Nanoligand for promoting cell adhesion and regeneration of macrophages, and method for promoting cell adhesion and regeneration of macrophages using the same

Abstract

To provide a method for accelerating cell adhesion and reproducing polarization of a macrophage.SOLUTION: A nano ligand for accelerating cell adhesion and regeneration of a macrophage comprises: a core which includes a magnetic nanoparticle; and a coating layer which is arranged to envelope the core and includes an integrin binding ligand peptide that is negatively charged. A method for accelerating cell adhesion and regeneration of the macrophage applies a magnetic field to a basal plate including the nano ligand to be capable of performing reversible control of a nano ligand slide in addition to temporal and spatial control thereof, and cell adhesion and phenotype of the macrophage 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 regeneration of macrophages, and a method for promoting cell adhesion and regeneration of macrophages using the nanoligand, specifically, to a method for remotely controlling cell adhesion and regenerative polarization of macrophages using the nanoligand. [Background technology]

[0002] Macrophages are the main cells responsible for innate immunity. Most exist as fixed cells throughout the body, but some exist as monocytes in the blood. These monocytes can differentiate into dendritic cells and macrophages. Most macrophages are fixed, mainly alveolar macrophages, F microglia, Kupffer cells, and Langerhans cells. These cells are distributed throughout the body, and when an antigen invades, they either prey on it or secrete toxins to destroy the antigen and transmit the antigen to lymphocytes to trigger an immune response. When an enemy invades a wound, monocytes in the blood leave the blood vessels like neutrophils and differentiate into macrophages to remove bacteria. Macrophages are also divided into migratory (free form) cells that move to multiple locations in the body and phagocytose, and fixed (fixed form) cells that are fixed to a designated organ and phagocytose. Fixed macrophages include Kupffer cells in the liver, alveolar macrophages, connective tissue structures (histocytes), and microglia in the brain.

[0003] Thus, in vivo ligand presentation technology has been disclosed as a method for efficiently controlling the regenerative effect of M2 polarization of macrophages and the anti-inflammatory effect of M1 polarization. However, conventional nano-ligand presentation in the body is almost static, and even if it is dynamic, it is not controlled by remote control in real time. The drawback was that it was not possible to induce reversible changes in nano-ligand density on a macroscale. [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 nanoligand that can electrostatically bind to a substrate and move therewith, and to provide a method for using the nanoligand to induce reversible changes in macroscopic nanoligand density through real-time remote control, thereby promoting cell attachment and regenerative polarization of macrophages. [Means for solving the problem]

[0006] The present invention relates to a method for producing a magnetic nanoparticle-based nanoparticle comprising the steps of: (a) providing a magnetic nanoparticle-based nanoparticle core; and (b) providing a coating layer surrounding the magnetic nanoparticle core, the coating layer comprising an integrin adhesion ligand peptide. The integrin adhesion ligand peptide is characterized by being negatively charged, and provides a nano-ligand for promoting cell adhesion and regeneration of macrophages.

[0007] The present invention further provides a method for producing the above-mentioned nanoligand for promoting cell adhesion and regeneration of macrophages, comprising the steps of: preparing a core containing magnetic nanoparticles; mixing the core with a first suspension containing a linker to produce a core having a linker bound thereto; and mixing the core having the linker bound thereto with a second suspension containing an integrin adhesion ligand peptide (RGD).

[0008] Furthermore, the present invention provides a method for promoting cell adhesion and regeneration of macrophages, comprising the steps of: preparing a nanoligand-presenting substrate by supporting a surface-activated substrate in a solution containing the above-mentioned nanoligand for promoting cell adhesion and regeneration of macrophages; and, after treating the nanoligand-presenting substrate with culture medium, applying an external magnetic field to adjust the cell adhesion and regeneration polarization of macrophages. Effect of the Invention

[0009] The nanoligand for promoting macrophage cell adhesion and regeneration according to the present invention is a core containing magnetic nanoparticles coated with a negatively charged ligand, and can easily move (slide) on a substrate due to electrostatic binding with the substrate.

[0010] Furthermore, the method of the present invention for promoting macrophage cell attachment and regeneration not only controls the sliding of the nanoligand in time and space by applying a magnetic field to a substrate containing the nanoligand, but also enables reversible control, and the magnetic field-based spatiotemporal control can efficiently adjust the cell attachment and phenotypic polarization of macrophages in vitro and in vivo. [Brief description of the drawings]

[0011] [Figure 1] 1 is a schematic diagram illustrating a nanoligand for promoting cell adhesion and regeneration of macrophages according to one embodiment of the present invention, and a method for promoting cell adhesion and regeneration of macrophages using the same. [Diagram 2] FIG. 1 is a schematic diagram of a method for promoting cell attachment and regeneration of macrophages according to one embodiment of the present invention. [Diagram 3] FIG. 1 is a transmission electron microscope image (TEM) of a slidable nano-ligand according to one embodiment of the present invention (scale bar: 20 nm). [Figure 4]Characterization of a slidable nanoligand according to one embodiment of the present invention. (a) Dynamic light scattering images of magnetic nanoparticles (MNPs) with size distribution and amino-silica coated MNPs. (b) High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image of a representative amino-silica coated MNP (scale bar: 20 nm). [Diagram 5] 1 is a Fourier transform infrared spectrum image of a slidable nano-ligand according to one embodiment of the present invention. [Figure 6] 1 shows a vibrating sample magnetometer hysteresis of a slidable nano-ligand according to one embodiment of the present invention. [Figure 7] 1 is an image illustrating electrostatic coupling between a substrate and a slidable nano-ligand according to an embodiment of the present invention. [Figure 8] Scanning electron microscope (SEM) images and atomic force microscope (AFM) images of in situ reversible spatiotemporal manipulation of micro- and nano-scale nano-ligand slides in one embodiment of the present invention. (a) and (b) are images of a positively charged substrate and nano-ligand, (c) and (d) are the results of time-lapse SEM imaging, and (e) shows the nano-scale displacement of the nano-ligand slide by AFM scanning. [Figure 9] FIG. 11 is an atomic force microscope (AFM) image of an in situ nanoligand slide taken without a permanent magnet as a comparative example. [Figure 10]Modulation of integrin β1 binding in in situ control of sliding nanoligand according to one embodiment of the present invention: (a) Schematic of sliding nanoligand with a permanent magnet at the "bottom left" of the substrate, (b) Confocal microscopy images of immunofluorescence for integrin β1 clusters after binding to the sliding nanoligand on "magnetic", "medium" and "non-magnetic" substrates (scale bar: 50 μm), (c) Quantification of staining intensity of integrin β1 clusters on "magnetic", "medium" and "non-magnetic" substrates. [Figure 11] Figure 1 shows experimental results of in situ control of nano-ligand sliding according to one embodiment of the present invention: (a) is a schematic diagram of the manipulation of the slidable nano-ligand by placing a permanent magnet on the bottom ("magnet" side) of the substrate, together with immunofluorescent staining of vinculin, actin, and nuclei and corresponding confocal microscopy images after 24 hours of macrophage culture with a permanent magnet placed on the bottom ("magnet" side) of the substrate; (b) is a graph showing quantification of cell attachment density, cell area, and cell aspect ratio of macrophages. [Figure 12] Comparative example of macrophage cell adhesion experiment results. (a) Confocal microscopy images of immunofluorescent staining of vinculin, actin, and nuclei after 24 hours of macrophage culture in the absence of RGD peptide ligand or magnetic field. (b) Graph showing quantification of macrophage density, area, and aspect ratio. [Figure 13] Experimental results of controlling macrophage cell adhesion by tailoring macro-scale nano-ligands according to one embodiment of the present invention. (a) Confocal microscopy images of immunofluorescence for F-actin and nuclear vinculin after 12 or 24 hours of macrophage culture with a permanent magnet placed on the bottom of the substrate ("magnetic field (MF)") or with the permanent magnet removed from the substrate ("no magnetic field (NMF)"). (b) Graph of calculated macrophage density, cell area, and cell aspect ratio for the "left" surface of the substrate. [Figure 14]1 is a graph showing quantification of macrophage density, area, and aspect ratio on the "right" side of a substrate by time tuning of a nanoligand according to one embodiment of the present invention. [Figure 15] 1 shows (a) immunofluorescence confocal microscopy images and (b) calculated graphs of macrophage density, cell area, and cell aspect ratio with respect to temporal switching of macroscale nano-ligand presentation according to one embodiment of the present invention. [Figure 16] Experimental results of phenotyping based on timed magnetic attraction of nanoligands according to one embodiment of the present invention. (a) Graph showing quantitative gene expression of M2 phenotype markers (Arginase-1 and Ym1) in macrophages cultured in M2 polarizing medium or M1 phenotype markers (iNOS and TNF-α) in macrophages cultured in M1 polarizing medium. (b)-(c) Confocal microscopy images of immunofluorescence of Arg-1 and nuclear iNOS in cultured macrophages. [Figure 17] 1 is a graph showing the M2 phenotype of macrophages in an M1 polarizing medium of a slideable nano-ligand according to an embodiment of the present invention. [Figure 18] 1 is a graph showing the M1 phenotype of macrophages in an M2 polarizing medium of a slideable nano-ligand according to an embodiment of the present invention. [Figure 19] 13 shows confocal immunofluorescence microscopy images of an experiment on M2 phenotype of macrophages in response to magnetic and electrostatic attraction of a slidable nano-ligand according to one embodiment of the present invention. [Figure 20] FIG. 1 shows (a) immunofluorescence confocal microscopy images and (b) graphs showing the density of adherent macrophages, cell area, cell aspect ratio, and calculations of area, aspect ratio, and Arg-1 staining intensity after culturing in M2 medium from a self-culture experiment of a nanoligand according to one embodiment of the present invention. [Figure 21]Experimental results on the cytoadhesive and inflammatory M1 phenotype of host macrophages in vivo in response to the magnetic attraction of a slidable nanoligand according to one embodiment of the present invention. (a) is a schematic diagram of the magnetic control of the slidable nanoligand in vivo. (b) is a confocal microscopy image of immunofluorescence of iNOS, F-actin, and nuclei of cells attached to the "magnetic" and "non-magnetic" sides of the substrate. (c) is a graph showing the calculation of gene expression profile (n=3) of M1 phenotype markers (iNOS and TNF-α), density, cell area, and cell aspect ratio (n=30) of attached cells in vivo. [Figure 22] 1 shows the results of an experiment of host neutrophil in vivo adhesion to a slidable nanoligand according to an embodiment of the present invention. (a) shows confocal microscopy images of immunofluorescence staining for NIMP-R14, F-actin, and nuclei of host cells attached to the "magnetic side" and "non-magnetic side" of a substrate showing the slidable nanoligand 24 hours after subcutaneous injection of IL-4 and IL-13 onto the substrate. (b) shows a graph showing the quantification of the density of NIMP-R14-positive host neutrophils attached in vivo. [Figure 23] 4A and 4B show experimental results of cell adhesion and regeneration of M2 phenotype of host macrophages in vivo in response to magnetic attraction of a slidable nanoligand according to an embodiment of the present invention. (a) shows confocal microscopy images of immunofluorescence staining of Arg-1, F-actin, and nuclei of host cells attached to the "magnetic" and "non-magnetic" sides of a substrate with a slidable nanoligand 24 hours after subcutaneous implantation of a substrate injected with IL-4 and IL-13. (b) shows graphs showing gene expression profiles (n=3) of M2 phenotype markers (Arg-1 and Ym1) of attached cells in vivo, density, cell area, and cell aspect ratio (n=30). (c) shows graphs showing calculations of nuclear to cytoplasmic YAP fluorescence ratio under ROCK signal inhibition (with Y27632) and (d) cell area under myosin II formation inhibition (with blebbistatin) shown in FIG. 4B. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In the following, preferred embodiments of the present invention will be described in more detail with reference to the accompanying drawings, in order to more specifically explain the present invention. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms.

