Method for producing polymer-coated substrate for capturing cancer cell

Abstract

To provide a method for producing a polymer-coated substrate for capturing cancer cells that enables a polymer layer to be stably formed on the surface of a substrate over a long duration even in liquid environments.SOLUTION: A method for producing a polymer-coated substrate for capturing cancer cells includes the step of forming a polymer layer with a hydrophilic polymer represented by formula (I) on the surface of a substrate subjected to antireflection treatment. [Formula 1] (where R1 is a hydrogen atom or a methyl group, R2 is an alkyl group, m is 1-5, and n is the number of repetitions).SELECTED DRAWING: None

Description

[Technical field]

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

[0002] A technology has been proposed to coat the surface of a substrate with a special polymer in order to create devices for capturing specific cells in blood and body fluids (blood cells, cancer cells present in blood and body fluids, etc.).

[0003] However, for example, when a glass substrate is used as the substrate, some special polymers do not have a high affinity with glass. If the affinity is not high, the polymer film formed by the coating may peel off from the substrate in a liquid environment such as water after coating, and may partially float or peel off. Since floating or peeling affects the capture performance, such as causing cancer cells to separate after capture, there is a need to provide a polymer-coated substrate for capturing cancer cells in which a polymer layer is stably formed (coated) on the substrate surface for a long period of time even in a liquid environment. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention aims to solve the above-mentioned problems and provide a method for producing a polymer-coated substrate for capturing cancer cells, in which a polymer layer is stably formed on the surface of the substrate for a long period of time even in a liquid environment. [Means for solving the problem]

[0005] The present invention relates to a method for producing a polymer-coated substrate for capturing cancer cells, which comprises a step of forming a polymer layer from a hydrophilic polymer represented by the following formula (I) on the surface of a substrate that has been subjected to an antireflection treatment. [ka] (In the formula, R 1 is a hydrogen atom or a methyl group, R 2represents an alkyl group, m is 1 to 5, and n is the number of repetitions. Effect of the Invention

[0006] According to the present invention, the method for producing a polymer-coated substrate for capturing cancer cells includes a step of forming a polymer layer from the hydrophilic polymer represented by formula (I) on the surface of a substrate that has been subjected to an anti-reflection treatment, and therefore the polymer layer is firmly adsorbed and coated on the surface of the substrate even in a liquid environment such as water, thereby preventing the polymer layer from floating up or peeling off, and a polymer-coated substrate for capturing cancer cells in which the polymer layer is stably formed on the surface of the substrate for a long period of time can be provided. Therefore, the use of the produced polymer-coated substrate for capturing cancer cells can improve the capturing performance of cancer cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] The present invention relates to a method for producing a polymer-coated substrate for capturing cancer cells, which comprises a step of forming a polymer layer from the hydrophilic polymer represented by formula (I) on the surface of a substrate that has been subjected to an antireflection treatment.

[0008] Tumor cells (cancer cells, etc.) that appear in body fluids, such as circulating tumor cells in the blood (several to several hundred cells / mL of blood), are very few in number, and it is considered important to capture as many tumor cells present in the collected body fluid as possible in order to provide them for testing. Since the present invention is a manufacturing method including a step of forming a polymer layer using a hydrophilic polymer represented by the formula (I) on the surface of a substrate that has been subjected to an anti-reflection treatment, a polymer-coated substrate for capturing cancer cells can be manufactured in which the polymer is firmly adsorbed on the substrate surface and is stable for a long period of time without lifting or peeling. In addition, since instability such as lifting or peeling of the polymer layer affects the capturing ability of cancer cells, excellent capturing performance of cancer cells can be obtained by improving the stability (long-term stable covering ability). Therefore, by measuring the number of cancer cells captured in the polymer layer of the prepared polymer-coated substrate for capturing cancer cells, the number of cancer cells in the body fluid can be determined, and the effect of cancer treatment can be confirmed. In addition, by culturing the captured cancer cells and checking the effectiveness of anticancer drugs, etc. using the cultured cells, it is possible to check the effectiveness of anticancer drugs, etc. outside the body before administering them, and it is also useful for selecting anticancer drugs, etc. Furthermore, genetic analysis of the captured cancer cells is useful for selecting anticancer drugs, etc.

