Cell adhesion and migration regulation platform using integrin-mediated single-molecule binding force probe

Abstract

The present invention relates to a cell adhesion and migration regulation platform using an integrin-mediated single-molecule binding force probe. The cell adhesion and migration regulation platform according to an embodiment of the present invention includes: a substrate (10); a binding layer (20) formed on the surface of the substrate (10); a single-molecule force probe (30), which has a predetermined binding force, is adhered to the cell by selectively binding to integrins in the cell membrane, and is ruptured when an external force applied by the cell exceeds the binding force; and a linker part (40) for connecting the binding layer (20) and the single-molecule force probe (30).

Description

A platform for regulating cell adhesion and migration using integrin-mediated single-molecule binding probes.

[0001] The present invention relates to a platform for controlling cell adhesion and movement using an integrin-mediated single-molecule binding force probe, and more specifically, to a platform for controlling the adhesion and movement of cells cultured thereon by differently forming the binding force between an integrin protein and an extracellular matrix protein in a cell membrane through structural changes in a single-molecule binding force probe.

[0002]

[0003] Cells within the human body are connected to the extracellular matrix (ECM), which composes tissues, through membrane proteins called integrins. As it becomes known that this connection transmits changes in the physical properties and stimuli outside the cell to the inside of the cell, thereby perceiving physical changes (mechanosensing) and regulating cellular function (mechanoregulation), the importance of integrin-mediated coupling between cells and their surroundings is growing. Changes in the binding between integrin proteins and ECM proteins control cell morphology and the activation status of cellular organelles, directly influencing fundamental cellular physiological responses such as cell adhesion, migration, and differentiation.

[0004] Cell adhesion is an essential element for the survival and function of adherent cells and tissues that make up most of the tissues, and cell migration is a key physiological response in understanding various physiological phenomena such as tissue damage recovery, immune response, and cancer metastasis.

[0005]

[0006] The purpose of the present invention is to provide a platform for controlling cell attachment and cell mobility by coating a single-molecule force probe on the surface of a substrate, which attaches to cells via integrins and has binding strength controlled at the pN level.

[0007]

[0008] A cell adhesion and movement control platform according to an embodiment of the present invention comprises: a substrate; a bonding layer formed on a surface of the substrate; a single-molecule force probe having a predetermined bonding strength, selectively binding to an integrin in a cell membrane to attach to a cell, and rupturing when an external force applied by the cell exceeds the bonding strength; and a linker portion connecting the bonding layer and the single-molecule force probe.

[0009] In addition, in the cell adhesion and movement control platform according to an embodiment of the present invention, the single-molecule force probe may include a central portion formed of a polymer having the binding force and ruptured by the external force; a ligand connected to the central portion and specifically binding to the integrin; and a linker binding portion connected to the central portion and binding to the linker portion.

[0010] In addition, in the cell adhesion and movement control platform according to an embodiment of the present invention, the central portion may include a first single-stranded DNA to which the ligand is connected at one of the ends; and a second single-stranded DNA to which the linker binding portion is connected at one of the ends and which is complementarily connected to the first single-stranded DNA.

[0011] In addition, in the cell attachment and movement control platform according to an embodiment of the present invention, the binding force can be controlled depending on the binding position of the two ends of the second single-stranded DNA to which the linker binding portion is bound.

[0012] Additionally, in the cell adhesion and migration control platform according to an embodiment of the present invention, the ligand may include a Cyclo RGDfk ligand.

[0013] Additionally, in the cell adhesion and migration control platform according to an embodiment of the present invention, the linker binding portion may include biotin.

[0014] In addition, in the cell adhesion and movement control platform according to an embodiment of the present invention, the linker portion may include a first linker moiety that binds to biotin, including at least one selected from the group consisting of avidin, streptavidin, and neutravidin; and a second linker moiety that includes biotin at one end and is bound to the first linker moiety, and has the other end bound to the binding layer through an antigen-antibody reaction.

