Nanostructure capable of controlling cell adhesion and method for manufacturing same

Abstract

A nanostructure capable of controlling cell adhesion, according to the present invention, may comprise a topography pattern having a plurality of protrusions. Further, adhesion force A(r) of a cell provided on the topography pattern may follow <equation 1> below. <Equation 1> A(r) = A0 - α(1 / r) – βr (wherein A0 = intrinsic adhesion force of a cell, α = cell stimulation constant with respect to the curvature of a protrusion, β = cell stimulation constant with respect to a concave portion formed by three adjacent protrusions, and r = half of the size of a protrusion in a two-dimensional planar perspective)

Description

Nanostructure capable of controlling cell adsorption and method for manufacturing the same

[0001] The present invention relates to a nanostructure capable of controlling cell adsorption and a method for manufacturing the same, and more specifically, to a nanostructure having a topography pattern formed thereon having a plurality of convex portions and a method for manufacturing the same.

[0002] In the in vivo environment of multicellular organisms, cells are exposed to diverse chemical and physical microenvironments, and their morphology and size vary depending on intercellular interactions as well as interactions between cells and the extracellular matrix (ECM). These cell-matrix interactions are induced by focal adhesion, which is essential for maintaining tissue homeostasis and morphogenesis; this is triggered by the activation and assembly of transmembrane proteins such as integrins. Focal adhesion plays a role in signal transduction by connecting the extracellular matrix with the intracellular actin cytoskeleton, and the expression levels of this adhesion are closely linked to cell migration and proliferation.

[0003] Previously, research on cell regulation based on chemical signals (such as chemotherapy) was mainstream, but recently, research on the effects of physical factors such as the stiffness and surface topography of the ECM on cell behavior is actively underway. In particular, there is an increasing number of attempts to mimic and control cell-substrate interactions in an in vitro environment by utilizing biomimetic surfaces. For example, Korean Patent Publication No. 10-2020-0018524 ​​discloses a technology that provides a cell culture support having linear nano-patterns of various widths and spacings, wherein these structural features can influence cell adhesion, migration, alignment, and tissue regeneration.

[0004] Cell motility is closely related to tissue morphology and is also associated with abnormal cellular behavior in diseases such as cancer. During cell migration, focal adhesion complexes are stabilized by the actin cytoskeleton and are characterized by dynamic rearrangement during movement. Cells initiate membrane protrusion to form new adhesions for migration, and as these adhesions dissipate posteriorly, migration occurs. Furthermore, recent studies have revealed that membrane tension influences cell motility, and during cell division, mitotic cell rounding—a morphological change from a flattened to a rounded form—occurs through the dissipation of adhesions, thereby establishing geometric conditions suitable for cell division.

[0005] However, the impact of changes in focal adhesion dynamics induced by cell-ECM interactions on cell motility and morphological changes has not yet been fully elucidated, and technologies for controlling such cell behavior on biomimetic surfaces are also still in the early stages. Therefore, there is a growing technical need for a new concept of physical platform capable of precisely controlling cell adsorption and migration.

[0006] The technical problem that the present invention aims to solve is to provide a nanostructure that provides the ability to effectively induce or observe behaviors such as cell adsorption, mobility, and proliferation.

[0007] Another technical problem that the present invention aims to solve is to provide a nanostructure capable of quantitatively predicting and controlling cell behavior.

[0008] Another technical problem that the present invention aims to solve is to provide a nanostructure having the advantage of being able to control adsorption force according to cell type.

[0009] The technical problems that the present invention aims to solve are not limited to those described above.

[0010] To solve the above technical problem, the present invention provides a nanostructure.

[0011] According to one embodiment, the nanostructure comprises a topography pattern having a plurality of convex portions, and the adsorption force (A(r)) of a cell provided on the topography pattern may follow the following <Equation 1>.

[0012] <Mathematical Formula 1>

[0013] A(r) = A0 - α(1 / r) - βr (A0 = intrinsic cell adhesion force, α = cell stimulation constant for the curvature of the convex part, β = cell stimulation constant for the concave part formed by three adjacent convex parts, r = half the size of the convex part in a two-dimensional plane)

[0014] According to one embodiment, the cell is classified into a first cell which is a cancer cell and a second cell which is a normal cell, and in the above <Equation 1>, the relationship between the adsorption force of the first cell and the r value may be expressed by the following <Equation 1-1>.

[0015] <Mathematical Formula 1-1>

[0016] A(r) = A0 - βr (A0 = intrinsic cell adhesion force, β = cell stimulation constant for the depression formed by three adjacent convexities, r = half the size of the convexity in a two-dimensional plane)

[0017] According to one embodiment, the relationship between the adsorption force of the second cell and the r value may include following <Equation 1>.

[0018] According to one embodiment, the first cell may include a mouse melanoma cell line (B16-F10; mouse melanoma cell line), and the second cell may include a human skin fibroblast (WS1; Human Skin Fibroblast Cell Line).

[0019] According to one embodiment, the adsorption force of the cells provided on the topography pattern is controlled based on a change in the cell area of ​​the cells according to half the size of the convex portion of the topography pattern, and the cells are classified into a first cell which is a cancer cell and a second cell which is a normal cell, and the adsorption force of the first cell is linearly reduced as half the size of the convex portion of the topography pattern increases in a two-dimensional plane view, and the adsorption force of the second cell has a maximum value when half the size of the convex portion of the topography pattern is equal to √(α / β) in a two-dimensional plane view, and the α value is a cell stimulation constant for the curvature of the convex portion of the topography pattern, and the β value is a cell stimulation constant for the concave portion formed by three adjacent convex portions of the topography pattern.

[0020] According to one embodiment, the maximum cell area of ​​the first cell provided on the topography pattern of the nanostructure is 1,100 μm 2 It includes the following, wherein the maximum cell area of ​​the second cell provided on the topography pattern of the nanostructure is 1,100 μm 2 It may include exceeding.

[0021] To solve the above technical problem, the present invention provides a method for manufacturing the nanostructure described above.

