JP4691745B2 - Method for coating antibacterial antithrombotic material - Google Patents

Description

（式中、Ｒ¹ 、Ｒ² 、Ｒ³ は炭素数１〜１２のアルキル基、または炭素数６〜１２のアリール基、または炭素数７〜２０のアラルキル基、Ｒ⁴ は炭素数１〜２５のアルキル基で、それぞれ同じでも異なっていてもよい。）
【０００９】
本発明の抗血栓性抗菌性医用材料の必須成分である第４級オニウムは、前記化２の構造を有することを特徴としているが、この第４級ホスホニウムは１種類だけ使用しても、何種類かを同時に使用してもよい。第４級ホスホニウムのリン原子に結合する４つの炭化水素鎖のうち、ひとつは炭素数１〜２５、好ましくは３〜２０、さらに好ましくは６〜２０のアルキル基である。他の３つの炭化水素鎖は、炭素数１〜１２、好ましくは１〜８のアルキル基、または炭素数６〜１２、好ましくは６〜１０のアリール基、または炭素数７〜２０、好ましくは７〜１２のアラルキル基である。
【００１０】
第４級ホスホニウムとしては具体的に、トリブチルラウリルホスホニウム、トリブチルミリスチルホスホニウム、トリブチルセチルホスホニウム、トリブチルステアリルホスホニウム、トリフェニルラウリルホスホニウム、トリフェニルミリスチルホスホニウム、トリフェニルセチルホスホニウム、トリフェニルステアリルホスホニウム、ベンジルジメチルラウリルホスホニウム、ベンジルジメチルミリスチルホスホニウム、ベンジルジメチルセチルホスホニウム、ベンジルジメチルステアリルホスホニウムなどが例示されるが、化１によって示される構造の化合物であれば、これらに限定されない。
【００１１】
本発明の抗血栓性抗菌性医用材料は抗凝血作用を有するムコ多糖類の使用を必須としているが、このムコ多糖類としてはヘパリン、コンドロイチン硫酸、ヒアルロン酸、デルマタン硫酸、ケラタン硫酸等が挙げられ、なかでもヘパリンが特に好ましい。
【００１２】
抗凝血作用を有するムコ多糖類と第４級ホスホニウムとのイオン性複合体を得る方法は特に限定されないが、例えば、ムコ多糖類の弱酸性緩衝液溶液もしくは分散液と、第４級ホスホニウム塩の弱酸性緩衝液溶液もしくは分散液を混合し、得られた沈澱を回収、凍結乾燥する方法などが挙げられる。この際の緩衝液に使用される溶質としては、２−（Ｎ−モルホリノ）エタンスルホン酸、ピペラジン−１，４−ビス（２−エタンスルホン酸）、Ｎ−（２−アセトアミド）−２−アミノエタンスルホン酸、Ｎ，Ｎ−ビス（２−ヒドロキシエチル）−２−アミノエタンスルホン酸、３−（Ｎ−モルホリノ）プロパンスルホン酸、３−（Ｎ−モルホリノ）−２−ヒドロキシプロパンスルホン酸、２−［４−（２−ヒドロキシエチル）−１−ピペラジニル］エタンスルホン酸が好ましく、特に好ましくは２−（Ｎ−モルホリノ）エタンスルホン酸（以下ＭＥＳと略記する）、ピペラジン−１，４−ビス（２−エタンスルホン酸）（以下ＰＩＰＥＳと略記する）、３−（Ｎ−モルホリノ）プロパンスルホン酸（以下ＭＯＰＳと略記する）である。
【００１３】
本発明においては、抗凝血作用を有するムコ多糖類と第４級オニウムのイオン性複合体と、有機高分子材料とから成る組成物をコーティングすることが特徴である。脂溶化ムコ多糖のブレンドによって材料表面が不活性化すると同時に、一部は有機高分子材料から徐放することよって抗血栓性、抗菌性が発揮されるものと考えられる。本発明の抗血栓性抗菌性医用材料では、有機高分子材料と脂溶化ムコ多糖の親和性により脂溶化ムコ多糖の徐放が制御され、長期間の溶出後も非常に優れた抗血栓性を維持することが可能である。さらに、脂溶化剤として機能する第４級オニウムの効果によって、抗血栓性と同時に抗菌性をも材料に導入することが可能である。
【００１４】