[0013] Macrophages mediate interactions with the continuously remodeled extracellular matrix (ECM), thereby causing spatially and temporally heterogeneous macro-scale nano-ligand distribution in the body. The nano-ligand for promoting cell attachment and regeneration of macrophages according to the present invention can mimic extracellular matrix (ECM) remodeling by enabling reversible remote control by changing the macro-scale nano-ligand distribution spatially and temporally, and can adjust the cell attachment and polarization of macrophages for controlling host responses spatially and temporally. The nano-ligand of the present invention is bound to the negatively charged slide nano-ligand via electrostatic interaction with the positively charged substrate, and the possibility of nano-ligand sliding was optimized using a polymer linker and negatively charged RGD ligand-coated magnetic core nanoparticles. The characteristics of macro-scale and in situ nano-scale nano-ligand slides were confirmed under an external magnetic field that changes the macro-scale nano-ligand density spatiotemporally and reversibly. Furthermore, the present invention presents a novel in situ manipulation of magnetically attracted slidable nanoligands at macroscale nanoligand densities to modulate the cell attachment and polarization phenotype of host macrophages in vivo. Specifically, the time-controlled magnetic attraction of the slidable nanoligands suppresses the inflammatory M1 phenotype of macrophages while stimulating the regenerative M2 phenotype. The magnetic attraction of the slidable nanoligands of the present invention also promotes the assembly of adhesion structures within macrophages and stimulates the polarization of the M2 phenotype. Thus, the nanoligands of the present invention enable the spatiotemporal modulation of immune-modulating tissue regeneration responses of implants in vivo through remote, spatiotemporal, and reversible control of the macroscale nanoligand densities.

[0014] The present invention provides a nanoligand for promoting cell adhesion and regeneration of macrophages, comprising a core containing magnetic nanoparticles and a coating layer containing an integrin adhesion ligand peptide arranged to envelop the core, wherein the integrin adhesion ligand peptide is negatively charged.

[0015] FIG. 1 is a schematic diagram showing the nanoligand for promoting macrophage cell adhesion and regeneration according to the present invention and the method for promoting macrophage cell adhesion and regeneration using the same.

[0016] From FIG. 1, it can be seen that the nanoligand of the present invention comprises a core containing magnetic nanoparticles and a coating layer containing an integrin adhesive ligand peptide, which is provided so as to encase the core, and the integrin adhesive ligand peptide is a negatively charged integrin peptide. Specifically, the integrin adhesive ligand peptide bound onto the core may surround the core like a micelle structure. This allows the surface charge of the nanoligand to be negatively charged. For example, the sliding nanoligand can be produced by first synthesizing superparamagnetic core iron oxide nanoparticles, functionalizing the superparamagnetic core nanoparticles with a functional aminosilica shell, and then coating the nanoligands with a polyethylene glycol (PEG) linker to graft negatively charged integrin adhesive ligand peptides (RGD, CCDRGD), thereby improving the sliding property of the nanoligand.

[0017] Moreover, Fig. 3 is a transmission electron microscope (TEM) image of the nanoligand for promoting macrophage cell attachment and regeneration according to the present invention, from which the size of the nanoligand can be understood. Specifically, the nanoligand may have a diameter of 30 to 60 nm. If the diameter of the nanoligand is less than 30 nm, it is difficult to control the movement of the nanoligand, and if it exceeds 60 nm, the efficiency of macrophage cell attachment 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. If the diameter of the magnetic nanoparticles is less than 5 nm, the particle size is too small, causing a lot of loss and decreasing efficiency. If the diameter of the magnetic nanoparticles is more than 30 nm, the diameter of the nanoligand is large, which may cause a problem of decreasing the cell adhesion efficiency of macrophages. More specifically, the magnetic nanoparticles may have a diameter of 5 to 15 nm, 15 to 20 nm, or 10 to 20 nm. By including the above magnetic nanoparticles, the nanoligand of the present invention can promote cell adhesion and regenerative polarization of macrophages using a magnetic field.

[0019] The magnetic nanoparticles may also be bonded to a silica surface. Specifically, the magnetic nanoparticles may have amino-silica bonded to the surface. The type of silica may be one or more of tetraethylorthosilicate (TEOS) and (3-aminopropyl)triethoxysilane (APTES).

[0020] For example, the nanoligand of the present invention has a structure in which the core and the 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 such a linker, the binding strength 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 attached to the core or to a linker attached to the core, or may surround the core. Specifically, the coating layer includes an integrin adhesive ligand peptide (RGD), which may be negatively charged or may include a negatively charged thiolated integrin adhesive ligand peptide. By including a negatively charged integrin adhesive ligand peptide as described above, the surface of the nanoligand of the present invention is negatively charged, which allows it to move freely on the substrate by electrostatic binding with the substrate. Due to these characteristics, the nanoligand is also called a "slidable nanoligand", and can promote cell attachment and regenerative polarization of macrophages through the sliding of the nanoligand on the substrate.

[0022] The present invention further provides a method for producing a magnetic nanoparticle-containing core, comprising the steps of: mixing the core with a first suspension containing a linker to produce a linker-attached core; The present invention provides a method for producing the above-mentioned nanoligand for promoting cell adhesion and regeneration of macrophages, which includes a step of mixing the core to which the linker is bound with a second suspension containing an integrin adhesion ligand peptide (RGD).

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

[0024] Specifically, the step of producing a core bound to a linker may be performed by stirring a suspension containing the core and the linker under dark conditions for 10 to 20 hours, or 10 to 15 hours. This results in a core bound to a linker. At this time, the core may be obtained by washing with a solvent two or more times while using a permanent magnet. The solvent may include one or more of dimethylformaldehyde (DMF) and dimethylsulfoxide (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 such a linker to the core, the binding strength 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 in a suspension containing an integrin-adhering ligand peptide (RGD) for 10 to 20 hours or 10 to 15 hours under dark conditions. At this time, the core is washed with a solvent while using a permanent magnet to obtain magnetic nanoparticles (nanoligands) bound to a negatively charged integrin-adhering ligand peptide. The solvent may contain one or more of dimethylformaldehyde (DMF) and dimethylsulfoxide (DMSO).

