Composition for cancer cell adhesion, cancer cell trapping filter, and method for detecting cancer cells

Abstract

The present application provides a biocompatible polymer material having cancer cell adhesion. The present application is a cancer cell adhesion composition comprising a biocompatible copolymer containing at least one repeating unit (A) represented by the following formula (1) and at least one repeating unit (B) represented by the following formula (2). In formula (1), R 1 represents a methyl group or an ethyl group. In formula (2), R 2 represents an aliphatic hydrocarbon group.

Description

Technical Field

[0001] This invention relates to a composition for cancer cell adhesion. Furthermore, this invention relates to a cancer cell trapping filter having the cancer cell adhesion composition. Additionally, this invention relates to a method for detecting cancer cells using the cancer cell trapping filter. Background Technology

[0002] Previously, research has been conducted on medical materials utilizing various polymers. In the case of medical materials, since synthetic materials, which are foreign to the organism, are used in contact with tissues and blood within the body, biocompatibility is required. To date, poly(2-methoxyethyl acrylate) (PMEA) has been developed as a biocompatible material (see Patent Document 1). It is known that PMEA exhibits biocompatibility due to the presence of so-called intermediate water (water in a state where the pyrothermal peak formed by low-temperature crystals originating from water is stably observed near -40°C during a heating process from -100°C to the polymer chains, which is considered to be weakly bound to the polymer chains through interactions with them) which is considered to be water in a state where, in differential scanning calorimetry measurements, the pyrothermal peak formed by low-temperature crystals originating from water is stably observed near -40°C during a heating process from -100°C.

[0003] In recent years, copolymers of diethylene glycol monoethyl monovinyl ether (EOEOVE), a hydrophilic vinyl ether, and n-butyl vinyl ether, a hydrophobic vinyl ether, have been proposed as biocompatible materials with an oxyethylene chain structure on the side chain, similar to PMEA, and containing intermediate water (see Patent Document 2). However, it has been shown that this copolymer exhibits extremely low adhesion to cancer cells.

[0004] On the other hand, currently, in order to detect cancer cells, it is necessary to use biocompatible polymer materials that also have the ability to adhere to cancer cells.

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: Japanese Patent Application Publication No. 2004-161954

[0008] Patent Document 2: International Publication No. 2017 / 150000 Summary of the Invention

[0009] The problem that the invention aims to solve

[0010] Therefore, the object of the present invention is to provide a polymeric material that is biocompatible and has the ability to adhere to cancer cells. Furthermore, the object of the present invention is to provide a cancer cell trapping filter using a polymeric material with cancer cell adhesion properties, and a method for detecting cancer cells.

[0011] Methods for solving problems

[0012] In order to solve the above-mentioned problems, the inventors of this application conducted in-depth research and found that a copolymer containing a specific repeating unit (A) derived from a specific hydrophilic vinyl ether that is not diethylene glycol monoethyl monovinyl ether (EOEOVE) and a repeating unit (B) derived from a specific hydrophobic vinyl ether has biocompatibility and cancer cell adhesion, thus completing the present invention.

[0013] That is, according to the present invention, the following invention is provided.

[0014] [1] A composition for cancer cell adhesion, comprising a biocompatible copolymer containing at least one repeating unit (A) represented by formula (1) below and at least one repeating unit (B) represented by formula (2) below.

[0015] [Chemical Formula 1]

[0016]

[0017] (where R is in the formula) 1 (Indicates methyl or ethyl)

[0018] [Chemical Formula 2]

[0019]

[0020] (where R is in the formula) 2 (representing aliphatic hydrocarbon groups)

[0021] [2] The cancer cell adhesion composition as described in [1], wherein the aforementioned biocompatible copolymer is a random copolymer of segment A formed by the aforementioned repeating unit (A) and segment B formed by the repeating unit (B).

[0022] [3] The cancer cell adhesion composition as described in [1] or [2], wherein the composition ratio (molar ratio) of the aforementioned biocompatible copolymer, the aforementioned repeating unit (A) and repeating unit (B) is 90:10 to 10:90.

[0023] [4] The cancer cell adhesion composition as described in any one of [1] to [3], wherein R in the aforementioned repeating unit (B) of the aforementioned biocompatible copolymer 2 It is a straight-chain or branched alkyl or alkenyl group with 2 to 10 carbon atoms, or a monocyclic or polycyclic alkyl or alkenyl group with 3 to 20 carbon atoms.

