KR-102959277-B1 - magnetic bar-substrate complex to control adhesion and differentiation of stem cell and method for preparing thereof

Abstract

The present invention relates to a magnetic rod-substrate complex for controlling the attachment and differentiation of stem cells, comprising: a substrate portion having thiol groups formed thereon; a magnetic rod portion having magnetism and moving upon the application of a magnetic field; a first ligand portion having one or more ligands connected to the magnetic rod portion and including a ligand; a first linker portion connecting the magnetic rod portion and the first ligand portion by being coupled to a portion of the surface of the magnetic rod portion; a second linker portion connecting the substrate portion and the magnetic rod portion by being coupled to a portion of the thiol groups; and a second ligand portion including a ligands coupled to a portion of the thiol groups; wherein the ligands of the first ligand portion and the second ligand portion are ligands that can be detected by stem cells.

Inventors

• 강희민
• 탕감 라마
• 김성열
• 민선홍
• 김강현
• 홍현식

Assignees

• 고려대학교 산학협력단

Dates

Publication Date

20260507

Application Date

20230104

Claims (20)

1. A substrate portion on which thiol groups are formed; A magnetic rod part that possesses magnetism and moves according to the application of a magnetic field; A first ligand portion comprising one or more ligands connected to the magnetic rod portion above and ligands; A first linker portion that connects the magnetic rod portion and the first ligand portion by being coupled to a part of the surface of the magnetic rod portion; A second linker portion that connects the substrate portion and the magnetic rod portion by combining with a portion of the thiol group; and Gold particles combined with at least a portion of the above thiol groups; and A second ligand portion comprising the ligand that binds to the gold particle and includes the ligand; The ligands of the first and second ligand portions are ligands that can be detected by stem cells, and The above magnetic rod portion is, Silica is coated on the surface of a rod-shaped ferric oxyhydroxide, the silica-coated ferric oxyhydroxide is reduced to form magnetite, and amino groups are introduced on the surface of the magnetic rod. A compound of Formula 1 below is bonded to the amino group to have a first linker part or a second linker part, and At least one of the first linker part and the second linker part has a flexible characteristic, The above silica-coated iron oxyhydroxide has an average d-spacing of adjacent lattice stripes of atoms in the (200) plane of 5.23 Å, and The above silica-coated magnetite has an average d-spacing of adjacent lattice stripes of atoms in the (220) plane of 3.07 Å, Magnetic rod-substrate complex for regulating stem cell adhesion and differentiation. [Equation 1] Here, n is 10 to 1000, and R1 and R2 are each one of hydroxyl, amine, thiol, maleimide, methoxy, azide, carboxylic acid, acrylate, cyanine, n-hydroxysuccinimide, aldehyde, acrylamide, epoxide, hydrazide, halide, methacrylate, and silane.
2. In paragraph 1, Controlling the attachment and differentiation of stem cells by applying a magnetic field to control the movement of the magnetic rod portion. Magnetic rod-substrate complex for regulating stem cell adhesion and differentiation.
3. In paragraph 1, The length of the magnetic rod portion is 600 nm to 800 nm, Magnetic rod-substrate complex for regulating stem cell adhesion and differentiation.
4. In paragraph 1, The maximum width of the magnetic rod portion is 80 nm to 130 nm, Magnetic rod-substrate complex for regulating stem cell adhesion and differentiation.
5. In paragraph 1, The magnetic rod portion has a density of 55 particles/ 100μm² to 70 particles/ 100μm² , Magnetic rod-substrate complex for regulating stem cell adhesion and differentiation.
6. In paragraph 1, The second ligand portion has a density of 25 particles/ μm² to 45 particles/ μm² , Magnetic rod-substrate complex for regulating stem cell adhesion and differentiation.
7. In paragraph 1, The above ligand is a CDDRGD ligand, Magnetic rod-substrate complex for regulating stem cell adhesion and differentiation.
8. In paragraph 1, The diameter of the gold particles is 15 nm to 30 nm, Magnetic rod-substrate complex for regulating stem cell adhesion and differentiation.