[0009] (Method of manufacturing polymer-coated substrate for capturing cancer cells) The method for producing a polymer-coated substrate for capturing cancer cells of the present invention includes a step of forming a polymer layer from the hydrophilic polymer represented by formula (I) on the surface of a substrate that has been subjected to an antireflection treatment.

[0010] In this specification, anti-reflective treatment means a treatment that reduces surface reflection. Examples of treatments that reduce surface reflection include anti-reflection treatments (non-glare treatments) such as spraying fine glass particles onto the substrate surface to create irregularities, and applying a special coating to the surface.

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

[0012] The substrate that has been subjected to the anti-reflection treatment preferably has a 60 degree gloss (gloss at 60 degrees) of 150% or less, more preferably 130% or less, and preferably 50% or more, more preferably 70% or more. In this specification, the 60 degree gloss is a value obtained by measuring the 60 degree gloss of the central part of a substrate that has been subjected to an anti-reflection treatment using a glossmeter (IG320, manufactured by HORIBA) in accordance with JIS-K7150.

[0013] The substrate is not particularly limited, and examples thereof include glass, acrylic resin, methacrylic resin, polyester resin, cycloolefin resin, silicone resin, etc. Among these, a glass substrate is preferable. The type of glass constituting the glass substrate is not particularly limited, and examples thereof include soda-lime glass, non-alkali glass, borosilicate glass (SiO2-B2O3-ZnO-based glass, SiO2-B2O3-Bi2O3-based glass, etc.), potash glass, crystal glass (glass containing PbO, for example, SiO2-PbO-based glass, SiO2-PbO-B2O3-based glass, SiO2-B2O3-PbO-based glass, etc.), titanium crystal glass, barium glass, boron glass (B2O3-ZnO-PbO-based glass, B2O3-ZnO-Bi2O3-based glass, B2O3-Bi2O3-based glass, B2O3-ZnO-based glass, etc.), strontium glass, alumina silicate glass, soda zinc glass, soda barium glass (BaO-SiO2-based glass, etc.), etc. These glasses may be used alone or two or more types may be mixed.

[0014] The thickness of the substrate subjected to the antireflection treatment is not particularly limited, but the average thickness is preferably 100 μm to 5000 μm, more preferably 100 μm to 3000 μm. The average thickness is determined by measuring the thickness at any 10 points using a micrometer and averaging the measured values.

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

[0016] R 2 The number of carbon atoms in the alkyl group of R is preferably 1 to 10, and more preferably 1 to 5. 2 is particularly preferably a methyl group or an ethyl group. m is preferably an integer of 1 to 3. n (the number of repeating units) is preferably an integer of 70 to 2,500, and more preferably an integer of 90 to 1,500.

[0017] The hydrophilic polymer represented by the formula (I) may be, for example, a homopolymer or copolymer of one or more hydrophilic monomers. The hydrophilic polymer may be used alone or in combination of two or more kinds.

[0018] Examples of the homopolymers and copolymers of the hydrophilic monomers include polyalkoxyalkyl methacrylates such as polymethoxyethyl (meth)acrylate, etc. Among these, polyalkoxyalkyl acrylates are preferred, and poly(2-methoxyethyl acrylate) is more preferred.

[0019] Specific examples of the hydrophilic monomer constituting the hydrophilic polymer represented by the formula (I) include alkoxyalkyl (meth)acrylates such as methoxyethyl (meth)acrylate. Among them, 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 kinds.

[0020] The above process may be a process in which two or more polymer layers are formed on the surface of the substrate that has been subjected to the anti-reflection treatment by using the hydrophilic polymer represented by the above formula (I), and the two or more polymer layers may be formed by the same hydrophilic polymer or different hydrophilic polymers. Among these, from the viewpoint of obtaining a better effect, it is preferable that the two or more polymer layers are formed by using the same hydrophilic polymer.

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

[0022] The Mn of the hydrophilic polymer constituting the outermost layer (the polymer layer formed on the outermost (surface) side) of the two or more polymer layers is preferably 5,000 or more, more preferably 6,000 or more, even more preferably 7,000 or more, and is preferably 30,000 or less, more preferably 25,000 or less, even more preferably 20,000 or less.

[0023] Of the two or more polymer layers, the number average molecular weight of the hydrophilic polymer forming the polymer layer on the surface of the substrate that has been subjected to the anti-reflection treatment (the polymer layer formed in contact with the substrate that has been subjected to the anti-reflection treatment (hereinafter also referred to as the innermost layer)) is preferably larger than the number average molecular weight of the hydrophilic polymer forming the other polymer layers (one or more polymer layers other than the innermost layer among the two or more polymer layers) in order to obtain a greater effect.

