JP6156874B2 - Bioinert film, coating liquid, production method thereof, and bioinert-treated substrate - Google Patents

Description

  The present invention relates to a bioinert film, a coating liquid used for the production thereof, a production method thereof, and a bioinert-treated substrate.

  Substrates that come into contact with water are generally prone to undesirable accumulation of biologically derived organic species, due to protein adsorption, bacterial adsorption, and subsequent propagation, or thrombus formation. is there.

  Such biofouling can have serious consequences. For example, in the medical field, bacterial infection through a catheter may be caused by biofouling, and in the industry, problems such as filter clogging and accumulation of organic materials on the surface occur.

As a method for reducing or preventing biofouling, Patent Document 1 discloses a film obtained by crosslinking a plurality of polymer chain grafted fine particles with a divinyl compound or the like. Here, per unit area on the surface of the particle is disclosed. The number of macromolecules is as small as about 0.03 chain / nm ² .

For example, in PMMA, which is one of general-purpose polymers, those having a molecular weight of 0.01 nm ² or more and less than 0.1 chain / nm ² are also called semi-dilute polymer brushes. Random coil shape, stretched coil shape, etc. are used, but there is room for further improvement in the suppression of biofouling.

  On the other hand, in recent years, as a precise polymerization method having simplicity and versatility, the living radical polymerization method has made dramatic progress, and various new materials have been easily synthesized (Patent Documents 2 and 3). By applying this living radical polymerization method to surface graft polymerization as a breakthrough, it has become possible to graft polymers of uniform length at a significantly higher density.

  This high-density polymer brush having a surface occupancy of about 10% or more is expected to exhibit completely different properties from the semi-dilute polymer brush. To date, high-density polymer brushes have (i) large swelling film thickness (stretch orientation) and high compression resistance comparable to the extended chain length, and (ii) high osmotic pressure and highly stretched molecules. Derived from the chain form, it shows a clear size exclusion effect in the molten state and in the solvent (does not take in molecules larger than a certain size into the brush layer), and can further suppress the adsorption of biological substances such as proteins, (Iii) It was demonstrated that mutual penetration does not occur between the same type of brushes, and as a result, the friction coefficient between the swollen brushes becomes extremely low.

  For example, as a result of synthesizing high-density polymer brushes of various hydrophilic polymers and examining their protein adsorption characteristics, the size exclusion effect unique to high-density polymer brushes significantly suppresses nonspecific adsorption of proteins, As a result of studying the adhesion characteristics of mouse-derived L929 fibroblasts to this hydrophilic high-density polymer brush, it has been clarified that cell adhesion is dramatically suppressed as compared with a semi-dilute brush.

  However, in the case of forming a high-density polymer brush by directly performing graft polymerization on the surface of a substrate having a relatively simple shape, the results of suppressing nonspecific adsorption of proteins and cell adhesion as described above were obtained. However, the application of a biologically inert film to the surface of a practical material, for example, a medical material such as a catheter, has not yet been sufficiently studied with demonstration.

International Publication WO2006 / 016800 Pamphlet JP 2010-280594 A Japanese Patent No. 3422463

  However, when a bioinert film is applied to the surface of a practical material as described above, a method of grafting polymer chains on the material surface at high density using a biocompatible polymer by living radical polymerization is used. , (1) Initiating group introduced into material surface, (2) Living radical polymerization (heat treatment in vacuum or under inert gas, polymerization time from tens of minutes to tens of hours), (3) Cleaning, etc. Such several stages of work are necessary, and further improvements have been demanded from a practical point of view in consideration of mass production.

  In particular, the shape of medical materials is often complicated, such as flat plates, fibers, tubes, circles, spheres, etc., but the more complicated the shape, the more difficult it is to perform operations such as uniform introduction of initiator groups and living radical polymerization. There was a problem.

  In addition, Patent Document 2 discloses fine particles that have been densified to a structure in which the polymer graft chains are extended by steric repulsion between the polymer graft chains, and their use in DDS and the like is being studied. There is no description of forming a film on a substrate by reacting with a crosslinking agent.

  The present invention has been made in view of the circumstances as described above, and can be easily and quickly produced without requiring a complicated operation regardless of the shape of the base material. It is an object of the present invention to provide a bioinert film, a coating liquid, a production method thereof, and a bioinert-treated base material that can greatly suppress adsorption and cell adhesion.

  In order to solve the above-described problems, the bioinert membrane of the present invention has a steric repulsion between polymer graft chains, and the polymer graft chains have a high density until the polymer graft chains are extended. Is characterized in that a plurality of polymer chain graft fine particles bonded to the surface of the inorganic fine particles are cross-linked with each other by a cross-linking agent.

In this bioinert membrane, it is preferable that the graft density of the polymer graft chain is 0.1 chain / nm ² or more.

  In this bioinert membrane, the polymer graft chain is obtained by living radical polymerization of at least one raw material monomer selected from (meth) acrylic acid or a derivative thereof, (meth) acrylamide or a derivative thereof, and a styrene derivative. It is preferable that the hydrophilic polymer chain be formed.

  In this bioinert membrane, the polymer graft chain has the following formula (I):

(Wherein R ¹ represents a C _{1 to} C ₃ alkyl group, R ² represents a C ₁ or C ₂ alkyl group, X represents a halogen atom, and n represents an integer of 3 to 10). It is preferable that the polymer-initiated group-containing silane coupling agent is bonded to the inorganic fine particles, and then the raw material monomer of the polymer graft chain is obtained by living radical polymerization.

  In this bioinert film, it is preferable that the cross-linking agent crosslinks the polymer chain graft fine particles and the base material on which the bioinert film is formed.

  In this bioinert membrane, the cross-linking agent is preferably a compound having at least two groups selected from a phenyl azide group and a pyridine azide group.

  In this bioinert film, the inorganic fine particles preferably have a particle diameter of 1 nm or more and 1 μm or less.

  In the bioinert film, the inorganic fine particles are preferably made of any material selected from silicon oxide, metal oxide, metal, and metal sulfide.