[0015] Additionally, in the cell adhesion and migration control platform according to an embodiment of the present invention, the binding layer may include fibronectin.

[0016]

[0017] The features and advantages of the present invention will become more apparent from the following detailed description based on the attached drawings.

[0018] Prior to this, the terms and words used in this specification and claims should not be interpreted in their usual or dictionary meanings, but should be interpreted in the sense and concept that conforms to the technical idea of ​​the present invention based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0019]

[0020] According to the present invention, cells can be cultured by controlling the intermolecular binding force in the pN unit using a DNA-based single-molecule force probe.

[0021] Additionally, micropatterning of fibronectin allows cells to be attached in a desired shape, allowing for the control of intermolecular bonding in a controlled environment.

[0022] Furthermore, the activity and polarity formation of intracellular organelles can be controlled within the fibronectin-DNA-based single-molecule force probe environment, ultimately controlling cell motility.

[0023]

[0024] FIG. 1 is a drawing illustrating a cell attachment and movement control platform according to an embodiment of the present invention.

[0025] FIG. 2 is a diagram illustrating cell attachment on a cell attachment and migration control platform according to an embodiment of the present invention, wherein (A) is a schematic diagram of a DNA-based single-molecule force probe (12 pN and 56 pN) and a polymer-based single-molecule force probe (198 pN) bound on a glass substrate, and (B) is a schematic diagram of structural changes according to the binding force of the DNA-based single-molecule force probe.

[0026] Figure 3 is an image and graph showing the change in cell diffusion according to the concentration of a single-molecule binding probe. (A) is an image showing the change in cell diffusion according to the concentration of biotin-bound fibronectin antibody and the change in single-molecule binding force, (B) is a graph showing the change in cell size on the surface of a single-molecule binding probe according to the concentration of bound fibronectin antibody, and (C) is a graph showing the change in cell size according to single-molecule binding force on the surface of a single-molecule binding probe to which 1% fibronectin antibody is bound.

[0027] Figure 4 is an image and graph showing changes in cell diffusion on the surface of a single-molecule force probe having a microstructure. (A) is an image showing changes in cell morphology on the surface of a single-molecule force probe in a 1 μm circular structure, (B) is an image showing changes in cell morphology on the surface of a 3 μm circular structure, and (C) is an image showing changes in cell size on the surface of a single-molecule force probe having a micro-circular structure, and (E) is a graph comparing the degree of changes in cell size according to the strength of the single-molecule force probe.

[0028] Figure 5 shows the change in cell polarity on the surface of a single-molecule force probe having a linear microstructure. (A) to (C) are the change in MEF polarity on the surface of a single-molecule force probe coupled to a 20 μm linear structure. (A) is the change in the Cdc42 active site under a 12 pN, (B) is the change in the Cdc42 active site under a 56 pN, and (C) is the change in the Cdc42 active site under a 198 pN single-molecule force probe. (D) is the change in the position of the Cdc42 active site according to the strength of the single-molecule force probe. (E) is the change in the movement of the Cdc42 active site per unit time. (F) to (H) are the change in the polarity of MEF in which FAK phosphorylation was inhibited on the surface of a single-molecule force probe coupled to a 20 μm linear structure. (F) is the change in the Cdc42 active site under a 12 pN, (G) is the change in the Cdc42 active site under a 56 pN, and (H) is the change in the Cdc42 active site under a 198 pN single-molecule force probe. (I) shows the change in the position of the Cdc42 active site according to the intensity of the single-molecule force probe in MEFs with suppressed FAK phosphorylation, and (J) shows the change in the movement of the Cdc42 active site per unit time in MEFs with suppressed FAK phosphorylation.

[0029] Figure 6 shows changes in cell migration on the surface of a single-molecule force probe having a linear microstructure. (A) shows cell migration on the surface of a single-molecule force probe coupled to a 20 μm linear structure, and (B) shows cell migration on the surface of a single-molecule force probe when treated with a FAK phosphorylation inhibitor (PF573228). (C) to (F) show changes in cell mobility according to the strength of the single-molecule force probe and treatment with a FAK phosphorylation inhibitor. (C) shows the total migration distance of the cell, (D) the migration distance in a single direction, (E) the migration time in a single direction, and (F) the migration displacement of the cell during the migration time.

