KR20210110128A - A nano-ligand for promoting cell adhesion and regeneration of macrophage and a method for promoting cell adhesion and regeneration of macrophage using the same - Google Patents

Abstract

The present invention relates to a nano-ligand for promoting cell adhesion and regeneration of macrophages and a method for promoting cell adhesion and regeneration of macrophages using the nano-ligand. The method for promoting cell adhesion and regeneration of macrophages of the present invention not only temporally and spatially controls the sliding of the nano-ligand, but also allows reversible control by applying a magnetic field to a substrate containing the nano-ligand, and can efficiently regulate the cell adhesion and phenotype of macrophages in vitro and in vivo through magnetic field-based temporal and spatial control.

Description

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

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. about how to control it.

Macrophages are the main cells responsible for innate immunity. Most of them are settled throughout the body, but some exist in the form of monocytes in the blood. These monocytes can differentiate into dendritic cells or macrophages. Most macrophages are sessile and typically include dust cells, microglia, Kupffer cells, and Langerhans cells, which are distributed throughout the body. Delivers antigens and triggers an immune response. When an enemy invades a wound, the monocytes in the blood go out of the blood vessels like neutrophils and differentiate into macrophages, removing bacteria. In addition, macrophages are divided into a free form, which moves and phagocytoses several places in the body, and a fixed form, which is fixed in a designated organ and phagocytic. Adherent macrophages include liver Kupffer cells, alveolar macrophages, connective tissue structures (Histiocytes), and brain microglia (Microglia).

As such, as a method for efficiently controlling the regeneration and anti-inflammatory effects of macrophages, a technique through the presentation of ligands in vivo is used. However, although most of the existing nanoligand presentation in vivo is static or dynamic, it is impossible to reversibly change the density of nanoligands on a macroscopic scale through real-time remote control.

Republic of Korea Patent Publication No. 10-2014-0084043

The problem to be solved by the present invention is to provide a movable nanoligand by electrostatic bonding with a substrate, and reversibly change the macroscopic nanoligand density through real-time remote using the nanoligand to attach macrophages to cells And to provide a method for promoting regeneration.

The present invention includes a core comprising magnetic nanoparticles; and a coating layer provided to surround the core and comprising an integrin ligand peptide,

The integrin ligand peptide provides a nanoligand for promoting cell adhesion and regeneration of macrophages, characterized in that they are negatively charged.

In addition, the present invention comprises the steps of preparing a core comprising magnetic nanoparticles; preparing a core to which a linker is bound by mixing the core with a first suspension containing a linker; And mixing the linker-coupled core with a second suspension comprising an integrin ligand peptide (RGD); for promoting cell adhesion and regeneration of macrophages according to any one of claims 1 to 5, comprising a; A method for preparing a nanoligand is provided.

In addition, the present invention comprises the steps of preparing a nanoligand presentation substrate by supporting a substrate with an activated surface in a solution containing the nanoligand for promoting cell adhesion and regeneration of macrophages as described above; And it provides a method for promoting cell adhesion and regeneration of macrophages, comprising the step of controlling the cell adhesion and regeneration of macrophages by applying an external magnetic field after treating the culture medium to the nanoligand presentation substrate.

The nanoligand for promoting cell adhesion and regeneration of macrophages according to the present invention is a negatively charged ligand coated on a core containing magnetic nanoparticles, and it is easy to move (sliding) on the substrate through electrostatic bonding with the substrate .

In addition, in the method for promoting cell adhesion and regeneration of macrophages according to the present invention, by applying a magnetic field to the substrate including the nanoligand, not only temporally and spatially controlling the sliding of the nanoligand, but also reversible control is possible, and the magnetic field Based on spatiotemporal control, we can efficiently regulate cell adhesion and phenotypic polarization of macrophages in vitro and in vivo.

1 is a schematic diagram showing a nanoligand for promoting cell adhesion and regeneration of macrophages and a method for promoting cell adhesion and regeneration of macrophages using the same according to an embodiment of the present invention.
2 is a diagram schematically illustrating a method for promoting cell adhesion and regeneration of macrophages according to an embodiment of the present invention.
3 is a transmission electron microscope image (TEM) of a slidable nanoligand according to an embodiment of the present invention. Scale bar represents 20 nm.
4 shows the characteristics of a slidable nanoligand according to an embodiment of the present invention: (a) is a dynamic light scattering image of magnetic nanoparticles (MNP) having a size distribution and amino-silica-coated MNP, ( b) is a representative high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image of amino-silica-coated MNPs. Scale bar represents 20 nm.
5 is a Fourier transform infrared spectrum image of a slidable nanoligand according to an embodiment of the present invention.
6 is a vibrating sample magnetometer hysteresis of a slidable nanoligand according to an embodiment of the present invention.
7 is a schematic image of electrostatic coupling between a substrate and a slidable nanoligand according to an embodiment of the present invention.
8 is a scanning electron microscope (SEM) and an atomic force microscope (SEM) for in situ reversible spatiotemporal manipulation of sliding of the macroscale and nanoscale nanoligands according to an embodiment of the present invention; AFM): (a) and (b) are images of the positively charged substrate and nanoligand, (c) and (d) are the results of type-lapse SEM imaging (Time-lapse SEM imaging) , and (e) shows the nanoscale displacement of the nanoligand sliding through AFM scanning.
9 is an atomic force microscope (AFM) image of the sliding of the nanoligand in situ in the absence of a permanent magnet of a comparative example.
Figure 10 shows the regulation of integrin β1 binding of in situ control of a sliding nanoligand according to an embodiment of the present invention: (a) a sliding nanoligand with a permanent magnet under the "left" of the substrate Schematic diagram, (b) confocal image of integrin β1 immunofluorescence after binding to a slidable nanoligand in “magnet”, “middle” and “non-magnet” of the substrate, scale bar means 50 μm, (c) ) A graph showing the quantification of staining intensity of integrin β1 clusters on the “magnetic”, “middle” and “non-magnetic” sides of the substrate.
11 is an image showing the results of an in situ control experiment of sliding of a nanoligand according to an embodiment of the present invention: (a) is 24 hours performed by positioning a permanent magnet on the bottom (“magnet” side) of the substrate After macrophage culture of . (b) is a graph showing the quantification of the adherent cell density, cell area, and cell aspect ratio of macrophages.
12 is a macrophage adhesion test result according to Comparative Example: (a) is a confocal immunofluorescence staining for vinculin, actin and nucleus after 24 hours of macrophage culture in the absence of RGD peptide ligand or magnetic field It is an image. (b) is a graph showing the quantification of macrophage density, area and aspect ratio.
13 is an experimental result for controlling the adhesion of macrophages through the regulation of macro-scale nanoligands according to an embodiment of the present invention. (a) shows F after 12 h or 24 h macrophage incubation performed by placing a permanent magnet on the bottom of the substrate [“magnetic field (MF)”] or withdrawing the permanent magnet from the substrate [“no magnetic field (NMF)”] -Confocal images of immunofluorescence for actin and vinculin with nucleus. (b) is a graph in which the density of macrophages, cell area, and cell aspect ratio are calculated for the “left” side of the substrate.
14 is a graph showing the quantification of macrophage density, area, and aspect ratio for the "right" side of the substrate according to the time control of the nanoligand according to an embodiment of the present invention.
15 is a graph of (a) immunofluorescence confocal image and (b) macrophage density, cell area and cell aspect ratio for temporal transformation of macroscopic nanoligand presentation according to an embodiment of the present invention.
16 is a phenotypic experiment result according to time-controlled magnetic attraction of a nanoligand according to an embodiment of the present invention: (a) is an M2 phenotypic marker (Arginase-1 and Ym1) for macrophages cultured in M2 polarized medium Alternatively, it is a graph showing quantitative gene expression of M1 phenotypic markers (iNOS and TNF-α) for macrophages cultured under M1 polarization medium. (bc) is a confocal image of immunofluorescence for Arg-1 and nuclear iNOS of cultured macrophages.
17 is a graph showing the M2 phenotype of macrophages in M1 polarizing medium of a slidable nanoligand according to an embodiment of the present invention.
18 is a graph showing the M1 phenotype of macrophages in M2 polarizing medium of a slidable nanoligand according to an embodiment of the present invention.
19 is a confocal image of immunofluorescence in the M2 phenotype experiment of macrophages for miraculous attraction to magnetic attraction of a slidable nanoligand according to an embodiment of the present invention.
20 is a confocal image of (a) immunofluorescence and (b) density, cell area and cell aspect ratio of adherent macrophages or cultured in M2 medium of a magnetic attraction regulation experiment of a nanoligand according to an embodiment of the present invention. It is a graph showing the calculation of the area, aspect ratio, and Arg-1 staining intensity afterward.
Figure 21 is the result of the host macrophage adhesion and inflammatory M1 phenotype experiments in vivo against the magnetic attraction of the slidable nanoligand according to an embodiment of the present invention: (a) is a slidable nanoligand in vivo It is a schematic diagram of the self-control of (b) is a confocal image of immunofluorescence for 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 density, cell area and cell aspect ratio (n=30) as well as gene expression profile (n=3) of M1 phenotypic markers (iNOS and TNF-α) of adherent cells in vivo.
22 is an in vivo adhesion test result of host neutrophils to the slidable nanoligand according to an embodiment of the present invention. (a) NIMP-R14, F of host cells attached to the “magnetic” and “non-magnetic” side of the substrate, showing slidable nanoligands 24 hours after subcutaneous injection of IL-4 and IL-13 on the substrate. -Confocal images of immunofluorescence staining for actin and nuclei. (b) is a graph showing the quantification of the density of attached NIMP-R14-positive host neutrophils in vivo.
23 shows the results of the adhesion and regeneration M2 phenotype experiments of host macrophages in vivo for magnetic attraction of the slidable nanoligand according to an embodiment of the present invention: (a) IL-4 and Immunofluorescence for Arg-1, F-actin and nuclei of cells attached to the “magnetic” and “non-magnetic” sides of the substrate with slidable nanoligands 24 hours after subcutaneous implantation by injecting IL-13 onto the substrate It is a confocal image. (b) A graph showing the calculation of density, cell area and cell aspect ratio (n=30) as well as gene expression profiles (n=3) of M2 phenotypic markers (Arg-1 and Ym1) of adherent cells in vivo. Figure 4b is a graph in which (c) the ratio of nuclear to cytoplasmic YAP fluorescence under ROCK inhibition (with Y27632) and (d) the cell area of myosin II inhibition (with blevisstatin) were calculated.