  The coating liquid of the present invention is a polymer in which steric repulsion occurs between polymer graft chains, and the polymer graft chains are bonded to the surface of the inorganic fine particles at a high density until the polymer graft chains are extended. The chain graft fine particles and the cross-linking agent are dissolved or dispersed in a solvent.

  The method for producing a coating liquid according to the present invention is a method for producing the coating liquid, wherein the inorganic fine particles having a living radical polymerization-initiating group-containing silane coupling agent bound to the surface serve as a catalyst for living radical polymerization. A step of performing living radical polymerization by dissolving or dispersing a metal complex and a raw material monomer of the polymer graft chain in a solvent to obtain the polymer chain graft fine particles as a precipitate; the polymer chain graft fine particles and the crosslinking agent; Are dissolved or dispersed in a good solvent that can be dissolved or dispersed at room temperature, the polymer chain graft fine particles have a concentration of 1.0 to 20.0% by mass, and the concentration of the crosslinking agent is 0.1 to And a step of preparing the coating liquid in a range of 10.0% by mass.

  The method for producing a bioinert film of the present invention includes a step of applying the coating liquid to a substrate to form a coating film, and irradiating the coating film with ultraviolet light or heat-treating the coating film. The polymer chain graft fine particles are crosslinked with the crosslinking agent to form a bioinert film.

  In this method for producing a bioinert film, it is preferable that the coating liquid is applied to the substrate by any one of wet film forming methods selected from a dipping method, a spin coating method, and a casting method.

  In this method for producing a bioinert film, the cross-linking agent is a compound having at least two groups selected from a phenyl azide group and a pyridine azide group, and the polymer is irradiated with ultraviolet light. It is preferable that the chain graft fine particles are crosslinked with the crosslinking agent to form a bioinert film.

  In this method for producing a bioinert film, the coating liquid is applied to the metal base material that has been surface-treated with an amino group-containing silane coupling agent, and the base material and the polymer chain graft microparticles are combined with the cross-linking agent. It is preferable to crosslink by.

  In this method for producing a bioinert film, it is preferable that the coating film is irradiated with ultraviolet light of 100 W or more for 10 seconds or more.

  In this method for producing a bioinert film, the coating film is preferably heat-treated at a temperature of 120 ° C. or more for 18 hours or more.

  The bioinert-treated substrate of the present invention has the bioinert film formed on the surface of any substrate selected from cell culture dishes, cell culture flasks, contact lens cases, and catheters. It is characterized by.

  According to the bioinert film, the coating liquid, the production method thereof, and the bioinert-treated base material of the present invention, it can be produced simply and in a short time without requiring complicated work regardless of the shape of the base material. And non-specific adsorption of proteins and cell adhesion can be greatly suppressed.

It is sectional drawing which shows typically an example of the bioinert film of this invention. It is a NMR spectrum of TEGBA (crosslinking agent) synthesize | combined in the Example. It is a photograph of the ultraviolet irradiation film | membrane of Example 1 (Entry1), Example 2 (Entry2), and Example 3 (Entry3). It is an AFM image of the ultraviolet irradiation film of Example 1 (Entry 1), Example 2 (Entry 2), and Example 3 (Entry 3). Human vascular endothelial cells (10,000 cells / cm ² ) were seeded on the coating surface of the ultraviolet irradiation film of Example 1 (Entry 1), Example 2 (Entry 2), Example 3 (Entry 3), and Comparative Example 1 (Entry 4). It is a photograph of a cell adhesion image after 24 hours. Human vascular endothelial cells (10,000 cells / cm ² ) were seeded on the coating surface of the ultraviolet irradiation film of Example 1 (Entry 1), Example 2 (Entry 2), Example 3 (Entry 3), and Comparative Example 1 (Entry 4). It is a graph which shows the cell adhesion number to each coating surface after 24 hours passed. It is a graph which shows the result of having measured the adsorption amount of the contaminating protein contained in bovine serum (FBS) with the crystal oscillator using the sample of Example 2 (Entry2). It is a photograph of the ultraviolet irradiation film | membrane of Example 4 (Entry5,6). It is an AFM image of the ultraviolet irradiation film | membrane of Example 4 (Entry5, 6). It is a photograph of the ultraviolet irradiation film | membrane of Example 5 (Entry7,8). It is an AFM image of the ultraviolet irradiation film | membrane of Example 5 (Entry7,8). It is a graph which shows the result of having evaluated cell survival rate about each sample of Example 6 (Entry12) using the cell growth (viability) capability analysis kit (WST-1). The result of having coated PPEGMA-SiP of Example 7 (Entry 13, crosslinker concentration 0.5 wt%) on the FEP film and performing FT-IR measurement of the coating film before and after curing by ultraviolet irradiation is shown. The result of having coated PPEGMA-SiP of Example 7 (Entry 14, crosslinker concentration 1 wt%) on the FEP film and performing FT-IR measurement of the coating film before and after curing by ultraviolet irradiation is shown. The result of having coated PPEGMA-SiP of Example 7 (Entry 15, crosslinker concentration 2.5 wt%) on the FEP film and performing FT-IR measurement of the coating film before and after curing by ultraviolet irradiation is shown. The result of having carried out the FT-IR measurement of the coating film before and after hardening by PPEGMA-SiP of Example 7 (Entry16, crosslinking agent density | concentration of 5 wt%) on the FEP film is shown. It is an AFM image of the ultraviolet irradiation film of Example 8 (Entry 17, 23, 26). It is a graph which shows the number of cell adhesion to each coating surface after seed | inoculating a human vascular endothelial cell (10,000 piece / cm < ² >) with respect to each sample of Example 8, and having passed 24 hours.

  The present invention is described in detail below.

  In the present invention, “polymer graft chain” means a polymer chain formed by extending two or more raw material monomers from the surface of inorganic fine particles by a polymerization reaction.