[0030] Figure 7 shows the changes in cell migration on the surface of a single-molecule force probe. (A) to (B) are images of cell migration on the surface of a single-molecule force probe depending on whether FAK phosphorylation was inhibited. (A) is a representative image of normal MEF cell migration on the surface of a single-molecule force probe, and (B) is a representative image of cell migration on the surface of a single-molecule force probe when treated with a FAK activity inhibitor (PF573228). (C) to (F) are quantitative evaluations of cell migration depending on the strength of the single-molecule force probe and whether or not FAK phosphorylation inhibitor was treated. (C) represents the total migration distance of the cell, (D) represents the migration distance in a single direction, (E) represents the migration time in a single direction, and (F) represents the migration displacement of the cell during the migration time.

[0031]

[0032] The objects, specific advantages, and novel features of the present invention will become more apparent from the following detailed description and preferred embodiments taken in conjunction with the accompanying drawings. In this specification, when reference numerals are given to components in each drawing, it should be noted that, as far as possible, identical components are given the same numerals even if they are shown in different drawings. Furthermore, terms such as "first," "second," etc. are used to distinguish one component from another, and the components are not limited by these terms. In the following description of the present invention, detailed descriptions of related known technologies that may unnecessarily obscure the gist of the present invention will be omitted.

[0033] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings.

[0034]

[0035] FIG. 1 is a drawing illustrating a cell adhesion and migration control platform according to an embodiment of the present invention, and FIG. 2 is a drawing illustrating cell attachment in a cell adhesion and migration control platform according to an embodiment of the present invention, in which (A) is a schematic diagram of a DNA-based single-molecule force probe (12 pN and 56 pN) and a polymer-based single-molecule force probe (198 pN) bound on a glass substrate, and (B) is a schematic diagram of structural changes according to the binding force of the DNA-based single-molecule force probe.

[0036] As illustrated in FIGS. 1 and 2, the cell adhesion and movement control platform of the embodiment of the present invention includes a substrate (10), a bonding layer (20) formed on the surface of the substrate (10), a single-molecule force probe (30) having a predetermined bonding strength, selectively binding to integrins in a cell membrane to attach to a cell, and rupturing when an external force applied by the cell exceeds the bonding strength, and a linker portion (40) connecting the bonding layer (20) and the single-molecule force probe (30).

[0037]

[0038] The present invention relates to a platform for regulating cell adhesion and migration using an integrin-mediated single-molecule binding probe. The binding between the extracellular matrix and the cell membrane protein integrin transmits changes in the physical properties and stimuli outside the cell to the inside of the cell, thereby recognizing the physical changes and regulating cell functions. This binding directly influences fundamental physiological responses of the cell, such as cell adhesion, migration, and differentiation, by controlling the cell morphology and the activation state of cell organelles. The present invention was devised to control the adhesion and migration of cells cultured thereon by differently forming the binding force between the integrin protein in the cell membrane and the extracellular matrix protein through a single-molecule binding probe (30).

[0039] Specifically, the cell adhesion and migration control platform according to an embodiment of the present invention includes a substrate (10), a bonding layer (20), a single-molecule force probe (30), and a linker portion (40).

[0040]

[0041] The substrate (10) is a member that fixes or supports the single-molecule force probe (30). Examples of the substrate (10) include a glass substrate, a PDMS substrate, and the like, but are not necessarily limited thereto. The substrate (10) can be made of various materials and may be the surface of a device to be inserted into a living body. In addition, since the shape of the substrate (10) is not particularly limited, a microchannel of a microfluidic chip having microchannels, a micropattern member, and the like may also correspond to the substrate (10).