[0022] According to one embodiment, the method for manufacturing the nanostructure comprises forming a topography pattern having a plurality of convex portions, and the adsorption force (A(r)) of the cell provided on the topography pattern may follow the following <Equation 1>.

[0023] <Mathematical Formula 1>

[0024] A(r) = A0 - α(1 / r) - βr (A0 = intrinsic cell adhesion force, α = cell stimulation constant for the curvature of the convex part, β = cell stimulation constant for the concave part formed by three adjacent convex parts, r = half the size of the convex part in a two-dimensional plane)

[0025] According to one embodiment, the step of forming the topography pattern may include: manufacturing a pre-nanostructure having a topography pattern layer laminated on the upper surface of a base substrate; providing a molding polymer on the pre-nanostructure and drying it to manufacture a mold structure having a concave pattern which is the reverse phase of the surface pattern of the topography pattern layer; and, after separating the mold structure from the pre-nanostructure, providing a biocompatible polymer on the mold structure and drying it to manufacture the nanostructure having the topography pattern which is the reverse phase of the concave pattern and is the same pattern as the surface pattern of the topography pattern layer.

[0026] According to one embodiment, the method for manufacturing the preliminary nanostructure may include the steps of: preparing the base substrate and the transfer stamp; forming a fixed layer by coating an amine-based resin on the base substrate; providing a plurality of beads on the transfer stamp and rubbing with an elastomer slab to form the topography pattern layer in the form of a monolayer in which the plurality of beads are arranged in a two-dimensional hexagonal dense array; transferring the topography pattern layer on the transfer stamp onto the base substrate on which the fixed layer is formed to manufacture the base nanostructure in which the base substrate, the fixed layer, and the topography pattern layer are sequentially stacked; and heat-treating the base nanostructure to remove the fixed layer between the base substrate and the topography pattern layer, and then washing and drying to manufacture the preliminary nanostructure in which the topography pattern layer is stacked on the upper surface of the base substrate.

[0027] According to one embodiment, the heat treatment temperature of the base nanostructure may be controlled to 400°C to 600°C.

[0028] According to one embodiment, the amine-based resin comprises polyethyleneimine (PEI), the polymer for the transfer stamp, the elastomer slab, and the mold comprises polydimethylsiloxane (PDMS), the beads comprise silica beads, and the biocompatible polymer may comprise a silicone elastomer or TPU (thermoplastic polyurethane).

[0029] According to one embodiment, the size of the beads may be 340 nm to 5,000 nm.

[0030] According to one embodiment, the cell is classified into a first cell which is a cancer cell and a second cell which is a normal cell, and in the above <Equation 1>, the relationship between the adsorption force of the first cell and the r value may be expressed by the following <Equation 1-1>.

[0031] <Mathematical Formula 1-1>

[0032] A(r) = A0 - βr (A0 = intrinsic cell adhesion force, β = cell stimulation constant for the depression formed by three adjacent convexities, r = half the size of the convexity in a two-dimensional plane)

[0033] According to one embodiment, the relationship between the adsorption force of the second cell and the r value may include following <Equation 1>.

[0034] According to one embodiment, the first cell may include a mouse melanoma cell line (B16-F10; mouse melanoma cell line), and the second cell may include a human skin fibroblast (WS1; Human Skin Fibroblast Cell Line).

[0035] The nanostructure according to the present invention includes a topographic pattern in which a plurality of convex and concave portions are regularly arranged, thereby providing a function that can effectively induce or observe behaviors such as cell adsorption force, mobility, and proliferation.

[0036] Since the above topography pattern is manufactured with the same shape as the surface pattern of the topography pattern layer implemented on the base substrate during the preliminary nanostructure formation stage in the manufacturing process of the nanostructure, cell behavior can be quantitatively predicted and controlled by applying an adsorption force model (e.g., <Equation 1> and <Equation 1-1>) according to the type and characteristics of the cell (e.g., size, mobility, etc.) when designing the pattern.

[0037] In particular, the first cell (e.g., cancer cell (B16-F10)) is relatively small in size and has high mobility, so it has low sensitivity to curvature stimulation, and accordingly, the adsorption force may decrease linearly when the size of the convex part of the topography pattern is increased by half (r). On the other hand, the second cell (e.g., normal cell (WS1)) responds sensitively to the curvature of the convex part of the topography pattern and the stimulation of the concave part, so the maximum adsorption force can be induced by adjusting the value of r (e.g., r = √(α / β)).

[0038] As such, the above-mentioned nanostructure has the advantage of being able to control adsorption force according to cell type, and through this, it can be effectively utilized in various biomedical fields such as evaluating the invasiveness of cancer cells, designing scaffolds for tissue engineering, and developing high-sensitivity biosensors.

[0039] FIG. 1 is a flowchart illustrating a method for manufacturing a nanostructure according to an embodiment of the present invention.

[0040] FIG. 2 is a flowchart illustrating a method for manufacturing a preliminary nanostructure according to an embodiment of the present invention.

[0041] FIG. 3 is a drawing for explaining the step of preparing a base substrate and a transfer stamp according to an embodiment of the present invention.

[0042] FIG. 4 is a drawing for explaining the step of forming a fixed layer according to an embodiment of the present invention.

[0043] FIG. 5 is a drawing for explaining the step of forming a topography pattern layer according to an embodiment of the present invention.

[0044] FIG. 6 is a diagram illustrating the steps for manufacturing a base nanostructure according to an embodiment of the present invention.

[0045] FIG. 7 is a diagram illustrating the steps for manufacturing a preliminary nanostructure according to an embodiment of the present invention.

[0046] FIG. 8 is a drawing for explaining the steps of manufacturing a mold structure according to an embodiment of the present invention.

[0047] FIG. 9 is a diagram illustrating the steps for manufacturing a nanostructure according to an embodiment of the present invention.

[0048] FIG. 10 is a drawing for explaining a nanostructure according to an embodiment of the present invention.

[0049] Figure 11 is a graph for comparing the curvature of the topographic pattern layer of a pre-nanostructure according to an experimental example of the present invention according to the diameter of the silica beads.