[0027] Here, a coating layer may be formed on the core by the process of stirring with the integrin adhesive ligand peptide. Specifically, the above-mentioned integrin adhesive ligand peptide may be negatively charged or may be a negatively charged thiolated integrin adhesive ligand peptide. By forming a coating layer on the core with the negatively charged integrin adhesive ligand peptide as described above, the surface of the nanoligand of the present invention is negatively charged, which allows it to move freely on the substrate by electrostatic binding with the substrate. Due to these characteristics, the nanoligand is also called a "slidable nanoligand" and can promote cell adhesion and regeneration of macrophages through the sliding of the nanoligand on the substrate.

[0028] Furthermore, the present invention provides a method for promoting cell adhesion and regeneration of macrophages, comprising the steps of: preparing a nanoligand-presenting substrate by supporting a substrate having a positively charged surface in a solution containing the above-mentioned nanoligand for promoting cell adhesion and regeneration of macrophages; and, after treating the nanoligand-presenting substrate with culture medium, applying an external magnetic field to adjust the cell adhesion and regeneration polarization of macrophages.

[0029] 1 and 2 are schematic diagrams of a method for promoting cell attachment and regeneration of macrophages according to an embodiment of the present invention. As shown in FIG. 1 and FIG. 2, a nano-ligand with a negatively charged surface is electrostatically bonded to a positively charged substrate, and a magnetic field is applied to promote cell attachment of macrophages in the area where the magnetic field is applied, while suppressing the inflammatory M1 phenotype and activating the regenerative M2 phenotype. Specifically, since the substrate and the nano-ligand are bonded by electrostatic bonding, the nano-ligand moves (slides) depending on the position where the magnetic field is applied, and thus, there is an advantage that the density of the nano-ligand can be adjusted in the area where the magnetic field is applied to promote cell attachment and regenerative polarization of macrophages in the desired area.

[0030] Specifically, the steps of manufacturing a nanoligand-presenting substrate may include the steps of immersing a surface of the substrate in an acidic solution, supporting the immersed substrate in an aminosilane solution to positively charge the surface of the substrate, and treating the positively charged substrate with ultrasound at room temperature.

[0031] In the step of immersing the surface of the substrate in an acidic solution, the substrate may be immersed in an acidic solution containing at least one of hydrochloric acid and sulfuric acid for 30 minutes to 2 hours, or 30 minutes to 1 hour, which effectively activates the surface of the substrate so that hydroxyl groups are bonded to the surface of the substrate and can be easily bonded to amino groups.

[0032] The step of activating the surface of the substrate may include carrying the substrate in an aminosilane solution under dark conditions, and activating the surface of the substrate so that it is positively charged. The aminosilane solution may contain (3-aminopropyl)triethoxysilane (APTES). In this case, activating the surface of the substrate means that the surface of the substrate is positively charged, and specifically, the substrate can be activated by bonding an amine group to the substrate. When the substrate surface is activated by immersing in the aminosilane solution as described above and the substrate surface is positively charged, the substrate can be bonded to the nanoligand by electrostatic attraction.

[0033] In addition, in the step of performing the treatment with ultrasonic waves, a substrate with a positively charged surface may be supported in a solution containing a nanoligand to produce a nanoligand-presenting substrate. Specifically, the substrate with a positively charged surface is placed in purified water and subjected to ultrasonic treatment in a solution containing a nanoligand, and the substrate is supported at room temperature for 30 minutes to 2 hours or 30 minutes to 1 hour.

[0034] The step of adjusting the cell adhesion and regenerative polarization of macrophages may be performed by applying a magnetic field of 100 to 700 mT for 12 to 48 hours after placing a nanoligand-presenting substrate inside or outside the body. Specifically, the step of adjusting the cell adhesion and regenerative polarization of macrophages may be performed 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 placing a nanoligand-presenting substrate inside or outside the body. As described above, by applying a magnetic field to the nanoligand-presenting substrate, the cell adhesion of macrophages to the nanoligands present on the substrate can be promoted, the polarization of the attached macrophages to the inflammatory M1 phenotype can be suppressed, and the polarization of the attached macrophages to the regenerative M2 phenotype can be promoted.

[0035] The step of adjusting the cell adhesion and regenerative polarization of macrophages may be performed by changing the position of the magnetic field applied to the substrate. Specifically, the position of the magnetic field applied to the substrate may be changed while applying a magnetic field of 100 to 600 mT, 200 to 600 mT, or 300 to 550 mT, to spatially control the cell adhesion and regenerative polarization of macrophages. For example, a magnetic field may be applied to a portion of the substrate to adjust the density of the nanoligand on the substrate, promoting cell adhesion of macrophages only in an arbitrary portion of the substrate, suppressing the polarization of the inflammatory M1 phenotype of the attached macrophages, and promoting the polarization of the regenerative M2 phenotype.

[0036] The step of adjusting the cell adhesion and phenotype of macrophages may be performed by changing the position of the magnetic field applied to the lower end of the substrate. Specifically, the position of the magnetic field applied to the substrate may be changed according to time while applying a magnetic field of 100 to 600 mT, 200 to 600 mT, or 300 to 550 mT, thereby controlling the cell adhesion and phenotype of macrophages temporally and spatially. More specifically, the magnetic field may be applied individually to each part of the substrate to adjust the density of the nanoligands located on the substrate according to time, thereby adjusting the degree of promotion of cell adhesion and regenerative M2 polarization of macrophages on each part of the substrate. For example, when a magnetic field is applied to the left side of the substrate for 12 to 24 hours and to the right side of the substrate for 24 to 36 hours, the amount of macrophages that have adhered or undergone regenerative M2 polarization may differ between the left and right sides of the substrate.

[0037] Hereinafter, examples of the present invention will be described. However, the following examples are merely preferred examples of the present invention, and the scope of the present invention is not limited to the following examples.

[0038] [Production Example] Manufacturing Example 1 Fabrication of slidable nano-ligands 1) Manufacturing of magnetic cores (MNP) For magnetic control of the slidable nanoligand, the magnetic core of the slidable nanoligand was prepared as follows. Approximately 80 mL of ethanol, 60 mL of deionized water (DI), and 140 mL of heptane were first mixed. In this mixture, 120 mmol of sodium oleate and 40 mmol of iron(III) chloride hexahydrate were dissolved in a solvent mixture of 80 mL of ethanol, 60 mL of deionized water (DI), and 140 mL of heptane at 70 °C for 4 h under an inert environment. The heptane layer containing iron oleate was collected and washed with deionized water. The heptane was evaporated to collect the dried iron oleate, to which 20 mmol of oleic acid and 200 g of 1-octadecene were added to approximately 40 mmol of dried iron oleate. The solution was stirred at 100 °C for 5 min. The temperature was then increased to 320 °C and maintained for approximately 30 min. The above mixture solution was suspended under air and cooled to room temperature, then washed with ethanol three times using a permanent magnet to recover the nanoparticles. To preserve the magnetic core nanoparticles (MNPs), the solution was stored in heptane until use.

[0039] 2) Aminosilica functionalization of the magnetic core (aminosilica coated MNPs) Magnetic core nanoparticles were coated with aminosilica shells for nanoassembly of slidable nanoligands. Magnetic core nanoparticles (30 mg) were dispersed in cyclohexane (25 mL). Triton-X (5 mL), 1-hexanol (5 mL), NH4OH (0.5 mL), and deionized water (1 mL) were added to the above suspension in sequence and stirred for 30 min. After stabilizing the emulsion, tetraethyl orthosilicate (TEOS, 12.5 μL) was gradually mixed and stirred for 10 min. For amino-functionalization, (3-aminopropyl)triethoxysilane (APTES, 6.25 mL) was mixed and stirred overnight. After amino-functionalization, the amino-silica shell-coated MNPs were washed with acetone and dimethylformamide (DMF) three times each and collected using a magnet.

[0040] 3) Fabrication of sliding nanoligands A polyethylene glycol (PEG) linker was used to improve the sliding properties of the nanoligands and to graft a charged RGD peptide ligand onto MNPs whose surfaces were coated with an aminosilica shell. The PEG linker was used to prevent cell absorption from the nanoassembly of the sliding nanoligands. In DMF (1 mL), the aminosilica-coated MNPs were used to dissolve 5 mg of maleimide-poly(ethylene glycol)-NHS ester (Mal-PEG-NHS ester; Mn = 5000 Da, Biochempeg). N,N-diisopropylethylamine (DIPEA, 2 μL) was added and stirred overnight, after which the MNPs were washed three times with DMF and three times with dimethyl sulfoxide (DMSO) and collected using a permanent magnet. In DMSO (1 mL), the PEG-aminosilica-coated MNPs were used to dissolve a negatively charged thiolated RGD peptide (CDDRGD, GL Biochem, 0.5 mg). To prevent disulfide bond formation, DIPEA (2 μl) and Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 10 mM) were added to the suspension and stirred overnight in the dark. The suspension was washed with DMSO and collected using a permanent magnet before the substrates that bind via electrostatic interactions were attached.