[0024] In the two or more polymer layers, the Mn of the hydrophilic polymer constituting the other polymer layer is preferably 5,000 or more, more preferably 6,000 or more, even more preferably 7,000 or more, and is preferably 30,000 or less, more preferably 25,000 or less, even more preferably 20,000 or less.

[0025] In the two or more polymer layers, when the number average molecular weight of the hydrophilic polymer forming the innermost layer is larger than the number average molecular weight of the hydrophilic polymer forming the other 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, even more preferably 60000 or more, and is preferably 200000 or less, more preferably 150000 or less, even more preferably 120000 or less. Within the above ranges, more advantageous effects tend to be obtained.

[0026] In this specification, the number average molecular weight (Mn) can be determined by converting it into standard polystyrene based on the measured value by gel permeation chromatography (GPC) (GPC-8000 series manufactured by Tosoh Corporation, detector: differential refractometer, column: TSKGEL SUPERMALTPORE HZ-M manufactured by Tosoh Corporation).

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

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

[0029] The polymer layer can be produced by coating (covering) the whole or part of the surface of the anti-reflection-treated substrate with a polymer by known techniques such as (1) a method of injecting a polymer solution or dispersion obtained by dissolving or dispersing the hydrophilic polymer represented by the formula (I) in various solvents onto the surface (concave of the substrate) of the substrate that has been subjected to an anti-reflection treatment, holding the polymer solution or dispersion for a predetermined period of time, and drying the polymer for a predetermined period of time as required, etc. A polymer-coated substrate for capturing cancer cells, in which the polymer layer is formed on the whole or part of the surface of the substrate that has been subjected to an anti-reflection treatment, can then be produced by adding other components to the polymer-coated substrate for capturing cancer cells as required, thereby producing a device capable of capturing, culturing, inspecting, etc., cancer cells.

[0030] As the solvent, injection method, coating (spraying) method, and the like, conventionally known materials and methods can be applied. The holding and drying times of (1) and (2) may be appropriately set depending on 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 even more preferably 5 minutes to 2 hours. Drying is preferably carried out at room temperature (about 23°C) to 80°C, and more preferably room temperature to 60°C. Drying may also be carried out under reduced pressure. Furthermore, after a certain period of holding, excess polymer solution / dispersion may be appropriately discharged and dried.

[0031] The solvent is not particularly limited as long as it can dissolve the polymer, and may be appropriately selected according to the polymer used. For example, water, an organic solvent, or a mixture thereof may be used. Examples of the organic solvent include alcohols such as methanol, ethanol, n-propanol, i-propanol, and methoxypropanol; ketones such as acetone and methyl ethyl ketone; tetrahydrofuran, acetonitrile, ethyl acetate, and toluene.

[0032] The concentration of the polymer solution or dispersion is not particularly limited and may be appropriately selected taking into consideration injectability, coatability, sprayability, productivity, etc., but the concentration of the polymer in the polymer solution or dispersion (100 mass%) is preferably 0.1 to 10.0 mass%, more preferably 0.2 to 5.0 mass%.

[0033] By forming a polymer layer made of a hydrophilic polymer represented by formula (I) on the surface of a substrate that has been subjected to the anti-reflection treatment in the above-mentioned manufacturing method, the polymer layer is firmly adsorbed to and coated on the surface of the glass substrate even in a liquid environment such as water, thereby preventing the polymer layer from floating up or peeling off, and a polymer-coated substrate for capturing cancer cells in which the polymer layer is stably formed on the surface of the substrate for a long period of time can be provided.

[0034] Since it is possible to capture cancer cells, for example, such a capture effect can be obtained by contacting a sample (blood or body fluid) with a polymer-coated substrate for capturing cancer cells, in which a polymer layer made of a hydrophilic polymer represented by formula (I) is formed on the surface of a substrate that has been subjected to the antireflection treatment. Therefore, by measuring the number of captured cancer cells, the number of cancer cells in the collected blood or body fluid can be determined, and it is expected that the effectiveness of cancer treatment can be confirmed. EXAMPLES

[0035] The present invention will be specifically described below based on examples, but the present invention is not limited to these examples.