[0050] FIGS. 12 and 13 are graphs for comparing the degree of protein expression (Western blot; vinculin, β-actin) in relation to the cell area and adsorption force of a first cell (B16-F10) and a second cell (WS1) provided on a topography pattern layer according to the silica bead diameter of a preliminary nanostructure according to an experimental example of the present invention.

[0051] FIG. 14 is a Western blot result graph quantifying the protein expression amount regarding DNA Damage Response (DDR) for first cells (B16-F10) seeded on a topography pattern layer according to the silica bead diameter of a pre-nanostructure according to an experimental example of the present invention.

[0052] FIG. 15 is a graph showing the expression level of a protein related to DNA damage response (DDR) of a second cell (WS1) seeded under cell aging induction conditions on a topography pattern layer formed according to the silica bead diameter of a preliminary nanostructure according to an experimental example of the present invention, analyzed and quantified by Western blot.

[0053] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings. However, the technical concept of the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to ensure that the disclosed content is thorough and complete and to ensure that the concept of the present invention is sufficiently conveyed to those skilled in the art.

[0054] In this specification, when a component is described as being on another component, it means that it may be formed directly on the other component or that a third component may be interposed between them. Additionally, in the drawings, the thicknesses of the films and regions are exaggerated for the effective description of the technical content.

[0055] Additionally, although terms such as first, second, third, etc., have been used to describe various components in the various embodiments of this specification, these components should not be limited by such terms. These terms are used merely to distinguish one component from another. Accordingly, what is referred to as the first component in one embodiment may be referred to as the second component in another embodiment. Each embodiment described and illustrated herein also includes its complementary embodiment. Furthermore, in this specification, "and / or" is used to mean including at least one of the components listed before and after it.

[0056] In the specification, singular expressions include plural expressions unless the context clearly indicates otherwise. Furthermore, terms such as "include" or "have" are intended to specify the existence of the features, numbers, steps, components, or combinations thereof described in the specification, and should not be understood as excluding the existence or addition of one or more other features, numbers, steps, components, or combinations thereof. Additionally, in this specification, "connection" is used to include both indirectly connecting multiple components and directly connecting them.

[0057] Furthermore, in describing the present invention below, if it is determined that a detailed description of related known functions or configurations could unnecessarily obscure the essence of the invention, such detailed description will be omitted.

[0058]

[0059] FIG. 1 is a flowchart for explaining a method for manufacturing a nanostructure according to an embodiment of the present invention, FIG. 2 is a flowchart for explaining a method for manufacturing a preliminary nanostructure according to an embodiment of the present invention, FIG. 3 is a diagram for explaining the step of preparing a base substrate and a transfer stamp according to an embodiment of the present invention, FIG. 4 is a diagram for explaining the step of forming a fixed layer according to an embodiment of the present invention, FIG. 5 is a diagram for explaining the step of forming a topography pattern layer according to an embodiment of the present invention, FIG. 6 is a diagram for explaining the step of manufacturing a base nanostructure according to an embodiment of the present invention, FIG. 7 is a diagram for explaining the step of manufacturing a preliminary nanostructure according to an embodiment of the present invention, FIG. 8 is a diagram for explaining the step of manufacturing a mold structure according to an embodiment of the present invention, FIG. 9 is a diagram for explaining the step of manufacturing a nanostructure according to an embodiment of the present invention, and FIG. 10 is a diagram for explaining a nanostructure according to an embodiment of the present invention.

[0060] Referring to FIGS. 1 to 7, a pre-nanostructure (300) is manufactured by stacking a topography pattern layer (200) on the upper surface of a base substrate (100) (S100).

[0061] Referring to FIGS. 1 to 3, the base substrate (100) and the transfer stamp (102) are prepared (S110).

[0062] The base substrate (100) may be a substrate in which, as described below, an amine-based fixing layer is formed on the base substrate (100), and the topography pattern layer (200) comprising a plurality of beads is transferred to the fixing layer, and then the fixing layer is removed so that the topography pattern layer (200) is fixed. For example, the base substrate (100) may be a glass substrate. For example, the base substrate (100) may be prepared in a state (creation of a hydrophilic surface) after removing oxides in a piranha solution (sulfuric acid:hydrogen peroxide = 3:1 v / v) and then plasma treating (10 minutes).

[0063] And, as described below, the transfer stamp (102) can function as a stamp that transfers the topography pattern layer (200) to the base substrate (100) after forming the topography pattern layer (200) on the transfer stamp (102). For example, the transfer stamp (102) may be polydimethylsiloxane (PDMS).

[0064] Referring to FIGS. 1 to 4, an amine-based resin (201) is coated on the base substrate (100) to form a fixed layer (202) (S120).

[0065] According to one embodiment, the amine-based resin (201) may be spin-coated on the base substrate (100). For example, the amine-based resin (201) may be an aqueous solution containing polyethyleneimine (PEI, 30%). For example, the spin coating of the amine-based resin (201) may be performed at 5,000 rpm for 60 seconds.

[0066] Accordingly, the fixing layer (202) that fixes the topography pattern layer (200) including a plurality of beads described later can be formed.

[0067] Referring to FIGS. 1 to 5, a plurality of beads (210) are provided on the transfer stamp (102) and rubbed with an elastomer slab (104) to form the topography pattern layer (200) in the form of a monolayer in which the plurality of beads are arranged in a two-dimensional hexagonal dense array (S130).

[0068] When the plurality of beads (210) are provided on the transfer stamp (102) and rubbed using the elastomer slab, the topography pattern layer (200) in which the plurality of beads (210) are easily arranged in a single layer in a two-dimensional hexagonal dense arrangement on the transfer stamp (102) can be easily formed. For example, the beads (210) may be silica beads. For example, the size (diameter) of the beads (210) may be 340 nm to 5,000 nm. For example, the elastomer slab (104) may be polydimethylsiloxane (PDMS).

[0069] Referring to FIGS. 1 to 6, the topography pattern layer (200) on the transfer stamp (102) is transferred onto the base substrate (100) on which the fixing layer (202) is formed, thereby manufacturing a base nanostructure (301) in which the base substrate (100), the fixing layer (202), and the topography pattern layer (200) are sequentially stacked (S140).

