Method for manufacturing polymer-coated substrate for capturing cancer cells

Abstract

Provided is a method for manufacturing a polymer-coated substrate for capturing cancer cells, in which a polymer layer can be stably held on the surface of the substrate for a long period of time even in a liquid environment. The method of manufacturing a polymer-coated substrate for capturing cancer cells includes forming a polymer layer on an anti-reflective treated substrate surface using a hydrophilic polymer represented by the following formula (I): wherein R1 represents a hydrogen atom or a methyl group; r2 represents an alkyl group; m represents 1 to 5; and n represents a repetition number.

Description

Technical Field

[0001] The present invention relates to a method of making a polymer-coated substrate for capturing cancer cells. Background Art

[0002] It has been proposed to coat a substrate surface with a specific polymer to produce a device for capturing specific cells (blood cells, cancer cells, etc. present in the blood or biological fluid) from blood or biological fluid.

[0003] However, for example, in the case of a glass substrate, some special polymer materials do not have a high affinity for glass. When a polymer material that does not have a high affinity is coated on a substrate to form a polymer film, the coating film may peel off from the substrate in a liquid environment (e.g., in water), may partially lift up, or may separate. The lifting or separation may affect the capture ability. For example, the coating film may separate after it captures cancer cells. A polymer-coated substrate for capturing cancer cells is needed, wherein the polymer layer can be stably formed (applied) on the substrate surface for a long time even in a liquid environment. Summary of the invention Technical issues

[0004] The present invention aims to solve the above problems and provide a method for manufacturing a polymer-coated substrate for capturing cancer cells, wherein the polymer layer can be stably maintained on the substrate surface for a long time even in a liquid environment. Solution to the problem

[0005] The present invention relates to a method for manufacturing a polymer-coated substrate for capturing cancer cells, the method comprising forming a polymer layer on the surface of an anti-reflection treated substrate using a hydrophilic polymer, wherein the hydrophilic polymer is represented by the following formula (I): In the formula, R 1 Represents a hydrogen atom or a methyl group; R 2 represents an alkyl group; m represents 1 to 5; and n represents a repetition number. Advantageous Effects of the Invention

[0006] The method for manufacturing a polymer-coated substrate for capturing cancer cells of the present invention comprises forming a polymer layer on the surface of a substrate that has been anti-reflection treated using a hydrophilic polymer represented by formula (I). Even in a liquid environment (e.g., in water), the polymer layer is strongly adsorbed and coated on the surface of the substrate, thereby preventing the polymer layer from lifting or separating. Therefore, a polymer-coated substrate for capturing cancer cells can be provided, wherein the polymer layer is stably maintained on the surface of the substrate for a long time. The ability to capture cancer cells can be improved by using a polymer-coated substrate for capturing cancer cells manufactured by this method. DETAILED DESCRIPTION

[0007] The present invention relates to a method for manufacturing a polymer-coated substrate for capturing cancer cells, comprising forming a polymer layer on the surface of a substrate subjected to an anti-reflection treatment using a hydrophilic polymer represented by formula (I).

[0008] The number of tumor cells (cancer cells, etc.) present in biological fluids is very small, such as circulating tumor cells (several to hundreds of cells / 1 ml of blood), and it is considered important to capture as many tumor cells as possible present in biological fluid samples to analyze them. The manufacturing method of the present invention includes forming a polymer layer on the surface of a substrate treated with anti-reflection using a hydrophilic polymer represented by formula (I). The method is capable of manufacturing a polymer-coated substrate for capturing cancer cells, wherein the polymer is firmly adsorbed on the substrate surface to be stably maintained for a long time without lifting or separation. Instabilities such as lifting or separation of the polymer layer will affect the capture of cancer cells. By improving stability (long-term stable coating performance), excellent cancer cell capture ability can be achieved. The number of cancer cells in a biological fluid can be determined by measuring the number of cancer cells captured by the polymer layer of the polymer-coated substrate manufactured for capturing cancer cells, so that the cancer treatment effect can be confirmed, etc. In addition, the captured cancer cells can be cultured and then used to determine the effect of a drug (e.g., an anticancer drug). Therefore, the in vitro effect of a drug (e.g., an anticancer drug) can be confirmed before administration. This technology can also be used to screen drugs (e.g., anticancer drugs). In addition, genetic analysis of captured cancer cells can be used to screen drugs (e.g., anti-cancer drugs).