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

Macrophages constantly interact with the remodeled extracellular matrix (ECM), resulting in spatially and temporally heterogeneous macro-scale distribution of nanoligands in vivo. The nanoligand for promoting cell adhesion and regeneration of macrophages according to the present invention can mimic extracellular matrix (ECM) remodeling by enabling reversible remote control by spatially and temporally changing macro-scale nanoligand distribution, It can modulate the adhesion and polarization of macrophages for the control of temporal and spatial host responses. The nanoligand of the present invention binds to a negatively charged sliding nanoligand through an electrostatic interaction of a positively charged substrate, and magnetic core nanoparticles coated with a polymeric linker and negatively charged RGD ligand. was used to optimize the sliding potential of the nanoligand. Characteristics of macroscopic and in-situ nano-scale sliding of nanoligands under an external magnetic field that spatially and reversibly change macro-scale nanoligand density were confirmed. Furthermore, the present invention presents unprecedented in situ manipulation of macro-scale nanoligand densities by magnetically attracting slidable nanoligands to modulate the adhesion and polarization phenotype of host macrophages in vivo. Specifically, the time-controlled magnetic attraction of slidable nanoligands suppresses the inflammatory M1 phenotype of macrophages but stimulates the regenerative M2 phenotype. In addition, the magnetic attraction of the slidable nanoligands of the present invention promotes the assembly of adhesion structures within macrophages, thereby stimulating their polarization to the M2 phenotype. Through this, the nanoligand according to the present invention enables the spatiotemporal modulation of the immunomodulatory tissue-regenerative response to the implant in vivo through remote, spatiotemporal and reversible control of the nanoligand density on the macroscale.

The present invention includes a core comprising magnetic nanoparticles; and a coating layer provided to surround the core and including an integrin ligand peptide, wherein the integrin ligand peptide is negatively charged. It provides a nanoligand for promoting cell adhesion and regeneration of macrophages.

1 is a schematic diagram showing a nanoligand for promoting cell adhesion and regeneration of macrophages according to the present invention and a method for promoting cell adhesion and regeneration of macrophages using the same.

Referring to Figure 1, the nanoligand of the present invention is a core comprising magnetic nanoparticles; and a coating layer provided to surround the core and including an integrin ligand peptide (peptide), it can be seen that the integrin ligand peptide is a negatively charged integrin peptide. Specifically, the integrin ligand peptide bound to the core may have a form surrounding the core, such as a micelle structure. Accordingly, the surface charge of the nanoligand may represent a negative charge. For example, a slidable nanoligand is a negatively charged integrin in which superparamagnetic core iron oxide nanoparticles are first synthesized, the superparamagnetic core nanoparticles are functionalized with a functional amino-silica shell, and then coated with a polyethylene glycol (PEG) linker. Ligand peptides (RGD, CCDRGD) can be grafted to improve the sliding properties of nanoligands.

3 is a transmission electron microscope (TEM) image of the nanoligand for promoting cell adhesion and regeneration of macrophages according to the present invention, and the size of the nanoligand can be seen. 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 is more than 60 nm, the cell adhesion efficiency of macrophages is lowered, which may cause a problem. More specifically, the nanoligand may have a diameter of 30 to 50 nm or 35 to 45 nm.

The magnetic nanoparticles are not particularly limited as long as they are magnetic nanoparticles. For example, the magnetic nanoparticles 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, resulting in a large loss, and thus the efficiency is lowered. problems may arise. 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 magnetic nanoparticles as described above, the nanoligand of the present invention can promote cell adhesion and regeneration of macrophages using a magnetic field.

In addition, the magnetic nanoparticles may be silica bonded to the surface. Specifically, the magnetic nanoparticles may be amino-silica bonded to the surface. The type of silica may be at least one of tetraethylorthosilicate (TEOS) and (3-aminopropyl)triethoxysilane (APTES).

For example, the nanoligand of the present invention has a structure connected by a linker between the core and the coating layer, 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, it is possible to increase the binding force between the core and the coating layer to increase the durability of the nanoligand.

The coating layer is coupled to a core or a linker coupled to the core, and may have a form surrounding the core. Specifically, the coating layer may include an integrin ligand peptide (RGD), and the integrin ligand peptide may be in a negatively charged form, and may include a negatively charged thiolated integrin ligand peptide. As described above, the surface of the nanoligand of the present invention, including the negatively charged integrin ligand peptide, has a negatively charged form, and thus can be freely moved on the substrate through electrostatic bonding with the substrate. Due to these characteristics, the nanoligand is also referred to as a 'slidable nanoligand', and can promote cell adhesion and regeneration of macrophages through sliding of the nanoligand on the substrate.

In addition, the present invention comprises the steps of preparing a core comprising magnetic nanoparticles;

preparing a core to which a linker is bound by mixing the core with a first suspension containing a linker; and

It provides a method for preparing the nanoligand for promoting cell adhesion and regeneration of macrophages as described above, comprising the step of mixing the linker-bound core with a second suspension containing an integrin ligand peptide (RGD).

In the preparing of the core, magnetic nanoparticles may be stirred with a silane solution to form a silane-coated core. Specifically, preparing the core may include stirring the magnetic nanoparticles with an amino-silane solution to form an amino-silane-coated core. The type of silane included in the silane solution may be at least one of tetraethylorthosilicate (TEOS) and (3-aminopropyl)triethoxysilane (APTES).

Specifically, the step of preparing the core to which the linker is bound may be performed by stirring the core in a suspension containing the linker under dark conditions for 10 hours to 20 hours or 10 hours to 15 hours. Accordingly, a core to which a linker is bonded can be obtained. In this case, the core to which the linker is bound can be obtained by washing twice or more with a solvent while using a permanent magnet. The solvent may include any one or more of dimethylformaldehyde (DMF) and dimethylsulfoxide (DMSO).

In this case, 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 bonding the linker as described above to the core, it is possible to increase the binding force between the core and the coating layer to increase the durability of the nanoligand.

In addition, the step of mixing with the second suspension may be performed by stirring the linker-bound core in a suspension containing the integrin ligand peptide (RGD) under dark conditions for 10 to 20 hours or 10 to 15 hours. In this case, magnetic nanoparticles (nanoligands) to which negatively charged integrin ligand peptides are bound can be obtained by washing with a solvent while using a permanent magnet. The solvent may include any one or more of dimethylformaldehyde (DMF) and dimethylsulfoxide (DMSO).