[0041] Comparative Manufacturing Example 1 A "No RGD" nanoligand was prepared in the same manner as in Preparation Example 1, except that the negatively charged thiolated RGD peptide (CDDRGD, GL Biochem) was not added.

[0042] [Example] Example 1 Slidable nanoligand and combination of said nanoligand with substrate Culture-grade glass coverslips (22 mm x 22 mm) were used to reversibly bind the slideable nanoligands prepared in the above preparation example to the substrate. The substrate was immersed in a 1:1 mixture of hydrochloric acid and methanol for 30 minutes to confirm that organic contaminants on the surface had been removed, and then washed three times with deionized water. This was immersed in sulfuric acid for 1 hour and washed with deionized water. The substrate was immersed in a 1:1 mixture of APTES and ethanol for 1 hour under dark conditions to aminated the substrate. The substrate was then washed three times with ethanol and dried at 100°C for 1 hour. To promote electrostatic interactions, the negatively charged slideable nanoligands (magnetic nanoparticles coated with RGD-PEG-amino silica shell) were diluted in DMSO (1:20), sonicated, and incubated with the positively charged aminated substrate in 1 mL of DMSO. The substrate was washed with deionized water to obtain a substrate on which the slideable nanoligands were present.

[0043] [Experimental Example] Experimental Example 1 In order to confirm the morphology of the slidable nanoligand according to the present invention, the slidable nanoligand was subjected to transmission electron microscopy (TEM) and dynamic light scattering-high angle annular dark field scanning transmission electron microscopy (HAADF-STEM) analysis, and the results are shown in Figures 3 and 4.

[0044] In addition, to confirm the properties and chemical binding characteristics of the sliding nano-ligand, Fourier transform infrared spectroscopy (FTIR) and vibrating sample magnetic force measurements were performed on the sliding nano-ligand, and the results are shown in Figures 5 and 6.

[0045] Specifically, transmission electron microscopy (TEM) experiments were performed using a Tecnai 20 (FEI, USA) for TEM imaging to characterize the size and shape of the slideable nanoligands.

[0046] Additionally, high angle annular dark field scanning transmission electron microscopy (HAADF-STEM) was used to characterize the size and shape of representative slideable nanoligands. HAADF-STEM imaging was performed using a JEOL 2100F with a probe size of 1 nm, a condenser aperture of 20 μm, and a collection angle of 80-150 mrad for Z ratio.

[0047] Additionally, dynamic light scattering (DLS) analysis was performed 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.

[0048] In addition, Fourier transform infrared spectroscopy (FTIR) was performed using GX1 (Perkin Elmer Spectrum, USA) to confirm the chemical binding properties of the slideable nanoligands. Samples that underwent analysis of changes in chemical binding properties were freeze-dried and densely packed in KBr pellets before analysis.

[0049] Vibrating sample magnetometry (VSM) was used to confirm the superparamagnetic properties of the nanoligands. The magnetic nanoparticle cores with the slideable nanoligands were subjected to VSM measurements (EV9; Microsense) at room temperature under an applied magnetic field. The corresponding magnetic moment was displayed in the hysteresis loop after normalization to the dry weight of the magnetic cores with the slideable nanoligands.

[0050] Figure 3 is also a transmission electron microscope image of the slidable nanoligand, with the scale bar being 20 nm. Figure 4 (a) is an image of the dynamic light scattering results of magnetic nanoparticles (MNPs) and amino-silica coated MNPs with size distribution, and (b) is a high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) image of amino-silica coated MNPs, with the scale bar being 20 nm.

[0051] According to Figure 3, the slidable nanoligand spheres containing a superparamagnetic core (16 ± 2 nm) and a slidable nanoligand core-shell (42 ± 5 nm) show a uniform shape, and according to Figure 4, as determined by dynamic light scattering, the uniform diameter of the superparamagnetic core of 15 ± 4 nm and the slidable nanoligand core-shell of 41 ± 5 nm, which is consistent with the TEM and high-angle annular dark-field scanning TEM (HAADF-STEM) images.

[0052] Figure 5 shows Fourier transform infrared spectrum images of a sliding nanoligand according to one embodiment. Specifically, the Fourier transform infrared spectrum images are of MNP, silica-coated MNP, and RGD ligand-presenting PEG-grafted silica-coated MNP (RGD-PEG-silica-coated MNP, which corresponds to a sliding nanoligand). Figure 6 shows magnetometer hysteresis of a vibration sample of the sliding nanoligand.

[0053] According to Figure 5, the change in chemical bonds during the preparation of the sliding nano-ligands can be seen by FTIR. Specifically, the Fe-O bond is 699 cm in the superparamagnetic iron oxide core nanoparticles. -1 The Si–O bond was detected at an absorption peak of 1168 cm from the silica shell. -1 The slidable nanoligand was detected at an absorption peak of 100 nm. n = 5000 Da) not only improves slide properties but also inhibits cellular absorption as demonstrated in the prior art, and the CDDRGD is 1152 cm-1 The absorption peak of the C=O bond is at 1635 cm -1 The absorption peak of 1000 nm indicated amide bond. From this TIR analysis, it was confirmed that the assembly of the sliding nano-ligand was successful.

[0054] In Fig. 6, superparamagnetism was confirmed with Ms of 20 emu / g. This shows that the slidable nano-ligand of the present invention is capable of reversible sliding due to its superparamagnetic property. This property is very important for magnetic control of the sliding of the nano-ligand in a time-dependent and reversible manner.

[0055] Experimental Example 2 To demonstrate the in situ reversible and spatiotemporal control of the sliding nanoligand according to the present invention, the sliding nanoligand was photographed using a scanning electron microscope (SEM) and imaged using an atomic force microscope (AFM), and the results are shown in Figures 8 and 9.

[0056] Figure 7 is a schematic image of electrostatic binding of a slidable nanoligand on a substrate. According to Figure 7, a magnetic nanoparticle (core) is PEGylated, and a negatively charged RGD peptide ligand (CDDRGD) is coated on the magnetic nanoparticle to form a slidable nanoligand, which is bound to a positively charged substrate via electrostatic interaction. Specifically, as illustrated in Figure 7, the present invention binds the slidable ligand to a positively charged substrate for in situ spatiotemporal control of nanoligand sliding to electrostatically control the presentation of the macroscale nanoligand. The electrostatic binding between the nanoligand and the substrate allows the nanoligand to reversibly slide.

[0057] Here, scanning electron microscopy (SEM) was performed to confirm the negatively charged slidable nanoligands bound to the positively charged substrate via electrostatic interactions, and field emission SEM imaging (FE-SEM, FEI, Quanta250FEG) was performed on the dried platinum-coated substrate. The density of the slidable nanoligands bound to the substrate was measured with Image J using 10 different images. SEM imaging was also performed to confirm the characteristics of the reversible spatiotemporal manipulation of the macroscale nanoligand density. The position of the permanent magnet (150 mT) was switched from the bottom left to the bottom right of the substrate and then switched again to the bottom left every 12 h. The corresponding changes in the macroscale nanoligand density were calculated using Image J, and the results are illustrated in Figure 8.

[0058] In situ magnetic atomic force microscopy (AFM) has also been used to perform 3D imaging of the sliding nanoligands on the substrate, as well as characterize the in situ nanoscale motion of the sliding nanoligands. For this purpose, AFM imaging (Asylum Research, XE-100 System) was performed in AC atmospheric mode at 25 °C. An AFM cantilever (Nanosensors, SSS-SEIHR-20) with spring constant (5-3 N / m) and resonance frequency (96-175 kHz) was used. Static serial imaging without magnets in the vicinity of the substrate was performed in the same scan area of ​​the substrate to examine the magnet-mediated displacement of the slidable nanoligand. For in situ magnetic imaging, imaging was first performed and a permanent magnet was placed on the bottom of the substrate and on the opposite side of the scan area to characterize the in situ nanoscale nanoligand slide. The results are shown in Figures 8 and 9.

[0059] Figure 8 shows the in situ reversible spatiotemporal manipulation images of macro- and nano-scale nanoligand slides. According to Figure 8a-b, a positively charged amino-functionalized substrate is randomly and uniformly bound to the surface of the substrate, as shown by scanning electron microscopy (SEM) and 3D atomic force microscopy (AFM). The density of the macroscopic nanoligands is 20±3 nanoligand particles / μm. 2 It was calculated that the above density allows the sliding (mobility) of the nanoligand to be effectively controlled, and does not induce aggregation of the nanoligand. Therefore, the nanoligand of the present invention is capable of reversible sliding (movement) on the substrate, and can reversibly control the cell adhesion and phenotype of macrophages in a spatiotemporal and reversible manner.

[0060] Figure 8c-d shows the results of SEM imaging of the spatiotemporal control experiment, which shows that the sliding property can be controlled by adjusting the density of the macroscale nanoligand using magnetic control. Specifically, in SEM imaging, a permanent magnet was placed at the bottom left of the substrate to attract the sliding nanoligand to the left, then switched to the right, and returned to the left every 12 hours. Time-lapse SEM imaging showed that compared to the right side, the nanoligand density on the left side was higher at 81% after 12 hours, lower at 31% after 24 hours, and significantly higher at 82% after 36 hours.