[0009] (Method for producing a polymer-coated substrate for capturing cancer cells) The method of manufacturing a polymer-coated substrate for capturing cancer cells of the present invention comprises forming a polymer layer on the surface of a substrate subjected to an anti-reflection treatment using a hydrophilic polymer represented by formula (I).

[0010] As used herein, the term "anti-reflective treatment" refers to a treatment that suppresses reflection from a surface. Examples of the treatment for suppressing surface reflection include anti-reflection treatment (non-glare treatment) performed by forming unevenness on the surface of the substrate by spraying fine glass particles on the surface or applying a special coating.

[0011] Specific examples of the anti-reflection treatment include non-reflective treatment, low reflection treatment, polarizing treatment, and anti-glare treatment. These may be performed alone or in combination of two or more.

[0012] The 60 degree glossiness (glossiness at 60 degrees) of the antireflection treated substrate is preferably 150% or less, more preferably 130% or less, but is preferably 50% or more, more preferably 70% or more. Herein, the term "60 degree glossiness" is a value determined by measuring the 60 degree glossiness of the center portion of the antireflection treated substrate using a gloss meter (IG320, HORIBA) according to JIS-K 7150.

[0013] Non-limiting examples of the substrate include glass, acrylic resin, methacrylic resin, polyester resin, cycloolefin resin, and silicone resin. Among them, a glass substrate is preferred. The type of glass in the glass substrate is not limited. Examples include soda-lime glass, alkali-free glass, borosilicate glass (SiO2-B2O3-ZnO glass, SiO2-B2O3-Bi2O3 glass, etc.), potassium glass, crystal glass (glass containing PbO, such as SiO2-PbO glass, SiO2-PbO-B2O3 glass, SiO2-B2O3-PbO glass, etc.), titanium crystal glass, barium glass, boron glass (B2O3-ZnO-PbO glass, B2O3-ZnO-Bi2O3 glass, B2O3-Bi2O3 glass, B2O3-ZnO glass, etc.), strontium glass, aluminosilicate glass, sodium zinc glass, sodium barium glass (BaO-SiO2 glass, etc.). The glasses can be used alone or in combination of two or more.

[0014] Although the thickness of the antireflection treated substrate is not limited, the average thickness is preferably 100 μm or more and 5000 μm or less, more preferably 100 μm or more and 3000 μm or less. The term "average thickness" refers to the average value of thicknesses measured at ten arbitrary points using a micrometer.

[0015] The polymer layer is formed using a hydrophilic polymer (a polymer having hydrophilicity) represented by the following formula (I): In the formula, R 1 Represents a hydrogen atom or a methyl group; R 2 represents an alkyl group; m represents 1 to 5; and n represents a repetition number. The hydrophilic polymer represented by formula (I) may be used alone or in combination of two or more.

[0016] R 2 The number of carbon atoms of the alkyl group is preferably 1 to 10, more preferably 1 to 5. 2 Particularly preferred is a methyl group or an ethyl group. The symbol m is preferably 1 to 3. The symbol n (the number of repeating units) is preferably 70 to 2500, more preferably 90 to 1500.

[0017] Examples of the hydrophilic polymer represented by formula (I) include copolymers or homopolymers of one or two or more hydrophilic monomers. The hydrophilic polymer may be used alone or in combination of two or more.

[0018] Examples of the homopolymer or copolymer of the hydrophilic monomer include polyalkoxyalkyl methacrylate, such as polymethoxyethyl (meth)acrylate. Polyalkoxyalkyl acrylate is preferred, and among them, poly (2-methoxyethyl acrylate) is more preferred.

[0019] Specific examples of hydrophilic monomers constituting the hydrophilic polymer represented by formula (I) include alkoxyalkyl (meth) acrylates, such as methoxyethyl (meth) acrylate. Alkoxyalkyl acrylates are preferred, and 2-methoxyethyl acrylate is more preferred. The hydrophilic monomers may be used alone or in combination of two or more.