Here, a coating layer may be formed on the core through a process of stirring with the integrin ligand peptide. Specifically, the integrin ligand peptide may be in a negatively charged form, and may be a negatively charged thiolated integrin ligand peptide. By forming a coating layer on the core with the negatively charged integrin ligand peptide as described above, the surface of the nanoligand of the present invention has a negatively charged form, and thus, it can be freely moved on the substrate through electrostatic bonding with the substrate. . Due to these characteristics, the nanoligand is also referred to as a 'slidable nanoligand', and can promote cell adhesion and regeneration of macrophages through sliding of the nanoligand on the substrate.

In addition, the present invention comprises the steps of preparing a nanoligand presentation substrate by supporting a substrate with an activated surface in a solution containing the nanoligand for promoting macrophage adhesion and regeneration described above; And it provides a method for promoting cell adhesion and regeneration of macrophages, comprising the step of controlling macrophage adhesion and regeneration by applying an external magnetic field after treating the culture medium to the nanoligand presentation substrate.

1 and 2 are diagrams schematically illustrating a method for promoting cell adhesion and regeneration of macrophages according to an embodiment of the present invention. 1 and 2, the surface of the negatively charged nanoligand is electrostatically bound on the positively charged substrate to promote the cell adhesion of macrophages to the portion to which the magnetic field is applied by applying a magnetic field, and the inflammatory M1 phenotype is inhibit and activate the regenerative M2 phenotype. Specifically, since the substrate and the nanoligand are coupled through electrostatic bonding, the nanoligand moves (slides) depending on a position to which a magnetic field is applied, and accordingly, the density of the nanoligand is adjusted in a portion to which a magnetic field is applied. Thus, there is an advantage in that it can promote cell adhesion and regeneration of macrophages to a desired site.

Specifically, preparing the nanoligand-presenting substrate may include immersing the surface of the substrate in an acidic solution; activating the substrate surface by immersing the immersed substrate in an aminosilane solution; and processing the activated substrate using ultrasonic waves at room temperature.

The step of immersing the surface of the substrate in the acidic solution may include immersion 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. Through this, the surface activation of the substrate can be effectively performed by bonding a hydroxyl group to the surface of the substrate to facilitate bonding with the amino group.

In the step of activating the substrate surface, the surface of the substrate may be activated by immersing the substrate in an amino-silane solution under dark conditions. The amino-silane solution may include (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, it may be activated by bonding an amine group on the substrate. As described above, the surface of the substrate is activated by immersion in the amino-silane solution to positively charge the surface of the substrate, so that the substrate can be coupled to the nanoligand by electrostatic attraction.

In addition, in the processing using ultrasonic waves, a substrate having a surface activated thereon is supported in a solution containing the nanoligand to prepare a nanoligand-presenting substrate. Specifically, the surface-activated substrate was immersed in a solution containing the nanoligand at room temperature for 30 minutes to 2 hours or 30 minutes for 1 hour under ultrasonication in purified water.

The step of controlling the cell adhesion and regeneration of macrophages may be performed by applying a magnetic field of 100 to 700 mT for 12 hours to 48 hours after positioning the nanoligand presentation substrate in vivo and in vitro. Specifically, the step of controlling the cell adhesion and regeneration of macrophages in vivo and ex vivo after positioning the nanoligand presentation substrate 100 to 600 Mt, 200 to 600 mT or 300 to 550 mT magnetic field for 12 hours to 36 hours, 24 It can be carried out by applying for hours to 26 hours or 12 hours to 24 hours. As described above, by applying a magnetic field to the nanoligand-presenting substrate, the cell adhesion of macrophages to the nanoligand located on the substrate is promoted, and the polarization of the inflammatory M1 phenotype of the adhered macrophages is suppressed and the polarization of the regenerative M2 phenotype is promoted. can do.

In addition, the step of controlling the cell adhesion and regeneration of macrophages may be performed by changing the position of the magnetic field applied to the substrate. Specifically, by changing the position of the magnetic field applied to the substrate while applying a magnetic field of 100 to 600 mT, 200 to 600 mT, or 300 to 550 mT, cell adhesion and regeneration of macrophages can be spatially controlled. For example, by applying a magnetic field to a portion of the substrate to control the density of the nanoligand on the substrate, the cell adhesion of macrophages is promoted only on a desired portion on the substrate, and the polarization of the inflammatory M1 phenotype of the adhered macrophages is suppressed and regenerated. Polarization of the M2 phenotype can be facilitated.

In addition, the step of controlling the cell adhesion and regeneration of macrophages may be performed by changing the position of the magnetic field applied to the bottom of the substrate with time. 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 over time to temporally and spatially control cell adhesion and phenotype of macrophages. More specifically, by applying a magnetic field to each part of the substrate individually, the density of dissimilar nanoligands located on the substrate can be adjusted over time to control the degree of promotion of cell adhesion and regeneration of M2 polarization of macrophages in each part on the substrate. have. 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 hours to 36 hours, the macrophages attached to the left and right sides of the substrate or regenerating M2-polarized macrophages The amount may vary.

Examples of the present invention will be described below. However, the following examples are only preferred examples of the present invention, and the scope of the present invention is not limited by the following examples.

[Production Example]

Preparation Example 1

Preparation of slidable nano-ligands

1) Magnetic core manufacturing (MNP)

For magnetic control of slidable nanoligands, magnetic cores of slidable nanoligands were prepared as follows. About 80 mL of ethanol, 60 mL of deionized water (DI) and 140 mL of heptane were first mixed. To this mixture, under an inert environment, 120 mmol of sodium oleate and 40 mmol of iron (III) chloride hexahydrate were added to 80 mL of ethanol, 60 mL of deionized water (DI) and 140 mL of heptane. was dissolved in a solvent mixture of 70 °C for 4 hours. The heptane layer containing iron oleate was collected and washed with deionized water. Heptane was then evaporated to collect dried iron oleate, to which 20 mmol of oleic acid and 200 g of 1-octadecene were added to about 40 mmol of dried iron oleate. The solution was stirred at 100° C. for 5 minutes. The temperature was then raised to 320° C. and held for approximately 30 minutes. The mixture solution was suspended under the atmosphere, cooled to room temperature, and washed with ethanol three times using a permanent magnet to collect nanoparticles. To store these magnetic core nanoparticles (MNPs), the solution was stored in heptane until used.

2) amino-silica functionalization of magnetic core (amino-silica coated MNP)

Magnetic core nanoparticles were coated with amino-silica shells for nanoassembly of slidable nanoligands. Magnetic core nanoparticles (30 mg) were dispersed in cyclohexane (25 mL). To the suspension, triton-X (Triton-X) (5 mL), 1-hexanol (5 mL), NHOH (0.5 mL) and deionized water (1 mL) were sequentially added and stirred for 30 minutes. After stabilizing the emulsion, tetraethyl orthosilicate (TEOS, 12.5 μL) was mixed slowly and stirred for 10 minutes. (3-aminopropyl)triethoxysilane (APTES, 6.25 mL) was mixed for amino functionalization and stirred overnight. After amino functionalization, the amino-silica shell coated MNPs were washed with acetone and dimethylformamide (DMF) three times each, and collected with a magnet.

3) Preparation of slidable nanoligand

A polyethylene glycol (PEG) linker was used to improve the sliding properties of the nanoligand, and a negatively charged RGD peptide ligand was grafted onto the surface amino-silica shell-coated MNP. A PEG linker was used to prevent cellular uptake in nanoassemblies of slidable nanoligands. 5 mg of maleimide-poly(ethylene glycol)-NHS ester (Mal-PEG-NHS ester; Mn = 5000 Da, Biochempeg) was dissolved in DMF (1 mL) using amino-silica coated MNP. N,N-diisopropylethylamine (DIPEA, 2 μL) was added thereto, and the mixture was stirred overnight, then washed 3 times with DMF and 3 times with dimethyl sulfoxide (DMSO), and then collected using a permanent magnet. Negatively charged thiolated RGD peptide (CDDRGD, GL Biochem, 0.5 mg) was dissolved by addition of PEG-amino-silica coated MNP in DMSO (1 mL). To avoid the formation of disulfide bonds, DIPEA (2 μl) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 10 mM) were added to this suspension and overnight under dark conditions. stirred. The suspension was washed with DMSO and collected using a permanent magnet prior to bonding the bonding substrates via electrostatic interaction.

