JP2007302745A - Copolymer, surface modifier, material adsorption inhibitor and hydrophilization treatment agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new material excellent in a surface modifying effect and capability for a wide use and also excellent in biocompatibility. <P>SOLUTION: Provided are a copolymer having a repeating unit represented by general formula (I), a surface modifier consisting of the above copolymer, a hydrophilization treatment agent consisting of the above copolymer, and a material adsorption inhibitor consisting of the above copolymer. In general formula (I), R<SB>1</SB>is a hydrogen atom, methyl group or ethyl group; X is an amino acid residue containing an ampholyte ion moiety; Y is a divalent nonionic group; n and m are each independently a positive integer and n:m=1:9-9:1; and R<SB>1</SB>, X and Y, which exist in a plurality if n and m are an integer of ≥2, may be each the same or different. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an amino acid-derived zwitterionic copolymer excellent in biocompatibility, a surface modifier comprising the copolymer, a substance adsorption inhibitor, and a hydrophilic treatment agent.

  In recent years, in various fields including biochemical analysis and clinical diagnosis, development of surface modifiers that impart desired physical properties to a material surface has been promoted. For example, in order to prevent non-specific adsorption in immunoplates for immunoassay, DNA chips in which nucleic acids are immobilized on the chip, blocking with proteins such as bovine serum albumin (BSA) is performed. ing.

Moreover, a phosphorylcholine group-containing copolymer having effects such as surface hydrophilization and protein adsorption inhibition has been proposed, and such a copolymer is commercially available (see Patent Documents 1 and 2).
JP-A-3-39309 JP-A-7-83923

  Since the phosphorylcholine group-containing copolymer has a structure similar to a phospholipid derived from a biological membrane, it is known to have excellent biocompatibility. However, the phosphorylcholine-containing copolymer sometimes uses a reagent having a high risk for synthesis, and the commercially available phosphorylcholine group-containing copolymer is expensive, so that there is a problem that the versatility is poor.

  Under such circumstances, the present invention has been made for the purpose of providing a novel material that is excellent in surface modification effect and versatility and is excellent in biocompatibility.

As a result of intensive studies to achieve the above-mentioned object, the present inventor is superior in a zwitterionic copolymer containing a zwitterionic unit having an amphoteric electrolyte group derived from an amino acid and a nonionic unit at a predetermined ratio. As a result, the present invention has been completed.
That is, the means for achieving the above object is as follows.
[1] A copolymer having a repeating unit represented by the following general formula (I).
[In general formula (I), R ₁ is a hydrogen atom, a methyl group or an ethyl group, X is an amino acid residue containing a zwitterionic moiety, Y is a divalent nonionic group, and n and m Are each independently a positive integer, n: m = 1: 9 to 9: 1, and when n and m are integers of 2 or more, a plurality of R ₁ , X and Y are the same or different. May be. ]
[2] The copolymer according to [1], wherein the amino acid is at least one selected from the group consisting of lysine, arginine and histidine.
[3] In the general formula (I), Y is a copolymer according to [1] or [2] represented by the following general formula (II).
[In General Formula (II), R ₂ is a hydrogen atom or a linear or branched alkyl group having 1 to 20 carbon atoms, and R ₃ is a hydrogen atom, a methyl group or an ethyl group. ]
[4] The copolymer according to any one of [1] to [3], wherein the mass average molecular weight is in the range of 1,000 to 1,000,000.
[5] A surface modifier comprising the copolymer according to any one of [1] to [4].
[6] A hydrophilic treatment agent comprising the copolymer according to any one of [1] to [4].
[7] A substance adsorption inhibitor comprising the copolymer according to any one of [1] to [4].
[8] The substance adsorption inhibitor according to [7], wherein the substance is a cell and / or a protein.

  According to the present invention, a novel copolymer having excellent biocompatibility and a high surface modification effect can be provided. Furthermore, the copolymer of the present invention can be easily synthesized and has high versatility.

Hereinafter, the present invention will be described in more detail.

[Copolymer]
The copolymer of the present invention is a copolymer having a repeating unit represented by the following general formula (I).

