JP2011078706A - Method for improving quality of surface biocompatibility - Google Patents
Method for improving quality of surface biocompatibility Download PDFInfo
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- JP2011078706A JP2011078706A JP2009235723A JP2009235723A JP2011078706A JP 2011078706 A JP2011078706 A JP 2011078706A JP 2009235723 A JP2009235723 A JP 2009235723A JP 2009235723 A JP2009235723 A JP 2009235723A JP 2011078706 A JP2011078706 A JP 2011078706A
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Abstract
Description
本発明は生体または生体に由来する物質と接触する表面の生体適合性を改質、特に、向上せしめる方法に関する。より具体的には、本発明は生体内の血液と人工物である材料とが接触した時に生じる血液の変性を低減乃至防止する方法に関する。 The present invention relates to a method for modifying, in particular improving the biocompatibility of a surface in contact with a living body or a substance derived from a living body. More specifically, the present invention relates to a method for reducing or preventing blood degeneration that occurs when blood in a living body comes into contact with an artificial material.
近年の医療工学の発展に伴い医療現場では、人工血管や人工心肺などの人工臓器が使用されるようになってきた。現在使用されている人工臓器の多くは高分子材料からできており、血液や生体組織と直接接触するものについては、生体適合性、特に、血液適合性や組織適合性が要求される。例えば、材料に血液が接触すると生体液は活性化され、酸化ストレスの上昇、血液の凝固反応や炎症反応を引き起こす。このとき活性化された白血球などの食細胞は、活性酸素種（ＲＯＳ）を産生すると伴に炎症性サイトカインを放出する。 With the development of medical engineering in recent years, artificial organs such as artificial blood vessels and heart-lung machines have been used in the medical field. Many of the artificial organs currently used are made of a polymer material, and those that come into direct contact with blood or biological tissue are required to have biocompatibility, particularly blood compatibility and tissue compatibility. For example, when blood comes into contact with the material, the biological fluid is activated, causing an increase in oxidative stress, blood coagulation reaction, and inflammation reaction. At this time, activated phagocytes such as leukocytes produce reactive oxygen species (ROS) and release inflammatory cytokines.
現在までに血液の活性化を小さくするために親水性ポリマー、生体膜を模倣したポリマー、ミクロドメイン構造を持つポリマー、生理活性物質を固定化した表面など様々な研究がなされてきた（例えば、非特許文献１および非特許文献２参照）。最近ではアセチルコリン基を有する高分子（ＰＭＰＣ）やポリアクリル酸２−メトキシエチルなどの生体適合性の良い新規ポリマーが開発されて来ている（例えば、特許文献１および特許文献２参照）。これらの材料は比較的性能がよく、その一部は実用化されているものの、完全に問題が解決されているわけではない。たとえば親水性ポリマーは血小板の吸着は抑制するが血液の活性化反応については抑制することができないことが挙げられる。生理活性物質は動物由来物質であり安全面に問題があることや、生理活性の自然低下といった問題がある。最近、トコフェロールのような抗酸化剤を表面にコーティングしてＲＯＳの発生を抑える方法が一定の成果を上げているものの、高価で化学量論的反応のため、公算可能が継続しないという問題があった。また、医療用物品の表面を被覆するためのポリマー性キャリアとして、ポリ（ヒドロキアルカン酸エステル−ｃｏ−エステルアミド）の側鎖に活性酸素をはじめとするフリーラジカルの捕捉剤としての２，２’，６，６’−テトラメチル−１−ピペリジニルオキシフリーラジカル（ＴＥＭＰＯ）を担持させたポリマーも提案されている（特許文献３参照）。なお、特許文献３では、上記フリーラジカル捕捉剤の他に、多種多様な薬剤、例えば、増殖抑制薬、抗血小板薬もしくは抗凝固薬、診断用薬剤を該ポリマーの側鎖に担持させることが提案されている。しかし、これらの薬剤を担持するポリマーで被覆された表面が具体的にどのような挙動を示すかを明らかにするデータは何ら開示されていない。 To date, various studies have been conducted to reduce blood activation, including hydrophilic polymers, polymers that mimic biological membranes, polymers with microdomain structures, and surfaces on which bioactive substances are immobilized (for example, (See Patent Document 1 and Non-Patent Document 2). Recently, new polymers having good biocompatibility such as a polymer having an acetylcholine group (PMPC) and 2-methoxyethyl polyacrylate have been developed (for example, see Patent Document 1 and Patent Document 2). Although these materials have relatively good performance and some have been put into practical use, the problem has not been completely solved. For example, hydrophilic polymer suppresses the adsorption of platelets but cannot inhibit the activation reaction of blood. The physiologically active substance is an animal-derived substance, which has a problem in safety and has a problem of a natural decrease in physiological activity. Recently, a method of suppressing the generation of ROS by coating an antioxidant such as tocopherol has achieved a certain result, but there is a problem that the probable possibility does not continue due to the expensive and stoichiometric reaction. It was. Further, as a polymeric carrier for coating the surface of a medical article, 2,2 ′ as a scavenger for free radicals including active oxygen in the side chain of poly (hydroxyalkanoic acid ester-co-esteramide). , 6,6′-Tetramethyl-1-piperidinyloxy free radical (TEMPO) is also proposed (see Patent Document 3). In addition, Patent Document 3 proposes that in addition to the above-mentioned free radical scavenger, a wide variety of drugs, for example, growth inhibitors, antiplatelet drugs or anticoagulants, and diagnostic drugs are carried on the side chain of the polymer. Has been. However, no data is disclosed which reveals how the surface coated with the polymer carrying these drugs specifically behaves.
上記先行技術の問題点は、下記のようにまとめることができる。
１）親水性ポリマーのミクロ相分離膜は、一度活性酸素が発生するとその活性酸素を捕捉することができない。
２）生体活性物質を固定化した表面は、当該物質は、通常、動物由来物質であり、生体で使用する際の安全面に問題があり、また、一度発生した活性酸素を捕捉することもできない。また、表面へ固定化が困難であり、表面の物理的安定が低い。
３）生体膜模倣ポリマーは、一度活性酸素が発生するとその活性酸素を捕捉することができない。
４）フリーラジカル捕捉剤を担持させたポリマーから形成される塗膜が抗血小板薬もしくは抗凝固薬を担持させたポリマーと異なり血液や生体に対して具体的にどのように作用するのか推測できない。
The problems of the above prior art can be summarized as follows.
1) The microphase separation membrane of hydrophilic polymer cannot capture the active oxygen once it is generated.
2) The surface on which the bioactive substance is immobilized is usually an animal-derived substance, and there is a problem in safety when used in a living body, and once generated active oxygen cannot be captured. . Further, it is difficult to fix to the surface, and the physical stability of the surface is low.
3) A biomimetic polymer cannot capture active oxygen once it is generated.
4) Unlike a polymer in which an antiplatelet drug or an anticoagulant is supported, a coating film formed from a polymer in which a free radical scavenger is supported cannot be specifically estimated how it acts on blood or a living body.