  In addition, “high density” means the density of graft chains when the graft chains are dense enough to cause steric repulsion between the graft chains. In this case, the graft chains are substantially extended in a direction perpendicular to the surface. Take the form.

  The “bond” means a bond formed by a general chemical reaction, and examples thereof include a covalent bond and an ionic bond.

  In the present invention, the “biologically inert membrane” is understood from the following points. Hydrophilic polymers (biocompatible polymers) have been reported to inhibit non-specific adsorption of proteins and cell adhesion when they are made into high-density polymer brushes by living radical polymerization. The structure and chemistry of high-density polymer brushes are reported. Various studies have been conducted on the effects of composition on nonspecific adsorption of proteins and cell adhesion (Review: (a) Senaratne, W., Andruzzi, L., and Ober, CK 2005. Self-assembled monolayers and polymer brushes in biotechnology: Current applications and futre perspectives.Biomacromolecules6: 2427. (b) Fristrup, CJ, Jankova, K., and Hvilsted, S. 2009.Surface-initiated atom transfer radical polymerization-a technique to develop biofunctional coatings.Soft Matter5 : 4623. Paper: (a) Yoshikawa, C., Goto, A., Tsujii, Y., Fukuda, T., Kimura, T., Yamamoto, K., and Kishida, A. 2006. Protein repellency of well- defined, concentrated poly (2-hydroxyethyl methacrylate) brushes by the size-exclusion effect. Macromolecules 39: 2284. (b) Yoshikawa, C., Goto, A., Ishizuka, N., Nakanishi, K., Kishida, A., Tsujii, Y., and Fukuda, T. 2007. Size-Exclusion Effect and Protein Repellency of Concentrated Polymer Brushes Prepared by Surface-initiated Living Radical Polymerization. Macromol. Symp. 248: 189. (c) C. Yoshikawa, Y. Hashimoto, S. Hattori, T. Honda, K Zhang, D. Terada, A. Kishida, Y. Tsujii, H. Kobayashi, Suppression of Cell Adhesion on Well-defined Concentrated Polymer Brushes of Hydrophilic Polymers. Chem. Letters, 2010, 39, 142-143. (D) Feng, W., Brash, JL, and Zhu, S. 2006. Non-biofouling materials prepared by atom transfer radical polymerization grafting of 2-methacryloloxyethyl phosphorylcholine: Separate effect of graft density and chain length on protein repulsion.Biomaterials27: 847. (e) Iwata, R., Suk-In, P., Hoven, VP, Takahara, A., Akiyoshi, K., and Iwasaki, Y. 2004. Control of nanobiointerfaces generated from well-defined biomimetic polyme r brush for protein and cell manipulations. Biomacromolecules 5: 2308. (f) Mei, Y., Elliott, JT, Smith, JR, Langenbach, KJ, Wu, T., Xu, C., Beers, KL, Amis, EJ , and Henderson, L. 2006. Gradient substrate assembly for quantifying cellular response to biomaterials. J. Biomed. Mater. Res. 79A (4): 974.). Those skilled in the art will understand the range that can be grasped as a bioinert film from the results of such conventional studies.

  FIG. 1 is a cross-sectional view schematically showing an example of the bioinert film of the present invention. In addition, the bridge | crosslinking aspect of FIG. 1 is an illustration to the last, and an actual bridge | crosslinking aspect is not limited to this figure.

  In the bioinert film 31 of the present invention, a plurality of polymer chain graft fine particles 21 in which the polymer graft chains 23 are bonded to the surface of the inorganic fine particles 22 are cross-linked with a cross-linking agent 25.

  In the polymer chain graft fine particles 21, steric repulsion occurs between the polymer graft chains 23, and the polymer graft chains 23 are bonded to the surface of the inorganic fine particles 22 at a high density until the polymer graft chains 23 are extended. Yes.

  The bioinert film 31 is formed on the surface of the base material 41, and the polymer chain graft fine particles 21 are also cross-linked with the surface of the base material 41 with a cross-linking agent 25.

Patent Document 1 discloses a film in which a plurality of polymer chain graft fine particles are crosslinked with a divinyl compound or the like. Here, the number of polymers per unit area on the particle surface is about 0.03 chain. / Nm ² is low. In such a semi-diluted polymer brush, the polymer takes a random coil shape, a stretched coil shape, or the like. On the other hand, the high-density polymer brush used in the present invention takes a form in which the polymer graft chain is elongated due to steric repulsion between the polymer graft chains, and the non-specific adsorption of the protein or cell The size exclusion characteristic for adhesion is completely different from the technique of Patent Document 1.

  That is, by using polymer chain graft fine particles and a crosslinking agent, it can be produced easily and in a short time without requiring a complicated operation regardless of the shape of the base material, and also due to the high density polymer brush. Thus, the present inventors have found a biologically inactive membrane capable of greatly suppressing nonspecific adsorption of proteins and cell adhesion based on experimental facts as disclosed in the Examples, and have completed the present invention.

Graft density of the polymer graft chain is preferably from 0.1 stranded / nm ^2, more preferably 0.1 to 1.2 strands / nm ^2, 0.3 to 1.2 strands / nm ² Gayori preferable. By controlling the graft density, nonspecific adsorption of proteins and cell adhesion can be suppressed.

In the present invention, “graft density” or “density” means the number of graft chains bonded to the surface per unit area (nm ² ) of the fine particle surface. Graft density (σ) is determined by the amount of polymer graft chains (graft amount, w) formed by extending from the fine particle surface by elemental analysis, and the value and the surface area of core fine particles (S, nm ² ), graft polymer Alternatively, it is calculated as follows using the number average molecular weight Mn of the free polymer and the Avogadro number (Av).
Calculation method: σ = (w / Mn) Av / S
The number average molecular weight (Mn) of the polymer graft chain is preferably from 2,000 to 100,000. The degree of polymerization of the polymer graft chain is, for example, 10 to 10,000. The molecular weight distribution of the polymer graft chain is preferably 1 to 1.5 and more preferably 1 to 1.3 in view of having a homogeneous graft surface.