Comparative Preparation Example 1

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

[Example]

Example 1

Sliding nanoligand and binding of the nanoligand to the substrate

In order to reversibly bind the slidable nanoligand prepared in Preparation Example to the substrate, culture-grade glass coverslips (22 mm x 22 mm) were used. The substrate was immersed in a 1:1 mixture of hydrochloric acid and methanol for 30 minutes to confirm that organic contaminants were removed from the surface, and then washed with deionized water. It was immersed in sulfuric acid for 1 hour and washed with deionized water. Amination of the substrate was formed by immersing the substrate in a 1:1 mixture of APTES and ethanol for 1 hour under dark conditions. The substrate was then washed with ethanol and dried at 100° C. for 1 hour. To facilitate electrostatic interaction, negatively charged slidable nanoligand (RGD-PEG-amino silica shell-coated magnetic nanoparticles) in 1 mL of DMSO was diluted with DMSO (1:20), followed by Incubated with positively charged amination substrates for 1 h under sonication. The substrate was washed with deionized water to obtain a substrate with a slidable nanoligand.

[Experimental example]

Experimental Example 1

In order to confirm the shape of the slidable nanoligand according to the present invention, transmission electron microscopy (TEM), dynamic light scattering, and high-angle annular dark field scanning TEM (HAADF-STEM) analysis were performed on the slidable nanoligand. is shown in FIGS. 3 and 4 .

In addition, in order to confirm the properties and chemical bonding properties of the slidable nanoligand, Fourier transform infrared spectroscopy (FTIR) and vibration sample magnetometry were performed on the slidable nanoligand, and the results are shown in FIGS. 5 and 6 It was.

Specifically, transmission electron microscopy (TEM) experiments were performed using Tecnai 20 (FEI, USA) to confirm the size and shape characteristics of the slidable nanoligand.

In addition, high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) was used to characterize the size and morphology of representative slidable nanoligands. , and a JEOL 2100F with 80-150 mrad collection angle for Z contrast.

In addition, dynamic light scattering (DLS) analysis was performed to quantify the size distribution profile (hydrodynamic diameter) in the assembly process of sliding nanoligands, DLS measurement (Zetasizer Nano ZS90 Malvern Panalytical, Malvern, UK).

In addition, Fourier transform infrared spectroscopy (FTIR) was performed using GX1 (Perkin Elmer Spectrum, USA) to confirm the chemical binding properties of the slidable nanoligand. Samples subjected to analysis for changes in chemical binding properties were lyophilized prior to analysis and densely packed into KBr pellets.

Vibrating sample magnetometry (VSM) measurement was applied to VSM measurement (EV9; Microsense) at room temperature under an applied magnetic field to confirm the superparamagnetic properties of the nanoligand. . The corresponding magnetic moments were displayed in a hysteresis loop after normalization to dry weight from the slidable nanoligand to the magnetic core.

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

Referring to FIG. 3, the spheres of the slidable nanoligand including the superparamagnetic core (16 ± 2 nm) and the slidable nanoligand core-shell (42 ± 5 nm) showed a uniform morphology. Also, referring to FIG. 4, with uniform diameters of a superparamagnetic core of 15 ± 4 nm and a slidable nanoligand core-shell of 41 ± 5 nm, as determined by dynamic light scattering, TEM and high-angle annular dark-field scanning TEM (high-angle annular dark-field scanning TEM, HAADF-STEM) images were consistent.

5 is a Fourier transform infrared spectral image of a slidable nanoligand according to an embodiment. Specifically, it is a Fourier transform infrared spectral image of MNP, silica-coated MNP and RGD ligand-presenting PEG-grafted silica-coated MNP (RGD-PEG-silica coated MNP, corresponding to a slidable nanoligand). 6 is a vibratory sample magnetometer hysteresis of a slidable nanoligand.

Referring to FIG. 5 , changes in chemical bonding during the manufacturing process of the slidable nanoligand can be seen through FTIR. Specifically, Fe-O bonding was detected at an absorption peak value of ^{699 cm -1 in superparamagnetic iron oxide core nanoparticles.} Si-O bonding was detected at an absorption peak value of ^{1168 cm -1 in the silica shell.} In the slidable nanoligand, the PEG linker (M _n = 5,000 Da) not only enhances the sliding properties as demonstrated in previous literature but also inhibits uptake by cells, and CDDRGD has C= at the absorption peak ^{of 1152 cm −1 .} The O bond and ^{the absorption peak at 1635 cm -1} showed an amide bond. This FTIR analysis confirmed the successful assembly of the slidable nanoligand.

Referring to FIG. 6 , superparamagnetism was confirmed with an Ms of 20 emu/g. Through this, since the slidable nanoligand according to the present invention exhibits superparamagnetic properties and enables reversible sliding, this property is very important for self-control of the sliding of the nanoligand temporally and reversibly.

Experimental Example 2

In order to demonstrate the in situ reversible spatiotemporal control of the slidable nanoligand according to the present invention, the slidable nanoligand was photographed with a scanning electron microscope (SEM), and atomic force microscopy (AFM) imaging was performed. is shown in FIGS. 8 and 9 .

7 is a schematic image of electrostatic bonding of a slidable nanoligand on a substrate. Referring to FIG. 7 , magnetic nanoparticles (cores) are PEGylated and coated with negatively charged RGD peptide ligand (CDDRGD) on the magnetic nanoparticles to form slidable nanoligands, which through electrostatic interaction bound to a positively charged substrate. Specifically, as shown in FIG. 7 , the present invention electrostatically bound a slidable ligand to a positively charged substrate for in situ spatiotemporal control of the sliding of nanoligands to control macroscale nanoligand presentation. Electrostatic bonding between the nanoligand and the substrate enabled the reversible movement of the nanoligand sliding.

Here, scanning electron microscopy (SEM) was used to identify negatively charged slidable nanoligands bound to positively charged substrates through static-mechanical interactions, and field emission SEM imaging (FE-SEM, FEI, Quanta 250) FEG) was performed on dried platinum-coated substrates. The density of the slidable nanoligands bound to the substrate was measured with Image J using 10 different images. SEM isolating was also performed to characterize the reversible and spatiotemporal manipulation of the nanoligand density on the macroscale. The position of the permanent magnet (150 mT) was switched from the bottom left to the bottom right of the substrate, and back to the bottom left every 12 hours. Corresponding changes in nanoligand density on the macroscale were calculated using image J, and the results are shown in FIG. 8 .

In addition, in situ magnetic atomic force microscopy (AFM) is to confirm the characteristics of 3D imaging of the slidable nanoligand on the substrate and the in situ nanoscale movement of the slidable nanoligand, AFM imaging (Asylum Research, XE-100 System) was performed in AC in air mode at 25°C. An AFM cantilever (Nanosensors, SSS-SEIHR-20) having a spring constant (5-3 N/m) and a resonance frequency (96-175 kHz) was used. For static serial imaging without magnets near the substrate, the same scanning area of the substrate was performed to examine the magnet-mediated displacement of the slidable nanoligand. For in-situ magnetic imaging, imaging was performed first, then a permanent magnet was placed on the bottom of the substrate or opposite to the scanning area, and then imaging was performed in the same scanning area to characterize in-situ nanoscale nanoligand sliding. , the results are shown in FIGS. 8 and 9 .

8 is an image of in situ reversible spatiotemporal manipulation of sliding of nanoligands on the macroscale and nanoscale. Referring to Figures 8a-b, the positively charged amino-functionalized substrate was confirmed that the nanoligand was randomly and uniformly bound to the substrate surface as shown by scanning electron microscopy (SEM) and 3D atomic force microscopy (AFM). did. In addition, the macroscopic nanoligand density was calculated to be ^{20 ± 3 nanoligand particles/μm 2 .} It can be seen that by having the above density, the sliding properties (mobility) of the nanoligands can be effectively controlled, and aggregation of the nanoligands does not occur. Therefore, the nanoligand of the present invention is capable of reversible sliding (movement) on the substrate, so that the cell adhesion and phenotype of macrophages can be controlled spatio-temporal and reversible.