[0070] In the process of manufacturing the above base nanostructure (301), the topography pattern layer (200), in which a plurality of beads (210) on the transfer stamp (102) are arranged, can be easily transferred and fixed by the amine-based fixing layer (202) on the base substrate (100).

[0071] Referring to FIGS. 1 to 7, the base nanostructure (301) is heat-treated to remove the fixing layer (202) between the base substrate (100) and the topography pattern layer (200), and then washed and dried to produce a preliminary nanostructure (300) having the topography pattern layer (200) provided on the upper surface of the base substrate (100) (S100).

[0072] According to one embodiment, the base nanostructure (301) may be heat-treated at a temperature of 400°C to 600°C for 2 to 4 hours, washed with water (e.g., DI water), and dried in an inert gas atmosphere (e.g., N2). Accordingly, the fixing layer (202) of the base nanostructure (301) can be easily removed, and the preliminary nanostructure (300) with the topography pattern layer (200) stably fixed on the base substrate (100) can be easily manufactured.

[0073] Referring to FIGS. 1 to 8, a mold polymer (401) is provided on the pre-nanostructure (300) and dried to produce a mold structure (400) having a concave pattern (402) which is the reverse phase of the surface pattern of the topography pattern layer (200) (S200).

[0074] The surface pattern of the topography pattern layer (200) may be a pattern in which a plurality of convex portions corresponding to a surface shape in which a plurality of beads (200) are arranged in a two-dimensional hexagonal dense structure and a plurality of concave portions formed by three adjacent convex portions are regularly arranged.

[0075] And, by providing the mold polymer (401) on the surface pattern of the topography pattern layer (200) and drying it, the mold structure (400) including the intaglio pattern (402) having an inverse structure with the surface pattern of the topography pattern layer (200) can be manufactured. For example, the mold polymer (401) may be polydimethylsiloxane (PDMS).

[0076] Referring to FIGS. 1 to 9, after separating the mold structure (400) from the preliminary nano structure (300), a biocompatible polymer (501) is provided on the mold structure (400) and dried to produce a nano structure (500) having a topography pattern (510) which is the reverse phase of the intaglio pattern (402) and is the same pattern as the surface pattern of the topography pattern layer (200) (S300).

[0077] When the biocompatible polymer (501) is provided on the intaglio pattern (402) of the mold structure (400) and dried, the nanostructure (500) having the topography pattern (510) having an inverse structure with the intaglio pattern (402) can be manufactured. For example, the biocompatible polymer (501) may be a silicone elastomer or TPU (thermoplastic polyurethane).

[0078] Referring to FIG. 10, the nanostructure (500) manufactured by the above-described manufacturing method is described.

[0079] As illustrated in FIG. 10, the nanostructure (500) may include a topography pattern (510) in which a plurality of convex portions (502) and a plurality of concave portions (504) are regularly arranged.

[0080] Accordingly, when a cell is provided on the topography pattern (510) of the nanostructure (500), the behavioral patterns of the cell, such as adsorption, movement, and proliferation by the topography pattern (510), can be observed.

[0081] And, the adsorption force (A(r)) of the cell provided on the topography pattern (501) of the nanostructure (500) can satisfy the following <Equation 1>.

[0082] <Mathematical Formula 1>

[0083] A(r) = A0 - α(1 / r) - βr (A0 = intrinsic cell adsorption force, α = cell stimulation constant for the curvature of the convex portion (502), β = cell stimulation constant for the concave portion (504) formed by three adjacent convex portions (502), r = half the size of the convex portion (502) in a two-dimensional planar view (xy plane))

[0084] In the specification of this application, the cell may be classified into a first cell which is a cancer cell and a second cell which is a normal cell. For example, the first cell may be a mouse melanoma cell line (B16-F10; mouse melanoma cell line). For example, the second cell may be a human skin fibroblast (WS1; Human Skin Fibroblast Cell Line).

[0085] Accordingly, when the cell is the first cell, the first cell is relatively smaller in size and moves faster than the second cell, so the sensitivity to the curvature (= 1 / r) of the convex portion (502) of the topography pattern (510) of the nanostructure (500) in <Equation 1> can be ignored.

[0086] Accordingly, α(1 / r) in the above <Equation 1> is ignored, and the adsorption force of the first cell can be expressed by the following <Equation 1-1>.

[0087] <Mathematical Formula 1-1>

[0088] A(r) = A0 - βr (A0 = intrinsic cell adsorption force, β = cell stimulation constant for the concave portion (504) formed by three adjacent convex portions (502), r = half the size of the convex portion (502) in a two-dimensional planar view (xy plane))

[0089] Accordingly, when the first cell is provided on the topography pattern (510) of the nanostructure (500), the adsorption force of the first cell may decrease linearly as the half size (r) of the size of the convex portion (502) increases in a two-dimensional planar view (xy plane). For example, the maximum cell area of ​​the first cell provided on the topography pattern (510) of the nanostructure (500) is 1,100 μm 2 It may be less than.

[0090] In contrast, if the cell is the second cell, the second cell is relatively larger and moves slower than the first cell, so it may be sensitive to the curvature (=1 / r) of the convex portion (502) of the topography pattern (510) of the nanostructure (500) and the concave portion (504) formed by three adjacent convex portions (502).

[0091] Accordingly, the adsorption force of the second cell can follow <Equation 1>. Accordingly, differentiating <Equation 1> with respect to r yields dA(r) / dr = d / dr(A0 - α(1 / r) - βr) = (α / r 2 It is rearranged as )-β, and since the extremum condition is when dA(r) / dr is 0, α / r 2 It can be summarized as = β, and it can be seen that the adsorption force of the second cell is maximized when the above r is equal to √(α / β).

[0092] Accordingly, when the second cell is provided on the topography pattern (510) of the nanostructure (500), it can be seen that the adsorption force of the second cell has a maximum value when the size (r) of half the size of the convex portion (502) in a two-dimensional planar view (xy plane) is equal to √(α / β). For example, the maximum cell area of ​​the second cell provided on the topography pattern (510) of the nanostructure (500) is 1,100 μm 2 It can exceed.