  The polymer graft chain can be obtained, for example, by living radical polymerization of a raw material monomer based on a polymerization initiating group on the surface of inorganic fine particles.

  As living radical polymerization, primitive transfer radical polymerization (ATRP), nitrogen oxide living radical polymerization (NMP), reversible addition-fragmentation chain transfer (RAFT) polymerization, and the like are known. Any method may be used.

  The type of raw material monomer is not particularly limited as long as the polymer graft chain is a hydrophilic polymer chain and has biocompatibility, and can be appropriately selected within the scope recognized by those skilled in the art.

  The term “hydrophilic” as used herein refers to a range recognized by those skilled in the art based on various studies on the effects of the structure and chemical composition of high-density polymer brushes on non-specific protein adsorption and cell adhesion (see the above-mentioned literature). Although not particularly limited, one example is that the hydrostatic contact angle of the bioinert membrane is preferably less than 50 °, more preferably less than 40 °, and even more preferably less than 30 °. It is.

  As a kind of raw material monomer, (meth) acrylic acid or its derivative (s), (meth) acrylamide or its derivative (s), a styrene derivative, etc. can be used, for example.

  In the present specification, “(meth) acryl” means “acryl” and “methacryl”.

Preferred examples include an acrylic acid derivative or a methacrylic acid derivative represented by CH ₂ = CR ^a COOR ^b . Here, R ^a represents a hydrogen atom or a methyl group, and R ^b represents any one selected from the following group:
- alkali metal, C ₃ -C ₂₀ hydrophilic groups, at least including at least hydrophilic group-least ether oxygen of C ₂ -C ₂₀ containing a hydrophilic group, at least hydroxyl group of C ₆ -C ₃₀ containing oxyethylene units primary amino group, secondary amino group C ₃ -C ₂₀ hydrophilic groups · -SO ₃ that or a tertiary amino group, ^-, -PO ₄ ^- -, and -COO ^- or an anion group selected from C ₆ -C ₂₀ hydrophilic group having an internal salt structure of benzene and quaternary ammonium cation The polymer graft chain may contain one monomer species (homopolymer) or randomly or in regular blocks It may include a plurality of arranged species (copolymers).

  Examples of the inorganic fine particles of the polymer chain graft fine particles include silicon oxide such as silica; metal; metal oxide; metal sulfide. Examples of the metal include noble metals such as Au, Ag, Pt, and Pd; Ti, Zr, Ta, Sn, Zn, Cu, V, Sb, In, Hf, Y, Ce, Sc, La, Eu, Ni, Examples include transition metals such as Co and Fe.

  The particle diameter of the inorganic fine particles is preferably 1 nm or more and 5 μm or less, and more preferably 1 nm or more and 1 μm or less. By controlling the particle size of the inorganic fine particles, a dense particle film is formed, and nonspecific adsorption of proteins and cell adhesion can be suppressed.

  The particle size of the inorganic fine particles may be one type or a mixture of two or more types. When the particle size of the inorganic fine particles is one, the size of the polymer chain graft fine particles becomes uniform, and a dense coating film can be formed.

  The polymer chain graft fine particles can be produced, for example, as follows. First, a compound as a polymerization initiating group-containing coupling agent is fixed on the surface of inorganic fine particles by a chemical adsorption method.

  Then, one or more raw material monomers are supplied to perform living radical polymerization.

  By fixing the polymerization initiating group-containing coupling agent on the surface of the inorganic fine particles in advance, the graft polymerization can be advanced while maintaining a constant graft density on the surface of the inorganic fine particles. In other words, the graft amount can be increased in proportion to the Mn (number average molecular weight) of the graft chain, the polymerization can proceed in a living manner, and almost all the graft chains on the surface of the fine particles can be grown almost uniformly. .

The graft density on the surface of the inorganic fine particles can be changed by adjusting the ratio of the polymerization initiator-containing coupling agent to the coupling agent not containing the polymerization initiator group. Specifically, when the inorganic fine particles are silicon oxide particles, if the ratio of the polymerization initiator-containing coupling agent to the coupling agent containing no polymerization initiator is 1: 0, the graft density is 0.00. Single strand / nm ² or more.

  The compound as the polymerization initiating group-containing coupling agent can be selected in consideration of the affinity with the fine particles.

  When the inorganic fine particles are silicon oxide, metal oxide, or metal sulfide particles, the polymerization initiator group-containing coupling agent represented by the above formula (I) is preferable as the polymerization initiator group-containing coupling agent.

In the formula (I), R ¹ represents a C ₁ -C ₃ alkyl group, preferably a C ₁ or C ₂ alkyl group, R ² represents a C ₁ or C ₂ alkyl group, X represents a halogen atom, preferably bromine Indicates an atom or a chlorine atom. n shows the integer of 3-10.

  Examples of the compound represented by the formula (I) include (2-bromo-2-methyl) propionyloxypropyltriethoxysilane (BPE), (2-bromo-2-methyl) propionyloxyhexyltriethoxysilane (BHE), and the like. Can be mentioned.

  In addition, for example, when the fine particles are a metal such as gold, silver, or platinum, it is preferable to use, for example, a disulfide compound as the polymerization initiating group-containing coupling agent. As the disulfide compound, for example, a compound represented by the following formula (II) can be used.

  Polymer chain graft fine particles are dissolved in solvent with inorganic fine particles with living radical polymerization polymerization initiator group-containing silane coupling agent bound to the surface, metal complexes that serve as a catalyst for living radical polymerization, and polymer graft chain raw material monomers. Alternatively, it can be dispersed and subjected to living radical polymerization to obtain a precipitate.

  Those skilled in the art will be able to select the details of such living radical polymerization and the catalyst to be used as appropriate.

  After the completion of the living radical polymerization, the target polymer chain graft microparticles are obtained from the reaction solution by a method commonly used in the art, such as extraction, distillation, washing, concentration, precipitation, filtration, drying, adsorption, precipitation, chromatography, etc. These methods can be isolated singly or in combination.