8c-d are SEM imaging results of spatiotemporal control experiments. It can be seen that sliding properties are controlled by controlling the density of macro-scale nanoligands using magnetic control. Specifically, SEM imaging placed a permanent magnet on the lower left side of the substrate to attract the slidable nanoligand to the left, then switched to the right, and returned to the left every 12 h, respectively. In type-lapse SEM imaging, compared with the right, the nanoligand density on the left side was high as 81% after 12 hours, as low as 31% after 24 hours, but was significantly higher at 82% after 36 hours.

8e is an in-situ magnetic AFM image through continuous in-situ magnetic AFM scanning in the same scan area regardless of the presence or absence of an external magnetic field in the scanning area as a comparative example of a spatiotemporal control experiment, showing nanoscale displacement of sliding of nanoligands gave. A magnetic field was generated by placing a magnet at the bottom (opposite side of the scanning area) of the substrate showing the slidable nanoligand in FIG. The case disappeared from the scanning area.

9 is an in situ atomic force microscopy (AFM) image of the sliding of a nanoligand in the absence of a magnet in the same scanning as a comparative experiment. Referring to FIG. 9 , a black dotted line is drawn along the slidable nanoligand in two different images. Scale bar represents 50 nm. Characterized by repeatable and stable imaging in the absence of magnets near the substrate, we found that the motion of the slidable nanoligand was negligible within 4 nm displacement in continuous scanning in the same area of the substrate.

Through this, it can be seen that the nanoligand according to the present invention can reversibly control the sliding of the nanoligand by changing the density of the macroscopic nanoligand in space-time and reversibly using a magnetic field.

Experimental Example 3

In the method for promoting cell adhesion and regeneration of macrophages using a slidable nanoligand according to the present invention, the following experiment was performed to confirm the effect of controlling the density of macrophages on the macrophage nanoligand on cell adhesion regulation of macrophages. did.

The binding experiment of integrin β1 to the slidable nanoligand was performed as follows. The binding efficiency of integrin β1 to the slidable nanoligand was quantified using a slidable nanoligand presentation substrate. Immersion in integrin β1 (50 μg/mL) in phosphate-buffered saline (PBS) at 4°C for about 12 h by placing a permanent magnet on the “left” bottom of the substrate. The treated substrate was used for immunofluorescence staining for integrin β1 (Santa Cruz Biotechnology) and subsequent confocal imaging to quantify integrin β1 bound to the in situ slidable nanoligand, and the results are shown in FIG. 10 .

Figure 10 shows the temporal regulation of integrin β1 (integrin β1) binding of in situ control of sliding nanoligands. (a) is a schematic diagram of a sliding nanoligand with a permanent magnet under the “left” of the substrate, and (b) is an integrin β1 cluster bound to the sliding nanoligand on the “left”, “middle”, and “right” of the substrate. Confocal images of immunofluorescence for (c) shows the quantification of staining intensity of integrin β1 clusters on the “magnetic”, “middle” and “non-magnetic” sides of the substrate. Data are expressed as mean ± standard error (n=30). Statistically significant differences are indicated by different alphabetic characters.

11 is an image showing the experimental results of in situ control of sliding of nanoligands according to an embodiment of the present invention. By controlling the ligand density on a macroscopic scale by magnetic attraction of a slidable nanoligand, the adhesion of macrophages has been shown to promote (a) of the substrate with confocal images corresponding to immunofluorescence staining for vinculin, actin and nuclei after 24 h of macrophage incubation performed by placing a permanent magnet on the bottom (“magnet” side) of the substrate. This is a schematic image of manipulating a slidable nanoligand by placing a permanent magnet on the bottom (“magnet” side). Images of adherent macrophages were taken at the center of the “magnetic”, “middle” and “non-magnetic” sides of the substrate (red dashed squares in the schematic). The scale bar is 20 μm. The control group includes “No RGD” and “No magnet” groups. (b) A graph showing the quantification of adherent cell density, cell area, and cell aspect ratio of macrophages. Data are expressed as mean ± standard error (n=30). Statistical significance is indicated by different alphabetic characters.

12 is a macrophage adhesion test result according to a comparative example, (a) is a confocal immunofluorescence staining for vinculin, actin and nucleus after 24 hours of macrophage culture in the absence of RGD peptide ligand or magnetic field. It is an image. Images of adherent macrophages were taken from the center of the “left”, “middle” and “right” of the substrate. The scale bar is 20 μm. (b) is a graph showing the quantification of macrophage density, area and aspect ratio. Data are expressed as mean ± standard error (n=30). Statistically significant differences are indicated by different alphabetic characters.

Referring to Figure 10a, it was investigated whether ligation of integrin β1 can be manipulated by an in situ slidable nanoligand. A slidable nanoligand-presenting substrate was incubated with integrin β1 for 12 hours, while a permanent magnet was placed under the substrate. It was placed under the position (“magnet” side). 10b-c, the immunofluorescence confocal image showed that the binding efficiency of integrin β1 to the nanoligand was significantly high, which was compared to the medium and non-magnetic side of the substrate, toward the magnet of the substrate. attracted by 30% and 55%, respectively. Because integrin ligation facilitates the attachment of macrophages, these results suggest that macrophages can attach more strongly to slidable nanoligands, which migrate towards the magnet and the macroscale density of nanoligands towards the magnet. raise the In addition, the present invention investigated the adherence of macrophages under the control of ligand density in situ on a macroscopic scale.

Referring to FIG. 11A , a magnet was placed on the underside (“magnet” side) of the substrate during incubation, and the attachment of imaged macrophages was observed at the center of the “magnet”, “middle” and “non-magnet” side of the substrate. did. In addition, control experiments were performed with substrates exhibiting slidable nanoligands or with nanoparticles without magnets (“No magnet”) or with nanoparticles without conjugated RGD peptide ligands (“No RGD”).

The result is that macrophages are more robust to the "magnetic" side of the substrate at significantly higher densities with prominent actin filament assembly and expression of vinculin in a longer form than those attached to the "middle" and "non-magnetic" side of the substrate and the cell region and the substrate. It was shown to be strongly attached (FIGS. 11a-b). Quantitatively, the density of adherent macrophages on the “magnet” side was 59% and 82% higher than that on the “medium” and “magnet” side, respectively. Similarly, the area of adherent macrophages on the “magnetic” side was 57% and 60% higher than on the “middle” and “non-magnetic” side, respectively. In addition, the aspect ratio (elongation shape factor) of adherent macrophages was 54% and 50% higher on the “magnetic” side than on the “medium and non-magnetic” side, respectively.

12A-B , the regulation of macrophage adhesion based on nanoligand-mediated-control is “No RGD” and “No RGD” showing similar adherent macrophage density and area in “left”, “middle” and “right” of the substrate It can be seen that it is inefficient for the “No magnet” group. The “No RGD” group showed minimal macrophage adhesion, indicating effective blocking. These results demonstrate that increasing macro-scale nanoligand density with the magnetic attraction of in situ slidable nanoligands efficiently promotes integrin ligation-mediated adhesion of macrophages.

Experimental Example 4

An experiment on whether macrophage density control of the nanoligand according to the present invention controls the reversible adhesion of macrophages was performed as follows.

Macrophages exhibit dynamic adhesion and polarization on the ECM, which are continuously remodeling with spatially and temporally varying macro-scale ligand distributions. Thus, spatially, temporally and reversibly changing macroscale remote control of nanoligand distribution could mimic ECM remodeling that modulates macrophage adhesion. To this end, the present invention tested whether the temporal shift of macro-scale nanoligand presentation by attracting slidable nanoligands could alter the adhesion of macrophages, as shown in FIGS. 13 to 15 .

13 is an experimental result of controlling the adhesion of macrophages through the regulation of macro-scale nanoligands. (a) F after 12 h or 24 h macrophage incubation performed by placing a permanent magnet on the bottom of the substrate [“magnetic field (MF)”] or withdrawing the permanent magnet from the substrate [“no magnetic field (NMF)”] -Confocal images of immunofluorescence for actin and vinculin with nucleus. “MF” or “NMF” was applied during the incubation period of 24 hours or alternately after 12 hours. Images of adherent macrophages were taken from the center of the “left” or “right” of the substrate. The scale bar is 20 μm. (b) is a graph in which the density of macrophages, cell area, and cell aspect ratio are calculated for the “left” side of the substrate. Data are expressed as mean ± standard error (n=30). Statistical significance is indicated by different alphabetic characters.