[0042]

[0043] The bonding layer (20) is a layer that fixes the single-molecule force probe (30) to the substrate (10) via the linker portion (40), and is formed on the surface of the substrate (10). The bonding layer (20) may be formed on the entire surface area of ​​the substrate (10) or only on a portion thereof. At this time, the single-molecule force probe (30) may be concentratedly placed on the area of ​​the substrate (10) where the bonding layer (20) is formed. The bonding layer (20) may be patterned into a predetermined shape, and thus cells may be attached in a desired shape, and cells may be cultured in an environment where the intermolecular bonding force is controlled.

[0044] The bonding layer (20) may include a material that is bonded to the linker portion (40) through various chemical reactions. For example, the bonding layer (20) may include fibronectin and may bind to the linker portion (40) through an antigen-antibody reaction.

[0045]

[0046] A single-molecule force probe (30) is a structure that controls the binding force between a cell and its surrounding environment at the pN level. The single-molecule force probe (30) has a predetermined binding force and selectively binds to integrins in the cell membrane to attach to cells. Cells attached to the single-molecule force probe (30) via integrins can be cultured, moved, and differentiated here. An external force is generated by the attachment and movement of cells, and the external force is transmitted to the single-molecule force probe (30) via integrins. If the external force (tension) exceeds the binding force of the single-molecule force probe (30), the single-molecule probe ruptures.

[0047] The binding force of the single-molecule force probe (30) corresponds to the binding force between integrins and the extracellular matrix. Since the binding force between integrins and the extracellular matrix affects physiological responses of cells, such as cell adhesion, migration, and differentiation, cell adhesion and migration can be controlled by adjusting the binding force of the single-molecule force probe (30).

[0048] A single-molecule force probe (30) may include a central portion (31), a ligand (32), and a linker binding portion (33). The central portion (31) may be formed of a polymer having binding strength and capable of being broken by an external force. The binding strength of the single-molecule force probe (30) is determined by the material, structure, etc. of the central portion (31). The central portion (31) may be formed based on a polymer such as PEG (Polyethylene glycol). Additionally, it may be formed based on DNA. A DNA-based central portion (31) will be described later.

[0049] The ligand (32) is a substance that is connected to the center (31) and specifically binds to integrin. This ligand (32) may include a cyclo RGDfk ligand (32).

[0050] The linker binding portion (33) is connected to the central portion (31) and is combined with the linker portion (40). As a result, the central portion (31) to which the ligand (32) is combined is fixed or supported by the substrate (10).

[0051] The DNA-based core (31) may include a first single-stranded DNA and a second single-stranded DNA that complementarily bind to each other. A ligand (32) may be linked to either end of the first single-stranded DNA, i.e., either the 5th end or the 3rd end. A linker binding portion (33) may be linked to either end of the second single-stranded DNA. Here, the binding strength may be controlled depending on the binding position of the both ends of the second single-stranded DNA to which the linker binding portion (33) is linked. For example, when a ligand (32) is connected to the 5th terminal of the first single-stranded DNA and connected to the integrin at the 5th terminal, a linker binding portion (33) is connected to the 5th terminal of the second single-stranded DNA that is complementarily bound to the first single-stranded DNA and connected to the substrate (10) through the linker portion (40), and when a linker binding portion (33) is connected to the 3rd terminal of the second single-stranded DNA and connected to the substrate (10) through the linker portion (40), the connection structures of the integrin and the substrate (10) are formed differently centered on the DNA-based center (31), resulting in a difference in binding strength. In addition, the binding strength can also be controlled by modifying the type, sequence, etc. of the bases that are complementarily bound between the first single-stranded DNA and the second single-stranded DNA. Accordingly, the intermolecular binding strength can be controlled in the pN unit.