The copolymer represented by the general formula (I) has a zwitterionic part and a nonionic part. The surface of the substance to be treated is adsorbed by the nonionic part, and the substance surface can be modified by the amphoteric ion part exhibiting surfactant properties.
Details of the general formula (I) will be described below.

In general formula (I), X is an amino acid residue having a zwitterionic moiety. The zwitterionic moiety is a site containing a carboxyl group derived from an amino acid and an amino group or an imidazolyl group. As the amino acid, either a synthetic amino acid or a natural amino acid may be used, but it is preferable to use a natural amino acid from the viewpoint of biocompatibility. Moreover, in order to exhibit zwitterionicity in the copolymer, it is preferable that there is an amino group for bonding to the main chain and a basic group that exists free. From the above points, it is preferable to use a basic amino acid having at least one amino group and a carboxyl group as the amino acid. Specific examples thereof include natural basic amino acids lysine, arginine and histidine. The main chain may be bound by an amino group located on the side chain of the amino acid or may be bound by an amino group at the alpha position. In the present invention, any of the binding forms can be adopted. Specific examples of X are shown below.

In general formula (I), R < ₁ > is a hydrogen atom, a methyl group, or an ethyl group. R ₁ is preferably a hydrogen atom or a methyl group, particularly preferably a methyl group, from the viewpoint of ease of synthesis.

In general formula (I), Y is a divalent nonionic group, preferably a group derived from a vinyl monomer. Examples of the vinyl monomer include n-butyl (meth) acrylate, methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, pentyl (meth) acrylate, hexyl (meth) acrylate, (Meth) acrylic acid heptyl, (meth) acrylic acid octyl, (meth) acrylic acid tridecyl, 2-hydroxyethyl methacrylate, (meth) acrylate, styrene, α-methylstyrene, methyl nucleus substituted styrene, chloro nucleus substituted styrene, chloride Examples thereof include vinyl, vinylidene chloride, ethylene, propylene, isobutylene, vinyl acetate, vinyl propionate, ethyl vinyl ether, n-butyl vinyl ether, diethyl itaconate, and di-n-butyl itaconate. Among these, Y is preferably a unit derived from a (meth) acrylic acid ester represented by the following general formula (II). In the present invention, “(meth) acrylic acid” includes methacrylic acid and acrylic acid.

In general formula (II), R < ₂ > is a hydrogen atom or a C1-C20 linear or branched alkyl group. Specific examples of the alkyl group include methyl group, ethyl group, butyl group, n-butyl group, pentyl group, hexyl group, heptyl group, octyl group, and tridecyl group. R ₂ is preferably a butyl group, a hexyl group or a tridecyl group from the viewpoint of easy availability of raw materials and ease of synthesis. R ₃ is a hydrogen atom, a methyl group or an ethyl group. R ₃ is preferably a hydrogen atom or a methyl group, and most preferably a methyl group, from the viewpoint of ease of synthesis and the like.

  In general formula (I), n and m are each independently a positive integer. For example, n is an integer in the range of 1-9, preferably 3-7, and m is an integer in the range of 1-9, preferably 3-7. n and m can be set by the value of n: m described later and the weight average molecular weight of the copolymer.

In the general formula (I), n: m = 1: 9 to 9: 1. When n: m is less than 1: 9, it is difficult to obtain a sufficient surface modification effect because the ratio of the zwitterionic portion is small. On the other hand, when n: m exceeds 9: 1, since the ratio of the nonionic part is small, it is difficult to hold the copolymer of the present invention on the material surface for a long time. Preferably, n: m = 2: 8 to 8: 2, and more preferably n: m = 3: 7 to 7: 3. In general formula (I), when n and m are integers of 2 or more, a plurality of R ₁ , X and Y may be the same or different. Moreover, both ends of the repeating unit are not particularly limited, and may be, for example, a hydrogen atom, a hydroxyl group, an alkyl group, or the like.