したがって、本発明の目的は、生体または生体に由来する物質と接触する表面に対して容易、かつ、安定に固定できる材料を用い、生体適合性に関連して、広範にかつ、より具体的に有意な挙動を示すように該表面を改質するための方法を提供することにある。 Accordingly, an object of the present invention is to use a material that can be easily and stably fixed to a living body or a surface that comes into contact with a substance derived from a living body, and more broadly and more specifically in relation to biocompatibility. The object is to provide a method for modifying the surface to show significant behavior.
発明者らは先に、触媒的に酸化還元を司るニトロキシラジカルを高分子に担持することにより生体内での代謝を回避し、ＲＯＳ消去剤として機能することを見出し提案した（特願２００８−１２０６２６及び特願２００８−１７８１５０）。今ここに、ＴＥＭＰＯ等のニトロキシドラジカルを側鎖に担持した一定の反復単位を含むポリマーが、生体または生体に由来する物質と接触する表面に安定に固定でき、該表面上でＲＯＳを消去もしくは吸収できることのみならず、該表面が、とくに、生体内の血液と人工物である材料とが接触した時に生じる血液の変性を低減乃至防止できることを見出した。これは、特許文献３が具体的に血液に関連するものとして、抗血小板薬、抗凝固薬、及び抗トロンビン薬、例えば、ヘパリンナトリウム、低分子ヘパリン、へパリノイド、ヒルジン、アルガトロパン、フォルスコリン、プロスタサイクリン等を使用することと明確に異なる。 The inventors have previously found and proposed that a nitroxy radical that catalyzes redox catalytically is supported on a polymer to avoid metabolism in vivo and function as a ROS scavenger (Japanese Patent Application 2008- 120626 and Japanese Patent Application No. 2008-178150). Now, a polymer containing a certain repeating unit carrying nitroxide radicals such as TEMPO on its side chain can be stably immobilized on a living body or a surface in contact with a substance derived from the living body, and erases or absorbs ROS on the surface. In addition to being able to do so, the present inventors have found that the surface can reduce or prevent blood degeneration that occurs particularly when blood in a living body comes into contact with an artificial material. This is because Patent Document 3 specifically relates to blood, such as antiplatelet drugs, anticoagulants, and antithrombin drugs such as heparin sodium, low molecular weight heparin, heparinoids, hirudin, argatropane, forskolin, prosta It is clearly different from using cyclin.
したがって、本発明は、本発明は生体または生体に由来する物質と接触する表面の生体適合性を改質する方法であって、該表面に
環状ニトロキシドラジカル部分を担持する反復単位を、少なくとも、ポリマー主鎖の反復単位の１５％以上含んでなり、他の反復単位が存在する場合には、対応する環状ニトロキシラジカル部分が水素原子もしくは官能基である反復単位であるか、または該反復単位と一緒になってコポリマーを形成しうる別の反復単位を含み、かつ、環状ニトロキシドラジカル部分を担持する反復単位と存在する場合の他の反復単位は相互にランダムに存在するかまたはブロックを形成してもよい、ポリマーを固定する工程を含んでなる、上記方法が提供される。好ましくは、該ポリマーはフィルムまたは被膜として目的の表面に固定される。
Accordingly, the present invention is a method for modifying the biocompatibility of a surface that contacts a living body or a substance derived from the living body, and the surface has a repeating unit carrying a cyclic nitroxide radical moiety on at least a polymer. If it contains more than 15% of the repeating units of the main chain and other repeating units are present, the corresponding cyclic nitroxyl radical moiety is a hydrogen atom or a repeating unit that is a functional group, or The repeating unit containing another repeating unit that can form a copolymer together and carrying a cyclic nitroxide radical moiety and the other repeating unit, if present, are randomly present in each other or form a block There is provided the above method comprising the step of immobilizing the polymer. Preferably, the polymer is fixed to the target surface as a film or coating.
本発明によれば、生体または生体に由来する物質と接触する表面とし、限定されるものでないが、ダイアライザー、コンタクトレンズ、人工血管、人工臓器、注射器、培養シャーレ、ピペットチップ、といった直接血液や血漿などの体液や細胞、組織などと接触する医療用具等の表面の生体適合性、特に血液適合性を改質乃至向上できる。 According to the present invention, a surface that comes into contact with a living body or a substance derived from a living body, but is not limited to direct blood or plasma such as a dialyzer, a contact lens, an artificial blood vessel, an artificial organ, a syringe, a culture dish, or a pipette tip. It is possible to modify or improve the biocompatibility of the surface of a medical device or the like that comes into contact with body fluids such as cells, tissue, etc., particularly blood compatibility.
本発明にいう、生体適合性とは、長期間にわたって生体に悪影響も強い刺激も与えず、本来の機能を果たしながら生体と共存できる属性をいう。生体は、人体をはじめ哺乳動物，その他の動物、さらには、植物を包含する概念で使用しており、したがって、生体に由来する物質とは、動物の器官、臓器、細胞、体液（例えば、ヒトにおいては血液、涙、唾液）等を意味する。 The biocompatibility referred to in the present invention refers to an attribute that can coexist with a living body while performing its original function without giving adverse effects or strong stimulation to the living body for a long period of time. Living organisms are used in a concept that includes human bodies, mammals, other animals, and even plants. Therefore, substances derived from living organisms include animal organs, organs, cells, body fluids (for example, humans). Means blood, tears, saliva) and the like.
上記表面の改質に使用できる、ポリマーは、下記式（ａ）〜（ｐ）で表される反復単位を、少なくとも、ポリマー主鎖の反復単位の１５％以上、好ましくは２０％、より好ましくは３５％以上をランダムもしくはブロックを形成するように含むコポリマーであるか、またはすべての反復単位がそれらのいずれかの１種であるホモポリマーであることができる。 The polymer that can be used for the modification of the surface has a repeating unit represented by the following formulas (a) to (p) at least 15% or more of the repeating unit of the polymer main chain, preferably 20%, more preferably It can be a copolymer containing more than 35% so as to form a random or block, or it can be a homopolymer in which all repeating units are any one of them.
上式中、Ｒは環状ニトロキシドラジカル部分を表し、
ｎは、５〜１０，０００、好ましくは、５〜５，０００、より好ましくは５〜２，５００の整数を表す。
Wherein R represents a cyclic nitroxide radical moiety;
n represents an integer of 5 to 10,000, preferably 5 to 5,000, more preferably 5 to 2,500.
上記反復単位が、ポリマー主鎖の反復単位の１００％を占めない場合、存在し得る残りの反復単位は、上記環状ニトロキシドラジカル部分を表す、Ｒが水素原子であるか、または他の官能基を形成し得る基を有するランダムに存在するかもしくはブロックを形成するように存在する反復単位である。ポリハロメチルスチレン（ａ）において、他の官能基を形成し得る基は−（ＣＨ２）−ＯＲまたは−（ＣＨ２）−ＮＨＲの−ＯＲおよび−ＮＨＲがハロゲン原子、例えば、フッ素、塩素もしくは臭素原子であることができる。 If the repeat unit does not occupy 100% of the repeat units of the polymer backbone, the remaining repeat unit that may be present represents the cyclic nitroxide radical moiety, R is a hydrogen atom, or other functional group A repeating unit that is present randomly or has a group that can form to form a block. In the polyhalomethylstyrene (a), the group capable of forming another functional group is — (CH 2 ) —OR or —OR of — (CH 2 ) —NHR and —NHR are halogen atoms such as fluorine, chlorine or It can be a bromine atom.