[0052]

[0053] The linker portion (40) connects the binding layer (20) and the single-molecule force probe (30). One end of the linker portion (40) may be connected to the linker binding portion (33) of the single-molecule force probe (30), and the other end may be connected to the binding layer (20). To this end, the linker portion (40) may include a first linker moiety and a second linker moiety. Here, the first linker moiety is connected to the linker binding portion (33), and the second linker moiety connects the binding layer (20) and the first linker moiety. For example, the linker binding portion (33) may include (Biotin), and the first linker moiety may include at least one selected from the group consisting of avidin, streptavidin, and neutravidin, and may be connected to the linker binding portion (33). Here, the second linker moiety includes biotin at one end thereof, so that one end of the second linker moiety can be connected to the first linker moiety. In addition, the other end of the second linker moiety is connected to a binding layer (20), and at this time, the other end of the second linker moiety can be connected to the binding layer (20) through an antigen-antibody reaction. For example, the binding layer (20) may include fibronectin, and the second linker moiety may include a fibronectin antibody coupled to biotin included at one end thereof.

[0054]

[0055] Meanwhile, a plurality of linker portions (40) are arranged on the substrate (10), and a single-molecule force probe (30) can be connected to each linker portion (40). Here, the plurality of single-molecule force probes (30) arranged on the substrate (10) may all have the same binding force, but this is not necessarily limited to this, and they may have different binding forces.

[0056] In addition, by forming a bonding layer (20) at a selective location among the surface areas of the substrate (10), the location of the single-molecule force probe (30) can be modified. That is, cells can be attached in a desired pattern through micro-patterning of the bonding layer (20), and the intermolecular bonding force can be controlled in a controlled environment.

[0057]

[0058] In summary, according to the present invention, cell culture can be achieved by controlling intermolecular binding forces at the pN level using a DNA-based single-molecule force probe. Furthermore, micropatterning of fibronectin allows cells to be attached in a desired shape, thereby controlling intermolecular binding forces in a controlled environment. Furthermore, within the fibronectin-DNA-based single-molecule force probe environment, the activity and polarity formation of intracellular organelles can be controlled, ultimately controlling cell motility.

[0059]

[0060] Below, the present invention is described in more detail through experimental examples.

[0061]

[0062] 1. Fabrication of a single-molecule force probe

[0063] We fabricated DNA-based single-molecule force probes with binding forces of 12 pN and 56 pN. To this end, we prepared one upper single-stranded DNA and two lower single-stranded DNAs with biotin-bound sites at different locations.

[0064] Top single-stranded DNA: 5- / 5Thiol C6 SS / CAC AGC ACG GAG GCA CGA CAC-3

[0065] Bottom single-stranded DNA for 12 pN: 5- / 5'Biotin / GTG TCG TGC CTC CGT GCT GTG-3

[0066] Bottom single-stranded DNA for 56 pN: 5-GTG TCG TGC CTC CGT GCT GTG- / 3'Biotin / 3

[0067] Integrin α on top of single-stranded DNA v To bind cycloRGDfK-NH2 that specifically binds to β3, a heterobifunctional cross-linker, sulfo-SMCC, was used. Specifically, the upper single-stranded DNA was added to a mixed solution (50 mM TCEP, 50 mM EDTA, pH 7.2-7.4) and reacted at room temperature for 30 minutes. 250 μl of 10 mM RGDfK-NH and 10 μl of 23 mM sulfo-SMCC were mixed and reacted at room temperature for 20 minutes. Afterwards, the two solutions were mixed and reacted at room temperature for 1 hour. Afterwards, the upper single-stranded DNA and each of 12 pN and 56 pN lower-strand DNAs were combined and reacted at 4°C for more than 8 hours to produce the product.

[0068]

[0069] 2. Prolonectin - Fabrication of a single-molecule force probe surface

[0070] FIG. 2 is a diagram illustrating cell attachment on a cell attachment and migration control platform according to an embodiment of the present invention, wherein (A) is a schematic diagram of a DNA-based single-molecule force probe (12 pN and 56 pN) and a polymer-based single-molecule force probe (198 pN) bound on a glass substrate, and (B) is a schematic diagram of structural changes according to the binding force of the DNA-based single-molecule force probe.