[0061] As a comparative example of spatiotemporal control experiments, Figure 8e shows in situ magnetic AFM images from successive in situ magnetic AFM scans performed on the same scan area with and without an external magnetic field in the scan area, illustrating the nanoscale displacement of the nanoligand slide. In Figure 8e, a magnet was placed on the bottom edge (opposite side of the scan area) of the substrate representing the slidable nanoligand to generate a magnetic field. In the absence of a magnet, it was clearly identified, but in the presence of a magnet, representing the nanoscale nanoligand slide, it disappeared from the scan area.

[0062] Fig. 9 shows in situ atomic force microscopy (AFM) images of the nanoligand slide taken in the absence of a magnet in the same scan as a comparative experiment. In Fig. 9, a black dotted line is drawn along the slidable nanoligand in two different images. The scale bar is 50 μm. It was confirmed that the motion of the slidable nanoligand was negligible, with a displacement of about 4 nm when successively scanning the same area of ​​the substrate, featuring repeatable and stable imaging in the absence of a magnet, was characterized.

[0063] This demonstrates that the nanoligand according to the present invention can use a magnetic field to reversibly change the density of macro-scale nanoligands in a spatiotemporal manner and to reversibly control the sliding of the nanoligands.

[0064] Experimental Example 3 In the method of promoting macrophage cell adhesion and regeneration using a sliding nano-ligand according to the present invention, the following experiment was carried out to confirm the effect of adjusting the density of the macro-scale nano-ligand on regulating macrophage cell adhesion.

[0065] The binding experiment of slideable nanoligand to integrin β1 was carried out as follows. The slideable nanoligand substrate was used to quantify the binding efficiency of slideable nanoligand to integrin β1. The substrate was immersed in integrin β1 (50 μg / mL) in phosphate buffered saline (PBS) at 4°C for about 12 hours by placing a permanent magnet at the bottom left of the substrate. The treated substrate was subjected to immunofluorescence staining for integrin β1 (Santa Cruz Biotechnology) followed by confocal imaging to quantify the integrin β1 bound to the slideable nanoligand in situ, and the results are illustrated in Figure 10.

[0066] Figure 10. Temporal regulation of integrin β1 binding during in situ control of slide nanoligand. (a) Schematic of slide nanoligand with a permanent magnet at the "bottom left" of the substrate, (b) Confocal microscopy images of immunofluorescence for integrin β1 clusters bound to slide nanoligand at the "left", "middle" and "right" sides of the substrate. Scale bar is 50 μm, these clusters are indicated by green arrows. (c) Quantification of staining intensity of integrin β1 clusters at the "magnetic", "middle" and "non-magnetic" sides of the substrate. Data are expressed as mean ± standard error (n=30). Statistically significant differences are denoted by different alphabetical letters.

[0067] FIG. 11 shows images of an in situ control experiment of nanoligand sliding according to one embodiment of the present invention, in which the magnetic attraction of the slidable nanoligand is used to adjust the density of the macroscale ligand, promoting macrophage attachment. (a) Schematic of the manipulation of the slidable nanoligand by placing a permanent magnet on the bottom ("magnet" side) of the substrate, together with immunofluorescent staining of vinculin, actin, and nuclei and corresponding confocal microscopy images after 24 hours of macrophage culture with a permanent magnet placed on the bottom ("magnet" side) of the substrate. Images of adherent macrophages were taken at the center (red dotted frame in the schematic) of the "magnet", "middle", and "non-magnet" sides of the substrate. Scale bar is 20 μm. Control groups include the "No RGD" group and the "No magnet" group. (b) Graph showing quantification of macrophage cell-attached cell density, cell area, and cell aspect ratio. Data are expressed as mean ± standard error (n=30). Statistical significance is denoted by different alphabetical letters.

[0068] Figure 12 shows the results of macrophage cell adhesion experiments with comparison: (a) Confocal microscopy images of immunofluorescent staining of vinculin, actin, and nuclei after 24 h of macrophage culture in the absence of RGD peptide ligand or magnetic field. Images of adherent macrophages were taken at the center of the "left", "middle", and "right" sides of the substrate. Scale bar is 20 μm. (b) Graph showing quantification of macrophage density, area, and aspect ratio. Data are expressed as mean ± standard error (n=30). Statistically significant differences are denoted by different alphabetical letters.

[0069] FIG. 10a explores whether ligation of integrin β1 can be manipulated by in situ slideable nanoligands, where slideable nanoligand-presenting substrates were incubated with integrin β1 for 12 hours while a permanent magnet was positioned underneath the substrate (the "magnet side"). According to FIG. 10b-c, immunofluorescence confocal microscopy images revealed that the binding efficiency of integrin β1 to the nanoligands was very high, being attracted 30% and 55% toward the magnet of the substrate compared with the medium and non-magnetic sides of the substrate, respectively. Since integrin ligation facilitates macrophage cytoadhesion, these results imply that macrophages can be strongly attached to the slideable nanoligands, which migrate toward the magnet and increase the macroscale nanoligand density on the magnet side. Furthermore, the present invention investigated the macrophage cytoadhesion under the control of macroscale in situ nanoligand density.

[0070] According to Figure 11a, a magnet was placed underneath the substrate ("Magnet" side) during incubation, and macrophage cell attachment was observed, imaged from the center of the "Magnet", "Middle" and "Non-Magnet" sides of the substrate. Control experiments were also performed on substrates that presented a sliding nanoligand or had no magnet ("No magnet") or nanoparticles without conjugated RGD peptide ligand ("No RGD").

[0071] As a result, macrophages were more firmly attached to the “magnet side” of the substrate, with a significantly higher density of actin filament assemblies and vinculin expression in a longer form than those attached to the “middle” and “non-magnet” sides of the cell area and substrate (Figure 11a-b). Quantitatively, the density of adherent macrophages on the “magnet side” was 59% and 82% higher than those on the “middle” and “non-magnet sides,” respectively. Similarly, the area of ​​adherent macrophages on the “magnet side” was 57% and 60% higher than those on the “middle” and “non-magnet sides,” respectively. Furthermore, the aspect ratio (elongation shape factor) of adherent macrophages was 54% and 50% higher on the “magnet side” than those on the “middle” and “non-magnet sides,” respectively.

[0072] According to Figure 12a-b, the regulation of macrophage cell attachment based on nanoligand-mediated control is inefficient for the "No RGD" and "No magnet" groups, which show similar adherent macrophage density and area on the "left", "middle", and "right" sides of the substrate. The "No RGD" group showed minimal macrophage cell attachment and effectively blocked it. These results indicate that increasing the macroscale nanoligand density with the magnetic attraction of in situ slideable nanoligands efficiently promotes integrin ligation-mediated attachment of macrophages.

[0073] Experimental Example 4 An experiment to determine whether macroscale density adjustment of the nanoligand according to the present invention controls the reversible adhesion of macrophages was carried out as follows.

[0074] Macrophages exhibit dynamic adhesion and polarization in the ECM, which is continuously remodeled with spatially and temporally changing macroscale ligand distribution. Therefore, remote control of spatially, temporally, and reversibly changing macroscale nanoligand distribution can mimic ECM remodeling to regulate macrophage cell adhesion. Therefore, the present invention conducted an experiment to determine whether time-switching of macroscale nanoligand presentation can change macrophage cell adhesion by attracting a slidable nanoligand, and the results are shown in Figures 13 to 15.

[0075] Figure 13 shows experimental results of controlling macrophage cell adhesion by tuning macro-scale nanoligands. (a) Confocal microscopy images of immunofluorescence for F-actin and nuclear vinculin after 12 or 24 h of macrophage culture with a permanent magnet placed on the bottom of the substrate ("magnetic field (MF)") or with the permanent magnet removed from the substrate ("no magnetic field (NMF)"). "MF" or "NMF" was applied during the 24 h culture period or alternately after 12 h. Images of adherent macrophages were taken at the center of the "left" or "right" side of the substrate. Scale bar is 20 μm. (b) Graphs of calculated macrophage density, cell area, and cell aspect ratio on the "left" surface of the substrate. Data are expressed as mean ± standard error (n=30). Statistical significance is indicated by different alphabetical letters.

[0076] Figure 14 is a graph showing quantification of macrophage density, area, and aspect ratio on the "right" side of the substrate for the experiment in Figure 13. A permanent magnet was placed on the bottom of the "left" side of the substrate ("magnetic field (MF)") or removed from the substrate ("no magnetic field (NMF)") while macrophages were cultured for 12 or 24 hours. "MF" or "NMF" was applied during the 24 hour culture period or alternately after 12 hours. Data are presented as mean ± standard error (n=30). Statistically significant differences are denoted with different alphabetical letters.