これらの反復単位のうち、ポリハロメチルスチレンから誘導できる反復単位（ａ）： Of these repeating units, the repeating unit (a) derivable from polyhalomethylstyrene:
を好もしいものとして挙げることができる。このような反復単位を含むポリマーは、一般的に、これらの反復単位に起因して疎水性を示すが、それにもかかわらず、生体適合性を上記表面に付与することができる。 Can be cited as a good thing. Polymers containing such repeating units generally exhibit hydrophobicity due to these repeating units, but can nevertheless impart biocompatibility to the surface.
Ｒの環状ニトロキシラジカル部分は、必要により、連結基、−ＣＨ２ＣＨ２Ｏ−、−ＣＨ２ＣＨ２Ｓ−、−ＣＯＣＨ２ＣＨ２Ｏ−、−ＣＯＣＨ２ＣＨ２Ｓ−、−（ＣＨ２）３−Ｏ−、−（ＣＨ２）３−Ｓ−、−ＣＯ（ＣＨ２）３−Ｏ−、−（ＣＨ２）３−Ｏ−等を介して結合することができる、２，２，６，６−テトラメチルピペリジン−１−オキシル−４−イル、２，２，５，５−テトラメチルピロリジン−１−オキシル−３−イル、２，４，４，−トリメチル−１，３−チアゾリジン−３−オキシル−２−イル、および２，４，４−トリメチル−イミダゾリジン−３−オキシ−２−イル等から選ぶことができる。より具体的には、次式 The cyclic nitroxy radical part of R is optionally connected to a linking group, —CH 2 CH 2 O—, —CH 2 CH 2 S—, —COCH 2 CH 2 O—, —COCH 2 CH 2 S—, — (CH 2 ) 3— O—, — (CH 2 ) 3 —S—, —CO (CH 2 ) 3 —O—, — (CH 2 ) 3 —O—, etc. , 6,6-tetramethylpiperidin-1-oxyl-4-yl, 2,2,5,5-tetramethylpyrrolidin-1-oxyl-3-yl, 2,4,4, -trimethyl-1,3- It can be selected from thiazolidine-3-oxyl-2-yl, 2,4,4-trimethyl-imidazolidin-3-oxy-2-yl, and the like. More specifically, the following formula
（式中、Ｒ’はメチル基である。）
のいずれかで表される環状ニトロキシドラジカル化合物の残基であることができる。
(In the formula, R ′ is a methyl group.)
Or a residue of a cyclic nitroxide radical compound represented by
上記のポリマーは、例えば、下記の反復単位を含むそれ自体公知のポリマーに上記の環状ニトロキシドラジカル部分を導入することにより製造できる。なお、この導入は、下記の反復単位を有するそれ自体公知のポリマーを表面に塗布した後、ニトロキシラジカル部分を導入してもよい。塗布は、それ自体公知のポリマー溶液を用いる塗布等であることができる。
式：
The above polymer can be produced, for example, by introducing the above cyclic nitroxide radical moiety into a polymer known per se containing the following repeating unit. In this introduction, a nitroxy radical moiety may be introduced after a polymer known per se having the following repeating unit is applied to the surface. The application can be an application using a polymer solution known per se.
formula:
（ここで、ｐは１または２を表し、Ｒ１は非結合末端において１個のフェニル基もしくはベンズヒドリル基により置換されていてもよいＣ１−Ｃ１２アルキル基を表し、ｎは３〜１，０００の整数を表す。）で表されるポリアミノ酸エステル鎖セグメント（ｉ）。
式：
(Wherein p represents 1 or 2, R 1 represents a C 1 -C 12 alkyl group optionally substituted by one phenyl group or benzhydryl group at the non-bonding end, and n represents 3-1, A polyamino acid ester chain segment (i) represented by an integer of 000).
formula:
（ここで、Ｒ１は非結合末端において１個のフェニル基もしくはベンズヒドリル基により置換されていてもよいＣ１−Ｃ１２アルキル基を表し、Ｒ２は水素原子またはＣ１−５アルキル基を表し、ｎは３〜１，０００の整数を表す。）で表されるポリ（（メタ）アクリル酸エステル）鎖セグメント（ｉｉ）。
式：
(Here, R 1 represents a C 1 -C 12 alkyl group optionally substituted by one phenyl group or benzhydryl group at the non-bonding end, and R 2 represents a hydrogen atom or a C 1-5 alkyl group. , N represents an integer of 3 to 1,000.) Poly ((meth) acrylic acid ester) chain segment (ii).
formula:
（ここで、ｎは３〜１，０００の整数を表す。）で表されるスチレン−無水マレイン酸共重合体鎖セグメント（ｉｉｉ）。
式：
(Here, n represents an integer of 3 to 1,000). A styrene-maleic anhydride copolymer chain segment (iii).
formula:
（ここで、Ｒ１は非結合末端において１個のフェニル基もしくはベンズヒドリル基により
置換されていてもよいＣ１−Ｃ１２アルキル基を表し、ｎは３〜１，０００の整数を表す。）で表されるポリリンゴ酸エステル鎖セグメント（ｉｖ）。
式：
(Here, R 1 represents a C 1 -C 12 alkyl group which may be substituted with one phenyl group or benzhydryl group at the non-bonding end, and n represents an integer of 3 to 1,000). Polymalate chain segment (iv) represented.
formula:
（ここで、ｎは３〜１，０００の整数を表す。）で表されるポリアミック酸鎖セグメント（ｖ）。
式：
(Here, n represents an integer of 3 to 1,000.) Polyamic acid chain segment (v).
formula:
（ここで、Ｌ１は塩素、臭素またはヨウ素原子を表し、ｎは３〜１，０００の整数を表す。）で表されるポリ（ハロメチルスチレン）鎖セグメント（ｖｉ）。
式：
(Here, L 1 represents a chlorine, bromine or iodine atom, and n represents an integer of 3 to 1,000.) A poly (halomethylstyrene) chain segment (vi).
formula:
（ここで、ｎは３〜１，０００の整数を表す。）で表されるポリ（グリシジル メタクリレート）鎖セグメント（ｖｉｉ）。
式：
(Here, n represents an integer of 3 to 1,000.) Poly (glycidyl methacrylate) chain segment (vii).
formula:
（ここで、ｎは３〜１，０００の整数を表す。）で表されるポリ（２−ヒドロキシエチル
メタクリレート）鎖セグメント（ｖｉｉｉ）。
式：
(Here, n represents an integer of 3 to 1,000). Poly (2-hydroxyethyl methacrylate) chain segment (viii).
formula:
（ここで、ｎは３〜１，０００の整数を表す。）で表されるポリエピクロロヒドリン鎖セグメント（ｉｘ）。
式：
(Here, n represents an integer of 3 to 1,000.) Polyepichlorohydrin chain segment (ix).
formula:
（ここで、ｎは３〜１，０００の整数を表す。）で表されるポリ−３，３−ビスクロロメチルオキセタン鎖セグメント（ｘ）。 (Here, n represents an integer of 3 to 1,000.) A poly-3,3-bischloromethyloxetane chain segment (x) represented by:
環状ニトロキシドラジカルの上記反復単位を含むポリマーへの導入は、例えば、前者のラジカル以外の官能基（例えば、アミノ基、アミノメチル基、ヒドロキシル基、ヒドロキシメチル基、カルキシル基またはカルボメチル基）と上記反復単位における反応性基（ハロゲン原子、カルボキシル基、エステル基、酸無水物基、マレイミド基またはエポキシド基）を介して、それ自体公知の縮合または不可反応等を実施することにより達成できる。 The introduction of the cyclic nitroxide radical into the polymer containing the repeating unit may be performed by, for example, functional groups other than the former radical (for example, amino group, aminomethyl group, hydroxyl group, hydroxymethyl group, carboxyl group or carbomethyl group) and the repeating unit. It can be achieved by carrying out a per se known condensation or non-reaction through a reactive group (halogen atom, carboxyl group, ester group, acid anhydride group, maleimide group or epoxide group) in the unit.