脂溶化ムコ多糖を有機高分子材料にブレンドする際の添加量は、特に制限されるものではないが、有機高分子材料１００重量部に対して脂溶化ムコ多糖を好ましくは０．１重量部〜５０重量部、さらに好ましくは１重量部〜３０重量部程度の量で添加するのが推奨される（以下、有機高分子材料１００重量部に対して添加剤１重量部を加えた場合、添加剤添加量は１ｐｈｒと表現する）。
【００１５】
有機高分子材料の組成や、合成方法に関しては特に制限されないが、例えばポリ塩化ビニル（以下ＰＶＣと略記する）や、ポリウレタン、ポリウレタンウレアが利用され得る。特に、親水性セグメントが適度に伸び、しなやかな構造を有するセグメント化ポリエーテルウレタン（ウレア）が好ましい。これは第一に医用材料として要求される加工性、弾性、可撓性等の機械的特性を満足するためであり、第二に抗血栓性、抗菌性の発揮に有利だからである。詳細な機構は明かではないが、重付加反応体の化学構造に柔軟性が有る場合には脂溶化ムコ多糖のモービリティが向上し、より活性を発揮しやすいコンフォメーションを取りながら材料−生体成分界面に滲出するためであろうと考えられる。
【００１６】
本発明は、脂溶化ムコ多糖と有機高分子材料とから成る組成物である抗菌性抗血栓性材料を基材表面にコーティングする方法に関する。この基材となる他の構造体の素材としては特に限定されるものではなく、ポリエーテルウレタン、ポリウレタン、ポリウレタンウレア、ＰＶＣ、ポリ塩化ビニリデン、ポリエステル、ポリプロピレン、ポリエチレン、ポリカーボネート等、従来より使用されている材質、また、将来使用されるであろう材質が広く利用できる。また、既存、および新規の材質からなる血液透析膜、血漿分離膜、吸着剤等の血液処理剤に抗血栓性を付与する目的で導入することも可能である。
【００１７】
本発明の抗菌性抗血栓性材料のコーティング方法は、脂溶化ムコ多糖と有機高分子材料とから成る組成物である抗菌性抗血栓性材料を基材表面にコーティングする際に、該脂溶化ムコ多糖および該有機高分子材料の良溶媒（以下単に良溶媒と略記する）と、該有機高分子材料の非溶媒もしくは貧溶媒（以下単に貧溶媒と略記する）との混合溶媒をコーティング溶媒として用いることが特徴である。
【００１８】
本発明に使用される良溶媒は脂溶化ムコ多糖の原料となる第４級ホスホニウムや有機高分子材料によって適宜選択する必要があるが、有機高分子材料がポリウレタンの場合はテトラヒドロフラン（以下ＴＨＦと略記する）、高分子材料がＰＶＣの場合にはジオキサン、ＴＨＦなどが好ましい。
【００１９】
また貧溶媒は、有機高分子材料を溶解させないものであればすべて使用できるが、脂溶化ムコ多糖の良溶媒であることが好ましく、メタノール、エタノール、アセトンなどが好ましい。
【００２０】
良溶媒と貧溶媒の好ましい混合比は、良溶媒、貧溶媒、有機高分子材料および基材の種類により異なる。例えば、有機高分子材料がポリウレタン、基材がポリウレタンの場合には良溶媒としてＴＨＦ、貧溶媒としてメタノールもしくはアセトンを１０：１〜１：３、好ましくは５：１〜１：２の重量比で混合した溶媒を使用するのが好ましい。貧溶媒添加量がこれより多い場合には良溶媒−貧溶媒の混合溶媒に対する有機高分子材料の溶解性が十分でなくコーティングが困難になってしまい、好ましくない。また、貧溶媒添加量がこれより少ない場合には長期にわたる抗菌性、抗血栓性が得られにくくなってしまう。
【００２１】
コーティング溶媒として良溶媒−貧溶媒の混合溶媒を利用することで長期にわたる抗菌性、抗血栓性が実現される詳細な機構については明らかではないが、コーティング、乾燥のプロセスにおいて、脂溶化ムコ多糖の有機高分子材料中における分布が最適化され、長期にわたって適当量が徐放されやすくなるものと推定される。
【００２２】
コーティング溶液は、まず脂溶化ムコ多糖と有機高分子材料を良溶媒に溶解し、貧溶媒を加えて調製するのが好ましいが、良溶媒と貧溶媒の混合溶媒に脂溶化ムコ多糖と有機高分子材料を溶解してコーティング溶液としてもよい。コーティング溶液中の固形分濃度は、０．０１〜３０重量％、好ましくは０．１〜１５重量％である。
【００２３】