14 is a graph showing the quantification of macrophage density, area, and aspect ratio for the “right” side of the substrate in the experiment shown in FIG. 13 . A permanent magnet was placed on the bottom of the "left" of the substrate during macrophage incubation for 12 or 24 hours ["magnetic field (MF)"] or removed from the substrate ["no magnetic field (NMF)"]. "MF" or "NMF" was applied over 24 hours of incubation or alternately after 12 hours. Data are expressed as mean ± standard error (n=30). Statistically significant differences are indicated by different alphabetic characters.

15 shows the “left” and “right” of the substrate by switching the position of the permanent magnet between the two opposite faces from the bottom of the “left” of the substrate to the bottom of the “right” and then the bottom of the “left” every 12 hours. (a) Time-resolved confocal images of immunofluorescence staining for vinculin, actin and nuclei after 12 h, 24 h and 35 h of macrophage incubation in ”. Sliding nanoligands were imaged at the center of the “left” and “right” of the substrate (red dashed squares in the schematic). The scale bar is 20 μm. (b) Calculated macrophage density, cell area and cell aspect ratio for the “left” side of the substrate. Data are expressed as mean ± standard error (n=30). Statistical significance is indicated by different alphabetic characters.

Referring to FIG. 13 , a permanent magnet was continuously positioned on the lower left side of the substrate (magnetic field “MF”) for 24 hours. A group with no magnets near the substrate for 24 h was also included (No magnetic field “NMF”). In addition, the present invention included a group (“NMF-MF”) in which no magnet was placed nearby during the initial 12 hours, but the left side magnet of the substrate was placed after 12 hours. It was found that after 24 h, the “MF” group exhibited significantly greater macrophage adhesion on the left side (magnet side) than the “NMF” group, especially with a high adherent cell area of 63% and an aspect ratio of 66% (Fig. 13a-b). ). This trend was also reflected in the “NMF-MF” group, which exhibited low levels of macrophage adhesion at 12 h without magnets, but markedly macrophages 24 h after magnet-mediated attraction to the left (magnet) of the slidable nanoligand. Adhesion was observed. When these slidable nanoligands are attracted to the magnet (left) side, they appear to remain after the magnet is removed after 12 h, as evidenced by maintaining macrophage adherence after 24 h in the “MF-NMF” group. This dramatic time-controlled switching of nanoligand sliding was effective in regulating macrophage adhesion on the left (magnetic side), but looking at Figure 14, it was not effective in regulating macrophage adhesion on the right (non-magnetic side), Confirm the magnet-mediated in situ control of nanoligand sliding.

In addition, the present invention tested the effect of spatiotemporal tuning of nanoligand sliding on the reversible attachment of macrophages. Referring to FIG. 15 , a permanent magnet was disposed on the lower left side of the substrate to attract the slidable nanoligand to the left for 12 hours. Then, the position of the magnet was maintained at the bottom right of the substrate for 12 hours and then at the bottom left for 12 hours. Time-resolved macrophage adhesion corresponding to the left and right was investigated (Fig. 15a). Referring to FIG. 15 , it can be seen that temporal regulation of nanoligand sliding controls the reversible adhesion of macrophages. At 12 h, the left side of the substrate showed a significant increase in the area of adherent macrophages by 60% and the aspect ratio by 44% compared to the right side (Fig. 15a-b). At 24 hours, the area and aspect ratio of adherent macrophages were slightly higher on the right side of the substrate than on the left side, 67% and 70%, respectively. After 36 h, reversibly, the left side of the substrate exhibited higher density, cell area and aspect ratio of adherent macrophages than the right side by 51%, 67% and 68%, respectively. Thus, this remote and spatiotemporal regulation of ninoligand distribution on the macroscale represents a strong control in the reversible regulation of macrophage adhesion.

Experimental Example 5

To confirm that the slidable nanoligand according to the present invention regulates the adhesion-dependent polarization of macrophages through time-controlled tuning, the following experiment was conducted, and the results are shown in FIGS. 16 to 20 It was.

Dynamic ECM remodeling exhibits a temporally diverse heterologous ligand distribution, which regulates the development of adherent structures of macrophages that regulate their functionally polarized phenotypes, thereby spatially and temporally regulating the host response to implants. Macrophages that give rise to cytoskeletal actin assembly and elongated conformational attachment structures are known to be functionally activated to the regenerative M2 phenotype. These previous reports suggest that previous findings suggest that temporal modulation of nanoligand presentation on the macroscale may alter the adhesion-dependent polarization of macrophages.

Figure 16 (a) is the M2 phenotypic markers (Arginase-1 and Ym1) for macrophages cultured in M2 polarized medium for 36 hours in the “left and right” of the substrate performed with a magnet located on the “left” of the substrate. ) or a graph showing quantitative gene expression of M1 phenotypic markers (iNOS and TNF-α) for macrophages cultured under M1 polarization medium. Data are expressed as mean ± standard error (n=3). (bc) Arg-1 and Arg-1 of macrophages cultured by placing the permanent magnet at the bottom of the substrate on the “left” [(magnetic field “MF”)] or not [(no magnetic field “NMF”)] Confocal images of immunofluorescence for iNOS in the nucleus. “MF” or “NMF” was applied during an incubation period of 36 hours or after 12 hours. Images of polarized macrophages were taken from the center of the “left” or “right” of the substrate. The scale bar is 20 μm. Other alphabetic characters indicate statistical significance.

17 is a graph showing the M2 phenotype of macrophages in M1 polarizing medium of a slidable nanoligand according to an embodiment of the present invention. Ineffective control of the M2 phenotype of macrophages under slidable nanoligands in M1 polarizing medium. M2 markers for macrophages cultured in M1-polarized medium (Arginase-1 and Quantitative gene expression of Ym1) is shown. Data are expressed as mean ± standard error (n=3).

18 is a graph showing the M1 phenotype of macrophages in M2 polarizing medium of a slidable nanoligand according to an embodiment of the present invention. Control of the M1 phenotype of macrophages is inefficient under slidable nanoligands in M2 polarized medium. M1 markers (iNOS and TNF-) for macrophages cultured in M2 polarized medium on the “left” and “right” of the substrate exposed to a permanent magnet placed on the “left” bottom of the substrate (the “magnet” side) for 36 h. The gene expression profile of α) is shown. Data are expressed as mean ± standard error (n=3).

19 is a result of the M2 phenotype experiment of macrophages for the miraculous attraction to the magnetic attraction of the slidable nanoligand according to an embodiment of the present invention. The magnetic attraction of the slidable nanoligand promotes ROCK2 expression when macrophage adhesion structures arise, thus promoting the M2 phenotype. After culturing macrophages in basal medium or M2 polarized medium for 36 hours in “magnet” and “non-magnet” of the substrate exposed to a permanent magnet placed on the “left” bottom of the substrate (the “magnet” side), ROCK2 and Confocal images of immunofluorescence staining for nuclei are shown. The scale bar is 20 μm.

20 is a confocal image of (a) immunofluorescence and (b) density, cell area and cell aspect ratio of adherent macrophages or cultured in M2 medium of a magnetic attraction regulation experiment of a nanoligand according to an embodiment of the present invention. It is a graph showing the calculation of the area, aspect ratio, and Arg-1 staining intensity afterward. Specifically, magnetic manipulation of slidable nanoligand attraction promotes the assembly of adherent structures in macrophages that stimulate the M2 phenotype. (a) Phenotypes on the “magnetic” and “non-magnet” side of the substrate, performed with a magnet located at the bottom of the substrate (the “magnetic” side), were ROCK inhibition (as in 50 μM Y27632), myosin II inhibition (10 μM blevista) Incubate macrophages in M2-polarized medium for 36 h under inhibition of actin polymerization (as in 2 µg/mL cytochalasin D) or actin polymerization inhibition (as in 2 µg/mL cytochalasin D) followed by culturing macrophages in basal medium for 36 h with Arg-1 for actin and nuclei. Confocal images of immunofluorescence for actin and nuclei after incubation. The scale bar is 20 μm. (b) Calculation of the density, cell area and cell aspect ratio of adherent macrophages after basal medium culture on the “magnetic” and “non-magnetic” side of the substrate or area, aspect ratio and Arg-1 staining intensity after culture in M2 medium is shown. Data are expressed as mean ± standard error (n=30). Statistical significance is indicated by different alphabetic characters.