本発明で使用できるポリマーが環状ニトロキシドラジカル部分を担持する反復単位（（ａ）〜（ｐ））以外の反復単位を有するコポリマーである場合、該コポリマーは、上記（ａ）〜（ｐ）で表される反復単位のＲが水素原子を表す反復単位、上記（ｉ）〜（ｘ）、あるいはまた、より具体的には、下記式（ｎは３〜１，０００の整数を表し、ｐは３〜５，０００の整数を表す。）で表される反復単位よりなる群から選ばれる反復単位であることができる。環状ニトロキシドラジカル部分を担持する反復単位（（ａ）〜（ｐ））のいずれかと、それ以外の反復単位は、ランダムに存在するかもしくはブロックを形成して存
在することができるが、好ましくはランダムに存在することができる。
When the polymer that can be used in the present invention is a copolymer having a repeating unit other than the repeating unit ((a) to (p)) carrying a cyclic nitroxide radical moiety, the copolymer is represented by the above (a) to (p). Wherein the repeating unit R represents a hydrogen atom, the above (i) to (x), or more specifically, the following formula (n represents an integer of 3 to 1,000, p is 3 Represents an integer of ˜5,000.) And a repeating unit selected from the group consisting of repeating units represented by: Any of the repeating units ((a) to (p)) carrying the cyclic nitroxide radical moiety and the other repeating units may be present at random or in a block form, but preferably at random. Can exist.
本発明にいう、生体適合性は、上記の定義の範疇内のものであれば、限定されるものでないが、血液適合性に注目している。本発明にいう、かような血液適合性の改質とは、より具体的には、血液と対応する未処理の表面が接触した場合に生じる該表面への血球吸着が上記ポリマーの塗布された表面では抑制され（表面と接触する血液の血小板および／または白血球の減少が抑制される）、または、血液中の活性酸素量の増加が抑制されることを意図している。 The biocompatibility referred to in the present invention is not limited as long as it is within the above definition, but attention is paid to blood compatibility. More specifically, the improvement of blood compatibility referred to in the present invention means that the above-mentioned polymer is applied to adsorb blood cells on the surface when blood and a corresponding untreated surface come into contact with each other. It is intended to be suppressed at the surface (inhibition of blood platelets and / or leukocyte reduction in contact with the surface) or to increase the amount of active oxygen in the blood.
したがって、本発明に従えば、ポリ（クロロメチルスチレン）（ＰＣＭＳ）のような反応性高分子に任意の割合でＴＥＭＰＯを導入し、残りの反応基を介して、例えば、ポリエチレングリコール（ＰＥＧ）等をさらに導入した表面を形成することができる。これにより血液適合性だけでなく、ＲＯＳが関与していることが考えられているコンタクトレンズや培養シャーレなど様々な機器表面の改質が可能である。この残りの反応性基がハロゲン原子である場合、さらには発明者らが先に提案した、特願２００８−２３８１３３に開示したような大気圧プラズマにより該ハロゲン原子を介して対応するポリマーを表面に固定することもできる。 Therefore, according to the present invention, TEMPO is introduced into a reactive polymer such as poly (chloromethylstyrene) (PCMS) at an arbitrary ratio, and the remaining reactive groups are used, for example, polyethylene glycol (PEG) or the like. Can be formed. As a result, not only blood compatibility but also various device surface modifications such as contact lenses and culture dishes that are considered to be involved in ROS are possible. When this remaining reactive group is a halogen atom, the corresponding polymer is introduced to the surface via the halogen atom by atmospheric pressure plasma as disclosed in Japanese Patent Application No. 2008-238133 previously proposed by the inventors. It can also be fixed.
本発明では、安定なニトロキシドラジカルを有するホモポリマー、ランダムコポリマー（ＮＲＰ）の被膜を表面に固定することで血液と材料が接触した際の活性酸素を捕捉することが可能である。また、本発明によれば、上記ポリマーから本質的になり、遊離したＴＥＭＰＯＬやその他のヘモグロビン、アルブミン等を含めることなく形成したコーティングが上記の生体適合性を向上できるが、本発明の目的に悪影響を及ぼさない範囲でアルブミン等を併用してもよい。 In the present invention, it is possible to capture active oxygen when blood and material come into contact with each other by fixing a coating of a homopolymer or random copolymer (NRP) having a stable nitroxide radical on the surface. In addition, according to the present invention, a coating consisting essentially of the above polymer and formed without including free TEMPOL, other hemoglobin, albumin, etc. can improve the above biocompatibility, but adversely affects the purpose of the present invention. Albumin or the like may be used in combination as long as it does not affect the range.
以下、本発明を、具体例を挙げて説明するが、本発明をこれらに限定することを意味しない。 Hereinafter, the present invention will be described with specific examples, but it is not meant to limit the present invention thereto.