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

[0037] (Examples 1 to 4) The surface of a plate glass (made of soda-lime glass, average thickness 1.35 mm) was subjected to a non-glare treatment, washed with methanol and dried to obtain a non-glare glass substrate as described in Table 1. A methanol solution of PMEA having a concentration shown in Table 1 was poured into the recessed portion of the obtained non-glare glass substrate. Then, the substrate was immediately vacuum-dried (coated) in an oven at 40° C. for 5 minutes to prepare a polymer-coated glass substrate.

[0038] Comparative Example 1 A methanol solution of PMEA with the concentration shown in Table 1 was poured into the recess of a plate glass (soda-lime glass, average thickness 1.35 mm) substrate. Immediately after that, it was vacuum dried (coated) in an oven at 40°C for 5 minutes to prepare a polymer-coated glass substrate.

[0039] (Examples 5 and 6, Comparative Example 2) According to Table 2, the Mn of PMEA, the concentration and injection amount of the methanol solution of PMEA, and the anti-glare treatment were changed, and the same procedure as in Example 1 was repeated to prepare a polymer-coated glass substrate. Since PMEA of polymer 2 (Mn 80,000) is poorly soluble in methanol at room temperature, it was heated to 40° C. to dissolve it, and a polymer solution was prepared. In Comparative Example 2 and Example 5, only one hydrophilic polymer layer was formed.

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

[0041] [Spiked blood test with added cancer cells (Examples 1 to 4, Comparative Example 1, Table 1)] Stained human colon cancer-derived epithelial cells (HT-29) were spiked into blood to prepare spiked blood (1000 HT29 cells / 1 mL of blood). 10% RosetteSep Human CD45 Depletion Cocktail was added to this spiked blood, and then it was centrifuged and concentrated using Lypmhoprep. The mononuclear layer was separated, PBS was added, and centrifugation was performed twice more. The mass formed at the bottom layer after centrifugation was suspended in liquid medium (the same volume as the initial whole blood volume) supplemented with 10% FBS (fetal bovine serum). 1.3 ml of this suspension was injected into a chamber (polymer-coated glass substrate) and allowed to adhere for 21 hours at 37°C. After that, non-adhered cells were washed with PBS solution. Next, the number of attached cancer cells (HT-29) and the remaining white blood cells were counted using a fluorescent microscope. From the obtained values, the number of cancer cells and the number of white blood cells / number of cancer cells x 100 (%) were calculated.

[0042] [Spiked blood test with added cancer cells (Examples 5 and 6, Comparative Example 2, Table 2)] Stained human colon cancer-derived epithelial cells (HT-29) were spiked into blood to prepare spiked blood (500 HT29 cells / 1 mL of blood). 10% RosetteSep Human CD45 Depletion Cocktail was added to this spiked blood, and then it was centrifuged and concentrated using Lypmhoprep. The mononuclear layer was separated, PBS was added, and centrifugation was performed twice more. The mass formed at the bottom layer after centrifugation was suspended in liquid medium (the same volume as the initial whole blood volume) supplemented with 10% FBS (fetal bovine serum). 1.3 ml of this suspension was injected into a chamber (polymer-coated glass substrate) and allowed to adhere for 24 hours at 37°C. Unadhered cells were then washed with PBS solution. The number of attached cancer cells (HT-29) was then counted using a fluorescent microscope. The number of cancer cells (relative value) was calculated from the obtained value.

[0043] [Evaluation of peeling of polymer layer in PBS] The polymer-coated glass substrates thus prepared were immersed in phosphate-buffered saline (PBS) and the state of peeling 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 rose over time and eventually peeled off.

[0047] In contrast, in Examples 1 to 4, no lifting of the polymer layer was observed even after 48 hours. Furthermore, the values ​​of white blood cell count / cancer cell count × 100 (%) were smaller in the Examples than in Comparative Example 1, and cancer cells were selectively captured. Examples 1 to 4, which used substrates with a 60-degree gloss of 150 to 50%, showed good selective capture. Furthermore, in Examples 5 and 6, which used a substrate with a 60 degree gloss of 90%, the number of cancer cells (relative value) was greater than in Comparative Example 2, which was not subjected to anti-glare treatment, and the number was particularly large in Example 6, which had two layers.