  The bioinert film of the present invention can be prepared using a coating solution in which the polymer chain graft fine particles and the crosslinking agent as described above are dissolved or dispersed.

  When preparing this coating solution, first, for example, inorganic fine particles in which a living radical polymerization-initiating group-containing silane coupling agent is bound to the surface, a metal complex serving as a catalyst for living radical polymerization, and a polymer graft chain The raw material monomer is dissolved or dispersed in a solvent, and living radical polymerization is performed to obtain the polymer chain graft fine particles as a precipitate. Thereafter, the polymer chain graft fine particles and the cross-linking agent are dissolved or dispersed in a good solvent that can be dissolved or dispersed at room temperature, and the concentration of the polymer chain graft fine particles is within the range of 1.0 to 20.0% by mass. The coating liquid can be prepared by setting the concentration of the agent within the range of 0.1 to 10.0% by mass.

  Examples of good solvents in which the polymer chain graft fine particles and the crosslinking agent can be dissolved or dispersed at room temperature include, for example, 1,4-dioxane, acetone, acetonitrile, chloroform, chlorophenol, cyclohexane, cyclohexanone, cyclopentanone, dichloromethane, and diethyl acetate. , Diethyl ketone, dimethyl carbonate, dimethylformamide, dimethyl sulfoxide, ethanol, ethyl acetate, m-cresol, mono- and di-alkyl substituted glycols, N, N-dimethylacetamide, diethyl ether, p-chlorophenol, 1,2- Propanediol, 1-pentanol, 1-propanol, 2-hexanone, 2-methoxyethanol, 2-methyl-2-propanol, 2-octanone, 2-propanol, 3-pentanone, 4-methyl -2-pentanone, hexafluoroisopropanol, methanol, methyl acetate, methyl acetoacetate, methyl ethyl ketone, methyl propyl ketone, n-methylpyrrolidone-2, n-pentyl acetate, phenol, tetrafluoro-n-propanol, tetrafluoroisopropanol, tetrahydrofuran , Toluene, xylene, water and the like. These may be used alone or in combination of two or more.

  The coating liquid of this invention can be apply | coated to a base material by the method already known as a wet film-forming method, for example. For example, a dipping method, a spin coating method, a casting method, or the like can be used.

  This coating liquid can coat the whole base material, or can also coat it partially. For example, the coating can be partially crosslinked and uncrosslinked portions can be removed by photolithography.

  The bioinert film of the present invention can be formed by applying a coating solution to a substrate to form a coating film, and then crosslinking the polymer chain graft microparticles with a crosslinking agent.

  Methods for initiating crosslinking include, for example, electron beam radiation, electromagnetic radiation (ultraviolet light (UV), visible light and near infrared), heat, and the addition of moisture when moisture curable compounds are used. It is done.

  In a preferred embodiment, the crosslinking is performed by ultraviolet light irradiation or heat. In this case, as the crosslinking agent, for example, a compound having at least two groups selected from a phenyl azide group and a pyridine azide group can be used.

  Preferred examples of the compound having at least two groups selected from a phenyl azide group and a pyridine azide group include those having a linear molecular weight in the range of 200 to 1,000.

  Examples of such a compound include polyethylene glycol bisazidobenzoate having 2 to 6 oxyethylene units. Among these, triethylene glycol bisazidobenzoate (TEGBA) has the following structure.

  The crosslinking agent as described above is bonded to the polymer graft chain by irradiation with ultraviolet light or heat. That is, the coated coating film is irradiated with ultraviolet light, or the coated coating film is heated (heat treatment) to crosslink the polymer chain graft microparticles with the crosslinking agent, thereby forming a bioinert film. be able to.

  As conditions for ultraviolet light irradiation at this time, it is preferable to irradiate the coating film with ultraviolet light of 100 W or more for 10 seconds or more.

  In the present invention, polymer chain graft microparticles having a biologically inert graft surface are prepared in advance, so that the solution containing the polymer chain is coated, and simple crosslinking reaction (for example, ultraviolet irradiation for 30 seconds) is simply performed. A bioinert membrane can be produced. And, by irradiation with ultraviolet rays, a bioinert film can be produced in a short time.

  Moreover, when forming a bioinert film | membrane by heat processing, as conditions for heat processing, it is preferable to heat-process a coating film at 120 degreeC or more for 18 hours or more.

  When a compound having at least two groups selected from the phenyl azide group and the pyridine azide group is used as a crosslinking agent, the azide group of the crosslinking agent reacts with and binds to the substrate surface, and the substrate and the polymer chain graft The fine particles can also be crosslinked with a crosslinking agent. For example, when there is an alkyl group, amino group, hydroxyl group or the like on the substrate surface, the crosslinking reaction between the crosslinking agent and the substrate surface proceeds.

  Furthermore, the phenyl azide group that becomes the cross-linking part reacts with many general-purpose polymer materials (PP, PE, PET, PMMA, PS, PU, cellulose, etc.) and glass materials (—OH groups) that are generally used as medical materials. (Langmuir 1991, 7, 2010-2012 Biomacromolecules 2007, 8, 679-685 Macromolecules 1994,27, 7809-7814 Langmuir 1996,11, 2267-2271 Chemical Reviews, Volume 88, Number 2, March / April , 1988), almost any substrate can be crosslinked regardless of the material.

  In addition, when a metal substrate is used, the metal substrate is surface-treated with an amino group-containing silane coupling agent or an alkyl group-containing silane coupling agent in advance, and this surface-treated metal substrate is The base material and the polymer chain graft microparticles can be cross-linked by a cross-linking agent by applying a coating liquid using a compound having at least two groups selected from the phenyl azide group and the pyridine azide group as a cross-linking agent. it can.

  As the amino group-containing silane coupling agent, for example, a compound represented by the following formula (III) can be used.

In the formula, R ¹ represents a C ₁ -C ₃ alkyl group, preferably a C ₁ or C ₂ alkyl group, and R ² represents a C ₁ or C ₂ alkyl group. n shows the integer of 3-10.

