells were fixed with 4% (w / v) paraformaldehyde at room temperature for 10 minutes and washed with PBS. The fixed cells were blocked with 3% (w / v) BSA and 0.1% (v / v) Triton-X (Triton-X, Sigma Aldrich) in PBS at room temperature for 30 minutes. The blocked cells were incubated with the primary antibody (integrin JPEG0007683149000002.jpg Cells were treated with vinculin, RUNX2, YAP, TAZ, p-FAK, FAK, RhoA, HuNu at 4°C for 16 hours and washed with PBS. The above cells were treated with secondary antibody, phalloidin, and DAPI at room temperature for 30 minutes and washed with PBS. Cells subjected to immunofluorescent staining were imaged with a confocal microscope (LSM700, Carl Zeiss) under the same exposure conditions for all comparison groups, and then analyzed with ImageJ software as described above.

[0103] In addition, to quantify the adhesion, differentiation, and mechanosensitivity of stem cells under the nanoligand slides, images subjected to immunofluorescent staining were analyzed using ImageJ software. In the case of integrin β1, the staining intensity was calculated from five different images using the histogram function. In the case of adherent cell density, the number of cell nuclei was calculated from five different DAPI-stained images. In the case of the area and aspect ratio (major axis, minor axis) of adherent cells, five different phalloidin-stained images were used for the calculation. In the case of the number of focal adhesions, as previously reported, five vinculin-stained images were used to calculate clusters with a size larger than 1 μm 2 For the differentiation (RUNX2) and mechanotransduction (YAP) of stem cells, the fluorescence ratio of the nuclei and cytoplasm of stem cells from five different images was used for the calculation.

[0104] Furthermore, reverse transcription polymerase chain reaction (qRT-PCR)-based assays for the differentiation of stem cells under in situ control of the nanoparticle slides were performed as follows. After culturing in osteogenic differentiation medium, the stem cells were harvested by applying Trizol (1 mL per group) to the substrates (separated left and right) for RNA extraction. For each group, 1 μg of RNA was used for reverse transcription into cDNA using a high-capacity RNA-to-cDNA kit. A StepOne Plus real-time PCR system (Applied Biosystems) was used for the real-time PCR reaction by Sybr Green assays. The expression of the target genes (RUNX2 and ALP) was normalized to the expression of GAPDH and then presented as fold expression.

Claims

1. A core containing magnetic nanoparticles, A coating layer provided so as to wrap the core and containing an integrin-binding ligand peptide, A linker provided between the core and the coating layer, A nanoligand for promoting cell adhesion and differentiation of stem cells, comprising: The integrin-binding ligand peptide contains a negatively charged thiolated integrin ligand peptide, and the nanoligand is electrostatically bound to a positively charged substrate via the negatively charged thiolated integrin ligand peptide, The density of the nanoligand is adjusted by moving the nanoligand on the substrate by applying a magnetic field. A nanoligand for promoting cell adhesion and differentiation of stem cells, characterized in that.

2. It is a structure in which the core and the coating layer are connected by a linker, The nanoligand according to claim 1, wherein the linker is a polyethylene glycol (PEG)-based linker.

3. The nanoligand according to claim 1, wherein the nanoligand has a diameter of 30 nm to 60 nm, and the magnetic nanoparticles have a diameter of 5 nm to 30 nm.

4. Preparing a core containing magnetic nanoparticles; Mixing the core with a first suspension containing a linker to produce a core to which the linker is bound; Mixing the core to which the linker is bound with a second suspension containing an integrin-binding ligand peptide (RGD) to bind the linker and the integrin-binding ligand peptide (RGD). The integrin-binding ligand peptide (RGD) contains a negatively charged thiolated integrin ligand peptide, and the core is electrostatically bound to a positively charged substrate via the negatively charged thiolated integrin ligand peptide. A method for producing a nanoligand for promoting cell adhesion and differentiation of stem cells according to any one of claims 1 to 3.

5. Manufacturing a nanoligand-presenting substrate by supporting a substrate with an activated surface on a solution containing the nanoligand for promoting cell adhesion and differentiation of stem cells according to any one of claims 1 to 3; A method for promoting cell adhesion and differentiation of stem cells, comprising the step of treating the stem cells on the nano-ligand presenting substrate in vitro and then applying an external magnetic field to regulate the adhesion and differentiation of the stem cells.

6. The step of manufacturing the nano-ligand presenting substrate comprises: immersing the surface of the substrate in an acidic solution; supporting the immersed substrate in an amino-silane solution to activate the surface of the substrate; treating the activated substrate using ultrasonic waves at room temperature. The method for promoting cell adhesion and differentiation of stem cells according to claim 5.

7. The method for promoting cell adhesion and differentiation of stem cells according to claim 5, wherein the substrate with the activated surface is obtained by supporting the substrate in an amino-silane solution to activate the surface.

8. The step of regulating the adhesion and differentiation of the stem cells is performed by applying a magnetic field of 100 to 700 mT for 12 to 48 hours after positioning the nano-ligand presenting substrate in vitro. The method for promoting cell adhesion and differentiation of stem cells according to claim 5.

9. The step of regulating the adhesion and differentiation of the stem cells is performed by changing the position of the magnetic field applied to the substrate. The method for promoting cell adhesion and differentiation of stem cells according to claim 5.

10. The step of regulating the adhesion and differentiation of the stem cells is performed by changing the position of the magnetic field applied to the substrate according to time. The method for promoting cell adhesion and differentiation of stem cells according to claim 5.

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