[0024] [5] The cancer cell adhesion composition as described in any one of [1] to [3], wherein R in the aforementioned repeating unit (B) of the aforementioned biocompatible copolymer 2It is a straight-chain or branched alkyl group having 2 to 10 carbon atoms.

[0025] [6] The composition for cancer cell adhesion as described in any one of [1] to [5], wherein the ratio of the weight-average molecular weight (Mw) to the number-average molecular weight (Mn) of the aforementioned biocompatible copolymer is 1.0 to 3.0.

[0026] [7] The cancer cell adhesion composition as described in any one of [1] to [6], wherein the number average molecular weight (Mn) of the aforementioned biocompatible copolymer is 3,000 to 30,000.

[0027] [8] A cancer cell trapping filter having, on at least a portion of its surface, a composition for cancer cell adhesion as described in any one of [1] to [7].

[0028] [9] A method for detecting cancer cells, comprising the step of filtering blood using the cancer cell trapping filter described in [8].

[0029]

[10] The method for detecting cancer cells as described in [9] further includes a step of culturing the cancer cells captured in the filtration step.

[0030] Invention Effects

[0031] According to the present invention, a polymeric material that is biocompatible and has the ability to adhere to cancer cells can be provided. Furthermore, according to the present invention, a cancer cell trapping filter using a polymeric material with cancer cell adhesion and a method for detecting cancer cells can be provided. Detailed Implementation

[0032] <Composition for Cancer Cell Adhesion>

[0033] The cancer cell adhesion composition based on the present invention comprises a biocompatible copolymer containing at least one repeating unit (A) and at least one repeating unit (B).

[0034] (Biocompatible copolymer)

[0035] The biocompatible copolymer of the present invention contains at least one repeating unit (A) represented by the following formula (1) and at least one repeating unit (B) represented by the following formula (2).

[0036] [Chemical Formula 3]

[0037]

[0038] (where R is in the formula) 1 (Indicates methyl or ethyl)

[0039] [Chemical Formula 4]

[0040]

[0041] (where R is in the formula) 2 (representing aliphatic hydrocarbon groups)

[0042] As a monomer that provides the repeating unit (A) of the aforementioned formula (1), a hydrophilic vinyl ether represented by the following formula (3) can be cited.

[0043] [Chemical Formula 5]

[0044] CH2=CH-O-CH2-CH2-OR 1 (3)

[0045] (where R is in the formula) 1 R in equation (1) 1 same.)

[0046] More specifically, examples of hydrophilic vinyl ethers represented by formula (3) include 2-methoxyethyl vinyl ether (MOVE) and 2-ethoxyethyl vinyl ether (EOVE). Among these, MOVE is preferred from the perspective of superior adhesion to cancer cells.

[0047] In addition, as a monomer that provides repeating unit (B), a hydrophobic vinyl ether represented by the following formula (4) can be cited.

[0048] [Chemical Formula 6]

[0049] CH2=CH-OR 2 (4)

[0050] (where R is in the formula) 2 R in equation (2) 2 same.)

[0051] Here, R 2 It is an aliphatic hydrocarbon group, specifically, a straight-chain or branched alkyl or alkenyl group, or a monocyclic or polycyclic alkyl or alkenyl group.

[0052] The aforementioned straight-chain or branched alkyl or alkenyl groups preferably have 2 to 10 carbon atoms, more preferably 3 to 8 carbon atoms, and even more preferably 4 to 6 carbon atoms.

[0053] Specific examples of straight-chain or branched alkyl or alkenyl groups include, for example, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, 1-(2-methyl)-butyl, 2-(2-methyl)-butyl, 1-(3-methyl)-butyl, 2-(3-methyl)-butyl, (2,2-dimethyl)-propyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, Alkyl groups such as 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, and 1-(2-ethyl)hexyl are straight-chain or branched; alkenyl groups such as vinyl, 1-propenyl, allyl, 2-butenyl, 3-butenyl, isopropenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, and 5-hexenyl are straight-chain or branched.

[0054] In addition, the monocyclic or polycyclic alkyl or alkenyl groups preferably have 3 to 20 carbon atoms, more preferably 4 to 15 carbon atoms, and even more preferably 5 to 12 carbon atoms.

[0055] Specific examples of monocyclic or polycyclic alkyl or alkenyl groups include, for example, monocyclic alkyl or alkenyl groups such as cyclopentyl, cyclopentylmethyl, methylcyclopentyl, dimethylcyclopentyl, cyclohexyl, cyclohexylmethyl, methylcyclohexyl, dimethylcyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, cyclotridecyl, cyclotetradecyl, cyclopentadecanyl, cyclooctadecyl, and cycloeicosyl; and polycyclic alkyl or alkenyl groups such as dicyclohexyl, decahydronaphthyl, norbornyl, methylnorbornyl, isobornyl, adamantyl, tricyclodecyl, tricyclodecenyl, and tetracyclododecyl.