[0020] The step of forming a polymer layer on the surface of the substrate treated with anti-reflection using a hydrophilic polymer represented by formula (I) may include forming two or more polymer layers. The two or more polymer layers may be formed using the same hydrophilic polymer or different hydrophilic polymers. In order to better achieve a favorable effect, it is desirable to use the same hydrophilic polymer to form two or more polymer layers.

[0021] The hydrophilic polymer represented by formula (I) constituting the two or more polymer layers may be appropriately selected. The hydrophilic polymer represented by formula (I) may be used alone or in combination of two or more in each polymer layer.

[0022] In two or more polymer layers, the Mn of the hydrophilic polymer constituting the outermost layer (the outermost (surface side) polymer layer) is preferably above 5000, more preferably above 6000, and further preferably above 7000. At the same time, it is preferably below 30000, more preferably below 25000, and further preferably below 20000.

[0023] In order to better achieve the beneficial effects, the number average molecular weight of the hydrophilic polymer constituting the polymer layer on the surface of the anti-reflection treated substrate (the polymer layer in contact with the anti-reflection treated substrate (hereinafter also referred to as the innermost layer)) among the two or more polymer layers is preferably greater than the number average molecular weight of the hydrophilic polymer constituting any other polymer layer (one or more polymer layers other than the innermost layer among the two or more polymer layers).

[0024] In the two or more polymer layers, the Mn of the hydrophilic polymer constituting the other polymer layer is preferably 5000 or more, more preferably 6000 or more, further preferably 7000 or more, and is preferably 30000 or less, more preferably 25000 or less, further preferably 20000 or less.

[0025] When the number average molecular weight of the hydrophilic polymer constituting the innermost layer among the two or more polymer layers is larger than the number average molecular weight of the hydrophilic polymer constituting the polymer layers other than the innermost layer, the Mn of the hydrophilic polymer constituting the innermost layer is preferably 32000 or more, more preferably 40000 or more, further preferably 60000 or more, and is preferably 200000 or less, more preferably 150000 or less, further preferably 120000 or less. When Mn is within the above range, the advantageous effects tend to be better achieved.

[0026] Herein, the number average molecular weight (Mn) can be measured using a gel permeation chromatograph (GPC) (GPC-8000 series, available from Tosoh Corporation; detector: differential refractometer; column: TSKgel SuperMultipore HZ-M available from Tosoh Corporation). The obtained value is calibrated using a polystyrene standard.

[0027] The total thickness (total film thickness) of one or two or more polymer layers (one or more hydrophilic polymer layers formed using the hydrophilic polymer represented by formula (I)) is preferably 10 to 1000 nm, more preferably 30 to 700 nm, and further preferably 50 to 350 nm. When the thickness is controlled within the above range, low adsorption of normal proteins and cells and selective capture of cancer cells can be expected.

[0028] The surface of the polymer layer (the surface of the polymer layer of the polymer-coated substrate for capturing cancer cells) preferably has at least partially (partially or entirely) a water contact angle of 60 degrees or less, more preferably 50 degrees or less. The lower limit is not limited, and the smaller the angle, the better.

[0029] A polymer-coated substrate for capturing cancer cells, wherein the polymer layer formed in whole or in part on the surface of the substrate treated for anti-reflection can be manufactured by coating (covering) the whole or in part of the surface of the substrate treated for anti-reflection with a polymer using known techniques. Examples of this technique include method 1): a polymer solution or dispersion obtained by dissolving or dispersing a hydrophilic polymer represented by formula (I) in a solvent is introduced onto the surface of the substrate treated for anti-reflection (the concave portion of the substrate), then maintained for a predetermined time, and optionally dried; and method 2): a polymer solution or dispersion is applied (sprayed) to the surface of the substrate treated for anti-reflection, and optionally subsequently dried for a predetermined time. The polymer-coated substrate for capturing cancer cells can then be combined with other components as needed. Thus, a device capable of, for example, capturing, culturing and / or analyzing cancer cells can be manufactured.