製造例１：
（１）ＭＥＡ（２−メトキシメチルアクリレート）／ＣＭＳ（クロロメチルスチレン）のランダムコポリマー（ＰＣＭ）の合成
Production Example 1:
(1) Synthesis of MEA (2-methoxymethyl acrylate) / CMS (chloromethylstyrene) random copolymer (PCM)
反応容器に撹拌子と反応開始剤としてのアゾビスイソブチロニトリル（ＡＩＢＮ）を加え、窒素置換を行った。そこに２０分窒素バブリングを行った１，４−ジオキサン、２−メトキシメチルアクリレート（ＭＥＡ）、クロロメチルスチレン（ＣＭＳ）、ガスクロマトグラフィー（ＧＣ）測定用の内部標準物質デカンを加えた。スターラーで撹拌しながら６５℃に加熱したオイルバスに反応容器を入れ重合を開始した。ＧＣ測定のため１時間毎にサンプルを０．５ｍＬ採取し、氷浴で冷却した後ＧＣ測定を行った。重合の停止はＧＣ測定によるＭＥＡ、ＣＭＳモノマーどちらかの転化率が８割程度になったところで行った。重合反応の停止は反応容器をオイルバスから取り出し、氷浴に浸けることにより行った。生成物は、冷却した大過剰のイソプロピルアルコール（ＩＰＡ）に沈殿させ、デカンテーションを行った。この過程を３回繰り返すことで精製した。沈殿物をナスフラスコに回収し、ＩＰＡをエバポレーターにより蒸発させた後、少量のベンゼンに溶解させ、液体窒素で凍結させ、減圧下で凍結乾燥を行った。ベンゼン凍結乾燥後ＧＰＣ、およびＣＤＣｌ３を用いて１Ｈ ＮＭＲ測定を行った。ＧＰＣ測定に関しては、ＰＳ検量線を用いて分子量を決定した。表１に実際に行った重合の仕込み量を示す。 A stirrer and azobisisobutyronitrile (AIBN) as a reaction initiator were added to the reaction vessel, and nitrogen substitution was performed. 1,4-Dioxane, 2-methoxymethyl acrylate (MEA), chloromethylstyrene (CMS), and internal standard substance decane for gas chromatography (GC) measurement, which were subjected to nitrogen bubbling for 20 minutes, were added thereto. While stirring with a stirrer, the reaction vessel was placed in an oil bath heated to 65 ° C. to initiate polymerization. A 0.5 mL sample was taken every hour for GC measurement, cooled in an ice bath, and then GC measurement was performed. The polymerization was stopped when the conversion rate of either MEA or CMS monomer by GC measurement was about 80%. The polymerization reaction was stopped by removing the reaction vessel from the oil bath and immersing it in an ice bath. The product was precipitated into a cooled large excess of isopropyl alcohol (IPA) and decanted. This process was purified three times. The precipitate was collected in an eggplant flask, IPA was evaporated by an evaporator, dissolved in a small amount of benzene, frozen with liquid nitrogen, and lyophilized under reduced pressure. After lyophilization of benzene, 1 H NMR measurement was performed using GPC and CDCl 3 . For GPC measurement, the molecular weight was determined using a PS calibration curve. Table 1 shows the amount of polymerization actually performed.
得られたＰＣＭの１Ｈ ＮＭＲおよびＰＣ測定結果を表２に示す。組成比については１Ｈ ＮＭＲ測定より求めた値である。 Table 2 shows the 1 H NMR and PC measurement results of the obtained PCM. The composition ratio is a value determined from 1 H NMR measurement.
（２）反応容器に撹拌子とＰＣＭ ２０ｍｇを加え窒素置換を３回行った（容器[1]）。これと並行して別の容器に撹拌子、４−ヒドロキシ−ＴＥＭＰＯ（ＴＥＭＰＯＬ）、ＮａＨ（ＴＥＭＰＯＬに対し２当量）を加え窒素置換を３回行った（容器[2]）。続いて両容器にそれぞれジメチルホルムアミド（ＤＭＦ）２ｍｌを加え、撹拌した。試薬が完全に混ざり合ったところで容器[2]の混合液を容器[1]に加え、一晩撹拌し反応させた。反応後、ＮａＨにより生じた塩を取り除くため反応溶液を吸引濾過した。濾液として得られた反応溶液はＴＥＭＰＯ含有ポリマー（ＮＲＰランダムコポリマー）溶液としてガラスビーズへのポリマーコーティングに用いた。 (2) A stirring bar and 20 mg of PCM were added to the reaction vessel, and nitrogen substitution was performed three times (container [1]). In parallel with this, a stir bar, 4-hydroxy-TEMPO (TEMPOL), and NaH (2 equivalents to TEMPOL) were added to another container, and nitrogen substitution was performed three times (container [2]). Subsequently, 2 ml of dimethylformamide (DMF) was added to both containers and stirred. When the reagents were completely mixed, the mixed solution in the container [2] was added to the container [1] and stirred overnight to react. After the reaction, the reaction solution was filtered with suction in order to remove the salt generated by NaH. The reaction solution obtained as a filtrate was used for polymer coating on glass beads as a TEMPO-containing polymer (NRP random copolymer) solution.
表面処理例１：ＮＲＰのガラスビーズへのコーティング
ガラスビーズへのポリマーのコーティングは溶媒留去法により行った。ＮＲＰランダムコポリマー溶液２ｍＬに７ｇガラスビーズを加え、浸潤させた。このビーズをシャーレに均一に広げデシケータにより真空下完全に乾燥を行った。次にこのポリマーコートビーズ７ｇを１５０ｍＬの蒸留水でソックスレー洗浄した。ＮＲＰ溶液中には、未反応のＴＥＭＰＯＬが存在するためポリマーコーティング後、未反応ＴＥＭＰＯＬを除去するためにソックスレー洗浄を行った。ソックスレー洗浄に関しては１時間毎に抽出液を０．５ｍＬ採取し、ＥＳＲ測定を行った。抽出液のＥＳＲシグナルがプラトーに達した（つまりビーズよりＴＥＭＰＯＬが流れでなくなった）のを確認できたところで洗浄を終了した。洗浄後、ビーズをゆっくりと吸引濾過した。ビーズがほぼ乾燥したら、シャーレへ均一に広げデシケータにより真空下完全に乾燥させた。以上の操作によりＮＲＰコートビーズを得た。
Surface Treatment Example 1: Coating of NRP on Glass Beads Polymer coating on glass beads was performed by a solvent distillation method. 7 g glass beads were added to 2 mL of NRP random copolymer solution and infiltrated. The beads were uniformly spread on a petri dish and completely dried under vacuum using a desiccator. Next, 7 g of this polymer-coated bead was Soxhlet washed with 150 mL of distilled water. Since there was unreacted TEMPOL in the NRP solution, Soxhlet washing was performed after the polymer coating to remove unreacted TEMPOL. For Soxhlet washing, 0.5 mL of the extract was collected every hour and ESR measurement was performed. Washing was terminated when it was confirmed that the ESR signal of the extract reached a plateau (that is, TEMPOL stopped flowing from the beads). After washing, the beads were slowly filtered with suction. When the beads were almost dry, they were spread evenly on a petri dish and completely dried under vacuum using a desiccator. NRP-coated beads were obtained by the above operation.
ソックスレー洗浄の抽出液を採取し、ＥＳＲ測定を行った結果を図１に示す。またＥＳＲ強度を縦軸、洗浄時間を横軸にプロットしたグラフを図２に示す。図２から７時間洗浄したところでＥＳＲ強度はプラトーに達していることが明らかとなった。したがって８時間のソックスレー洗浄でビーズ表面に存在するフリーのＴＥＭＰＯＬは、完全に除去されたことが示された。さらに抽出液のシグナル強度から、ガラスビーズ表面に存在するＴＥＭＰＯ量を求めた。その結果ＴＥＭＰＯはＣＭＳ基に対し１００％近く導入できていることが確認された。 FIG. 1 shows the result of collecting the extract of Soxhlet washing and performing ESR measurement. FIG. 2 is a graph in which the ESR intensity is plotted on the vertical axis and the cleaning time is plotted on the horizontal axis. From FIG. 2, it was revealed that the ESR intensity reached a plateau after 7 hours of washing. Therefore, it was shown that the free TEMPOL present on the bead surface was completely removed by the Soxhlet wash for 8 hours. Furthermore, the amount of TEMPO present on the glass bead surface was determined from the signal intensity of the extract. As a result, it was confirmed that TEMPO was introduced almost 100% with respect to the CMS group.