[0056] Among these aliphatic hydrocarbon groups, linear or branched alkyl groups are preferred. More specifically, as hydrophobic vinyl ethers represented by formula (4), n-butyl vinyl ether (NBVE) and 2-ethylhexyl vinyl ether (EHVE) are preferred. Among these, NBVE is preferred from the perspective of superior adhesion to cancer cells.

[0057] The composition ratio (molar ratio) of repeating unit (A) and repeating unit (B) in the biocompatible copolymer (hereinafter, sometimes referred to as "copolymer") of the present invention is preferably in the range of 90:10 to 10:90, more preferably in the range of 85:15 to 15:85, and particularly preferably in the range of 80:20 to 20:80.

[0058] Regarding the molecular weight of the copolymer in this invention, for example, the number-average molecular weight (Mn) determined from the standard curve of standard polystyrene using gel permeation chromatography (GPC) is preferably 1,000 to 100,000, more preferably 2,000 to 50,000, and even more preferably 3,000 to 30,000. If Mn is within the above range, the adhesion of the copolymer to cancer cells becomes more excellent.

[0059] The ratio of weight-average molecular weight (Mw) to number-average molecular weight (Mn) of the copolymer in this invention (molecular weight distribution, Mw / Mn) is preferably 1.0 to 3.0, more preferably 1.0 to 2.0, even more preferably 1.0 to 1.5, and even more preferably 1.0 to 1.3. If the molecular weight distribution (Mw / Mn) is within the above range, the adhesion of the copolymer to cancer cells becomes more excellent.

[0060] The copolymer in this invention can be any copolymer, including random copolymers and block copolymers. However, from the perspective of cancer cell adhesion, a random copolymer comprising segment A formed by repeating unit (A) and segment B formed by repeating unit (B) is preferred. The compositional distribution of segments A and B in the random copolymer is not particularly limited; for example, examples include completely random copolymers with a composition close to statistical randomness and conical (gradient) random copolymers with a gradient compositional distribution. By making the copolymer a random copolymer, sufficient adhesion to cancer cells can be obtained regardless of molecular weight.

[0061] The copolymer of the present invention can be prepared by polymerizing the hydrophilic vinyl ether represented by formula (3) and the hydrophobic vinyl ether represented by formula (4) using conventional methods. As this polymerization method, living cationic polymerization is particularly preferred in order to obtain copolymers with the desired composition ratio and molecular weight with good reproducibility. In living cationic polymerization, the molecular weight of the copolymer is almost entirely determined by the molar ratio of monomer to polymerization initiator; therefore, by changing the amount of monomer and polymerization initiator used, the molecular weight of the copolymer can be arbitrarily controlled over a wide range.

[0062] The polymerization initiator used in the polymerization of copolymers is not particularly limited as long as it enables the cationic polymerization to proceed in an active manner. For example, as an active cationic polymerization initiator for vinyl ethers, HI / I2-based initiators (e.g., Japanese Patent Application Publication No. 60-228509) and polymerization initiators composed of Lewis acid catalysts (organoaluminum compounds, etc.) and additives such as bases (ethers, etc.) (e.g., Japanese Patent No. 3096494, Japanese Patent Application Publication No. 7-2805, Japanese Patent Application Publication No. 62-257910, Japanese Patent Application Publication No. 1-108202 and Japanese Patent Application Publication No. 1-108203) are preferred.

[0063] The amount of polymerization initiator used relative to the total amount of raw material monomers is preferably 0.001 to 20 mol%, more preferably 0.01 to 10 mol%, and particularly preferably less than 1 mol%.

[0064] Furthermore, the active cationic polymerization reaction is preferably carried out in the presence of a suitable organic solvent, but it can also be carried out in the absence of a suitable organic solvent. Examples of usable organic solvents include aromatic hydrocarbon solvents such as benzene, toluene, and xylene; aliphatic hydrocarbon solvents such as propane, n-butane, isobutane, n-pentane, isopentane, n-hexane, n-heptane, n-octane, isooctane, decane, hexadecane, and cyclohexane; halogenated hydrocarbon solvents such as dichloromethane, dichloroethane, and carbon tetrachloride; and ether solvents such as diethyl ether, dibutyl ether, tetrahydrofuran (THF), dioxane, and ethylene glycol diethyl ether. These organic solvents can be used alone or in combination of two or more, as needed. Among these organic solvents, aromatic hydrocarbon solvents and aliphatic hydrocarbon solvents are preferred, with toluene or cyclohexane being particularly preferred.