9. delete
10. In paragraph 1, The lengths of the first linker part and the second linker part are formed to be longer than the height of the second ligand part, Magnetic rod-substrate complex for regulating stem cell adhesion and differentiation.
11. In paragraph 1, By applying a magnetic field, the movement of the magnetic rod portion is restricted, and Moving the magnetic rod portion toward the substrate portion to increase the density of the ligand, thereby promoting the attachment and differentiation of stem cells. Magnetic rod-substrate complex for regulating stem cell adhesion and differentiation.
12. In paragraph 1, Since no magnetic field is applied, the magnetic rod part moves freely, and Lowering the density of the above ligand to inhibit stem cell attachment and inhibit differentiation, Magnetic rod-substrate complex for regulating stem cell adhesion and differentiation.
13. A method for manufacturing a magnetic rod-substrate composite of any one of claims 1 to 8 and claims 10 to 12, A step of cleaning the substrate to remove impurities and then introducing thiol groups to the surface; A step of incubating the substrate portion into which thiol groups have been introduced in a solution containing gold particles to bind gold particles to a portion of the thiol groups; A step of bonding the second linker portion of a magnetic rod portion comprising a first linker portion and a second linker portion to at least a portion of the thiol group to which the gold particle is not bonded; A step of incubating in a solution containing a ligand to bind the ligand to the gold particles to form a second ligand portion, and binding the ligand to the first linker portion to form a first ligand portion; and The method comprising the step of inactivating thiol groups that are not bonded to the gold particles or magnetic rods. Method for preparing a magnetic rod-substrate composite for controlling the attachment and differentiation of stem cells.
14. In Paragraph 13, The above magnetic rod portion is, A step of coating the surface of the iron oxyhydroxide with silica by washing the rod-shaped iron oxyhydroxide and then stirring it in a silica-containing solution; A step of reducing silica-coated iron oxyhydroxide to form silica-coated magnetite; A step of introducing amino groups to the surface of the magnetic rod; and A step comprising: bonding a compound of Formula 1 below to the amino group to form a first linker portion or a second linker portion; Method for preparing a magnetic rod-substrate composite for regulating the attachment and differentiation of stem cells; [Equation 1] Here, n is 10 to 1000, and R1 and R2 are each one of hydroxyl, amine, thiol, maleimide, methoxy, azide, carboxylic acid, acrylate, cyanine, n-hydroxysuccinimide, aldehyde, acrylamide, epoxide, hydrazide, halide, methacrylate, and silane.
15. In Paragraph 13, Controlling the density of the first ligand portion by controlling the concentration of the solution containing the gold particles, Method for preparing a magnetic rod-substrate composite for controlling the attachment and differentiation of stem cells.
16. delete
17. A magnetic rod-substrate complex for controlling the attachment and differentiation of stem cells according to either Article 1 or Article 12, and a magnetic field for controlling the attachment of stem cells to control differentiation, Method for controlling stem cell adhesion and differentiation.
18. In Paragraph 17, A magnetic field is applied to one surface of the substrate to pull the magnetic rod portion in the direction of the substrate, thereby restricting the movement of the magnetic rod portion, and By forming the distance between the first ligand portion and the second ligand portion close to increase the density of the ligand, the cell attachment and differentiation are promoted. Method for controlling stem cell adhesion and differentiation.
19. In Paragraph 17, The magnetic rod part moves freely when the above magnetic field is not applied, and Lowering the density of the above ligand to inhibit cell adhesion and inhibit differentiation, Method for controlling stem cell adhesion and differentiation.
20. In Paragraph 17, The above magnetic field is applied at 200mT to 350mT, Method for controlling stem cell adhesion and differentiation.