  The mass average molecular weight (Mw) of the copolymer of the present invention is not particularly limited, but is preferably 1,000 to 1,000,000 from the viewpoint of adsorptivity to the material surface. The mass average molecular weight (Mw) is more preferably 3,000 to 100,000, still more preferably 5,000 to 100.000. Further, the polydispersity (Mw / Mn) represented by the ratio of the mass average molecular weight (Mw) to the number average molecular weight (Mn) is not particularly limited but is, for example, about 1 to 3.

  The copolymer of the present invention can be easily synthesized by a known method. The polymerization method is not particularly limited, and any method such as random polymerization or block polymerization may be used, but it is preferable to synthesize by random polymerization from the viewpoint of easy synthesis. An example of the synthesis method is shown below, but the present invention is not limited to the following example.

First, an amino acid is introduced into the side chain of the vinyl monomer to obtain a compound represented by the following general formula (III). The reaction can be performed by a known method in a suitable solvent.
[In general formula (III), X and R ₁ have the same definitions as in general formula (I). ]

  Next, the compound represented by the general formula (III) and the raw material polymerizable monomer are copolymerized in the presence of a radical polymerization initiator. At this time, a copolymer containing a zwitterionic part and a nonionic part can be obtained at a desired ratio by adjusting the charging ratio of the compound represented by the general formula (III) and the raw material polymerizable monomer. it can.

  The radical polymerization initiator used in the polymerization reaction is not particularly limited. For example, 2,2′-azobisisobutyronitrile, benzoyl peroxide, diisopropyl peroxydicarbonate, t-butylperoxy-2-ethylhexa Known radical initiators such as noate, t-butylperoxypivalate, t-butylperoxydiisobutyrate, persulfate or persulfate-bisulfite can be used. The amount of the polymerization initiator used is, for example, 100,000 to 10,000,000 parts by mass, preferably 100,000 with respect to 100 parts by mass in total of the compound represented by the general formula (III) and the raw material polymerizable monomer. It can be set to -500,000 mass parts.

The reaction conditions for the polymerization reaction are not particularly limited. For example, the reaction temperature may be 50 to 150 ° C., the reaction time may be about 1 to 168 hours, and the reaction solvent may be ethanol or methanol. , Acetone, dioxane, N, N-dimethylformamide and other known radical polymerization solvents may be used.
In addition, it can confirm that the target copolymer was obtained by the above reaction by well-known methods, such as NMR.

The copolymer of the present invention has the advantage of having an excellent surface modification effect, being excellent in biocompatibility because it is derived from amino acids, and being able to be synthesized at a relatively low cost.
In addition, since the phosphorylcholine group-containing copolymer contains a strong acid (phosphate group) and a strong base (quaternary ammonium group) in the zwitterionic part, the same surface modification as that of the conventional phosphorylcholine group-containing copolymer. In order to obtain an effect, it was considered necessary to use a strong acid / strong base type zwitterion. However, strong acid / strong base type zwitterions have a concern about safety to living bodies.
On the other hand, in the copolymer of the present invention, the zwitterionic part is constituted by a weak acid (carboxyl group) and a weak base (amino group or imidazolyl group). Thus, according to the present invention, an excellent surface modification effect such as a non-specific adsorption inhibiting effect can be obtained by a highly safe weak acid / weak base type amphoteric ion moiety.

[Surface modifier, hydrophilizing agent, substance adsorption inhibitor]
The surface modifier of the present invention is composed of the copolymer of the present invention, and is a copolymer having a nonionic part that can be adsorbed on the surface according to the physical properties of the surface of the substance to be treated. It is preferable. Thereby, the nonionic part is adsorbed on the surface of the substance, whereby the copolymer can be stably held on the surface of the substance for a long time. For example, when treating a hydrophobic surface, the nonionic part is preferably hydrophobic. The surface modifier of the present invention can modify the surface by exhibiting the characteristics of the zwitterionic portion of the copolymer on the surface of the substance. In addition, the modification effect can be maintained for a long time by being adsorbed to the surface of the substance by the nonionic part of the copolymer and being stably held.