[0065] The polymerization temperature varies depending on the type of polymerization initiator, monomer, and solvent used, but is typically -80 to 150°C, preferably -50 to 100°C, and particularly preferably -20 to 80°C. The polymerization time also varies depending on the polymerization initiator, monomer, solvent, and reaction temperature used, but is typically around 10 minutes to 100 hours. The polymerization reaction can be carried out appropriately by either batch or continuous methods. After the polymerization reaction, unreacted monomers can be purified using known methods, if necessary.

[0066] <Cancer Cell Capture Filter>

[0067] The cancer cell trapping filter (hereinafter, sometimes simply referred to as "filter") of the present invention has the above-described cancer cell adhesion composition on at least a portion of its surface. By using this filter to filter blood, cancer cells in the blood can be trapped. As a filter, there is no particular limitation as long as it is a filter for blood filtration, and conventionally known filters can be used. It is sufficient that at least a portion of the filter's surface has the cancer cell adhesion composition, but preferably, at least 50% of the total area of ​​the surface on the side in contact with the filtered blood is covered by the cancer cell adhesion composition, more preferably at least 80%, and even more preferably, the entire surface on the side in contact with the filtered blood is covered by the cancer cell adhesion composition.

[0068] For the filter, from the perspective of more efficiently capturing cancer cells, the average pore size of the through-hole is preferably 5 to 30 μm, more preferably 5 to 15 μm, and even more preferably 5 to 10 μm.

[0069] The average opening ratio of the filter is preferably 5-50%, more preferably 10-40%, and even more preferably 20-40%. Here, the average opening ratio of the filter refers to the proportion of the area of ​​the through holes to the total area of ​​the filter.

[0070] Various methods can be used to maintain the cancer cell adhesion composition on the filter surface: methods that coat the filter surface with the cancer cell adhesion composition; methods that use active energy rays such as radiation, electron beams, and ultraviolet rays to bond the filter surface with a biocompatible polymer; methods that cause the functional groups on the filter surface to react and bond with the cancer cell adhesion composition; and so on. When using a coating method, any method such as coating, spraying, or dipping can be used to coat the cancer cell adhesion composition. Coating can be performed by adhering the cancer cell adhesion composition to the substrate surface using methods such as dipping, spraying, or spin coating, and then removing the solvent (drying). The film thickness after drying is preferably 0.01 μm to 1.0 mm, more preferably 0.1 to 100 μm, and even more preferably 0.5 to 50 μm. If the film thickness is within the above range, sufficient adhesion of cancer cells can be achieved.

[0071] To ensure a more secure adhesion of the cancer cell adhesion composition to the substrate, the substrate can be heated after coating with the cancer cell adhesion composition. Alternatively, the biocompatible polymer can be cross-linked. Examples of cross-linking methods include, for instance, adding cross-linking monomers to the polymer material beforehand. Electron beams, gamma rays, or light irradiation can be used during cross-linking.

[0072] There are no particular restrictions on the material and shape of the filter; for example, porous materials, fibers, non-woven fabrics, membranes, sheets, and tubes can be used. As for the substrate material, examples include natural polymers such as cotton and linen, nylon, polyester, polyacrylonitrile, polyolefins, halogenated polyolefins, polyurethane, polyamide, polysulfone, polyethersulfone, poly(meth)acrylate, halogenated polyolefin ethylene-polyvinyl alcohol copolymer, butadiene-acrylonitrile copolymer, and other synthetic polymers or mixtures thereof. Additionally, examples include metals, ceramics, and their composite materials, and the filter can also be composed of multiple substrates.

[0073] There are no particular restrictions on the metals mentioned above; for example, precious metals such as gold and silver, base metals such as copper, aluminum, tungsten, nickel, chromium, and titanium, and alloys of these metals can be used. The metals can be used in their elemental form or, for functional purposes, in the form of alloys with other metals or metal oxides. From the viewpoint of price and availability, nickel, copper, and metals with these as their main components are preferred. Here, the main component refers to a component that accounts for more than 50% by weight of the material forming the substrate. Through-holes can also be formed on these metals using methods such as photolithography to create a mesh filter.

[0074] <Methods for detecting cancer cells>

[0075] The method for detecting cancer cells of the present invention includes a step of filtering blood using the cancer cell trapping filter described above, and preferably further includes a step of culturing the cancer cells trapped in the filtration step. The steps are described in detail below.