[0071] Referring to Fig. 2, a DNA-based single-molecule force probe surface was manufactured by combining fibronectin, a biotin-conjugated fibronectin antibody, neutravidin, and a DNA-based single-molecule force probe each having a single structure of DNA conjugated to it on a glass substrate.

[0072] First, 50 μg / ml of fibronectin was coated on a glass substrate at 4°C for 8 hours. After washing the substrate three times with phosphate-buffered saline (PBS), a 1 / 10 dilution of biotin-conjugated fibronectin antibody was coated at room temperature for 1 hour. After washing the substrate three times with PBS, neutraavidin was coated at room temperature for 30 minutes. After washing the substrate three times with PBS, a PEG polymer-based single-molecule force probe and a DNA single-molecule force probe with different pN single-molecule forces depending on the structure were coated at 4°C for 30 minutes to create a single-molecule force probe surface.

[0073]

[0074] 3. Fabrication of a microstructured single-molecule force probe surface

[0075] A micro-sized micropattern surface was fabricated using a micropattern chrome mask with circular structures of 1, 3, and 5 μm and linear structures of 20 μm. After coating a silicon wafer with a negative photosensitizer, SU8, to a height of 1 μm, the micropattern structure was formed using a photolithography process and UV irradiation and SU8 developer. A poly-dimethylsiloxane (PDMS) solution was placed on the structure and cured at 70°C for 10 hours. The PDMS stamp thus fabricated was separated from the silicon wafer, and 20 μl of a 50 μg / ml fibronectin solution was placed on the PDMS stamp and coated at room temperature for 30 minutes. The fibronectin on the PDMS stamp was removed using PBS and placed on an O2 plasma-treated polymer substrate and removed after 1 minute. To passivate the portion other than fibronectin, 0.1 mg / ml Poly(L-Lysine)(20)-grafted[3.5]-PEG(2) was coated at room temperature for 30 minutes. The subsequent process was the same as the process of coating the biotin-conjugated fibronectin antibody in the fibronectin-single molecule force probe surface fabrication process above.

[0076]

[0077] 4. Fibroblast culture

[0078] To observe cell adhesion and migration on the surface of a single-molecule force probe, mouse embryonic fibroblasts (MEFs) were used. 10,000 MEFs were seeded onto the surface of the single-molecule force probe and cultured for 6 hours for each experiment.

[0079]

[0080] 5. Comparison of proliferation of embryonic fibroblasts

[0081] Fibronectin - The degree of cell attachment according to the number of single-molecule force probes on the surface of a single-molecule force probe was compared. The degree of cell attachment and spreading was confirmed under each single-molecule force probe condition in which the concentration of biotin-conjugated fibronectin antibody was varied at 0%, 0.2%, 1%, 4%, 10%, and 100%, and the results are shown in Figure 3.

[0082] Figure 3 is an image and graph showing the change in cell diffusion according to the concentration of a single-molecule binding probe. (A) is an image showing the change in cell diffusion according to the concentration of biotin-bound fibronectin antibody and the change in single-molecule binding force, (B) is a graph showing the change in cell size on the surface of a single-molecule binding probe according to the concentration of bound fibronectin antibody, and (C) is a graph showing the change in cell size according to single-molecule binding force on the surface of a single-molecule binding probe to which 1% fibronectin antibody is bound.

[0083] Referring to Figure 3, it was confirmed that the degree of cell adhesion and proliferation decreased as the concentration of biotin-conjugated fibronectin antibody increased. As the concentration of fibronectin antibody decreased, the sensitivity of cells to the single-molecule force probe increased, and it was confirmed that the difference in cell proliferation according to the strength of the single-molecule force acting on the integrin was most evident under the 1% condition.

[0084]

[0085] 6. Comparison of the diffusion of embryonic fibroblasts on the surface of a microcircular structured single-molecule force probe.