[0093] In summary, the topography pattern (510) of the nanostructure (500) according to the embodiment of the present invention is implemented in the same shape as the surface pattern of the topography pattern layer (200) formed on the base substrate (100) during the step of forming the preliminary nanostructure (300) during the manufacturing process of the structure, so <Equation 1> and <Equation 1-1> can be consistently applied according to the cell type even during the manufacturing design stage.

[0094] Accordingly, the adsorption force for each cell type can be quantitatively controlled by adjusting the r value of the convex portion (502) as a design variable based on characteristics such as cell size and mobility.

[0095] This structure enables the precise induction or observation of cell behaviors such as adsorption, migration, and proliferation, and can be effectively utilized in various biomedical fields, including the evaluation of cancer cell motility, the design of scaffolds for tissue engineering, and the enhancement of biosensor sensitivity.

[0096] In addition, the nanostructure (500) according to the embodiment of the present invention is not limited to a silica bead-based structure and can be applied to nanostructures of various materials (e.g., polymer, metal, ceramic, hydrogel, etc.) capable of realizing the same surface topography pattern, and accordingly, a similar cell response control effect can be expected.

[0097]

[0098] Hereinafter, experimental examples and characteristic evaluation results of preliminary nanostructures according to embodiments of the present invention are described.

[0099]

[0100] Preliminary nanostructures according to experimental examples

[0101] A glass slide (1x1) was prepared as a base substrate by immersing it in a piranha solution (sulfuric acid:hydrogen peroxide = 3:1 v / v) for more than 30 minutes to remove oxides from the surface, washing it with DI water, and plasma treating it (10 minutes).

[0102] Then, a fixed layer was formed by spin-coating (5,000 rpm, 60 seconds) an aqueous PEI (Polyethyleneimine) solution (30%) on the base substrate.

[0103] Then, silica beads (340 nm to 5,000 nm) were provided on a transfer stamp (PDMS) and the elastomer slab (PDMS) was rubbed to form a monolayer topographic pattern layer in which a plurality of silica beads were arranged in a two-dimensional hexagonal dense array.

[0104] Then, the topography pattern layer on the transfer stamp was transferred onto the base substrate on which the fixing layer was formed to manufacture a base nanostructure.

[0105] Then, the base nanostructure was heat-treated (500°C, 3 hours) to remove the fixing layer, thereby producing a pre-nanostructure in which the topography pattern layer is fixed on the base substrate.

[0106]

[0107] Cell seeding according to experimental example

[0108] First cell (B16-F10), which is a cancer cell, and second cell (WS1), which is a normal cell, were used, and said cells were prepared to be used in the range of passage 3 to passage 6.

[0109] For cell culture, a medium containing 1% penicillin / streptomycin (P / S) in DMEM (Dulbecco's Modified Eagle Medium) was used, and the cells were set to be cultured at 37°C and 5% CO2 conditions.

[0110] As substrates for seeding cells, a pre-nanostructure according to the experimental example and a glass slide (control group) were used, and the pre-nanostructure according to the experimental example underwent sterilization and drying pretreatment before seeding. Specifically, the pre-nanostructure according to the experimental example was treated to be sterilized by immersing it in 70% ethanol (EtOH) for about 30 minutes, then washed with DI water, and then dried with nitrogen (N2) gas.

[0111] Subsequently, 2x10 cells are placed on the above-mentioned preliminary nanostructure or the glass slide according to the experimental example. 4 cells / cm 2 It was inoculated at a seeding density and maintained for 1 day (24 hours) after inoculation.

[0112] After the completion of the above culture, subsequent analysis was performed for cell adsorption and area analysis or protein analysis.

[0113]

[0114] Measurement of cell area according to experimental example

[0115] (1) Fixation and permeabilization

[0116] The preliminary nanostructures and glass slides according to the experimental example in which cell culture was completed were washed with PBS, and the cells were treated to be fixed with 4% paraformaldehyde (PFA) dissolved in PBS at room temperature for 15 minutes. After the fixation treatment, PBS washing was performed three times to remove residual fixative, and then 0.2% Triton X-100 was treated at room temperature for 10 minutes to enable cell membrane permeability.

[0117] (2) Blocking

[0118] To inhibit non-specific binding, cells were treated with a blocking solution containing 3% BSA in PBS-T for 30 minutes at room temperature.

[0119] (3) Primary antibody reaction

[0120] A primary antibody against vinculin was applied for the detection of focal adhesion proteins. Specifically, mouse anti-vinculin (MilliporeSigma, MAB3574)** was prepared by diluting it in PBS-T + 3% BSA at a dilution ratio of 1:200, and the primary antibody was reacted overnight (approximately 12 to 16 hours) at 4°C.

[0121] (4) Secondary antibody & F-actin staining

[0122] After the primary antibody reaction described above, a wash was performed to remove residual antibodies, and subsequently, a secondary antibody was applied. Goat Anti-Mouse IgG H&L (Alexa Fluor 488) was used as the secondary antibody, and the reaction was performed at room temperature for 2 hours after being prepared at a dilution ratio of 1:200. In addition, TRITC-phalloidin was applied to clarify the boundaries of the cytoskeleton. The TRITC-phalloidin was prepared at a dilution ratio of 1:300 and configured to perform co-staining according to the conditions of the Actin Cytoskeleton and Focal Adhesion Staining Kit (FAK100).

[0123] (5) Nuclei staining

[0124] For nuclear staining, DAPI was applied. Specifically, DAPI was prepared at a dilution ratio of 1:500 in PBS-T + 3% BSA and reacted for 10 minutes under dark conditions, after which residual staining solution was removed by PBS washing.

[0125] (6) Imaging and area quantification (Imaging & analysis)

[0126] Fluorescence imaging was performed using a Lionheart FX fluorescence microscope (BioTek, Agilent). Cell area was quantified using ImageJ. Specifically, cell boundaries were established based on phalloidin signals, and after an ROI was designated based on the cell boundaries (cell ROI setting), the area of ​​the corresponding ROI was calculated as the cell area.