  Surfaces to be treated include polystyrene, polypropylene, polyethylene, polysulfonic acid, polymethyl acrylate (ethyl), polymethyl methacrylate (ethyl), polymeric materials such as polyurethane, inorganic materials such as silicon and glass, titanium, Various surfaces, such as metal materials, such as stainless steel and aluminum, Preferably a hydrophobic surface can be mentioned. Further, the shape of the surface is not particularly limited, but specific examples include a substrate surface, a test tube inner wall, a particle surface, a porous surface, and the like.

  The surface modifier of the present invention can be applied to the material surface as it is, but it is preferable to use it by dissolving it in a suitable volatile solvent from the viewpoint of operability. The solvent can be appropriately selected from those capable of dissolving the copolymer of the present invention, but methanol, ethanol, acetone, and a mixed solvent thereof as a solvent that does not swell the material when applied to a polymer material Alternatively, it is preferable to use a mixed solvent with water. The concentration of the copolymer in the solution can be appropriately set so as to obtain a desired surface modification effect. The said density | concentration can be 0.01-5 mass%, for example, Preferably it can be 0.1-3 mass%.

  A first preferred embodiment of the surface modifier of the present invention is a hydrophilic treatment agent. The hydrophilic treatment agent of the present invention can hydrophilize the hydrophobic surface by exerting the hydrophilicity imparting effect by the zwitterionic portion of the copolymer on the surface. Further, since the non-ionic part (preferably hydrophobic part) of the copolymer is adsorbed on the hydrophobic surface and stably maintained, the hydrophilic effect can be maintained for a long time.

  A second preferred embodiment of the surface modifier of the present invention is a substance adsorption inhibitor. The substance adsorption inhibitor of the present invention is used in immunoplates for immunoassay in which antibodies or antigens are immobilized on a plate, DNA chips in which nucleic acids are immobilized on a chip, etc. After the target substance is bound to the solid phase surface, it can be used to prevent nonspecific adsorption of proteins, cells, etc. to the region not covered with the target substance.

  In the following, the present invention will be further illustrated by examples. However, this invention is not limited to the aspect shown in the Example.

[Example 1]
Synthesis of copolymer Synthesis of N-methacryloyl-L-histidine (MHis) 10 g (64 mmol) of L-histidine (manufactured by Wako) was dissolved in 40 mL of 2N NaOH and sufficiently cooled in an ice bath. Methacryloyl chloride (manufactured by Wako) 7.3 mL (76 mmol, 1.2 eq.) Diluted with 20 mL of dioxane was slowly added dropwise using a dropping funnel under a nitrogen atmosphere. It was. Dioxane in the reaction solution was removed with a rotary evaporator, and the pH of the solution was adjusted to 2 with 6N HCl to perform ether extraction to remove unreacted products and by-products. Thereafter, the aqueous phase was adjusted to pH 5 with 2N NaOH and concentrated, and ethanol was added to extract the target product (unreacted L-histidine and generated NaCl were removed by this operation). The extracted ethanol solution was once concentrated and added dropwise to an excessive amount of acetone that was vigorously stirred to precipitate the target product, which was then suction filtered. The obtained solid was dissolved again in ethanol and precipitated again in acetone, and then the precipitate was collected by filtration and dried overnight under reduced pressure to obtain N-methacryloyl-L-histidine (MHis) hydrochloride ( Yield 65%). FIG. 1 shows the ¹ H-NMR spectrum (1.0% (wt / v) in D ₂ O) of the obtained N-methacryloyl-L-histidine (MHis).

2. Synthesis of poly (MHis / BMA) MHis and n-butyl methacrylate (BMA) (manufactured by Kanto Chemical Co., Inc.) synthesized in step 1 were weighed in a reaction vessel at the composition ratio shown in Table 1, and dissolved in 20 mL of EtOH so that the monomer concentration was 0.5M. As a polymerization initiator, azoisobutyronitrile (AIBN) was added to 1 mol% with respect to the monomer, dissolved oxygen in the reaction system was removed by N ₂ bubbling, and then sealed and polymerized at 70 ° C. for 20 hours. Went. Purification of the polymer after polymerization was performed by dialysis using methanol and distilled water in this order using a MWCO 3500 dialysis membrane, and the polymer suspension was freeze-dried to obtain the desired product.