１００ｍｇのＮＲＰコートビーズを１ｍＬクロロホルムに浸すことでコーティング層を抽出し、ＥＳＲ測定を行った結果を図３に示す。クロロホルム溶液からは、ＥＳＲシグナルが観察されソックスレー洗浄後もガラスビーズ表面にラジカルが存在することを確認した。通常、低分子ＴＥＭＰＯは希薄溶液中において、窒素核と不対電子の相互作用により３本線のスペクトルを示すが、検出されたＥＳＲシグナルは、溶液のラジカル（ＴＥＭＰＯ）濃度が低いにも関わらずブロードな１本線のスペクトルを示した。これは、ＰＣＭＳにＴＥＭＰＯが固定化されたことによってＴＥＭＰＯの運動性が低下し、スペクトルの線幅が増大した結果、スペクトルが３本線から１本線に変化したものと考えられる。したがってＴＥＭＰＯはＰＣＭＳポリマーに結合しており、ガラスビーズ表面にはＮＲＰランダムコポリマーがコートされていることが示唆された。 FIG. 3 shows the results of extracting the coating layer by immersing 100 mg of NRP-coated beads in 1 mL of chloroform and performing ESR measurement. From the chloroform solution, an ESR signal was observed, and it was confirmed that radicals were present on the glass bead surface even after Soxhlet washing. Normally, low-molecular-weight TEMPO shows a three-line spectrum due to the interaction between nitrogen nuclei and unpaired electrons in a dilute solution, but the detected ESR signal is broad despite the low radical (TEMPO) concentration in the solution. A single-line spectrum was shown. This is presumably because the TEMPO mobility was lowered by the TEMPO being immobilized on the PCMS, and the spectrum line width was increased. As a result, the spectrum was changed from three lines to one line. Therefore, it was suggested that TEMPO was bonded to the PCMS polymer, and the NRP random copolymer was coated on the glass bead surface.
試験例１：ＮＲＰランダムコポリマーの全血接触試験
１００ｍｇのポリマーコートビーズを５００μＬチューブへ入れたもの及びコントロールとしてビーズの入っていないチューブに生理食塩水４０μＬを添加。ＳＤラット（オス、５−６週齢）から心臓採血により得たヘパリン濃度５ ＩＵ／ｍＬの血液を、各ポリマーコートビーズが入ったチューブへ４００μＬ分注し、室温にて２０分間混和を行った。混和はロータリーミキサーを用いてチューブが１分間に１回の割合で回転するように行った。所定時間混和後、直ちにｃｅｌｌｔａｃ α（ＮＩＨＯＮ ＫＯＨＤＥＮ）にて血球数を測定した。血球数については、コントロール（容器のみ）の値を１００％とし計算を行った。
Test Example 1: Whole Blood Contact Test of NRP Random Copolymer 40 μL of physiological saline was added to a tube containing 100 mg of polymer-coated beads in a 500 μL tube and a tube without beads as a control. 400 μL of heparin concentration of 5 IU / mL obtained by heart blood sampling from SD rats (male, 5-6 weeks old) was dispensed into tubes containing each polymer-coated bead and mixed at room temperature for 20 minutes. . Mixing was performed using a rotary mixer so that the tube was rotated once per minute. After mixing for a predetermined time, the blood cell count was immediately measured with celltac α (NIHON KOHDEN). The number of blood cells was calculated with the value of the control (container only) being 100%.
図４にビーズと血液を２０分間接触混和させた際の血小板減少率示す。この結果から、ＰＣＭについてはＭＥＡの組成比が増加することで血小板減少が抑制されていくことが明らかとなった。ＴＥＭＰＯの導入効果については、全ての組成比のサンプルについてもＴＥＭＰＯを導入したＰＣＭＴの方がＰＣＭに比べ血小板減少が抑制できていることが図より明らかとなった。またＣＭＳの組成比が大きなポリマーほどＴＥＭＰＯ導入後の血小板減少抑制の効果が大きいことが分かった。これはＣＭＳの組成比が大きなポリマーほどより多くのＴＥＭＰＯが導入されたためだと考えられる。 FIG. 4 shows the platelet reduction rate when the beads and blood are mixed for 20 minutes. From this result, it was clarified that platelet reduction is suppressed by increasing the composition ratio of MEA for PCM. Regarding the effect of introducing TEMPO, it was clear from the figure that PCMT in which TEMPO was introduced was able to suppress thrombocytopenia as compared with PCM in all the composition ratio samples. It was also found that a polymer having a higher composition ratio of CMS has a greater effect of suppressing platelet reduction after introduction of TEMPO. This is considered to be because more TEMPO was introduced into a polymer having a higher composition ratio of CMS.
製造例２：
（１）ポリクロロメチルスチレン（ＰＣＭＳ）の合成
反応容器に撹拌子とＡＩＢＮ（１ｍｍｏｌ、１６４．２ｍｇ）を加えた。次に反応容器中を真空にした後、窒素雰囲気下とした。この操作を３回繰り返すことにより、反応容器内を窒素雰囲気にした。そこに２０分間窒素バブリングを行ったジオキサン（５０ｍＬ）、ＣＭＳ（１００ｍｍｏｌ、１４．２ｍｇ）、ガスクロマトグラフィー（ＧＣ）測定用の内部標準物質ｎ−デカン（３ｍＬ）を加えた。スターラーで撹拌しながら６５℃に熱したオイルバスに反応容器を入れ重合を開始した。ＧＣ測定のため１時間毎にサンプルを０．５ｍＬ採取し、氷浴で冷却した後ＧＣ測定を行った。１８時間攪拌後、反応容器を氷浴につけ、重合反応を停止させた。生成物はメタノールを用いた再沈精製を行った。沈殿物をナスフラスコに回収し、メタノールをエバポレーターにより蒸発させた後、少量のベンゼンに溶解させ、液体窒素で凍結し、減圧下で凍結乾燥を行った。凍結乾燥後、白色の粉末状のポリマーが９．６グラム得られ、収率は６３．２％となった。得られたポリマーはゲル透過クロマトグラフィー（ＧＰＣ）測定およびＣＤＣｌ３を用いて１Ｈ ＮＭＲ測定を行った。ＧＰＣ測定に関しては、ＰＳ検量線を用いて分子量を決定した。ラジカル重合により得られたＰＣＭＳホモポリマーの１Ｈ ＮＭＲ測定結果よりＰＣＭＳホモポリマーが合成されていることが確認された（図５）。またＧＰＣ測定結果より数量平均分子量Ｍｎは１３０００、多分散度Ｍｗ／Ｍｎは２．１と決定された（図６）。
Production Example 2:
(1) Synthesis of polychloromethylstyrene (PCMS) A stirring bar and AIBN (1 mmol, 164.2 mg) were added to a reaction vessel. Next, the reaction vessel was evacuated and then placed in a nitrogen atmosphere. By repeating this operation three times, the inside of the reaction vessel was made a nitrogen atmosphere. Dioxane (50 mL), nitrogen gas bubbled for 20 minutes, CMS (100 mmol, 14.2 mg), and internal standard substance n-decane (3 mL) for gas chromatography (GC) measurement were added thereto. While stirring with a stirrer, the reaction vessel was placed in an oil bath heated to 65 ° C. to initiate polymerization. A 0.5 mL sample was taken every hour for GC measurement, cooled in an ice bath, and then GC measurement was performed. After stirring for 18 hours, the reaction vessel was placed in an ice bath to stop the polymerization reaction. The product was purified by reprecipitation using methanol. The precipitate was collected in an eggplant flask, methanol was evaporated by an evaporator, dissolved in a small amount of benzene, frozen with liquid nitrogen, and lyophilized under reduced pressure. After lyophilization, 9.6 grams of white powdery polymer was obtained, yielding 63.2%. The obtained polymer was subjected to gel permeation chromatography (GPC) measurement and 1 H NMR measurement using CDCl 3 . For GPC measurement, the molecular weight was determined using a PS calibration curve. From the 1 H NMR measurement result of the PCMS homopolymer obtained by radical polymerization, it was confirmed that the PCMS homopolymer was synthesized (FIG. 5). From the GPC measurement results, the number average molecular weight Mn was determined to be 13000 and the polydispersity Mw / Mn was determined to be 2.1 (FIG. 6).