[0076] (Filtration process)

[0077] Methods for detecting cancer cells utilize blood filtration to capture cancer cells in the blood with a high recovery rate. Examples of blood filtration methods include installing filters within the blood circulation pathway. In such methods, blood accumulated in bone marrow, spleen, liver, etc., umbilical cord blood, as well as lymph and tissue fluid, can be used as samples, but using circulating peripheral blood is the simplest. Detecting the presence of circulating cancer cells (hereinafter referred to as "CTCs") in peripheral blood is a useful means of assessing cancer progression.

[0078] When using the method described above, which involves filtering blood through a filter in the blood circulation path to detect the presence of cancer cells, this can be implemented, for example, by: assembling the filter into the flow path, introducing peripheral blood into the flow path, thereby capturing cells containing CTCs, and confirming the presence of CTCs in the captured cells. Examples of methods for introducing blood into the flow path include: pressurizing from the inlet direction of the flow path; depressurizing from the outlet direction of the flow path; using a peristaltic pump; etc. Furthermore, regarding the area of ​​the filter used, for example, in the case of concentrating CTCs from 1 mL of blood, 1–10 cm² is suitable. 2 It is suitable.

[0079] When CTCs are captured using the methods described above, not only CTCs but also blood cells such as white blood cells are captured simultaneously. Therefore, it is necessary to confirm whether the recovered cells contain cancer cells. For example, after capturing CTCs using the methods described above, they can be identified as cancer cells by staining with fluorescently labeled antibodies against cancer markers. Examples of antibodies against cancer markers include anti-EpCAM antibodies.

[0080] Alternatively, cancer cells can be identified by analyzing the genes of cells captured using the methods described above. For example, mutations in genes such as p53, K-RAS, H-RAS, N-RAS, BRAF, and APC can be analyzed to confirm cancer cell status. Furthermore, the results of these gene analyses can be used to determine subsequent treatment strategies for patients. Alternatively, cancer cells can be identified by measuring telomerase activity and other parameters in cells captured using the methods described above.

[0081] (Cultivation process)

[0082] The method for detecting cancer cells also includes a step of culturing cancer cells, thereby enabling the detection of trace amounts of cancer cells. There are no particular limitations on the method for culturing cancer cells; methods commonly used for cell culture can be used.

[0083] Example

[0084] The present invention will now be described in more detail by way of examples and comparative examples, but the present invention is not limited to these examples, etc.

[0085] It should be noted that, in the embodiments, the composition ratio of the copolymer is respectively determined by... 1 The molecular weight average (Mw), number average (Mn), and molecular weight distribution (Mw / Mn) were obtained from the ¹H NMR analysis results, and were calculated from the molecular weight analysis results of GPC (converted to polystyrene). The analytical apparatus and measurement conditions are described below.

[0086] (NMR measurement conditions)

[0087] • Device: JEOL manufactured, model: AL-400

[0088] Solvent: Deuterated chloroform

[0089] • Measurement temperature: 30℃

[0090] (GPC determination conditions)

[0091] ·Device: Made by Tosoh Corporation, "HLC-8320GPC"

[0092] • Detector: RI detector

[0093] • Mobile phase: Tetrahydrofuran

[0094] • Flow rate: 1 mL / min

[0095] • Chromatographic column: 3 Shodex LF-804 columns manufactured by Showa Denko Corporation

[0096] Column temperature: 40℃

[0097] [Example 1]

[0098] Synthesis of 2-methoxyethyl vinyl ether / n-butyl vinyl ether random copolymer (MOVE-ran-NBVE) A

[0099] Under a dry nitrogen atmosphere, 76.0 mL of toluene as a solvent, 32.6 mL of ethyl acetate as an added base, 2.3 mL of 2-methoxyethyl vinyl ether (MOVE) as a hydrophilic vinyl ether, 10.3 mL of n-butyl vinyl ether (NBVE) as a hydrophobic vinyl ether, and 494.0 μL of 1-isobutoxyethyl acetate (IBEA) as an initiator were added to a Schlenk tube equipped with a three-way stopcock valve that had been heated and dehydrated at above 300°C for 10 minutes. The mixture was stirred thoroughly.

[0100] Next, while maintaining the temperature at 0°C, Et was added as a Lewis acid catalyst. 1.5 AlCl 1.5 Polymerization was initiated with 0.93M (3.7mL) and the reaction was allowed to proceed for 90 minutes.