  Examples of the compound represented by the formula (III) include AMP (3-aminopropyltrimethoxysilane).

  As the alkyl group-containing silane coupling agent, for example, an alkyl group, preferably a linear alkyl group having 1 to 8 carbon atoms, substituted with an aminoalkyl group bonded to Si in the above formula (III) is used. it can.

  The bioinert membrane of the present invention can be prepared with any suitable thickness. Usually it has a thickness in the range between 50 nm and several tens of micrometers. Preferably, the thickness of the coating is 50 nm to 1000 nm.

  The substrate to which the bioinert film of the present invention is applied is not particularly limited. For example, a flat or curved, rigid or flexible substrate can be used, and various metallic substrates and resins can be used. A substrate, a glass substrate, or the like can be used.

  Examples of bioinert-treated substrates on which the bioinert film of the present invention is formed include, for example, cell culture dishes, cell culture flasks, contact lens cases, catheters, implants, stents, and biological samples (eg, blood). A tube, a microtiter plate, a microfluidic device, a biosensor, a microtube, a PCR tube, a separation filter, a pipette chip and the like can be mentioned. Among them, it is suitable for cell culture dishes, cell culture flasks, contact lens cases, catheters and the like.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
(Example 1: Entry 1)
[Sample preparation]
1. Preparation of polymer chain graft microparticles Synthesis of polymerization initiation group-containing silane coupling agent (2-bromo-2methyl) propionyloxypropyltriethoxysilane (BPE)

(1) Synthesis of BPE Performed by a two-step reaction. As a first step, a mixed solution of allyl alcohol (42 g), triethylamine (88 g) and tetrahydrofuran (THF) (1 L) was ice-cooled, and 2-bromoisobutyryl bromide (100 g) was added dropwise thereto.

  Thereafter, the reaction solution was stirred at 0 ° C. for 3 hours, and further stirred at room temperature for 10 hours. The reaction solution was concentrated, THF was added to precipitate a salt, filtered, and the filtrate was concentrated. The obtained solution was diluted with chloroform (1 L), and diluted with 1N hydrochloric acid aqueous solution (3 × 500 ml), saturated carbonic acid. Washing was carried out in the order of aqueous sodium hydrogen solution (3 × 500 ml) and pure water (3 × 500 ml).

  The organic phase was dried, concentrated and purified by distillation under reduced pressure to obtain 1- (2-bromo-2-methyl) propionyloxy-2-propene (BPP) in a yield of 50%.

  As the second stage, BPP (34 g), triethoxysilane (81.5 g), toluene (170 ml) and calsted catalyst (500 μL) were sequentially added into the flask, and the mixture was stirred at room temperature for 12 hours under an argon atmosphere. did.

After the reaction, it was purified by distillation under reduced pressure, and BPE was synthesized with a yield of 20%.
(2) Introduction of initiation group into silica fine particles <BPE immobilization on silica fine particles>
Silica fine particles (manufactured by Nippon Shokubai Co., Ltd., product name “Seahoster (registered trademark) KE-E10” (average particle size 130 nm)) were used as the silica fine particles.

  A dispersion of silica fine particles in ethanol (7.7 wt%, 30 mL) was added to a mixed solution of 28% aqueous ammonia (13.9 g) and ethanol (200 mL).

  The mixture was stirred at room temperature for 2 hours, BPE (2 g) dissolved in ethanol (10 mL) was added dropwise, and the mixture was stirred at room temperature for 18 hours.

  Thereafter, the silica fine particles were collected by a centrifuge and washed with ethanol to obtain silica fine particles having an initiating group.

Silica microparticles having this initiating group were stored in ethanol.
(3) Silica fine particles (200 mg) having living radical polymerization initiating groups using silica fine particles, poly (ethylene glycol) methyl ether methacrylate (PEGMA, number of oxyethylene units: 8 to 9) (5.0 g), Cu (I) A solution of Br (7.6 mg), 2,2-bipyridine (16.4 mg) and ethyl 2-bromoisobutyrate (EBIB) (7.3 mg) in methanol (5 g) in a glove box with Pyrex (registered) (Trademark) and then polymerized at 30 ° C. for 3 hours to obtain composite particles in which polymer graft chains were bonded to the surface of silica fine particles.

  The composite particles were purified by repeating centrifugation and redispersion in methanol.

  The monomer conversion rate in the polymer graft chain was determined by NMR.

  The molecular weight and molecular weight distribution of the free polymer (PPEGMA) produced from EBIB were determined by gel filtration chromatography (GPC).

The graft density is calculated from the graft amount determined by thermogravimetric analysis (TG-DTA) and the molecular weight of the free polymer, and all samples have a high graft density of 0.2 chain / nm ² or more. confirmed.

  The polymerization conditions and polymerization results used here are summarized in Table 1.

2. Synthesis of cross-linking agent (triethylene glycol bisazidobenzoate: TEGBA) First, 4-azidobenzoic acid (3.0 g), triethylene glycol (1.9 g), N, N-dimethyl-4-aminopyridine (0. 26 g) was dissolved in 60 ml of dichloromethane.

  1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) (6.04 g) was added to this, and after stirring for 19 hours, 0.1 M hydrochloric acid (200 ml) was further added and stirred for 15 minutes.

  Next, the aqueous layer and the organic layer were separated from each other, and then the organic layer was washed with pure water (200 ml), a saturated aqueous sodium hydrogen carbonate solution (200 ml), and pure water (200 ml).

  The organic layer was concentrated to obtain TEGBA in 74% yield.

  The NMR spectrum (FIG. 2) of this TEGBA (crosslinking agent) was examined to confirm the molecular structure.

  According to the reaction shown below.