[0127]

[0128] Western blot (vinculin, β-actin) according to experimental example

[0129] For Western blot analysis to compare protein expression, first cells (B16-F10) and second cells (WS1) were cultured for 24 hours on a glass slide and a pre-nanostructure according to the experimental example.

[0130] (1) Protein extraction (Lysis)

[0131] For cell protein extraction, ice-cold RIPA buffer was used, and the RIPA buffer was prepared to contain a protease / phosphatase inhibitor (Thermo Fisher). Under these conditions, cells were lysed, and the process was carried out to recover proteins from the cell lysate.

[0132] (2) Protein quantification

[0133] The recovered protein was quantified using a BCA assay (Thermo Fisher), and each sample was adjusted to equal protein amount.

[0134] (3) Sample preparation

[0135] 5× SDS loading buffer was mixed with the quantified protein and treated by heating at 100°C for 5 minutes to denature the protein.

[0136] (4) Electrophoresis and Transfer (SDS-PAGE & Transfer)

[0137] Protein separation was performed on a 10% SDS-polyacrylamide gel, and after electrophoresis, the proteins were transferred to a 0.45 μm nitrocellulose membrane (GE Healthcare).

[0138] (5) Antibody incubation and detection

[0139] The primary antibody reaction was performed at room temperature for 2 hours, and vinculin (CST) was used as the target protein and β-actin (Santa Cruz, sc-47778) as the loading control. Subsequently, an HRP-conjugated secondary antibody (Santa Cruz) was used as the secondary antibody, and the secondary antibody reaction was performed at room temperature for 2 hours. Detection was performed using ECL (Thermo Fisher), and the chemiluminescence signal was acquired via ChemiDoc XRS (Bio-Rad).

[0140]

[0141] Figure 11 is a graph for comparing the curvature of the topographic pattern layer of a pre-nanostructure according to an experimental example of the present invention according to the diameter of the silica beads.

[0142] Referring to Fig. 11, the curvature of the convex portion (K = 1 / r) of pre-nanostructures prepared by controlling the diameter of the silica beads to 400 nm to 5,000 nm in the method for preparing pre-nanostructures according to the experimental example was compared.

[0143] As can be seen in Figure 11, as the diameter of the silica beads increases, the curvature of the convex portion of the surface pattern of the topography pattern layer decreases.

[0144]

[0145] FIGS. 12 and 13 are graphs for comparing the degree of protein expression (Western blot; vinculin, β-actin) in relation to the cell area and adsorption force of a first cell (B16-F10) and a second cell (WS1) provided on a topography pattern layer according to the silica bead diameter of a preliminary nanostructure according to an experimental example of the present invention.

[0146] Referring to FIG. 12, in the method for preparing a preliminary nanostructure according to an experimental example, first cells (B16-F10) were seeded into the preliminary nanostructure according to the experimental example prepared according to the diameter of the silica beads (control group (flat), SB1200 (1,200 nm), SB1600 (1,600 nm), SB2000 (2,000 nm), SB3000 (3,000 nm), SB5000 (5,000 nm)) using the cell seeding method according to the experimental example, and the cell area was measured using the cell area measurement method according to the experimental example (Fig. 12 (a)). Then, the adsorption force of the first cells (B16-F16) provided on the topography pattern layer of the preliminary structure according to the experimental example was measured using the Western blot method according to the experimental example (Fig. 12 (b)).

[0147] As can be seen in FIG. 12, in the case of the first cell, as the diameter of the silica bead increases, the cell area of ​​the first cell spread on the topography pattern layer decreases. Also, as the diameter of the silica bead increases, the adsorption force of the first cell on the topography pattern layer decreases.

[0148] In addition, for the first cell, when the diameter of the silica bead is SB1200 (1,200 nm, r = 0.6 μm), the adsorption force on the first cell (the cell area spread by the first cell) is 1,000 μm 2 And, when the diameter of the silica bead is SB5000 (5,000 nm, r = 2.5 μm), the adsorption force on the first cell (cell area spread by the first cell) is 700 μm 2 It can be seen that it is.

[0149] According to an embodiment of the present invention, the adsorption force for the first cell (cell area where the first cell is spread on the topographic pattern of the nanostructure) is not affected by the value of the cell stimulation constant (α) for the curvature of the convex portion of the nanostructure due to the inherent characteristics of the first cell, so the adsorption force of the first cell provided on the topographic pattern of the nanostructure can be expressed by the following <Equation 1-1>.

[0150] <Mathematical Formula 1-1>

[0151] A(r) = A0 - βr (A0 = intrinsic cell adhesion force, β = cell stimulation constant for the depression formed by three adjacent convexities, r = half the size of the convexity in a two-dimensional plane)

[0152] thus,

[0153] β = (1,000μm 2 -700μm 2 ) / (2.5μm-0.6μm) = 158μm ≈ 160μm, and

[0154] A0 = A(r) + βr = 1,000μm 2 + (160μm x 0.6μm) ≒ 1,100μm 2 It can be seen that it is.

[0155] Accordingly, the above <Mathematical Formula 1-1> can finally be expressed as A(r) = 1100 - 160r.

[0156] Referring to FIG. 13, in the method for manufacturing a preliminary nanostructure according to an experimental example, a second cell (WS1) was seeded into the preliminary nanostructure according to the experimental example, which was prepared according to the diameter of the silica beads (control group (flat), SB340 (340 nm), SB800 (800 nm), SB1200 (1,200 nm), SB1600 (1,600 nm), SB2000 (2,000 nm)), using the cell seeding method according to the experimental example, and the cell area was measured using the cell area measurement method according to the experimental example (Fig. 13 (a)). Then, the adsorption force of the second cell (WS1) provided on the topography pattern layer of the preliminary structure according to the experimental example was measured using the Western blot method according to the experimental example (Fig. 13 (b)).