[0086] In order to compare the degree of cell proliferation according to changes in fibronectin, the change in the degree of MEF proliferation was confirmed on the surface of fibronectin microcircular structures (1 ㎛, 3 ㎛, 5 ㎛) manufactured using a PDMS stamp, on which a single-molecule force probe was bound, and the results are shown in Fig. 4.

[0087] Figure 4 is an image and graph showing changes in cell diffusion on the surface of a single-molecule force probe having a microstructure. (A) is an image showing changes in cell morphology on the surface of a single-molecule force probe in a 1 μm circular structure, (B) is an image showing changes in cell morphology on the surface of a 3 μm circular structure, and (C) is an image showing changes in cell size on the surface of a single-molecule force probe having a micro-circular structure, and (E) is a graph comparing the degree of changes in cell size according to the strength of the single-molecule force probe.

[0088] Referring to Figure 4, in the 5 μm micro-spherical structures, the degree of cell diffusion change with single-molecule binding increased. In contrast, in the 1 μm micro-spherical structures, the degree of cell diffusion did not change significantly even with increasing strength of the single-molecule binding probe. This indicates that the size of the attachment area of ​​cells binding via fibronectin is an important factor in cell adhesion and diffusion.

[0089]

[0090] 7. Comparison of the mobility of embryonic fibroblasts on the surface of a linear, single-molecule binding probe.

[0091] To determine the change in the mobility of MEFs according to the binding force between integrin proteins and ligand proteins, the change in polarity and mobility of cells according to the change in the intensity of a single-molecule force probe in a 20 μm linear microstructure were tracked in real time to confirm the change in cell mobility.

[0092] Figure 5 shows the change in cell polarity on the surface of a single-molecule force probe having a linear microstructure. (A) to (C) are the change in MEF polarity on the surface of a single-molecule force probe coupled to a 20 μm linear structure. (A) is the change in the Cdc42 active site under a 12 pN, (B) is the change in the Cdc42 active site under a 56 pN, and (C) is the change in the Cdc42 active site under a 198 pN single-molecule force probe. (D) is the change in the position of the Cdc42 active site according to the strength of the single-molecule force probe. (E) is the change in the movement of the Cdc42 active site per unit time. (F) to (H) are the change in the polarity of MEF in which FAK phosphorylation was inhibited on the surface of a single-molecule force probe coupled to a 20 μm linear structure. (F) is the change in the Cdc42 active site under a 12 pN, (G) is the change in the Cdc42 active site under a 56 pN, and (H) is the change in the Cdc42 active site under a 198 pN single-molecule force probe. (I) shows the change in the position of the Cdc42 active site according to the intensity of the single-molecule force probe in MEFs with suppressed FAK phosphorylation, and (J) shows the change in the movement of the Cdc42 active site per unit time in MEFs with suppressed FAK phosphorylation.

[0093] Referring to Figure 5, it was confirmed that as the intensity of the single-molecule force probe increased, the expression of the Cell division cycle 42 (Cdc42) protein, which indicates cell polarity, was formed at the front of the cell.

[0094] Additionally, to confirm cell mobility, cells were tracked in real time on a surface to which single-molecule force probes were bound and compared.

[0095] Figure 6 shows changes in cell migration on the surface of a single-molecule force probe having a linear microstructure. (A) shows cell migration on the surface of a single-molecule force probe coupled to a 20 μm linear structure, and (B) shows cell migration on the surface of a single-molecule force probe when treated with a FAK phosphorylation inhibitor (PF573228). (C) to (F) show changes in cell mobility according to the strength of the single-molecule force probe and treatment with a FAK phosphorylation inhibitor. (C) shows the total migration distance of the cell, (D) the migration distance in a single direction, (E) the migration time in a single direction, and (F) the migration displacement of the cell during the migration time.

[0096] Referring to Figure 6, we confirmed that MEFs migrate in a single direction as the single-molecular force increases. This increase in cell polarity and mobility due to the increase in single-molecular force was confirmed to be rapidly reduced when phosphorylation of Focal adhesion kinase (FAK), which is activated through integrin signaling, was inhibited.