製造例および表面処理例２：ＰＣＭＳ−ＴＥＭＰＯ（ＮＲＰホモポリマー）の合成とコーティング
ＰＣＭＳ−ＴＥＭＰＯは、製造例１の（２）に記載の方法に準じて、以下のスキームにより合成した
Production Example and Surface Treatment Example 2: Synthesis and Coating of PCMS-TEMPO (NRP Homopolymer) PCMS-TEMPO was synthesized according to the following scheme according to the method described in (2) of Production Example 1.
ガラスビーズへのポリマーのコーティングは上記の表面処理例１と同様の方法を用いた。 The glass bead was coated with the polymer in the same manner as in the surface treatment example 1 described above.
ガラスビーズ表面へポリマーがコートされているかを確認するためＸＰＳ測定を行った。その結果を図７に示す。図７よりコーティングされていないガラスビーズに比べＰＣＭＳコートビーズ、ＰＣＭＳ−ＴＥＭＯ（ＮＲＰ）コートビーズのＣ元素ピークが大きいことが確認できる。このことからガラスビーズ表面に確かにポリマーがコートされていることが確認された。またＰＣＭＳコートビーズではＣｌ元素ピークが観察され、目的のポリマーがコート出来ていることが明らかとなった。 XPS measurement was performed to confirm whether the polymer was coated on the glass bead surface. The result is shown in FIG. From FIG. 7, it can be confirmed that the C element peak of the PCMS coated beads and the PCMS-TEMO (NRP) coated beads is larger than the uncoated glass beads. From this, it was confirmed that the polymer was surely coated on the glass bead surface. In addition, a Cl element peak was observed in the PCMS coated beads, which revealed that the target polymer was coated.
さらにＰＣＭＳ−ＴＥＭＰＯ（ＮＲＰ）コートビーズでは、Ｃｌ元素ピークは見られず、ＴＥＭＰＯ由来のＮ元素ピークが現れた。これはＴＥＭＰＯＬとＣＭＳ基が反応し、ポリマーからＣｌ元素が抜け、ＴＥＭＰＯが導入されたことを示す結果である。したがってＴＥＭＰＯはポリマーに結合した状態でガラスビーズ表面に存在していることが明らかとなった。以上の結果よりガラスビーズ表面にＮＲＰがコートされたことが確認された。 Further, in the PCMS-TEMPO (NRP) coated beads, no Cl element peak was observed, and an N element peak derived from TEMPO appeared. This is a result showing that TEMPOL and CMS group reacted, Cl element was removed from the polymer, and TEMPO was introduced. Therefore, it was revealed that TEMPO was present on the glass bead surface in a state of being bonded to the polymer. From the above results, it was confirmed that NRP was coated on the glass bead surface.
各ＴＥＭＰＯ仕込み比のＮＲＰについてもＸＰＳ測定を行い、Ｎ元素とＣｌ元素の比より、ＰＣＭＳへのＴＥＭＰＯの導入率を求めた。その結果を表３にまとめる。 XPS measurement was also performed for NRP at each TEMPO charging ratio, and the introduction rate of TEMPO into PCMS was determined from the ratio of N element to Cl element. The results are summarized in Table 3.
表３より、ＴＥＭＰＯ仕込み比の増加に従いＴＥＭＰＯ導入量も増加していることがわかる。またＥＳＲ測定より求めたＴＥＭＰＯ導入率とＸＰＳ測定より求めた導入率は同等の値を示し、目的のポリマーがガラスビーズにコート出来ていることが明らかとなった。 From Table 3, it can be seen that the amount of TEMPO introduced increases as the TEMPO preparation ratio increases. In addition, the TEMPO introduction rate determined from the ESR measurement and the introduction rate determined from the XPS measurement showed the same value, and it was revealed that the target polymer was coated on the glass beads.
試験例２：ＰＣＭＳ−ＴＥＭＰＯ（ＮＲＰホモポリマー）コートビーズの全血接触試験
１００ｍｇのポリマーコートビーズを５００μＬチューブへ入れたもの及びコントロールとしてビーズの入っていないチューブに生理食塩水４０μＬを添加。ＳＤラット（オス、５−６週齢）から心臓採血により得たヘパリン濃度５ ＩＵ／ｍＬの血液を、各ポリマーコートビーズが入ったチューブへ４００μＬ分注し、室温にて３０分間混和を行った。混和はロータリーミキサーを用いてチューブが１分間に１回の割合で回転するように行った。所定時間混和後、直ちにｃｅｌｌｔａｃ α（ＮＩＨＯＮ ＫＯＨＤＥＮ）にて血球数を測定した。血球数については、コントロール（容器のみ）の値を１００％とし計算を行った。
Test Example 2: Whole blood contact test of PCMS-TEMPO (NRP homopolymer) -coated beads 100 mg of polymer-coated beads was placed in a 500 μL tube, and 40 μL of physiological saline was added to a tube without beads as a control. 400 μL of heparin concentration 5 IU / mL obtained by heart blood sampling from SD rats (male, 5-6 weeks old) was dispensed into tubes containing each polymer-coated bead and mixed at room temperature for 30 minutes. . Mixing was performed using a rotary mixer so that the tube was rotated once per minute. After mixing for a predetermined time, the blood cell count was immediately measured with celltac α (NIHON KOHDEN). The number of blood cells was calculated with the value of the control (container only) being 100%.