3. GPC measurement GPC measurement was performed on each of the polymers synthesized in step 1 to determine the mass average molecular weight (Mw) and polydispersity (Mw / Mn). The analysis conditions are as follows.
Each copolymer weighed in an Eppendorf tube was suspended in MeOH, dissolved by adding 0.1 N HClaq until the suspension was completely transparent, MeOH was removed with a centrifugal evaporator, and then freeze-dried. It was dissolved in a carrier (DMF (10 mM LiBr)) and then injected through a syringe filter having a pore size of 0.45 μm.
(Detector; refractive index, column; TOSOH TSKgel a-M + TSKguardcolumn, carrier; DMF (10 mM LiBr), 0.6 mL / min, rt, standard polymer; polystyrene)

4). NMR measurement Each polymer synthesized in (1) was suspended in MeOH, dissolved by adding 0.1 N NaOHaq until the suspension became completely transparent, MeOH was removed by a centrifugal evaporator, freeze-dried, and deuterium When MeOH or MeOH was not completely dissolved, a small amount of heavy water was added for complete dissolution, and NMR measurement was performed at room temperature (measuring device; JEOL EX300 manufactured by JEOL Ltd.). The NMR spectrum of each polymer is shown in FIG.
The composition ratio of the copolymer was calculated from the relative ratio of the MHis imidazole-derived peak (2H) near 7 to 9 ppm and the methyl group-derived peak (2H) immediately below the BMA carbonyl group near 4 ppm in the obtained NMR spectrum. The results are shown in Table 2.

5). Adsorption test 1.0 g of polystyrene resin (200-400 mesh) manufactured by Tokyo Chemical Industry Co., Ltd. was put in a test tube, and a 0.1% by weight (wt / v) sample or bovine serum albumin (BSA) solution was added thereto, and vortexed.・ Shake with a mixer. Thereafter, centrifugation (1000 rpm, 5 minutes, room temperature) was performed, and the absorbance at 210 nm of the supernatant solution was measured to quantify the concentration. The amount of adsorption was determined from the decrease from the original solution concentration. The results are shown in FIG.
As shown in FIG. 3, the histidine homopolymer not containing the hydrophobic part was poor in the adsorptivity to the surface, whereas each copolymer exhibited an adsorptivity equivalent to or better than that of BSA.

[Example 2]
Surface hydrophilization test Preparation of polymer solution It was dissolved in MeOH containing 10% of 0.1N NaOHaq synthesized in Example 1 so that each would be 0.5% (wt / v), and filtered using a syringe filter with a pore size of 0.45 μm. A polymer solution was prepared.

2. Contact angle measurement The polymer solution prepared in (1) was spin coated on a polystyrene (Pst) substrate surface (1000 rpm, 30 sec). After thoroughly washing in the order of PBS and Milli-Q, the contact angle in liquid of bubbles (air) in water was measured using a three-phase kit (measuring device: Kyowa Interface Science DM300). In addition, untreated substrate and commercially available phosphorylcholine group-containing copolymer (Nippon Yushi Lipidure; 2-methacryloyloxyethyl phosphorylcholine (MPC) and butyl methacrylate (BMA) copolymer (MPC: BMA = 3: 7)) The underwater contact angle was measured in the same manner for the base material subjected to the same treatment. The results are shown in Table 3.

  As shown in Table 3, it was confirmed that the surface treatment with each polymer synthesized in Example 1 resulted in a smaller contact angle on the polystyrene surface and the surface was hydrophilized. However, as shown in FIG. 3, the histidine homopolymer has a low surface adsorbability and it is difficult to obtain a sufficient hydrophilic retention ability (durability).