Description

Magnetic bar-substrate complex to control adhesion and differentiation of stem cell and method for preparing thereof The present invention relates to a magnetic rod-substrate composite for controlling the attachment and differentiation of stem cells and a method for manufacturing the same, and more specifically, to a technology for controlling the attachment and differentiation of stem cells by applying a magnetic field to the magnetic rod-substrate composite to control the magnetic rod. Physical screens occurring in the extracellular matrix (ECM) separate various compartments of tissues to aid in regulating homeostasis and tissue regeneration by controlling biomolecule transport and cell infiltration. Certain tissues can act as physical screens to regulate tissue repair mechanisms involving the interactions of various cells. However, artificial materials that mimic the ECM and can dispersively and dynamically regulate bioactive surfaces are uncommon. Integrins dynamically form connections with the ECM where bioactive ligands are expressed, and RGD ligands mediate focal adhesion and mechanotransduction of cells. Light or magnetic fields can regulate cell adhesion by remotely controlling ligand blockade, and conventionally, light such as ultraviolet (UV), visible light, and near-infrared (NIR) has been used to photochemically control ligand blockade. For example, UV has been applied to chemically degrade photosensitive polyethylene glycol-based brushes that control the blockade of gold nanoparticles grafted with ligands to activate cell adhesion. Using photoisomers such as azobenzene derivatives, ligand blockade via self-assembled brushes can be controlled by illuminating with UV and visible light or a single wavelength capable of stimulating mechanotransduction. Furthermore, magnetic fields have the characteristic of easily penetrating tissues within the body, enabling non-invasive control of physical screens. For example, cell adhesion can be remotely controlled by manipulating ligand density through the control of particles with magnetic properties. The prior art study *Anisotropic Ligand Nanogeometry Modulates the Adhesion and Polarization State of Macrophages* (Nano Lett. 2019, 19, 3, 1963-1975) discloses the regulation of macrophage adhesion, but differs in that it discloses a technique regarding macrophages and does not induce cell adhesion by regulating magnetic particles and ligands. FIG. 1 is a schematic diagram illustrating the process of manufacturing a magnetic rod-substrate composite according to one embodiment of the present invention and controlling the attachment and differentiation of stem cells by controlling the manufactured magnetic rod-substrate composite. FIG. 2 schematically illustrates the process of manufacturing a magnetic rod-substrate composite capable of controlling the attachment and differentiation of stem cells according to one embodiment of the present invention. Figure 3 shows a TEM image of a bare UMLN according to one embodiment of the present invention. Figures 4 to 6 show the characteristics of bare UMLN, UMLN, and MLN according to one embodiment of the present invention. Figure 7 analyzes the characteristics of gold nanoparticles of different sizes manufactured according to one embodiment of the present invention. Figures 8 and 9 show the zeta potential measured for MLN after amiro-silica coating, PEGylation, and RGD ligand coating, and for gold nanoparticles after citrate capping and RGD coating, according to one embodiment of the present invention. FIG. 10 is the result of an experiment to see if movement is controlled by applying a magnetic field to a substrate combined with a magnetic rod and gold particles according to one embodiment of the present invention. FIG. 11 shows the strength of the magnetic field exerted according to the distance of the magnet in accordance with one embodiment of the present invention. Figure 12 is the result of observing that the height of a magnetic rod changes depending on the application of a magnetic field according to one embodiment of the present invention. Figure 13 is the result of an experiment to see if the height of the magnetic rod changes depending on the application of a magnetic field in an IMLN according to one embodiment of the present invention. FIGS. 14 and 15 show the degree of attachment of stem cells in "Mov.DC.", "Mov.C.", and "Immov.C." states according to one embodiment of the present invention. FIG. 16 is the result of an experiment to determine whether the attachment of stem cells can be controlled solely by the substrate itself, according to one embodiment of the present invention. FIGS. 17 to 19 are the results of experiments on stem cell attachment by attaching ligands only to MLN or gold nanoparticles according to one embodiment of the present invention. Figures 20 and 21 are the results of an experiment to determine whether the attachment of stem cells is affected by the strength of the magnetic field ac