Referring to FIG. 16 , it can be seen that the time-controlled magnetic attraction of the slidable nanoligand stimulates the M2 phenotype while suppressing the M1 phenotype of macrophages. The phenotypic presentation of macrophages in time-controlled manipulation of slidable nanoligands was examined by examining a substrate in which the slidable nanoligand was present and the substrate was continuously placed with a permanent magnet on the lower left side of the substrate for 36 h (“MF” group). and were not placed near the substrate continuously for 36 hours (“NMF” group), or switched from “NMF” to “MF” at 12 hours. After culturing macrophages in M1 polarized medium, gene expression profiles were obtained from the “MF” group, the M1 markers iNOS (inducible nitric oxide synthase) and TNF-α (tumor necrosis factor-α, tumor necrosis). factor-α) was significantly lower at 192% and 231%, respectively, on the left side (magnetic side) than on the right side (non-magnetic side) (FIG. 16a). In contrast, after culturing in M2 polarized medium in the “MF” group, the expression of the M2 markers Arg-1 (arginasae-1) and Ym1 (chitinase-like 3) was higher on the left side (magnetic side) than on the right side (non-magnetic side), respectively. increased significantly to 715% and 383%. Referring to FIGS. 17 and 18 , in the “MF” group, the expression of either the M1 marker after culturing in M2 polarized medium or the M2 marker after culturing in M1 polarized medium did not differ between the left and the right, which is a nanoligand for regulating macrophage polarization. It represents the necessary condition for polarizable lysis stimulation with self-control of sliding.

Referring to Figures 16b-c, confocal images of immunofluorescence provided discovery of gene expression profiles. In the “MF” group, iNOS fluorescence was significantly lower after incubation in M1 polarized medium, but Arg-1 immunofluorescence was significantly higher after incubation in M2 polarized medium on the left side (magnetic side) compared to the right side (non-magnetic side). . This trend was also consistent in the “NMF-MF” group, which showed low iNOS immunofluorescence after M1 polarization medium culture, but high Arg-1 immunofluorescence after M2 polarization medium culture on the magnet side, which activates M2 polarization. This suggests that the slidable nanoligand can be attracted towards the magnet at any defined time. In the “NMF” group, no significant differences were observed in iNOS and Arg-1 immunofluorescence between both sides. Through this, it can be seen that the magnetic attraction of the slidable nanoligand, which improved the development of a pervasive attachment structure on the magnet side, inhibited inflammatory M1 polarization while polarizing macrophages to a regenerating M2 phenotype.

Next, we experimented with a method for facilitating polarization to the M2 phenotype by magnetic attraction of a slidable nanoligand in which the assembly of adherent structures in macrophages is facilitated. Referring to FIG. 19 , it is known that the elongated shape, the organ and contractility of the actin skeleton, and the adhesion structure of macrophages including ROCK mediate the M2 phenotype. In basal medium and M2 polarized medium cultures, macrophages displayed significantly higher ROCK2 immunofluorescence on the magnetic side than on the non-magnetic side. In particular, referring to FIG. 20, during culture in M2 polarized medium, ROCK inhibition by Y27632 significantly reduced adherent macrophage area and aspect ratio by 32% and 33%, respectively, as well as Arg-1 immunofluorescence by 29% on the magnet side. decreased. Consistently, the area, aspect ratio and Arg-1 immunofluorescence of adherent macrophages under myosin II inhibition by blavistatin decreased by 33%, 31% and 29% on the magnet side, respectively, during culture in M2 polarized medium. Reductions in area, aspect ratio and Arg-1 immunofluorescence of adherent macrophages were also clearly observed by inhibition of actin polymerization by cytochalasin D. On the non-magnetic side, inhibition of ROCK, myosin II actin polymerization did not result in significant changes in the adhesion structure and production of the M2 phenotype. Taken together, these results suggest that the slidable nanoligand effectively promotes the assembly of the adherent structure of macrophages in promoting the development of the M2 phenotype through self-regulation.

Experimental Example 6

The following experiments were performed to confirm that the slidable nanoligand according to the present invention spatially regulates the attachment and phenotype of host macrophages in vivo through in situ control, and the results are shown in FIGS. 21 to 23 It was.

Material implants are of paramount importance to control the adherent and functional phenotype of macrophages to elicit a host response and elicit a host response to the implant. Remote control of spatiotemporal, reversible and macroscale nanoligand mutations can mimic dynamic and heterogeneous ECM remodeling to spatially modulate host responses to implants. In particular, it is very advantageous to mediate tissue repair while suppressing inflammation by regulating macrophage adhesion and regeneration and production of the anti-inflammatory M2 phenotype. To this end, the present invention explored the in vivo translation of the adherent structure assembly-mediated M2 phenotype of adherent macrophages in spatially diverse regulation of regenerative and anti-inflammatory immune responses to implants. Recently, it has been shown that spatially non-uniform regulation of cell adhesion by UV light is possible. However, in the present invention, we used a tissue permeability and cell compatibility alternative using magnetic fields to modulate macrophage adhesion as well as the functional phenotype of adherent macrophages.

Figure 21 is the result of the host macrophage adhesion and inflammatory M1 phenotype experiments in vivo against the magnetic attraction of the slidable nanoligand according to an embodiment of the present invention: (a) is a slidable nanoligand in vivo It is a schematic diagram of the self-control of After subcutaneous implantation, both IL-4 and IL-13 were injected onto a substrate having a slidable nanoligand substrate. (b) is a confocal image of immunofluorescence after 24 h for iNOS, F-actin and nuclei of cells attached to the “magnetic” and “non-magnetic” sides of the substrate. A permanent magnet was continuously placed on the bottom of the substrate (the "magnet" side) for 24 hours. Images of cells were taken from the center of the "magnetic" or "non-magnetic" side of the substrate. The scale bar is 20 μm. (c) is a graph showing the calculation of density, cell area and cell aspect ratio (n=30) as well as gene expression profile (n=3) of M1 phenotypic markers (iNOS and TNF-α) of adherent cells in vivo. Data are presented as mean ± standard error. Other alphabetic characters indicate statistical significance.

22 is an in vivo adhesion test result of host neutrophils to the slidable nanoligand according to an embodiment of the present invention. (a) shows the slidable nanoligands 24 hours after subcutaneous injection of IL-4 and IL-13 on the substrate. NIMP-R14, F of host cells attached to the “magnetic” and “non-magnetic” sides of the substrate -Confocal images of immunofluorescence staining for actin and nuclei. A permanent magnet was continuously placed on the bottom of the substrate (the “magnet” side) for 24 hours. Images of cells were taken from the center of the “left” or “right” of the substrate. The scale bar is 20 μm. (b) is a graph showing the quantification of the density of attached NIMP-R14-positive host neutrophils in vivo. Data are expressed as mean ± standard error (n=30). The different alphabetic characters represent statistically significant differences.

23 shows the results of the M2 phenotype test for adhesion and regeneration of host macrophages in vivo for magnetic attraction of a slidable nanoligand according to an embodiment of the present invention: ((a) IL-4 and immunofluorescence for Arg-1, F-actin and nuclei of cells attached to the “magnetic” and “non-magnetic” sides of the substrate with slidable nanoligands 24 hours after subcutaneous implantation by injecting IL-13 onto the substrate. is a confocal image of. A permanent magnet is continuously placed on the bottom (“magnet” side) of the substrate. The image of the cell is taken from the center of the “magnetic” side or “non-magnet” side of the substrate. The scale bar is (b) Gene expression profiles (n=3) of the M2 phenotypic markers (Arg-1 and Ym1) of adherent cells in vivo, as well as calculations of density, cell area and cell aspect ratio (n=30). Figure 4b shows (c) the ratio of nuclear to cytoplasmic YAP fluorescence under ROCK inhibition (with Y27632) and (d) the graph of calculating the cell area of myosin II inhibition (with blevisstatin). It is expressed as ± standard error Different alphabetic letters indicate statistical significance.