[0077] Figure 15: Time-resolved confocal microscopy images of (a) immunofluorescence staining for vinculin, actin, and nuclei after 12, 24, and 36 h of macrophage culture on the "left" and "right" sides of the substrate, with the permanent magnet position switched between the opposing sides every 12 h from the bottom of the "left" side to the bottom of the "right" side to the bottom of the "left" side of the substrate. The slideable nanoligand was imaged at the center of the "left" and "right" sides of the substrate (red dotted frame in the schematic). Scale bar is 20 μm. (b) Calculations of macrophage density, cell area, and cell aspect ratio on the "left" side of the substrate. Data are expressed as mean ± standard error (n = 30). Statistical significance is indicated by different alphabetical letters.

[0078] According to FIG. 13, a permanent magnet was placed at the bottom left of the substrate (magnetic field, “MF”) for 24 hours. A group without a magnet was also placed near the substrate for 24 hours (No magnetic field, “NMF”). Additionally, the present invention includes a group where no magnet was placed near the substrate for the first 12 hours, but after 12 hours, a magnet was placed on the left side of the substrate (“NMF-MF”). After 24 hours, the “MF” group showed significantly greater macrophage attachment from the left side (magnet side) than the “NMF” group, especially the area of ​​the highly attached cells was 63% higher and the aspect ratio was 66% higher (FIG. 13a-b). This trend was reflected in the “NMF-MF” group, which showed low levels of macrophage cell attachment at 12 hours in the absence of a magnet, but significant macrophage cell attachment was observed after 24 hours from magnetically mediated attraction to the left side (magnet) of the slidable nanoligand. Once the slidable nanoligand is attracted to the magnet (left) side, it will remain even after the magnet is removed after 12 hours, as already demonstrated by maintaining macrophage adhesion after 24 hours in the "MF-NMF" group. Although such a dramatic time-controlled switching of the nanoligand slide was effective in regulating macrophage cell adhesion on the left side (magnet side), it cannot effectively regulate macrophage adhesion on the right side (non-magnet side) according to Figure 14. Thus, the magnet-mediated in situ control of the nanoligand slide can be confirmed.

[0079] Furthermore, the present invention conducted an experiment on the effect of spatiotemporal tuning of nano-ligand slide on reversible macrophage adhesion. According to FIG. 15, a permanent magnet was placed at the bottom left of the substrate to attract the slidable nano-ligand to the left side for 12 hours. Then, the magnet was positioned at the bottom right of the substrate for 12 hours and left at the bottom left for 12 hours. Time-resolved macrophage adhesion was investigated on the left and right sides (FIG. 15a). According to FIG. 15, the temporal tuning of nano-ligand slide controls reversible macrophage cell adhesion. At 12 hours, the left side of the substrate showed a significant increase in the area of ​​adherent macrophages, 60%, and the aspect ratio, 44%, compared with the right side (FIG. 15a-b). At 24 hours, the right side of the substrate showed a 67% and 70% increase in the area and aspect ratio of adherent macrophages, respectively, compared with the left side. After 36 h, the left side of the substrate reversibly exhibited higher adherent macrophage density, cell area, and aspect ratio than the right side, which increased by 51%, 67%, and 68%, respectively. Thus, remote and spatiotemporal regulation of macroscale nanoligand distribution demonstrates powerful control in the reversible regulation of macrophage cell adhesion.

[0080] Experimental Example 5 In order to confirm that the slidable nanoligand according to the present invention can regulate the cell adhesion-dependent polarization of macrophages by time-controlled tuning, the following experiments were carried out, and the results are shown in FIGS.

[0081] Dynamic ECM remodeling temporally exhibits various heterogeneous ligand distributions, which modulate the development of cell attachment structures in macrophages that regulate their functional polarized phenotype, spatially and temporally modulating the host response to implants. It is well-known that macrophages that generate cytoskeletal actin assemblies and extended morphology of attachment structures are functionally activated as regenerative M2 phenotypes. Existing reports suggest that previous findings suggest that temporal modulation of macroscale nano-ligand presentation can modify cell attachment-dependent polarization of macrophages.

[0082] FIG. 16(a) is a graph showing quantitative gene expression of M2 phenotype markers (Arginase-1 and Ym1) in macrophages cultured in M2 polarizing medium or M1 phenotype markers (iNOS and TNF-α) in macrophages cultured in M1 polarizing medium for 36 hours on the "left" and "right" sides of the substrate, with the magnet positioned on the "left" side of the substrate. Data are presented as mean ± SEM (n=3). (b)-(c) are confocal microscopy images of immunofluorescence for Arg-1 and nuclear iNOS in macrophages cultured with ("magnetic field (MF)") or without ("no magnetic field (NMF)") a permanent magnet placed on the bottom of the plate. "MF" or "NMF" was applied during the 36-hour culture period or after 12 hours. Images of polarized macrophages were taken from the center of the "left" or "right" sides of the substrate. Scale bars are 20 μm. Different alphabetic letters indicate statistical significance.

[0083] FIG. 17 is a graph showing the M2 phenotype of macrophages in M1 polarizing medium of slideable nanoligand according to one embodiment of the present invention. Under slideable nanoligand in M1 polarizing medium, the control of M2 phenotype of macrophages was not efficient. Quantitative gene expression of M2 markers (Arginase-1 and Ym1) of macrophages cultured in M1 polarizing medium on the "left" and "right" sides of the substrate for 36 hours, exposed to a permanent magnet placed on the bottom of the "left" side of the substrate ("magnet side"). Data are expressed as mean ± standard error (n=3).

[0084] FIG. 18 is a graph showing the M1 phenotype of macrophages in M2 polarizing medium of slideable nanoligand according to one embodiment of the present invention. The control of M1 phenotype of macrophages was inefficient under slideable nanoligand in M2 polarizing medium. Genetic profiles of M1 markers (iNOS and TNF-α) of macrophages cultured in M1 polarizing medium on the "left" and "right" sides of the substrate for 36 hours exposed to a permanent magnet placed on the bottom of the "left" side of the substrate ("magnet side"). Data are expressed as mean ± standard error (n=3).

[0085] 19 shows the experimental results of M2 phenotype of macrophages against magnetic and electrical attraction of slideable nanoligand according to one embodiment of the present invention. Magnetic attraction of slideable nanoligand promotes expression of ROCK2 and promotes M2 phenotype when cell attachment structure of macrophages is generated. Confocal microscopy images of immunofluorescence staining for ROCK2 and nuclei are shown after macrophages were exposed to a permanent magnet placed on the bottom of the "left side" of the substrate ("magnetic side") and cultured in basal medium or M2 polarizing medium for 36 hours on the "magnetic side" and "non-magnetic side" of the substrate. Scale bar is 20 μm.

[0086] 20 shows (a) immunofluorescence confocal microscopy images and (b) graphs showing the density, cell area, cell aspect ratio of adherent macrophages, or the area, aspect ratio, and Arg-1 staining intensity after incubation in M2 medium, from a self-adherent nanoligand conditioning experiment according to one embodiment of the present invention. Specifically, magnetic manipulation of the slidable nanoligand attraction promotes the assembly of cell adhesion structures in macrophages that stimulate the M2 phenotype. (a) Confocal microscopy images of immunofluorescence for actin and nucleus Arg-1 after culturing macrophages in M2 polarizing medium for 36 hours, and for actin and nucleus after culturing macrophages in basal medium for 36 hours, with the magnet located at the bottom (the "magnetic" side) of the substrate, and with inhibition of ROCK signaling (as with 50 μM Y27632), ROCK signaling formation (as with 10 μM blebbistatin), or actin polymerization (as with 2 μg / mL cytochalasin D) in experiments with the magnet located at the bottom (the "magnetic" side). Scale bar is 20 μm. (b) Calculations of adherent macrophage density, cell area, and cell aspect ratio after culturing in basal medium on the "magnetic" and "non-magnetic" sides of the substrate, and area, aspect ratio, and Arg-1 staining intensity after culturing in M2 medium. Data are presented as mean ± SEM (n = 30). Statistical significance is denoted by different alphabetical letters.

[0087] According to Figure 16, the time-controlled magnetic attraction of the slideable nanoligand suppresses the M1 phenotype of macrophages while stimulating the M2 phenotype. In the time-controlled manipulation of the slideable nanoligand, the substrates with slideable nanoligands were examined for macrophage phenotype presentation by placing a permanent magnet on the bottom left of the substrate for 36 consecutive hours ("MF" group) or removing it from the vicinity of the substrate for 36 consecutive hours ("NMF" group), switching from "NMF" to "MF" every 12 hours. After culturing macrophages in M1-polarizing medium, the gene expression profile showed that the expression of M1 markers iNOS (inducible nitric oxide synthase) and TNF-α (tumor necrosis factor-α) was significantly lower on the left side (magnetic side) than on the right side (non-magnetic side), by 192% and 231%, respectively, in the "MF" group (Figure 16a). In contrast, after culturing with M2 polarizing medium in the "MF" group, the expression of M2 markers Arg-1 (arginasae-1) and Ym1 (chitinase-like 3) was significantly increased by 715% and 383%, respectively, on the left side (magnetic side) compared with the right side (non-magnetic side). According to Figures 17 and 18, in the "MF" group, the expression of M1 markers after culturing with M2 polarizing medium, or M2 markers after culturing with M1 polarizing medium, was not different between the left and right sides, which is a prerequisite for polarized lytic stimulation with magnetic control of the nanoligand slide to regulate macrophage polarization.