[0030] As the solvent, the introducing method, the applying (spraying) method, etc., conventionally known materials or methods can be applied. The holding / drying time in method 1) or 2) can be appropriately selected according to the size of the substrate, the type of liquid introduced, etc. The holding time is preferably 10 seconds to 10 hours, more preferably 1 minute to 5 hours, and further preferably 5 minutes to 2 hours. Drying is preferably carried out at room temperature (about 23°C) to 80°C, more preferably at room temperature to 60°C. Drying can be carried out under reduced pressure. After the predetermined holding time, the excess polymer solution or dispersion can be appropriately discharged before drying.

[0031] The solvent can be any solvent that can dissolve the polymer, and can be appropriately selected according to the polymer used. The solvent can be water, an organic solvent or a mixture thereof. Examples of organic solvents include alcohols (e.g., methanol, ethanol, n-propanol, isopropanol and methoxypropanol), ketones (e.g., acetone and methyl ethyl ketone), tetrahydrofuran, acetonitrile, ethyl acetate and toluene.

[0032] The concentration of the polymer solution or dispersion is not limited and can be appropriately selected in consideration of ease of introduction, application and spraying, productivity, etc. The concentration of the polymer is preferably 0.1-10.0% by mass, more preferably 0.2-5.0% by mass, based on 100% by mass of the polymer solution or dispersion.

[0033] When a polymer layer is formed on the surface of a substrate subjected to anti-reflection treatment using a hydrophilic polymer represented by formula (I) according to the manufacturing method, the polymer layer is strongly adsorbed and coated on the surface of the glass substrate even in a liquid environment (e.g., in water), thereby preventing the polymer layer from being lifted or separated. Therefore, a polymer-coated substrate for capturing cancer cells can be provided, wherein the polymer layer is stably maintained on the surface of the substrate for a long time.

[0034] Since the polymer-coated substrate for capturing cancer cells can capture cancer cells, when the sample (blood or biological fluid) contacts the polymer-coated substrate for capturing cancer cells (wherein a polymer layer is formed on the surface of the substrate after anti-reflection treatment using a hydrophilic polymer represented by formula (I)), a capture effect can be exhibited. By measuring the number of captured cancer cells, the number of cancer cells in the blood or biological fluid sample can be determined, thereby expecting to confirm the cancer treatment effect, etc. Example

[0035] The present invention is specifically described based on the embodiments. However, the present invention is not limited to the embodiments.

[0036] (Preparation of Hydrophilic Polymer) Poly(2-methoxyethyl acrylate) (PMEA) was prepared by thermally polymerizing 2-methoxyethyl acrylate (25 wt % in toluene solution) at 60° C. for 7 hours using a 12.5 mg / mL solution of azobisisobutyronitrile (AIBN) in toluene.

[0037] (Examples 1 to 4) The surface of a flat glass (made of soda lime glass and having an average thickness of 1.35 mm) was subjected to a non-glare treatment. Then, the flat glass was washed with methanol and dried, thereby obtaining a non-glare glass substrate as shown in Table 1. A PMEA methanol solution having a concentration shown in Table 1 was introduced into the concave portion of the obtained non-glare glass substrate, and then immediately dried in a 40° C. oven under vacuum for 5 minutes (coating). Thus, a polymer-coated glass substrate was prepared.

[0038] (Comparative Example 1) A PMEA methanol solution having a concentration shown in Table 1 was introduced into the concave portion of a flat glass substrate (made of soda lime glass, with an average thickness of 1.35 mm), and then immediately vacuum dried in an oven at 40° C. for 5 minutes (coating). Thus, a polymer-coated glass substrate was prepared.

[0039] (Examples 5 and 6, Comparative Example 2) The preparation of the polymer-coated glass substrate was the same as in Example 1, except that the Mn of PMEA, the concentration and feed amount of the PMEA methanol solution, and the anti-glare treatment were changed according to Table 2. Polymer 2 (Mn: 80000) as PMEA is hardly soluble in methanol at room temperature. Therefore, the polymer was heated to 40° C. to dissolve, thereby preparing a polymer solution. In Comparative Example 2 and Example 5, only one hydrophilic polymer layer was formed.