ポリマーコートビーズに５ＩＵ／ｍＬ濃度ヘパリン化全血を接触させ、血小板数・白血球数を測定した結果を図８に示す。血小板減少はＰＣＭＳへのＴＥＭＰＯ導入率が増すにつれ、抑制されることが観察された。同様に白血球についてもＴＥＭＰＯ導入率が増すことで減少が抑制されることが観察された。これはビーズ表面に存在するＴＥＭＰＯにより、材料への血球吸着が抑制できることを示唆する結果である（図９）。図９のＳＥＭ画像からもＰＣＭＳ−ＴＥＭＰＯコートビーズは、ＰＣＭＳコートビーズよりも明らかに血球吸着が少なく、ＴＥＭＰＯ導入が表面と生体適合性を向上する効果をしめすことが確認された。 FIG. 8 shows the results of contacting the polymer-coated beads with 5 IU / mL concentration heparinized whole blood and measuring the platelet count and white blood cell count. It was observed that thrombocytopenia was suppressed as the TEMPO introduction rate into PCMS increased. Similarly, it was observed that the decrease in leukocytes was suppressed by increasing the TEMPO introduction rate. This is a result that suggests that TEMPO present on the bead surface can suppress blood cell adsorption to the material (FIG. 9). Also from the SEM image of FIG. 9, it was confirmed that the PCMS-TEMPO coated beads had significantly less blood cell adsorption than the PCMS coated beads, and that the introduction of TEMPO showed the effect of improving the surface and biocompatibility.
試験例３：活性酸素測定
ビーズと血液が接触した時に生じる活性酸素は、化学発光法により測定した。化学発光物質としては、スーパーオキシド特異的に発光を示す試薬ＭＰＥＣを用いた。９６ウェルプレートに各ポリマーコートビー５０ｍｇを加え、そこにエタノールに溶解させた１ｍＭ
ＭＰＥＣ溶液５０μＬを分注した。次に終濃度が１％となるようにＰＢＳ（１ｍＭ ｐＨ７．４）で２％に薄めた血液（５−６週齢ラットより心臓採血で得た全血）を５０μＬ分注し、直ちに発光量をマイクロプレートリーダーにより３０秒のインターバルで測定した。ビーズと血液との接触によって生じた活性酸素産生量を求めるため、ビーズを入れていない血液のみの時に生じる発光量を目的のサンプルの発光量から差し引いてバックグラウンドを補正した。その結果を図１０に示す。図１０より、ＰＣＭＳコートビーズ（Ｔ０−ｂｅａｄｓ）では、血液と接触後、発光量は徐々に増加していき活性酸素が産生されていることが観察された。一方ＰＣＭＳ−ＴＥＭＰＯコートビーズ（Ｔ９４−ｂｅａｄｓ）では、血液を接触後も発光量の増加が見られず、ほとんど活性酸素が産生されていなことが確認された。
Test Example 3: Active Oxygen Measurement Active oxygen generated when the bead and blood contacted was measured by a chemiluminescence method. As the chemiluminescent substance, a reagent MPEC that emits light specifically for superoxide was used. 50 mM of each polymer coated bee was added to a 96 well plate, and 1 mM dissolved in ethanol there.
50 μL of MPEC solution was dispensed. Next, 50 μL of blood diluted to 2% with PBS (1 mM pH 7.4) so that the final concentration is 1% (whole blood obtained by heart blood sampling from 5-6 week-old rats) was dispensed, and the amount of luminescence immediately Was measured at 30 second intervals with a microplate reader. In order to determine the amount of active oxygen produced by the contact between the beads and blood, the background was corrected by subtracting the amount of luminescence generated when only blood without beads was added from the amount of luminescence of the target sample. The result is shown in FIG. From FIG. 10, it was observed that in PCMS coated beads (T0-beads), the amount of luminescence gradually increased after contact with blood and active oxygen was produced. On the other hand, in PCMS-TEMPO coated beads (T94-beads), no increase in the amount of luminescence was observed after contact with blood, and it was confirmed that almost no active oxygen was produced.
次に異なるＴＥＭＰＯ導入率のＮＲＰコートビーズを用いて活性酸素産生量を測定した。９６ウェルプレートに各ＮＲＰコートビーズを加え、そこに１ｍＭ ＭＰＥＣ ５０ｍｌを分注した。次に終濃度が１％になるように二％希釈血液５０μＬを分注し、マイクロプレートリーダーを用いて１分間の発光量積算値を測定した（図１１）。図１１より、ＴＥＭＰＯの導入率が増すほど、発光量は小さくなり活性酸素の産生が抑制されていることが確認された。 Next, the amount of active oxygen produced was measured using NRP-coated beads with different TEMPO introduction rates. Each NRP-coated bead was added to a 96-well plate, and 50 ml of 1 mM MPEC was dispensed therein. Next, 50 μL of 2% diluted blood was dispensed so that the final concentration was 1%, and the integrated amount of luminescence for 1 minute was measured using a microplate reader (FIG. 11). From FIG. 11, it was confirmed that the amount of luminescence decreased and the production of active oxygen was suppressed as the introduction rate of TEMPO increased.
本発明より安定ニトロキシラジカルを含むポリマーで一定の表面を処理することにより、無処理表面が血液と接触した際に生じる可能性のある血液の変性、例えば、活性酸素発生を抑制し血液の活性化を抑制することができる。したがって、本発明は、血液の吸着を防ぐため、血液と直接接触する人工血管や人工臓器などの表面コーティング剤に関連する技術分野の産業で利用できる。 By treating a certain surface with a polymer containing a stable nitroxy radical from the present invention, blood denaturation that may occur when an untreated surface comes into contact with blood, for example, the generation of active oxygen is suppressed. Can be suppressed. Therefore, the present invention can be used in industries in the technical field related to surface coating agents such as artificial blood vessels and artificial organs that are in direct contact with blood in order to prevent blood adsorption.
Claims (7)
下記式で表される、環状ニトロキシドラジカル部分を担持する反復単位よりなる群から選ばれるいずれか１種の単位を、少なくとも、ポリマー主鎖の反復単位の１５％以上含んでなり、他の反復単位が存在する場合には、対応する単位の環状ニトロキシドラジカル部分が水素原子もしくは他の官能基を形成し得る基である反復単位または該反復単位と一緒になってコポリマーを形成しうる反復単位を含む、ポリマーを固定する工程を含んでなる、上記方法：
ｎは、５〜１０，０００の整数を表す。 A method for modifying the biocompatibility of a living body or a surface that comes into contact with a substance derived from a living body, which is selected from the group consisting of repeating units carrying a cyclic nitroxide radical moiety represented by the following formula on the surface: Or a single unit containing at least 15% or more of the repeating units of the polymer main chain, and when other repeating units are present, the cyclic nitroxide radical portion of the corresponding unit is a hydrogen atom or other functional group The method comprising the step of immobilizing a polymer comprising a repeating unit that is a group capable of forming a repeating unit or a repeating unit that can form a copolymer together with the repeating unit:
n represents an integer of 5 to 10,000.
−Ａ−Ｂ−
で表され、かつ、
Ａが、式
で表され、かつ、Ｒが式
Ｂが、式
で表される反復単位よりなる群から選ばれる反復単位
を含んでなるランダムコポリマー。 Formula I:
-A-B-
And
A is the formula
And R is the formula
A random copolymer comprising repeating units selected from the group consisting of repeating units represented by:
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| WO2017094691A1 (en) | 2015-11-30 | 2017-06-08 | 国立大学法人筑波大学 | Nitroxy-radical-containing copolymer having phosphate residue, and use thereof |
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| JP2018113928A (en) * | 2017-01-19 | 2018-07-26 | 国立大学法人 筑波大学 | Contact type materials with biological substance in which surface thereof is modified by biocompatible polymer |
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