[0157] As can be seen in FIG. 13, in the case of the second cell (WS1), it can be seen that the cell area of ​​the second cell (WS1) spread on the topography pattern layer, where the diameter of the silica beads is controlled to 1,200 nm, has a maximum value. Also, it can be seen that the adsorption force of the second cell (WS1) has a maximum value on the topography pattern layer, where the diameter of the silica beads is controlled to 1,200 nm.

[0158] In addition, in the case of the second cell, when the diameter of the silica bead is SB340 (340 nm, r1 = 0.17 μm), the adsorption force on the second cell (the cell area spread by the second cell, A1) is 300 μm 2 And, when the diameter of the silica bead is SB1200 (1,200 nm, r2 = 0.6 μm), the adsorption force on the second cell (cell area spread by the second cell, A2) is 1,200 μm 2 And, when the diameter of the silica bead is SB1000 (1,000 nm, r3 = 0.5 μm), the adsorption force on the second cell (cell area spread by the second cell, A3) is 1,000 μm 2 It can be seen that it is.

[0159] The adsorption force on the second cell (cell area where the second cell is spread on the topography pattern of the nanostructure) according to an embodiment of the present invention can be expressed by the following <Equation 1>.

[0160] <Mathematical Formula 1>

[0161] A(r) = A0 - α(1 / r) - βr (A0 = intrinsic cell adhesion force, α = cell stimulation constant for the curvature of the convex part, β = cell stimulation constant for the concave part formed by three adjacent convex parts, r = half the size of the convex part in a two-dimensional plane)

[0162] thus,

[0163] A2-A1=-α(1 / r2-1 / r1)-β(r2-r1) = 900 = -α(-4.2157)-β(0.43)

[0164] A3-A2=-α(1 / r3-1 / r2)-β(r3-r2) = -200 = -α(-0.6666)-β(0.40).

[0165] Accordingly, α = 319μm 3 , β = 1,031μm, A0= 2,350μm 2 It can be seen that it is.

[0166] Therefore, the above <Mathematical Formula 1> can ultimately be expressed as A(r) = 2,350 - 319 / r - 1,031r.

[0167]

[0168] FIG. 14 is a Western blot result graph quantifying the protein expression amount regarding DNA Damage Response (DDR) for first cells (B16-F10) seeded on a topography pattern layer according to the silica bead diameter of a pre-nanostructure according to an experimental example of the present invention.

[0169] Referring to FIG. 14, in the method for preparing a preliminary nanostructure according to an experimental example, the preliminary nanostructure according to the experimental example was prepared according to the diameter of the silica beads (control group (flat), SB1200 (1,200 nm), SB1600 (1,600 nm), SB2000 (2,000 nm), SB3000 (3,000 nm), SB5000 (5,000 nm)), and the first cells (B16-F10) were seeded on a Ctrl (plastic for general tissue culture, TC plate) using the cell seeding method according to the experimental example. Subsequently, the expression levels of p53 (Tumor Protein p53), p21 (Cyclin-Dependent Kinase Inhibitor 1A, CDKN1A), and p16 (Cyclin-Dependent Kinase Inhibitor 2A, CDKN2A), which are associated with DNA damage response, were measured using the Western blot method and quantified using GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) as a loading control.

[0170] As can be seen in Fig. 14, it can be seen that as the size of the silica beads within the pre-nanostructure according to the experimental example increases, that is, as the spread area (adsorption force) of the first cell on the topographic pattern layer of the pre-nanostructure decreases, the expression levels of proteins p53, p21, and p16 significantly increase. Accordingly, it can be seen that as the cell adsorption force decreases, a stress response is induced, and DDR pathway activation and cellular aging phenotypes are promoted.

[0171]

[0172] FIG. 15 is a graph showing the expression level of a protein related to DNA damage response (DDR) of a second cell (WS1) seeded under cell aging induction conditions on a topography pattern layer formed according to the silica bead diameter of a preliminary nanostructure according to an experimental example of the present invention, analyzed and quantified by Western blot.

[0173] Referring to Fig. 15, cell aging was induced in second cells (WS1) by treating them with Paclitaxel (PTX, 100 nM), and then the second cells were seeded using the cell seeding method according to the experimental example on different substrate (preliminary nanostructure) conditions, including a control group (Flat), SB1200 (1,200 nm), SB2000 (2,000 nm), and CTR (condition without PTX treatment). Subsequently, the expression levels of DDR-related proteins p53 (Tumor Protein p53) and p16 (Cyclin-Dependent Kinase Inhibitor 2A, CDKN2A) were measured by Western blot, and β-actin (Beta-actin) was used as an internal control (loading control) for quantification.

[0174] As shown in Fig. 15, when the second cells, aging induced by PTX, are seeded onto the topographic pattern layer of the SB1200 pre-nanostructure (condition with the highest adsorption capacity), it can be seen that the expression levels of p53 and p16 proteins are significantly reduced compared to the Flat or SB2000 conditions. Accordingly, it can be seen that as cell adsorption capacity increases, the stress response and activation of the DDR pathway are alleviated, and the cell aging phenotype is suppressed.

[0175]

[0176] For reference, the same experimental results can be reproduced in a nanostructure having a topography pattern identical to the surface pattern of the topographene pattern layer of the pre-nanostructure according to the experimental example of the present invention. This supports the fact that the effect of the present invention is not limited to the material of the pattern but is applicable to all nanostructures having the same topography pattern.

[0177]

[0178] Although the present invention has been described in detail using preferred embodiments, the scope of the invention is not limited to specific embodiments and should be interpreted by the appended claims. Furthermore, those skilled in the art will understand that many modifications and variations are possible without departing from the scope of the invention.

[0179] The nanostructure according to an embodiment of the present invention enables precise induction or observation of cell behaviors such as adsorption, migration, and proliferation, and can be effectively utilized in various biomedical fields, such as evaluating cancer cell motility, designing scaffolds for tissue engineering, and improving biosensor sensitivity.