[Example 3]
Protein adsorption inhibition test A polymer solution prepared in the same manner as in Example 2 was filled in each well of a non-surface polystyrene 96-well plate manufactured by IWAKI, immediately removed and air-dried, and then PBS, Milli-Q Washed in order.
The following treatment was performed using the commercially available phosphorylcholine group-containing copolymer and BSA used in Example 2 as comparative samples.
(i) The phosphorylcholine group-containing copolymer was treated in the same manner as described above.
(ii) As for BSA, 300 μl of 1% (wt / v) PBS solution was added to each well, allowed to stand at room temperature for 1 hour, and then washed 5 times with the attached wash solution (0.001% Tween solution).
Subsequently, 0.2 μg / mL of HRP-labeled immunoglobulin (IgG (H + L)) in PBS was added to 100 μl of each well and allowed to stand at room temperature for 1 hour. After washing 5 times with Wash solution, add 100 μl of substrate (ABTS; 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), Kirkegaard & Perry Laboratories, Inc.) solution to each well. After performing the enzyme reaction for a predetermined time, absorbance at 415 nm was measured with a plate reader (Bio-rad Model 680). The results are shown in FIG.
As shown in FIG. 4, the adsorption of IgG was suppressed on the surface treated with each copolymer synthesized in Example 1 as compared with the amount adsorbed on the untreated surface and the surface treated with the histidine homopolymer. Its effect is equivalent to or better than bovine serum albumin (BSA) or a phosphorylcholine group-containing copolymer usually used as a blocking agent in the ELISA method. As the MHis content increases, the adsorption suppression effect increases and the MHis content increases. At about 50%, IgG adsorption was almost completely suppressed.

[Example 4]
Cell adsorption inhibition test 50 μL of the polymer solution prepared in the same manner as in Example 2 was added to a nunc surface untreated polystyrene 4-well plate, uniformly applied, air-dried, and then PBS, Milli-Q. Washed in order. After sterilization overnight with a UV lamp, each well was seeded with 1 × 10 ⁴ cells / cm ² of mouse osteoblasts (MC3T3) and incubated at 37 ° C. with 5% CO ₂ . A photograph of the well surface after 24 hours of incubation is shown in FIG.
As shown in FIG. 5, on the untreated surface, cells adhered and stretched, whereas on the surface treated with each copolymer, adhesion and stretching were observed when the MHis content was low, but the ratio was The surface treated with a small copolymer with an MHis content of about 50% could almost completely inhibit cell adhesion.

  The copolymer of the present invention has an excellent surface modification effect, and can be suitably used for hydrophilizing a hydrophobic surface and suppressing nonspecific adsorption.

2 is a ¹ H-NMR spectrum of N-methacryloyl-L-histidine (MHis) synthesized in Example 1. FIG. It is a ¹ H-NMR spectrum of the synthesized copolymer in Example 1. The result of an adsorptivity test is shown. The result of a protein adsorption | suction suppression test is shown. The result of a cell adsorption | suction suppression test is shown.

Claims (8)

The copolymer which has a repeating unit represented by the following general formula (I).
[In general formula (I), R ₁ is a hydrogen atom, a methyl group or an ethyl group, X is an amino acid residue containing a zwitterionic moiety, Y is a divalent nonionic group, and n and m Are each independently a positive integer, n: m = 1: 9 to 9: 1, and when n and m are integers of 2 or more, a plurality of R ₁ , X and Y are the same or different. May be. ] The copolymer according to claim 1, wherein the amino acid is at least one selected from the group consisting of lysine, arginine and histidine. The copolymer according to claim 1 or 2, wherein Y in the general formula (I) is represented by the following general formula (II).
[In General Formula (II), R ₂ is a hydrogen atom or a linear or branched alkyl group having 1 to 20 carbon atoms, and R ₃ is a hydrogen atom, a methyl group or an ethyl group. ] The copolymer according to any one of claims 1 to 3, wherein the weight average molecular weight is in the range of 1,000 to 1,000,000. The surface modifier which consists of a copolymer of any one of Claims 1-4. The hydrophilic treatment agent which consists of a copolymer of any one of Claims 1-4. The substance adsorption inhibitor which consists of a copolymer of any one of Claims 1-4. The substance adsorption inhibitor according to claim 7, wherein the substance is a cell and / or a protein.