3. Preparation of Coating Solution Using Fine Particles A PPEGMA-SiPs concentration of 2.0 to 9.0 wt% and a TEGBA concentration of 0.5 wt% were used to prepare a toluene solution containing polymer chain grafted fine particles and TEGBA (Example) 1 coating solution).
4). Using a coating spin coater (MIKASA, MS-A100), the above-mentioned coating solution was coated on a silicon substrate modified with aminopropyltriethoxysilane (AMP) under conditions of 1500 rpm and 30 seconds. A coating coating film was formed.
5. Irradiation of UV to the coating solution coating film Using an ultraviolet lamp (As One, handy type UV curing device), the coating surface of the coating coating film of Example 1 was irradiated with UV light at 100 W for 30 seconds, and then the UV irradiation of Example 1 was performed. A film was formed.

  After irradiation, the coating was ultrasonically cleaned using a good solvent such as toluene or chloroform.

In addition, when not irradiating with ultraviolet rays, it was confirmed that the coating coating film of Example 1 was peeled off from the silicon substrate by ultrasonic cleaning.
(Example 2: Entry 2)
A coating solution of Example 2 was prepared in the same manner as Example 1 except that the concentration of the PPEEGMA graft silica fine particles (PPEGMA-SiPs) was 5.0 wt%.

A coating coating film of Example 2 was formed in the same manner as in Example 1 except that the coating liquid of Example 2 was used, and an ultraviolet irradiation film of Example 2 was formed.
(Example 3: Entry 3)
A coating solution of Example 3 was produced in the same manner as Example 1 except that the concentration of PPEGMA-SiPs was 9.0 wt%.

A coating application film of Example 3 was formed in the same manner as in Example 1 except that the coating liquid of Example 3 was used, and an ultraviolet irradiation film of Example 3 was formed.
[Sample evaluation of Examples 1 to 3]
6). Photograph and AFM Image Evaluation FIG. 3 is a photograph of the ultraviolet irradiation film of Example 1 (Entry 1), Example 2 (Entry 2), and Example 3 (Entry 3), and FIG. 4 is an AFM image. A uniform film was formed without peeling off from the silicon substrate.
7). Cell adhesion evaluation Example 1 (Entry 1), Example 2 (Entry 2), Example 3 (Entry 3), and Comparative Example 1 (Entry 4) were coated with human vascular endothelial cells (10,000 cells / cm ² ) on the coating surface. Sowing. In Comparative Example 1 (Entry 4), a coating solution containing only TEGBA was used.

  The cell adhesion image after 24 hours is shown in FIG. 5 (photo of HUVEC adhesion test on the VS-like coating), and the number of cell adhesion to each coating surface is shown in FIG. 6 (the vertical axis in FIG. 6 is normalized by the number of cell seeding). ) It was confirmed that cells hardly adhered to the coating surface containing the composite fine particles. From the comparison with Comparative Example 1, it can be said that the presence of PPEGMA-SiPs significantly suppressed cell adhesion.

  Table 2 shows the coating compositions of Examples 1 to 3 and the HUVEC adhesion test results.

8). Protein Adsorption Evaluation Using the sample of Example 2 (Entry 2), the adsorption of coexisting proteins contained in bovine serum (FBS) was examined by quartz crystal measurement.

The results of examining the amount of adsorption for 1 hour using undiluted FBS are shown in FIG. 7 (Adsorption measurement of bovine serum protein (FBS)). After 1 hour, when the phosphate buffer was washed, the baseline returned to the original position, and the adsorption amount was almost 0 ng / cm ² , confirming that protein adsorption was inhibited.
(Example 4: Entry 5, 6)
9. Preparation of coating film on polymer substrate Using the same coating solution as in Example 2 , a coating film was prepared on a PET film and an FEP film, and an ultraviolet irradiation film was similarly formed (Entry 5, 6: see Table 3). A photograph is shown in FIG. 8, and an AFM image is shown in FIG. The coating film was obtained on the polymer film as well as on the silicon substrate.

(Example 5: Entry 7, 8)
10. Heat treatment film of coating liquid coating film A coating coating film is prepared on the AMP-immobilized silicon substrate and the untreated silicon substrate using the same coating liquid as in Example 2, and heated at 120 ° C. for 20 hours to be a heat treated film (Entry 7, 8: see Table 4).

  The heat-treated film was returned to room temperature and then ultrasonically cleaned using a good solvent such as toluene or chloroform. An optical photograph is shown in FIG. 10, and an AFM image is shown in FIG.

(Example 6: Entry 12)
11. Cytotoxicity evaluation (dissolution test)
When an unreacted cross-linking agent is present, the cross-linking agent elutes during cell culture, which may affect (toxic) cells. Therefore, the following dissolution test was performed.

First, samples shown in Table 5 were formed on a silicon substrate. Those samples were arranged in a 24-well plate, 1 ml of serum medium was added, and the plate was allowed to stand for 24 hours in an incubator. Human vascular endothelial cells (1.5 × 10 ⁴ cells / well) are prepared in a 96-well plate (preculture), and 50 μl of serum medium in which the sample is immersed is added to 96-well cells for 24 hours. Cultured.

  Thereafter, cell viability was evaluated using a cell proliferation (viability) ability analysis kit, WST-1 (Takara Bio). The results are shown in FIG. The absorbance on the vertical axis indicates the proportion of living cells, and all samples were almost the same as the absorbance (survival rate) on the silicon substrate. From this, it is considered that there is almost no unreacted cross-linking agent or even if it is eluted, it does not affect the cells.

(Example 7: Entry 13, 14, 15, 16)
12 Confirmation of cross- linking reaction by UV In order to examine the degree of cross-linking of the cross-linking agent by ultraviolet rays, PPEGMA-SiP was coated on the FEP film by changing the concentration of the cross-linking agent (Table 6). FT-IR measurement of the coating film was performed before and after curing by ultraviolet irradiation (FIGS. 13 to 16). Regardless of the concentration of the crosslinking agent, it can be seen that the peak of the azido group disappears after the ultraviolet irradiation, and that almost all of the azide group is consumed in the crosslinking reaction by the ultraviolet irradiation.