[0040] The polymer-coated glass substrates prepared in Examples and Comparative Examples were evaluated by the following methods.

[0041] [Analysis of blood mixed with cancer cells (Examples 1 to 4, Comparative Example 1, Table 1)] Dyed human colon adenocarcinoma cells (HT-29) were spiked into the blood to prepare spiked blood (1000 HT29 cells / 1 mL of blood). 10% RosetteSep human CD45 depletion composition (Depletion Cocktail) was added to the spiked blood, and the mixture was concentrated by centrifugation using Lypmhoprep. Then, the mononuclear cell layer was separated. PBS was added to the mononuclear cell layer, and then centrifuged twice. After centrifugation, the aggregates in the lower layer were suspended in a liquid culture medium containing 10% fetal bovine serum (FBS), and the liquid volume was equal to the initial whole blood volume. 1.3 mL of the suspension was introduced into a chamber (polymer-coated glass substrate) and placed at 37°C for 21 hours to cause adhesion. Thereafter, non-adherent cells were washed away with a PBS solution. Next, the number of adhered cancer cells (HT-29) and residual leukocytes was counted using a fluorescent microscope. The number of cancer cells and the percentage (%) of "number of leukocytes / number of cancer cells × 100" were determined based on the count.

[0042] [Analysis of blood mixed with cancer cells (Examples 5 and 6, Comparative Example 2, Table 2)] Dyed human colon adenocarcinoma cells (HT-29) were spiked into the blood to prepare spiked blood (500 HT29 cells / 1 mL of blood). 10% RosetteSep human CD45 depletion composition was added to the spiked blood, and the mixture was concentrated by centrifugation using Lypmhoprep. Then, the mononuclear cell layer was separated. PBS was added to the mononuclear cell layer and then centrifuged twice. After centrifugation, the aggregates in the lower layer were suspended in a liquid culture medium containing 10% fetal bovine serum (FBS) in a liquid volume equal to the initial whole blood volume. 1.3 mL of the suspension was introduced into a chamber (polymer-coated glass substrate) and placed at 37°C for 24 hours to cause adhesion. Thereafter, non-adherent cells were washed away with a PBS solution. Next, the number of adhered cancer cells (HT-29) was counted using a fluorescence microscope. The number of cancer cells (relative value) was determined based on the count.

[0043] [Evaluation of polymer layer separation in PBS] The polymer-coated glass substrate fabricated above was immersed in phosphate buffered saline (PBS), and separation was observed after 1 hour, 24 hours, and 48 hours.

[0044] [Table 1]

[0045] [Table 2]

[0046] In Comparative Example 1, when immersed in PBS, the polymer layer gradually lifted up over time and finally separated.

[0047] In contrast, in Examples 1 to 4, lifting of the polymer layer was not observed even after 48 hours. The percentage (%) of "number of white blood cells / number of cancer cells × 100" in Examples was smaller than that in Comparative Example 1, and thus cancer cells could be selectively captured. In Examples 1 to 4, the selective capture was particularly good using a substrate having a 60 degree glossiness of 150% to 50%. In Examples 5 and 6 using a substrate having a 60-degree glossiness of 90%, the number of adhered cancer cells (relative value) was greater than that in Comparative Example 2 using a substrate not subjected to a non-glare treatment. The number was particularly large in Example 6 in which two polymer layers were formed.

[0048] Exemplary embodiments of the present invention include the following. Embodiment 1: A method for making a polymer-coated substrate for capturing cancer cells, the method comprising A hydrophilic polymer is used to form a polymer layer on the surface of the substrate after anti-reflection treatment, and the hydrophilic polymer is represented by the following formula (I): In the formula, R 1 Represents a hydrogen atom or a methyl group; R 2 represents an alkyl group; m represents 1 to 5; and n represents a repetition number.

[0049] Embodiment 2: The method for manufacturing a polymer-coated substrate for capturing cancer cells according to embodiment 1, The anti-reflection treatment is selected from at least one of non-reflection treatment, low-reflection treatment, polarization treatment and non-glare treatment.