Referring to FIG. 21 , the nanoligand-presenting substrate was used for subcutaneous implantation in balb/c mice. After implantation, M2 polarized soluble factors (interleukin-4 and -13) were injected into the substrate (Fig. 21a). A permanent magnet was attached to the bottom of the substrate (“magnet” side) from the ventral side of the mouse to facilitate sliding of the nanoligand toward the magnet side of the substrate. The substrates were collected after 24 h for confocal imaging of immunofluorescence and gene expression analysis. Confocal images of immunofluorescence for iNOS and F-actin showed that distinct colocalization was more prevalent on the non-magnetic side than on the magnetic side (Fig. 21b). At the same time, superior development of adherent structures with significantly higher density, area and aspect ratio (67%, 36% and 53%, respectively) of macrophages on the magnet side was observed, indicating that both iNOS and TNF in host macrophages than on the non-magnetic side. was promoted to a remarkably low expression of 33% and 60%, respectively (FIGS. 21b-c).

Referring to FIG. 22 , host neutrophils as well as host macrophages were observed through NIMP-R14 immunofluorescence during acute inflammation. At the same time, referring to FIG. 23A , confocal images of immunofluorescence for Arg-1 and F-actin showed a very distinct assembly of attachment structures by F-actin, indicating that the magnetic side was much more numerous than the non-magnetic side. It resulted in colocalization with regenerating Arg-1 expression. Referring to FIGS. 23A-B , it was also confirmed that both Arg-1 and Ym1 (regenerative M2 marker) were significantly higher in expression of 27% and 60% in host macrophages on the magnet side compared to the non-magnet side. This suggests that remote control through magnetic attraction of slidable nanoligands promotes the regenerative M2 phenotype of host macrophages, but suppresses the inflammatory M1 phenotype in vivo in a spatially controlled manner. Tight control of early acute inflammatory and regenerative responses is known to govern long-term host responses to implants. Experiments on magnetic field-mediated spatiotemporal and reversible organ regulation in promoting tissue repair and inhibiting inflammation and fibrous capsule formation in deep internal tissues suggest that immunomodulation of implants is possible in a clinical environment.

In the above experimental example, the adhesion and phenotype of macrophages in culture under in situ control of nanoligand sliding were tested as follows. The effect of nanoligand sliding in vitro on the adhesion and phenotype of macrophages was investigated. Substrates with nanoligands were sterilized under UV light illumination for 1 hour. Sterile substrates were blocked with 1% bovine serum albumin (BSA) to minimize non-nano-ligand-specific adhesion of macrophages. 50 k cells/cm2 of macrophages (RAW 264.7, passage 5, ATCC) were plated on the treated substrate. Macrophages were basal with 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. Cultured in medium. Cells were applied to a magnet (270 mT) placed on the bottom of the substrate (“magnet” or “left”) and their adhesion was examined at the center of the various sides (magnetic, middle and non-magnetic side). Nanoparticles without RGD peptide ligands or nanoligands or substrates with nanoligands without application of magnets were used as controls. Adherence of macrophages under temporal manipulation of slidable nanoligands can be achieved by placing a permanent magnet on the bottom of the substrate [“magnetic field (MF)”] or withdrawing the permanent magnet from the substrate [“no magnetic field (NMF)”]. was tested with a substrate in which Temporal switching of the permanent magnet was performed between the two opposite sides from the bottom of the “left” of the substrate to the bottom of the “right” and then back again to the bottom of the “left”. The polarization phenotype of macrophages under time-controlled manipulation of slidable nanoligands is dependent on placing a permanent magnet on the bottom of the substrate [“magnetic field (MF)”] or withdrawing the permanent magnet from the substrate [“no magnetic field (NMF)”]. Substrates in which nanoligands that are dependent on "NMF" and "MF" were tested were used. M1 polarization medium contained 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). Adherent structure assembly-mediated M2 phenotype of macrophages substrates under ROCK inhibition (as 50 μM Y27632), myosin II inhibition (as 10 μM blevisstatin) or actin polymerization inhibition (as 2 μg/mL cytochalasin D) After applying to the magnet placed on the bottom (“magnet” side) of the

In addition, macrophage adhesion analysis under in situ sliding nanoligand by immunofluorescence and confocal imaging was tested as follows.

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

In addition, macrophage polarization analysis under an in situ sliding nanoligand having a reverse transcription polymerase chain reaction was tested as follows.

Macrophages were cultured in either M1 or M2 polarized media and harvested in 1 mL of Trizol per sample. The substrate was split into two halves (magnetic and non-magnetic side). For each sample, 900 ng of extracted RNA was reverse transcribed into cDNA using a high capacity RNA-to-cDNA kit. Real-time PCR reactions (StepOne Plus Real-Time PCR System, Applied Biosystems) were run using cDNA with Sybr Green assay. After normalization for GAPDH expression, relative fold expressions of target genes (iNOS, TNF-α, Arginase-1, and Ym1) were presented.

In addition, in vivo remote control of nanoligand sliding to regulate the adherence and phenotype of host macrophages was tested as follows.

To investigate the effect of nanoligand sliding in vivo on the adherence and phenotype of host macrophages, nanoligand-presenting silicon substrates were used for subcutaneous implantation. Two-month-old balb/c mice approved by the Korea University Animal Care and Use Committee were used. A mixture of 2 mL of alfaxan and 1 mL of rompun was used for intraperitoneal injection. The dorsal side of the mice was incised with a length of approximately 2.5 cm. After injection, IL-4 (50 ng) and IL-13 (50 ng) were injected onto the substrate. The adhesion of mouse macrophages to the substrate was tested. A permanent magnet (“magnet” side) attached to the bottom (ventral side) of the substrate promoted the sliding of the nanoligand toward the magnet. Twenty-four hours after implantation, the substrates were recovered for confocal imaging of immunofluorescence and RT-PCR.

Claims (12)

a core comprising magnetic nanoparticles; and a coating layer provided to surround the core and comprising an integrin ligand peptide,
The integrin ligand peptide is a nanoligand for promoting cell adhesion and regeneration of macrophages, characterized in that negatively charged. According to claim 1,
The magnetic nanoparticles are nanoligands for promoting cell adhesion and regeneration of macrophages, characterized in that silica is bound to the surface. According to claim 1,
Further comprising a linker connecting the core and the coating layer,
The linker is a polyethylene glycol (PEG)-based linker, a nanoligand for promoting cell adhesion and regeneration of macrophages. According to claim 1,
The nanoligand has a diameter of 30 nm to 60 nm, and the magnetic nanoparticles have a diameter of 5 nm to 30 nm. A nanoligand for promoting cell adhesion and regeneration of macrophages. According to claim 1,
wherein the integrin ligand peptide comprises a negatively charged thiolated integrin ligand peptide;
A nanoligand for promoting cell adhesion and regeneration of macrophages, wherein the surface of the nanoligand is provided in a negatively charged form. Preparing a core containing magnetic nanoparticles;
preparing a core to which a linker is bound by mixing the core with a first suspension containing a linker; and
Nano for promoting cell adhesion and regeneration of macrophages according to any one of claims 1 to 5, including the step of mixing the linker-bound core with a second suspension containing an integrin ligand peptide (RGD) A method for preparing a ligand. A method for promoting cell adhesion and regeneration of macrophages according to any one of claims 1 to 5, comprising: preparing a nanoligand presentation substrate by supporting a surface activated substrate with a solution containing the nanoligand; and
A method for promoting cell adhesion and regeneration of macrophages, comprising the step of controlling the cell adhesion and regeneration of macrophages by applying an external magnetic field after treating the culture medium to the nanoligand presentation substrate. 8. The method of claim 7,
The manufacturing of the nanoligand presentation substrate comprises:
immersing the surface of the substrate in an acidic solution;
activating the substrate surface by immersing the immersed substrate in an aminosilane solution; and
A method for promoting cell adhesion and regeneration of macrophages comprising; treating the activated substrate using ultrasound at room temperature. 8. The method of claim 7,
A method for promoting cell adhesion and regeneration of macrophages, characterized in that the surface-activated substrate is activated by supporting the substrate in an aminosilane solution. 8. The method of claim 7,
The step of controlling the cell adhesion and regeneration of macrophages is performed by applying a magnetic field of 100 to 700 mT for 12 hours to 48 hours after positioning the nanoligand presentation substrate in vivo and in vitro to promote cell adhesion and regeneration of macrophages. Way. 8. The method of claim 7,
The step of controlling the cell adhesion and regeneration of macrophages is a method for promoting cell adhesion and regeneration of macrophages performed by changing the position of a magnetic field applied to the substrate. 8. The method of claim 7,
The step of controlling cell adhesion and regeneration of macrophages is a method for promoting cell adhesion and regeneration of macrophages, wherein the position of a magnetic field applied to a substrate is changed with time.

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