[0088] From Figure 16b-c, it can be seen that the immunofluorescence confocal microscopy images illustrate the findings of gene expression profiles. In the "MF" group, iNOS fluorescence was considerably lower after M1 polarized batch culture, while Arg-1 immunofluorescence was higher after culture in M2 polarized medium on the left side (magnet side) compared with the right side (non-magnet side). Such a trend was further consistent with the "NMF-MF" group, which showed low iNOS immunofluorescence after culture in M1 polarized medium, but high Arg-1 immunofluorescence after culture in M2 polarized medium on the magnet side, suggesting that the slideable nanoligand could be attracted on the magnet at any defined time to activate M2 polarization. No significant difference in both iNOS and Arg-1 immunofluorescence was observed in the "NMF" group. This demonstrates that the magnetic attraction of sliding nanoligands, enhanced by the development of pervasive attachment structures on the magnetic side, polarized macrophages towards a regenerative M2 phenotype while suppressing the proinflammatory M1 polarization.

[0089] Next, we performed an experiment to determine how the magnetic attraction of the sliding nanoligand facilitated the polarization of macrophages to the M2 phenotype in the assembly of adhesion structures. As shown in Figure 19, the cell adhesion structures of macrophages, which include elongated shape, actin cytoskeleton organization and contractility, and ROCK, mediate the M2 phenotype. In cultures in basal medium and M2 polarization medium, macrophages showed significantly higher ROCK2 immunofluorescence on the magnet side than on the non-magnetic side. In particular, as shown in Figure 20, while cultured in M2 polarization medium, inhibition of ROCK signaling by Y27632 reduced the area and aspect ratio of adherent macrophages by 32% and 33%, respectively, and further significantly reduced Arg-1 immunofluorescence by about 29% on the magnet side. Consistently, under inhibition of ROCK signaling by blebbistatin during culture in M2 polarization medium, the area, aspect ratio, and Arg-1 immunofluorescence of adherent macrophages were reduced by 33%, 31%, and 29%, respectively, on the magnet side. The reduction in the area, aspect ratio, and Arg-1 immunofluorescence of adherent macrophages was also clearly observed by inhibition of actin polymerization with cytochalasin D. On the non-magnetic side, inhibition of ROCK and myosin II actin polymerization did not significantly change the adhesion structure and production of M2 phenotype. This indicates that the sliding nanoligand effectively promotes the assembly of cell adhesion structures in macrophages in the process of promoting the development of M2 phenotype by magnetic control.

[0090] Experimental Example 6 In order to confirm that the sliding nanoligand according to the present invention spatially regulates the cell attachment and phenotype of host macrophages in vivo through in situ control, the following experiment was carried out and the results are shown in FIGS. 21 to 23.

[0091] It is paramount for material implants to control the cell adhesion and functional phenotype of macrophages to induce host responses of the implant. The spatiotemporal, reversible, and remote control of macroscale nano-ligand mutations can mimic dynamic and heterogeneous ECM remodeling to spatially regulate the host response of the implant. In particular, it is highly advantageous to regulate the cell adhesion and production of the regenerative and anti-inflammatory M2 phenotype of macrophages to mediate tissue repair while suppressing inflammation. For this reason, the present invention explored the in vivo translation of the M2 phenotype to spatially regulate the regenerative and anti-inflammatory immune response of the implant and mediate the cell adhesion structure assembly of adherent macrophages. Recently, it has been revealed that spatially heterogeneous regulation of macrophage cell adhesion by UV light is possible. However, in the present invention, we exploited tissue permeability and cell compatibility using a magnetic field to regulate not only the cell adhesion of macrophages but also the functional phenotype of adherent macrophages.

[0092] FIG. 21 shows the results of an experiment on the cell adhesion and inflammatory M1 phenotype of host macrophages in vivo in response to the magnetic attraction of a slidable nanoligand according to one embodiment of the present invention. (a) shows a schematic diagram of the magnetic control of the slidable nanoligand in vivo. Both IL-4 and IL-13 were injected onto the substrate with the slidable nanoligand substrate after subcutaneous implantation. (b) shows the immunofluorescence confocal microscopy images of iNOS, F-actin, and nuclei of cells attached to the "magnetic" and "non-magnetic" sides of the substrate after 24 hours. A permanent magnet was placed on the bottom ("magnetic" side) of the substrate for 24 hours. Images of cells were taken at the center of the "magnetic" or "non-magnetic" side of the substrate. The scale bar is 20 μm. (c) shows the calculation of the gene expression profile (n=3) of M1 phenotype markers (iNOS and TNF-α), density, cell area, and cell aspect ratio (n=30) of attached cells in vivo. Data are expressed as mean±SEM. Different alphabetical letters indicate statistical significance.

[0093] FIG. 22 shows the results of a host neutrophil in vivo adhesion experiment for a slideable nanoligand according to one embodiment of the present invention. (a) Confocal microscopy images of immunofluorescence staining for NIMP-R14, F-actin, and nuclei of host cells attached to the "magnetic" and "non-magnetic" sides of a substrate representing a slideable nanoligand 24 hours after subcutaneous injection of IL-4 and IL-13 on the substrate. A permanent magnet was kept on the bottom ("magnetic side") of the substrate for 24 hours. Images of cells were taken at the center of the "left" or "right" side of the substrate. Scale bar is 20 μm. (b) Quantification of the density of NIMP-R14 positive host neutrophils attached in vivo. Data are expressed as mean ± standard error (n=30). Different alphabetical letters indicate statistically significant differences.

[0094] FIG. 23 illustrates the results of an experiment on cell adhesion and regeneration M2 phenotype of host macrophages in vivo in response to magnetic attraction of slideable nanoligands according to one embodiment of the present invention. (a) Confocal microscopy images of immunofluorescence staining for Arg-1, F-actin, and nuclei of host cells attached to the “magnetic” and “non-magnetic” sides of the slideable nanoligand-bearing substrate 24 hours after subcutaneous implantation of the substrate infused with IL-4 and IL-13. A permanent magnet was positioned consecutively on the bottom (“magnetic” side) of the substrate. Images of cells were taken from the center of the “magnetic” or “non-magnetic” side of the substrate. Scale bar is 20 μm. (b) Graph showing the calculation of gene expression profile (n=3) of M2 phenotype markers (Arg-1 and Ym1), density, cell area, and cell aspect ratio (n=30) of attached cells in vivo. Figure 4b shows the calculated nuclear to cytoplasmic YAP fluorescence ratio under ROCK signal inhibition (with Y27632) (c) and cell area under ROCK signal inhibition (with blebbistatin) (d). Data are expressed as mean ± standard error. Different alphabetical letters indicate statistical significance.

[0095] According to Figure 21, nanoligand-presenting substrates were subcutaneously implanted into BALB / c mice. After implantation, M2-polarizing soluble factors (interleukin-4 and -13) were injected into the substrate (Figure 21a). A permanent magnet was attached to the bottom ("magnet side") of the substrate from the ventral side of the mouse to facilitate sliding of the nanoligands with the magnet side of the substrate facing up. The above substrates were retrieved after 24 hours for immunofluorescence confocal imaging and gene expression analysis. Immunofluorescence confocal microscopy images of iNOS and F-actin showed clear colocalization, with the magnet side predominating over the non-magnetic side (Figure 21b). At the same time, we observed a superior development of adhesion structures with significantly higher macrophage density, area, and aspect ratio (67%, 36%, and 53%, respectively) on the magnetic side, which promoted significantly lower expression of both iNOS and TNF, by 33% and 60%, respectively, in host macrophages than on the nonmagnetic side ( Figure 21 b–c).

[0096] According to FIG. 22, not only host macrophages but also host neutrophils were observed by NIMP-R14 immunofluorescence during acute inflammation. At the same time, according to FIG. 23a, confocal microscopy images of immunofluorescence for Arg-1 and F-actin showed a very clear assembly of adhesion structures by F-actin, which resulted in much higher expression and colocalization of regenerative Arg-1 on the magnet side than on the non-magnetic side. According to FIG. 23a-b, both Arg-1 and Ym1 (regenerative M2 marker) were expressed 27% and 60%, respectively, higher in host macrophages on the magnet side compared to the non-magnetic side. This indicates that remote control by magnetic attraction of the slideable nanoligand promotes the regenerative M2 phenotype of host macrophages while suppressing the inflammatory M1 phenotype in vivo in a spatially coordinated manner. It is known that tight control of early acute inflammation and regenerative responses affect the long-term host response to implants. We conducted experiments on spatiotemporal, reversible, and long-term magnetic field-mediated modulation of the implant's immune response in deep internal tissues, promoting tissue repair and inhibiting inflammation and fibrous capsule formation, and confirmed that immune modulation of the implant is possible in a clinical environment.