[0097]

[0098] 8. Comparison of the mobility of embryonic fibroblasts on the surface of a single-molecule binding probe.

[0099] To investigate the change in the mobility of MEFs according to the binding force between integrin proteins and ligand proteins on a fibronectin surface with uncontrolled directionality, the mobility of cells was confirmed on a planar structure without a microstructure.

[0100] Figure 7 shows the changes in cell migration on the surface of a single-molecule force probe. (A) to (B) are images of cell migration on the surface of a single-molecule force probe depending on whether FAK phosphorylation was inhibited. (A) is a representative image of normal MEF cell migration on the surface of a single-molecule force probe, and (B) is a representative image of cell migration on the surface of a single-molecule force probe when treated with a FAK activity inhibitor (PF573228). (C) to (F) are quantitative evaluations of cell migration depending on the strength of the single-molecule force probe and whether or not FAK phosphorylation inhibitor was treated. (C) represents the total migration distance of the cell, (D) represents the migration distance in a single direction, (E) represents the migration time in a single direction, and (F) represents the migration displacement of the cell during the migration time.

[0101] Referring to Figure 7, as in the fine linear structure, MEFs showed increased unidirectional cell motility as the single-molecule binding force increased. In cells where FAK phosphorylation was inhibited, cell motility was significantly reduced, showing cell motility similar to that on a surface coated with a 12 pN single-molecule force probe. These results demonstrated that the strength of integrin-mediated single-molecule force controls cell motility by modulating the activation of FAK, an integrin-mediated signal transduction pathway.

[0102]

[0103] Although the present invention has been described in detail through specific examples, this is intended to specifically explain the present invention, and the present invention is not limited thereto, and it is clear that modifications and improvements can be made by those skilled in the art within the technical spirit of the present invention.

[0104] All simple modifications or changes of the present invention fall within the scope of the present invention, and the specific scope of protection of the present invention will be made clear by the appended claims.

[0105]

[0106] The present invention is recognized as having industrial applicability as a platform for controlling cell attachment and cell mobility by coating a single-molecule force probe on the surface of a substrate, which attaches to cells via integrin and has a binding force controlled at the pN level.

Claims

1. Substrate; A bonding layer formed on the surface of the substrate; A single-molecule force probe that has a predetermined binding strength, selectively binds to integrins within the cell membrane, attaches to cells, and ruptures when an external force applied by the cell exceeds the binding strength; and A cell attachment and migration control platform comprising a linker portion connecting the above bonding layer and the single-molecule force probe.

2. In claim 1, The above single molecule force probe is, A central portion formed of a polymer having the above bonding strength and being ruptured by the above external force; A ligand that is connected to the central portion and specifically binds to the integrin; and A cell attachment and migration control platform comprising a linker binding portion connected to the central portion and bonded to the linker portion.

3. In claim 2, The above center is, A first single-stranded DNA having said ligand linked to one of the ends of the two ends; and A cell attachment and migration control platform comprising a second single-stranded DNA complementarily linked to the first single-stranded DNA, wherein the linker binding portion is connected to one of the ends of the sockliner.

4. In claim 3, A cell attachment and migration control platform in which the binding force is controlled depending on the binding position of both ends of the second single-stranded DNA to which the linker binding portion is bound.

5. In claim 2, The above ligand is, Cell adhesion and migration control platform comprising Cyclo RGDfk ligand.

6. In claim 2, The above linker binding portion is, A cell adhesion and migration control platform containing biotin.

7. In claim 6, The above linker part, A first linker moiety binding to biotin, comprising at least one selected from the group consisting of avidin, streptavidin, and neutravidin; and A cell attachment and migration control platform comprising a second linker molecule, the second linker molecule including biotin at one end and bound to the first linker molecule at the other end by an antigen-antibody reaction with the binding layer.

8. In claim 8, The above bonding layer is, Cell adhesion and migration control platform containing fibronectin.

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