[0101] The polymerization was terminated using methanol containing a small amount of sodium methoxide (1M). 3g of ion exchange resin (trade name: AMBERLYST MSPS2-1·DRY, manufactured by ORGANO Co., Ltd.) was added to the terminated solution, and the mixture was stirred overnight. The solution was then passed through diatomaceous earth and a 1μm pore size filter. After solvent evaporation, the mixture was dried under reduced pressure to obtain the target random copolymer A. The composition ratio, weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular weight distribution (Mw / Mn) of the obtained random copolymer A are shown in Table 1.

[0102] [Example 2]

[0103] Synthesis of 2-methoxyethyl vinyl ether / n-butyl vinyl ether random copolymer (MOVE-ran-NBVE) B

[0104] The amount of IBEA added was changed to 185.0 μL, and otherwise, the random copolymer B was obtained using the same procedure as in Example 1. The composition ratio, weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular weight distribution (Mw / Mn) of the obtained random copolymer B are shown in Table 1.

[0105] [Example 3]

[0106] Synthesis of 2-methoxyethyl vinyl ether / n-butyl vinyl ether random copolymer (MOVE-ran-NBVE) C

[0107] Add 9.3 mL of MOVE as a hydrophilic vinyl ether, 2.6 mL of NBVE as a hydrophobic vinyl ether, and 300.0 μL of IBEA as an initiator. Otherwise, proceed with the same procedure as in Example 1 to obtain random copolymer C. The composition ratio, weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular weight distribution (Mw / Mn) of the obtained random copolymer C are shown in Table 1.

[0108] [Example 4]

[0109] Synthesis of 2-methoxyethyl vinyl ether / n-butyl vinyl ether random copolymer (MOVE-ran-NBVE) D

[0110] Add 5.8 mL of MOVE as a hydrophilic vinyl ether, 6.4 mL of NBVE as a hydrophobic vinyl ether, and 300.0 μL of IBEA as an initiator. Otherwise, proceed with the same procedure as in Example 1 to obtain random copolymer D. The composition ratio, weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular weight distribution (Mw / Mn) of the obtained random copolymer D are shown in Table 1.

[0111] [Example 5]

[0112] Synthesis of 2-ethoxyethyl vinyl ether / n-butyl vinyl ether random copolymer (EOVE-ran-NBVE)

[0113] 7.3 mL of 2-ethoxyethyl vinyl ether (EOVE) as a hydrophilic vinyl ether, 6.4 mL of NBVE as a hydrophobic vinyl ether, and 300.0 μL of IBEA as an initiator were added, and the random copolymer E was obtained using the same procedure as in Example 1. The composition ratio, weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular weight distribution (Mw / Mn) of the obtained random copolymer E are shown in Table 1.

[0114] [Example 6]

[0115] The synthesis of 2-methoxyethyl vinyl ether / 2-ethylhexyl vinyl ether random copolymer (MOVE-ran-EHVE) Cheng F

[0116] Add 5.8 mL of MOVE as a hydrophilic vinyl ether, 8.0 mL of 2-ethylhexyl vinyl ether (EHVE) as a hydrophobic vinyl ether, and 300.0 μL of IBEA as an initiator. Otherwise, proceed with the same procedure as in Example 1 to obtain random copolymer F. The composition ratio, weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular weight distribution (Mw / Mn) of the obtained random copolymer F are shown in Table 1.

[0117] [Example 7]

[0118] Synthesis of 2-methoxyethyl vinyl ether / n-butyl vinyl ether random copolymer (MOVE-ran-NBVE)

[0119] The amount of IBEA added was changed to 74.0 μL, and the random copolymer G was obtained using the same procedure as in Example 4. The composition ratio, weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular weight distribution (Mw / Mn) of the obtained random copolymer G are shown in Table 1.

[0120] [Comparative Example 1]

[0121] Synthesis of poly(n-butyl vinyl ether) (PNBVE) H

[0122] Without adding hydrophilic vinyl ethers, 12.9 mL of NBVE as a hydrophobic vinyl ether and 300.0 μL of IBEA as an initiator were added, and homopolymer H was obtained using the same procedure as in Example 1. The weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular weight distribution (Mw / Mn) of the obtained homopolymer H are shown in Table 1.