[0097] In the above example experiment, the cell attachment and phenotype of macrophages from cultures were examined under in situ control of nanoligand slides as follows: The effect of in vitro nanoligand slides on cell attachment and phenotype of macrophages was examined. Substrates with nanoligands were sterilized with UV light for 1 hour. Sterilized substrates were blocked with 1% bovine serum albumin (BSA) to minimize non-nano-ligand specific attachment of macrophages. 50kcells / cm 2Macrophages (RAW264.7, passage 5, ATCC) were plated on the treated substrates. Macrophages were cultured in basal medium containing high glucose DMEM supplemented with 10% (v / v) heat-inactivated fetal bovine serum and 50 U / mL penicillin / streptomycin at 37 °C and 5% CO2. Cells were examined for adhesion by applying a magnet (270 mT) placed on the bottom ("magnetic side" or "left side") of the substrate and at the center of the different sides (magnetic side, middle, non-magnetic side). Substrates with RGD peptide ligand or nanoparticles without nanoligand or nanoligand without magnet application served as control groups. Macrophage cell adhesion under temporal manipulation of the sliding nanoligand was examined on substrates with nanoligand by placing a permanent magnet on the bottom of the substrate ("magnetic field (MF)") or by removing the permanent magnet from the substrate ("no magnetic field (NMF)"). The permanent magnet was switched temporally between the two opposing surfaces by moving it from the bottom of the "left" side of the substrate to the bottom of the "right" side and then back to the bottom of the "left" side. Under time-controlled manipulation of the sliding nanoligand, the polarization phenotype of macrophages was dependent on the placement of a permanent magnet at the bottom of the substrate ("magnetic field (MF)") or the removal of the permanent magnet from the substrate ("no magnetic field (NMF)"), and was therefore examined using substrates with nanoligands that switched between "NMF" and "MF". M1 polarization medium included basal medium supplemented with lipopolysaccharide (LPS, 10 ng / mL) and recombinant interferon-gamma (IFN-γ, 10 ng / mL). M2 polarization medium included basal medium supplemented with interleukin-4 (IL-4, 20 ng / mL) and interleukin-13 (IL-13, 20 ng / mL). The cell adhesion structure assembly-mediated M2 phenotype of macrophages was examined after application of magnets placed on the bottom ("magnet side") of the substrate in the presence of ROCK signaling inhibition (as with 50 μM M Y27632), ROCK signaling inhibition (as with 10 μM blebbistatin), or actin polymerization inhibition (as with 2 μg / mL cytochalasin D).

[0098] Furthermore, the analysis of the cytoadhesion of macrophages under the in situ sliding nanoligands by immunofluorescence and confocal imaging was carried out as follows.

[0099] Macrophage cultures were immersed in a 4% (w / v) paraformaldehyde solution for 10 min and then washed with PBS. Cultures were incubated in a blocking buffer of 3% PSA and 0.1% Triton-X in PBS for 30 min. Cultures were immersed in solutions of primary antibodies against integrin β1, vinculin, iNOS, arginase-1, ROCK2, and NIMP-R14 overnight at 4 °C and washed with PBS. Cultures were immersed in a mixed solution of secondary antibodies, phalloidin, and DAPI for 30 min at room temperature and then washed with PBS. Cultures were mounted on microscope slides and imaged under a confocal microscope (LSM700, Carl Zeiss) applying the same exposure conditions for all comparison groups and examined with ImageJ software. Confocal microscopy images were used to quantify the cytoadherence of macrophages. The binding intensity of integrin β1 was calculated with the histogram function. The density of adherent macrophages was calculated by counting DAPI-stained nuclei, and the area and aspect ratio of adherent macrophages were calculated by analyzing F-actin staining as previously reported.

[0100] Furthermore, macrophage polarization analysis in situ slideable nanoligands with reverse transcription polymerase chain reaction was performed as follows.

[0101] Macrophages were cultured in M1 or M2 polarization medium and harvested with 1 mL of Trizol per sample. The substrate was divided into two (magnetic and non-magnetic sides). For each sample, 900 ng of extracted RNA was reverse transcribed into cDNA using a high-capacity RNA-cDNA kit. Real-time PCR reactions (StepOne Plus Real-Time PCR System, Applied Biosystems) were performed with the cDNA used for Sybr Green analysis. After normalization to GAPDH expression, the relative fold expressions of target genes (iNOS, TNF-α, Arginase-1, Ym1) were presented.

[0102] Furthermore, the following experiment was performed for the in vivo remote control of the nanoligand slide to regulate the adhesion and phenotype of host phagocytes. To investigate the effect of the in vivo nanoligand slide on the cell adhesion and phenotype of host macrophages, the nanoligand-presenting silicon substrate was used for subcutaneous implantation. Approximately 8-week-old BALB / c mice approved by the Animal Care and Use Committee of Korea University were used as the realization animals. A mixture of 2 mL of alfaxalone and 1 mL of rompun was administered by intraperitoneal injection. An incision was made on the back of the mouse with a length of approximately 2.5 cm. After the injection, IL-4 (50 ng) and IL-13 (50 ng) were injected onto the substrate. The cell adhesion of mouse macrophages to the substrate was tested. A permanent magnet (the "magnet side") attached to the bottom (ventral side) of the substrate promoted the sliding of the nanoligand toward the magnet. At 24 hours after implantation, the substrate was retrieved for confocal imaging of immunofluorescence and RT-PCR.

Claims

1. a core comprising magnetic nanoparticles; a coating layer that is provided so as to surround the core and that includes an integrin adhesion ligand peptide; A slideable nanoligand for promoting cell attachment and regeneration of macrophages, comprising: The integrin adhesion ligand peptide is negatively charged, whereby the nanoligand binds to a positively charged substrate via electrostatic interaction and is slidable on the substrate, and the adhesion and regeneration of macrophages is promoted by controlling the density of the nanoligand by applying a magnetic field to slide the nanoligand.

2. The slidable nano-ligand for promoting cell attachment and regeneration of macrophages according to claim 1, wherein the magnetic nanoparticles have silica bound to their surfaces.

3. Further comprising a linker connecting the core and the coating layer; The slidable nanoligand for promoting cell attachment and regeneration of macrophages according to claim 1 , wherein the linker is a polyethylene glycol (PEG)-based linker.

4. The slidable nano-ligand for promoting cell attachment and regeneration of macrophages according to claim 1, wherein the nano-ligand has a diameter of 30 nm to 60 nm, and the magnetic nanoparticles have a diameter of 5 nm to 30 nm.

5. the integrin adhesion ligand peptide comprises a negatively charged thiolated integrin adhesion ligand peptide; The slidable nanoligand for promoting cell attachment and regeneration of macrophages according to claim 1 , wherein the surface of the nanoligand is negatively charged.

6. Providing a core comprising magnetic nanoparticles; mixing the core with a first suspension containing a linker to produce a linker-attached core; mixing the linker-bound core with a second suspension containing an integrin adhesion ligand peptide (RGD); A method for producing a slidable nanoligand for promoting cell adhesion and regeneration of macrophages according to any one of claims 1 to 5, comprising:

7. A step of supporting a positively charged substrate in a solution containing a slidable nanoligand for promoting cell adhesion and regeneration of macrophages according to any one of claims 1 to 5, thereby producing a nanoligand-presenting substrate in which the nanoligand containing a negatively charged integrin adhesion ligand peptide is bound to the substrate via electrostatic interaction; After treating the nanoligand-presenting substrate with a culture medium in vitro, an external magnetic field is applied to slide the nanoligand on the nanoligand-presenting substrate, thereby controlling the density of the nanoligand, thereby adjusting the cell attachment and regenerative polarization of macrophages; 23. A method for promoting cell attachment and regeneration of macrophages, comprising:

8. The step of manufacturing the nanoligand-presenting substrate includes: Immersing a surface of the substrate in an acidic solution; placing the immersed substrate in an aminosilane solution to positively charge the surface of the substrate; treating the positively charged substrate with ultrasound at room temperature; The method for promoting cell attachment and regeneration of macrophages according to claim 7, comprising:

9. The method for promoting cell attachment and regeneration of macrophages according to claim 7, characterized in that the substrate having a positively charged surface is activated by supporting the substrate in an aminosilane solution so that the surface of the substrate is positively charged.

10. The method for promoting cell adhesion and regeneration of macrophages according to claim 7, wherein the step of adjusting the cell adhesion and regeneration polarization of the macrophages is performed by placing the nanoligand-presenting substrate outside the body and then applying a magnetic field of 100 to 700 mT for 12 to 48 hours.

11. The method for promoting cell attachment and regeneration of macrophages according to claim 7 , wherein the step of adjusting the cell attachment and regenerative polarization of the macrophages is performed by changing the position of a magnetic field applied to a substrate.

12. The method for promoting cell attachment and regeneration of macrophages according to claim 7 , wherein the step of adjusting the cell attachment and regenerative polarization of the macrophages is performed by changing the position of a magnetic field applied to a substrate over time.

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