Claims

1. A nanostructure having a topography pattern formed thereon having multiple convex portions, A nanostructure comprising a cell adsorption force (A(r)) provided on the above topography pattern, which follows <Equation 1> below. <Mathematical Formula 1> A(r) = A0 - α(1 / r) - βr (A0 = intrinsic cell adhesion force, α = cell stimulation constant for the curvature of the convex part, β = cell stimulation constant for the concave part formed by three adjacent convex parts, r = half the size of the convex part in a two-dimensional plane) 2. In Paragraph 1, The above cells include a first cell which is a cancer cell and a second cell which is a normal cell, and A nanostructure comprising, wherein in the above <Mathematical Formula 1>, the relationship between the adsorption force of the first cell and the r value is expressed by the following <Mathematical Formula 1-1>. <Mathematical Formula 1-1> A(r) = A0 - βr (A0 = intrinsic cell adhesion force, β = cell stimulation constant for the depression formed by three adjacent convexities, r = half the size of the convexity in a two-dimensional plane) 3. In Paragraph 2, A nanostructure comprising the relationship between the adsorption force of the second cell and the r value, wherein the relationship follows <Equation 1>.

4. In Paragraph 2, The first cell above includes a mouse melanoma cell line (B16-F10; mouse melanoma cell line), and The second cell above is a nanostructure comprising human skin fibroblasts (WS1; Human Skin Fibroblast Cell Line).

5. A nanostructure having a topography pattern formed in which a plurality of convex and oblong segments are regularly arranged, The adsorption force of the cell provided on the topography pattern is controlled based on a change in the cell area of ​​the cell according to half the size of the convex portion of the topography pattern, and The above cells include a first cell which is a cancer cell and a second cell which is a normal cell, and The adsorption force of the first cell includes that, from a two-dimensional plane perspective, it decreases linearly as the size of half the size of the convex portion of the topography pattern increases, and The adsorption force of the second cell includes having a maximum value when half the size of the convex portion of the topography pattern is equal to √(α / β) in terms of a two-dimensional plane, and A nanostructure comprising: the above α value is a cell stimulation constant for the curvature of the convex portion of the topography pattern, and the above β value is a cell stimulation constant for the concave portion formed by three adjacent convex portions of the topography pattern.

6. In Paragraph 5, The maximum cell area of ​​the first cell provided on the topography pattern of the nanostructure is 1,100 μm 2 Including the following, The maximum cell area of ​​the second cell provided on the topography pattern of the nanostructure is 1,100 μm 2 A nanostructure containing more than 7. A method for manufacturing a nanostructure comprising the step of manufacturing a nanostructure by forming a topography pattern having a plurality of convex portions, A method for manufacturing a nanostructure comprising the adsorption force (A(r)) of a cell provided on the above topography pattern following <Equation 1> below. <Mathematical Formula 1> A(r) = A0 - α(1 / r) - βr (A0 = intrinsic cell adhesion force, α = cell stimulation constant for the curvature of the convex part, β = cell stimulation constant for the concave part formed by three adjacent convex parts, r = half the size of the convex part in a two-dimensional plane) 8. In Paragraph 7, The step of forming the above topography pattern is, A step of manufacturing a pre-nanostructure having a topography pattern layer laminated on the upper surface of a base substrate; A step of providing a mold polymer on the above-mentioned pre-nanostructure and drying it to manufacture a mold structure having a concave pattern which is the reverse phase of the surface pattern of the topography pattern layer; and A method for manufacturing a nanostructure, comprising the step of separating the mold structure from the pre-nanostructure, providing a biocompatible polymer on the mold structure, and drying to produce the nanostructure having the topography pattern which is the reverse phase of the intaglio pattern and is the same pattern as the surface pattern of the topography pattern layer.

9. In Paragraph 8, The above method for manufacturing a preliminary nanostructure is, A step of preparing the above base substrate and transfer stamp; A step of forming a fixing layer by coating an amine-based resin on the above base substrate; A step of providing a plurality of beads on the transfer stamp and rubbing with an elastomer slab to form the topography pattern layer in the form of a monolayer in which the plurality of beads are arranged in a two-dimensional hexagonal dense array; A step of manufacturing a base nanostructure in which the base substrate, the fixing layer, and the topography pattern layer are sequentially stacked by transferring the topography pattern layer on the transfer stamp onto the base substrate on which the fixing layer is formed; and A method for manufacturing a nanostructure, comprising the step of heat-treating the base nanostructure to remove the fixing layer between the base substrate and the topography pattern layer, and then washing and drying to manufacture the pre-nanostructure having the topography pattern layer laminated on the upper surface of the base substrate.

10. In Paragraph 9, A method for manufacturing a nanostructure, comprising controlling the heat treatment temperature of the base nanostructure to 400℃ to 600℃.

11. In Paragraph 9, The above amine-based resin comprises polyethyleneimine (PEI), and The above transfer stamp, the above elastomer slab, and the above polymer for the mold comprise polydimethylsiloxane (PDMS), and The above beads include silica beads, A method for manufacturing a nanostructure, wherein the above-mentioned biocompatible polymer comprises a silicone elastomer or TPU (thermoplastic polyurethane).

12. In Paragraph 11, A method for manufacturing a nanostructure, comprising that the size of the beads is 340 nm to 5,000 nm.

13. In Paragraph 7, The above cells include a first cell which is a cancer cell and a second cell which is a normal cell, and A method for manufacturing a nanostructure comprising, wherein in the above <Mathematical Formula 1>, the relationship between the adsorption force of the first cell and the r value is expressed by the following <Mathematical Formula 1-1>. <Mathematical Formula 1-1> A(r) = A0 - βr (A0 = intrinsic cell adhesion force, β = cell stimulation constant for the depression formed by three adjacent convexities, r = half the size of the convexity in a two-dimensional plane) 14. In Paragraph 13, A method for manufacturing a nanostructure, comprising the relationship between the adsorption force of the second cell and the r value following <Mathematical Formula 1>.

15. In Paragraph 14, The first cell above includes a mouse melanoma cell line (B16-F10; mouse melanoma cell line), and The second cell above is a nanostructure comprising human skin fibroblasts (WS1; Human Skin Fibroblast Cell Line).

Images