(Example 8: Entry 17-31)
13. Coating on Various Polymer Films Various polymer films were coated at the composition concentrations shown in Table 7 and crosslinked by UV irradiation or heat treatment. The UV irradiation condition was 100 W for 30 seconds, and the heat treatment condition was 120 degrees 180 hours. After each crosslinking treatment, the sample was subjected to ultrasonic cleaning using an organic solvent such as toluene, chloroform, and ethanol.

As an example, AFM images of Entry 17, 23, and 26 are shown in FIG. 17 (the coating conditions are all the same). It can be seen that the surface is covered with PPEGMA-SiPs even after ultrasonic cleaning.
14 Cell adhesion The human vascular endothelial cells (10,000 cells / cm ² ) were seeded on the samples shown in Table 7. A cell culture plastic sheet (TCPS) (Wako) was used as a reference. The number of cell adhesion after 24 hours to each coating surface is shown in FIG. 18 (the vertical axis in FIG. 18 is normalized by the number of cell seeding). From FIG. 18, it was confirmed that vascular endothelial cells hardly adhered on any polymer substrate surface.

  As described above, in Examples 1 to 8, a coating film can be easily produced without requiring a complicated operation by ultraviolet irradiation or heat treatment on the coating liquid coating film, and particularly in a short time by ultraviolet irradiation. A coating film could be produced. The obtained coating film could significantly suppress non-specific protein adsorption and cell adhesion, and functioned as a biologically inactive film.

21 Polymer chain graft fine particle 22 Inorganic fine particle 23 Polymer graft chain 25 Crosslinker 31 Bioinert film 41 Base material

Claims (17)

A plurality of polymer chain graft fine particles in which steric repulsion occurs between the polymer graft chains and the polymer graft chains are bonded to the surface of the inorganic fine particles at a high density until the polymer graft chains are extended. In bioinert membranes that are cross-linked by a cross-linking agent ,
A bioinert film characterized in that the cross-linking agent crosslinks the polymer chain graft microparticles and a substrate on which the bioinert film is formed . The bioinert membrane according to claim 1, wherein a graft density of the polymer graft chain is 0.1 strand / nm ² or more.   The polymer graft chain is a hydrophilic polymer chain obtained by living radical polymerization of at least one raw material monomer selected from (meth) acrylic acid or a derivative thereof, (meth) acrylamide or a derivative thereof, and a styrene derivative. The bioinert membrane according to claim 1 or 2, wherein The polymer graft chain is represented by the following formula (I):

(Wherein R ¹ represents a C _{1 to} C ₃ alkyl group, R ² represents a C ₁ or C ₂ alkyl group, X represents a halogen atom, and n represents an integer of 3 to 10). 4. The product according to claim 1, wherein the polymer-initiated group-containing silane coupling agent is bonded to the inorganic fine particles, and then the raw material monomer of the polymer graft chain is obtained by living radical polymerization. The bioinert membrane according to any one of the above. The bioinert film according to any one of claims 1 to 4, wherein the inorganic fine particles are an object different from the base material .   The bioinert film according to any one of claims 1 to 5, wherein the crosslinking agent is a compound having at least two groups selected from a phenyl azide group and a pyridine azide group.   The bioinert film according to any one of claims 1 to 6, wherein a particle diameter of the inorganic fine particles is 1 nm or more and 1 µm or less.   The bioinert film according to any one of claims 1 to 7, wherein the inorganic fine particles are made of any material selected from silicon oxide, metal oxide, metal, and metal sulfide. .   Polymer chain graft fine particles in which steric repulsion occurs between polymer graft chains and the polymer graft chains are bonded to the surface of the inorganic fine particles at a high density until the polymer graft chains are extended, and a crosslinking agent A coating liquid, wherein the is dissolved or dispersed in a solvent.   It is a manufacturing method of the coating liquid of Claim 9, Comprising: The said inorganic microparticles | fine-particles couple | bonded with the surface of the living radical polymerization polymerization initiator group containing silane coupling agent, the metal complex used as the catalyst of living radical polymerization, and the said high Living radical polymerization is performed by dissolving or dispersing the raw material monomer of the molecular graft chain in a solvent to obtain the polymer chain graft microparticle as a precipitate, and the polymer chain graft microparticle and the crosslinking agent are dissolved or dispersed at room temperature. These are dissolved or dispersed in a possible good solvent, the concentration of the polymer chain graft fine particles is in the range of 1.0 to 20.0 mass%, and the concentration of the crosslinking agent is 0.1 to 10.0 mass%. And a step of preparing the coating liquid within the range.   Applying the coating liquid according to claim 9 to a substrate to form a coating film, and irradiating the coating film with ultraviolet light, or heat-treating the coating film, thereby the polymer chain graft fine particles And a step of cross-linking each other with the cross-linking agent to form a bio-inert film.   The method for producing a bioinert film according to claim 11, wherein the coating liquid is applied to the substrate by any one of a wet film forming method selected from a dipping method, a spin coating method, and a casting method.   The crosslinking agent is a compound having at least two groups selected from a phenyl azide group and a pyridine azide group, and the polymer chain graft fine particles are crosslinked by the crosslinking agent by irradiating the coating film with ultraviolet light. The method for producing a bioinert film according to claim 11 or 12, wherein a bioinert film is formed.   The coating liquid is applied to the metal base material surface-treated with an amino group-containing silane coupling agent, and the base material and the polymer chain graft fine particles are cross-linked by the cross-linking agent. 14. A method for producing a bioinert film according to 13.   The method for producing a bioinert film according to any one of claims 11 to 14, wherein the coating film is irradiated with ultraviolet light of 100 W or more for 10 seconds or more.   The method for producing a bioinert film according to any one of claims 11 to 14, wherein the coating film is heat-treated at a temperature of 120 ° C or more for 18 hours or more.   The bioinert membrane according to any one of claims 1 to 8 is formed on the surface of any substrate selected from a cell culture dish, a cell culture flask, a contact lens case, and a catheter. A bioinert-treated substrate characterized.