[0123] [Comparative Example 2]

[0124] Synthesis of poly(2-methoxyethyl acrylate) (PMEA) I

[0125] 15.00 g of 2-methoxyethyl acrylate (MEA) as a monomer was dissolved in 60.23 g of 1,4-dioxane, and bubbling was performed under argon atmosphere for 30 minutes. Then, 15.03 mg of 2,2'-azobisisobutyronitrile (AIBN) was added as an initiator, and polymerization was carried out at 75°C for 6 hours while bubbling under argon atmosphere. The resulting polymer solution was added dropwise to 1 L of n-hexane, thereby recovering the polymer as a precipitate. Purification was achieved by repeating the precipitation process three times using a THF / n-hexane system. After precipitation, the polymer was contacted with pure water and stirred for at least 24 hours to remove substances dissolved in the water. Solvent removal was then performed using an evaporator and vacuum drying (at 40°C for at least 72 hours). Homopolymer I was obtained as the final product. The weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular weight distribution (Mw / Mn) of the obtained homopolymer I are shown in Table 1.

[0126] [Comparative Example 3]

[0127] Diethylene glycol monoethyl monovinyl ether / n-butyl vinyl ether random copolymer (EOEOVE-ran-NBVE) Synthesis J

[0128] Under a dry nitrogen atmosphere, to a 300 mL three-necked flask equipped with a three-way stopcock valve and heated to dehydrate at above 300 °C for 10 minutes, 181.0 mL of toluene as a solvent, 76.4 mL of ethyl acetate as an added base, 4.0 mL of diethylene glycol monoethyl monovinyl ether (EOEOVE) as a hydrophilic vinyl ether, 28.2 mL of N-butyl vinyl ether (NBVE) as a hydrophobic vinyl ether, and 4 mM (450.0 μL) of the acetic acid adduct of isobutyl vinyl ether as an initiator were added, and the mixture was stirred thoroughly.

[0129] Next, while maintaining the temperature at 0°C, Et was added as a Lewis acid catalyst. 1.5 AlCl 1.5 Polymerization was initiated with 8 mM (8.8 mL) and the reaction was allowed to proceed for 90 minutes.

[0130] The polymerization was terminated using methanol containing a small amount of sodium methoxide (1M). 5% by mass of ion exchange resin (trade name: AMBERLYST MSPS2-1·DRY, manufactured by ORGANO Co., Ltd.) was added to the terminated solution, and the mixture was stirred at room temperature for 1 hour. The solution was then passed through diatomaceous earth and a filter with a pore size of 1 μm. After evaporation of the solvent, the mixture was dried under reduced pressure to obtain the target random copolymer J. The composition ratio, weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular weight distribution (Mw / Mn) of the obtained random copolymer J are shown in Table 1.

[0131] [Evaluation Test]

[0132] Platelet adhesion test

[0133] To investigate blood compatibility, platelet adhesion tests were conducted on polyethylene terephthalate (PET) plates (Examples 1-7, Comparative Examples 1-3) coated with the polymers A-J synthesized above.

[0134] The surface coating of PET sheets utilizing each polymer is achieved by spin-coating a 1.0 wt% ethanol solution of each polymer onto the surface of the PET sheet.

[0135] Whole blood purchased from the United States was brought to room temperature before the experiment and centrifuged at 400 rcf for 5 minutes. Approximately 0.5 mL of the supernatant was collected as platelet-rich plasma (PRP). Subsequently, it was further centrifuged at 2500 rcf for 10 minutes. Approximately 2 mL of the supernatant was collected as platelet-poor-rich plasma (PPP). The inoculation density was 4.0 × 10⁻⁶. 7 cells / cm 2Platelet suspensions were prepared by diluting PRP with PPP. 0.2 mL of the prepared platelet suspension was added dropwise to both polymer-coated and uncoated PET plates (blank), and incubated at 37°C for 60 minutes. Next, the plates were rinsed with phosphate buffer and incubated at 37°C in 1% glutaraldehyde solution for 120 minutes to fix the adhered platelets. Subsequently, the plates were washed with phosphate-buffered saline and water, and dried in a silica gel container for at least two days. After drying, the sample surface was observed using a scanning electron microscope, and measurements were taken at 1×10⁻⁶. 5 μm 2 The number of platelets adhering to the area. The relative number of platelets adhering to each example and comparative example was evaluated according to the following criteria, with the number of platelets adhering to the uncoated PET plate (blank) set to 1. The evaluation results are shown in Table 1.

[0136] (Evaluation Criteria)

[0137] ◎: The number of platelets adhering is less than 0.1.

[0138] ○: The number of platelets adhering is 0.1 or more but less than 0.2.

[0139] ×: The number of platelets adhering is 0.2 or more.