[0048] The present invention (1) is a method for producing a polymer-coated substrate for capturing cancer cells, which comprises a step of forming a polymer layer from a hydrophilic polymer represented by the following formula (I) on the surface of a substrate that has been subjected to an antireflection treatment. [ka] (In the formula, R 1 is a hydrogen atom or a methyl group, R 2 represents an alkyl group, m is 1 to 5, and n is the number of repetitions.

[0049] The present invention (2) is a method for producing a polymer-coated substrate for capturing cancer cells according to the present invention (1), wherein the antireflection treatment is at least one selected from the group consisting of a non-reflection treatment, a low-reflection treatment, a polarizing treatment, and a non-glare treatment.

[0050] The present invention (3) is a method for producing a polymer-coated substrate for capturing cancer cells according to the present invention (1) or (2), wherein the 60 degree gloss of the substrate subjected to the antireflection treatment is 150% to 50%.

[0051] The present invention (4) is a method for producing a polymer-coated substrate for capturing cancer cells according to the present invention (1) or (2), wherein the 60 degree gloss of the substrate subjected to the antireflection treatment is 130% to 70%.

[0052] The present invention (5) is a method for producing a polymer-coated substrate for capturing cancer cells, which is an optional combination of any of the present inventions (1) to (4) in which the substrate subjected to the antireflection treatment is a glass substrate.

[0053] The present invention (6) is a method for producing a polymer-coated substrate for capturing cancer cells, which is an optional combination with any of the present inventions (1) to (5), wherein the total thickness of the polymer layer is 10 to 1000 nm.

[0054] The present invention (7) is a method for producing a polymer-coated substrate for capturing cancer cells, which is any combination with any of the present inventions (1) to (6), in which the step of forming two or more polymer layers using the hydrophilic polymer represented by formula (I) on the surface of the substrate that has been subjected to the antireflection treatment.

[0055] The present invention (8) is a hydrophilic film comprising the two or more polymer layers formed of the same hydrophilic polymer, This is a method for producing a polymer-coated substrate for capturing cancer cells according to present invention (7), in which the number average molecular weight of the hydrophilic polymer forming the polymer layer on the substrate surface that has been subjected to the anti-reflection treatment, among the two or more polymer layers, is larger than the number average molecular weight of the hydrophilic polymer forming the other polymer layers.

[0056] The present invention (9) is a method for producing a polymer-coated substrate for capturing cancer cells according to the present invention (8), wherein the hydrophilic polymer forming the other polymer layer has a number average molecular weight of 30,000 or less.

Claims

1. A method for producing a polymer-coated substrate for capturing cancer cells, comprising the step of forming a polymer layer on a surface of a substrate that has been subjected to an anti-reflection treatment using a hydrophilic polymer represented by the following formula (I). 【Chemistry 1】 (In the formula, R １ is a hydrogen atom or a methyl group, R ２ represents an alkyl group, m is 1 to 5, and n is the number of repetitions.)

2. The method for producing a polymer-coated substrate for capturing cancer cells according to claim 1 , wherein the anti-reflection treatment is at least one selected from the group consisting of a non-reflection treatment, a low-reflection treatment, a polarizing treatment, and a non-glare treatment.

3. The method for producing a polymer-coated substrate for capturing cancer cells according to claim 1 or 2, wherein the substrate subjected to the anti-reflection treatment has a 60 degree gloss of 150% to 50%.

4. The method for producing a polymer-coated substrate for capturing cancer cells according to claim 1 or 2, wherein the substrate subjected to the anti-reflection treatment has a 60 degree gloss of 130% to 70%.

5. 3. The method for producing a polymer-coated substrate for capturing cancer cells according to claim 1, wherein the substrate to which the anti-reflection treatment has been applied is a glass substrate.

6. The method for producing a polymer-coated substrate for capturing cancer cells according to claim 1 or 2, wherein the total thickness of the polymer layer is 10 to 1000 nm.

7. 3. The method for producing a polymer-coated substrate for capturing cancer cells according to claim 1 or 2, wherein the step comprises forming two or more polymer layers using the hydrophilic polymer represented by formula (I) on the surface of the substrate subjected to the anti-reflection treatment.

8. the two or more polymer layers are formed of the same hydrophilic polymer, The method for producing a polymer-coated substrate for capturing cancer cells as described in claim 7, wherein, among the two or more polymer layers, the number average molecular weight of the hydrophilic polymer forming the polymer layer on the substrate surface subjected to the anti-reflection treatment is larger than the number average molecular weight of the hydrophilic polymer forming the other polymer layers.

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

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