[0050] Embodiment 3: The method for manufacturing a polymer-coated substrate for capturing cancer cells according to embodiment 1 or 2, Wherein, the 60-degree glossiness of the anti-reflection treated substrate is 150% to 50%.

[0051] Embodiment 4: A method for manufacturing a polymer-coated substrate for capturing cancer cells according to embodiment 1 or 2, The 60-degree glossiness of the anti-reflection treated substrate is 130% to 70%.

[0052] Embodiment 5: A method for manufacturing a polymer-coated substrate for capturing cancer cells according to any one of embodiments 1 to 4, Wherein, the substrate subjected to anti-reflection treatment is a glass substrate.

[0053] Embodiment 6: A method for manufacturing a polymer-coated substrate for capturing cancer cells according to any combination of any one of embodiments 1 to 5, Wherein, the total thickness of the polymer layer is 10 to 1000 nm.

[0054] Embodiment 7: A method for manufacturing a polymer-coated substrate for capturing cancer cells according to any combination of any one of embodiments 1 to 6, Wherein, the forming of the polymer layer comprises forming two or more polymer layers on the surface of the anti-reflection treated substrate using the hydrophilic polymer represented by formula (I).

[0055] Embodiment 8: The method for manufacturing a polymer-coated substrate for capturing cancer cells according to embodiment 7, wherein the two or more polymer layers are formed using the same hydrophilic polymer, and Among the two or more polymer layers, the number average molecular weight of the hydrophilic polymer constituting the polymer layer on the surface of the substrate subjected to the anti-reflection treatment is greater than the number average molecular weight of the hydrophilic polymer constituting the other polymer layers.

[0056] Embodiment 9: The method for manufacturing a polymer-coated substrate for capturing cancer cells according to embodiment 8, The number average molecular weight of the hydrophilic polymer constituting the other polymer layer is less than 30,000.

Claims

1. A method for making a polymer-coated substrate for capturing cancer cells, the method comprising: A hydrophilic polymer is used to form a polymer layer on the surface of the substrate after anti-reflection treatment, and the hydrophilic polymer is represented by the following formula (I): In the formula, R 1 Represents a hydrogen atom or a methyl group; R 2 represents an alkyl group; m represents 1 to 5; and n represents a repetition number.

2. The method for manufacturing a polymer-coated substrate for capturing cancer cells according to claim 1, in, The anti-reflection treatment is selected from at least one of a non-reflection treatment, a low-reflection treatment, a polarization treatment and a non-glare treatment.

3. The method for manufacturing a polymer-coated substrate for capturing cancer cells according to claim 1 or 2, in, The 60-degree glossiness of the anti-reflection treated substrate is 150% to 50%.

4. The method for manufacturing a polymer-coated substrate for capturing cancer cells according to claim 1 or 2, in, The 60-degree glossiness of the anti-reflection treated substrate is 130% to 70%.

5. A method for manufacturing a polymer-coated substrate for capturing cancer cells according to any one of claims 1 to 4, in, The substrate subjected to anti-reflection treatment is a glass substrate.

6. A method for manufacturing a polymer-coated substrate for capturing cancer cells according to any one of claims 1 to 5, in, The total thickness of the polymer layer is 10 to 1000 nm.

7. A method for manufacturing a polymer-coated substrate for capturing cancer cells according to any one of claims 1 to 6, in, The forming of the polymer layer includes forming two or more polymer layers on the surface of the substrate subjected to the anti-reflection treatment using the hydrophilic polymer represented by formula (I).

8. The method for manufacturing a polymer-coated substrate for capturing cancer cells according to claim 7, in, The two or more polymer layers are formed using the same hydrophilic polymer, and Among the two or more polymer layers, the number average molecular weight of the hydrophilic polymer constituting the polymer layer on the surface of the substrate subjected to the anti-reflection treatment is greater than the number average molecular weight of the hydrophilic polymer constituting the other polymer layers.

9. The method for manufacturing a polymer-coated substrate for capturing cancer cells according to claim 8, in, The number average molecular weight of the hydrophilic polymer constituting the other polymer layer is 30,000 or less.

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