[0140] <Cancer cell adhesion assay>

[0141] To investigate the adhesion of cancer cells, adhesion tests were conducted on polyethylene terephthalate (PET) plates (Examples 1-7, Comparative Examples 2 and 3) coated with the polymers A-G, I, and J synthesized above. It should be noted that for polymer H, due to the high number of platelets adhering and poor blood compatibility, cancer cell adhesion tests were not performed.

[0142] The procedure was the same as for the platelet adhesion test described above. PET plates coated with various polymers and uncoated PET plates (blank) were prepared. The prepared PET plates were then inserted into 24 wells (bottom area 1.9 cm²). 2 Then, using a pipette, 1.0 mL of cancer cell suspension (using culture medium supplemented with 10% serum, at a seeding density of 10,000 cells / cm²) was added to each well of the PET plate. 2(Adjusted by method), and incubated at 37°C for 60 minutes. Human fibrosarcoma cell lines HT-1080 and SW480 were used as cancer cells. Next, the cells were rinsed with phosphate-buffered saline, and the number of cancer cells adhering to the sample surface was counted. To facilitate the measurement, the cells were immobilized with 4% paraformaldehyde, and the nuclei were stained with 4',6-diamino-2-phenylindole (DAPI). F-actin was stained with phalloidin labeled with Alexa Fluor488. The number of nuclei was counted using an all-in-one fluorescence microscope (KEYENCE, BZ-X710) as the number of cancer cells. The relative number of cancer cells adhering to each example and comparative example was evaluated according to the following criteria, with the number of cancer cells adhering to an uncoated PET plate (blank) set to 1. The evaluation results are shown in Table 1. It should be noted that, for polymer J, adhesion to HT-1080 was not observed, therefore adhesion tests for SW480 were not performed.

[0143] (Evaluation Criteria)

[0144] ◎: The number of cancer cells adhering is greater than 3.0.

[0145] ○: The number of cancer cells adhering is greater than 2.5 and less than 3.0.

[0146] △: The number of cancer cells adhering is greater than 1.0 and less than 2.5.

[0147] ×: The number of cancer cells adhering is less than 1.0.

[0148] [Table 1]

[0149]

Claims

1. Use of a biocompatible copolymer in the manufacture of a composition for cancer cell adhesion, said biocompatible copolymer containing at least one repeating unit (A) represented by the following formula (1) and at least one repeating unit (B) represented by the following formula (2). [Chemical Formula 1] In equation (1), R 1 Indicates methyl or ethyl. [Chemical Formula 2] In equation (2), R 2 Indicates an aliphatic hydrocarbon group. The molar ratio of repeating unit (A) to repeating unit (B) in the biocompatible copolymer is 90:10 to 10:

90. The ratio of the weight-average molecular weight (Mw) to the number-average molecular weight (Mn) of the biocompatible copolymer is 1.0 to 1.

5. The number-average molecular weight (Mn) of the biocompatible copolymer is 3,000 to 30,000.

2. The use as described in claim 1, wherein, The biocompatible copolymer is a random copolymer of segment A formed by the repeating unit (A) and segment B formed by the repeating unit (B).

3. The use as described in claim 1 or 2, wherein, The molar ratio of repeating unit (A) to repeating unit (B) in the biocompatible copolymer is 85:15 to 15:

85.

4. The use as described in claim 1 or 2, wherein, R in the repeating unit (B) of the biocompatible copolymer 2 It is a straight-chain or branched alkyl or alkenyl group with 2 to 10 carbon atoms, or a monocyclic or polycyclic alkyl or alkenyl group with 3 to 20 carbon atoms.

5. The use as described in claim 1 or 2, wherein, R in the repeating unit (B) of the biocompatible copolymer 2 It is a straight-chain or branched alkyl group having 2 to 10 carbon atoms.

6. The use as described in claim 1 or 2, wherein, The ratio of the weight-average molecular weight (Mw) to the number-average molecular weight (Mn) of the biocompatible copolymer is 1.0 to 1.

3.

7. The use as described in claim 1 or 2, wherein, The number-average molecular weight (Mn) of the biocompatible copolymer is 5,300 to 24,000.

8. Use of the cancer cell adhesion composition in the manufacture of a cancer cell trapping filter having the cancer cell adhesion composition on at least a portion of its surface, said cancer cell adhesion composition comprising the biocompatible copolymer of any one of claims 1 to 7.

9. Use of the cancer cell trapping filter of claim 8 in the manufacture of an apparatus for a method of detecting cancer cells, the method of detecting cancer cells including a step of filtering blood.

10. The use as described in claim 9, wherein the method for detecting cancer cells further includes a step of culturing the cancer cells captured in the filtration step.

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