基材へのコーティング方法については特に限定されないが、塗布法、スプレー法、ディップ法等が例示される。
【００２４】
【発明の実施の形態】
このようにして、本発明の抗菌性抗血栓性材料のコーティング方法が実施される。詳細な機構は明かではないが、本発明によってコーティングされた抗菌性抗血栓性材料は生体成分との接触初期段階ではもちろん、接触が長期にわたった後も良好な抗凝血性が維持できる。また、第４級ホスホニウムの効果によって抗血栓性と同時に抗菌性をも導入することができる。
このような利点を活かして、本発明の抗菌性抗血栓性材料のコーティング方法は、各種の医療用器具あるいは機器類に広く適用できる。具体的には、血液透析膜や血漿分離膜、血液中老廃物の吸着用コーティング剤に抗菌性、抗血栓性が付与できる。また、人工肺用の膜素材（血液と酸素の隔壁）や人工心肺におけるシート肺のシート材料、大動脈バルーン、血液バッグ、カテーテル、カニューレ、シャント、血液回路等広範な分野に用いられ得る。また、抗菌性を同時に付与できる特長を利用し、従来生体−材料界面からの感染が問題であったＩＶＨなどに適用することも特に好ましい。
【００２５】
以下、実施例を用いて本発明を説明する。
〈実施例１〉
ヘパリンナトリウム塩１０．００ｇをイオン交換水に溶解させ、全量で１００ｍｌとした。塩化トリ−ｎ−ブチルラウリルホスホニウム（以下ＴＢＬＰ・Ｃｌと略記する）１６．７６ｇをイオン交換水に溶解させ、全量で１６８ｍｌとした。双方の溶液を氷冷下で混合し、そのまま４℃で１５時間静置して懸濁液を得た。この懸濁液を３３００ｒｐｍで遠心沈降させて沈殿を回収し、さらに蒸留水を加え懸濁させた後遠心分離によって沈殿を洗浄する操作を３回繰り返し、その後沈殿を乾燥させてＴＢＬＰ・Ｃｌとヘパリンの複合体（以下ＴＢＬＰ−Ｈｅｐと略記する）を得た。このＴＢＬＰ−Ｈｅｐはベンゼン、ＤＭＦ、ＴＨＦ、クロロホルム等の有機溶媒に可溶であった。
【００２６】
市販脂肪族系ポリウレタン（Ｔｅｃｏｆｌｅｘ（商品名）ＥＧ８０Ａ、以下Ｔｅｃｏと略記する）１４ｇと上記で得たＴＢＬＰ−Ｈｅｐ２．８ｇにＴＨＦ１２７．６８ｇを加えて一様な溶液とした。この溶液にアセトン１９１．５２ｇを加えてコーティング溶液Ａを得た。コーティング溶液Ａは固形分濃度５重量％、良溶媒／貧溶媒重量比２／３、有機高分子材料に対する脂溶化ムコ多糖添加量２０ｐｈｒである。
【００２７】
外径１８Ｇのポリウレタン製チューブを上記で得たコーティング溶液Ａに浸漬して引き揚げ、乾燥させることによってコーティングチューブＡを得た。
【００２８】
コーティングチューブＡの抗菌性を以下の方法で評価した。なお、一連の操作は全て無菌的に行った。
ブロース液（滅菌生理食塩水で５０倍希釈）により、およそ１×１０⁷ 個／ｍｌの濃度とした黄色ブドウ球菌液（以下この菌液を菌原液と呼ぶ）を調製した。この菌原液の濃度は、次のように測定した。菌原液を１０⁴ 倍に希釈した後１００μｌを普通寒天板にまき、２４時間後に形成された緑膿菌のコロニー数を計測した。このコロニー数をＮ個とすると、菌原液の濃度Ｃは
Ｃ＝１０⁴ ×Ｎ／０．１＝１０⁵ ×Ｎ［個／ｍｌ］
と示される。
この菌原液１００μｌをブロース液（滅菌生理食塩液で４０倍希釈）で希釈して全量で４０ｍｌに調製した（以下この液を浸漬原液と呼ぶ）。浸漬原液に、ＥＯＧ滅菌したコーティングチューブＡ４０ｃｍ分を適当な長さに切断して浸漬し、３７℃で２４時間培養した。培養後、浸漬原液を滅菌生理食塩水で１０倍系列で１０⁴ 倍まで希釈した（以下１０ⁿ 倍希釈液と略記する）。それぞれの希釈液１００μｌを普通寒天培地上にき、２４時間後普通寒天板上に形成された黄色ブドウ球菌のコロニー数が３０ないし３００個のプレートについて計測した。計測して得られたコロニー数をＮ_n 個とすると、コーティングチューブＡとの接触後の菌数Ｎ_a は次の式で与えられる。
Ｎa ＝４０×１０ⁿ ×Ｎ_n ／０．１
コーティングチューブＡと接触する前の菌原液の濃度は前記Ｃの通りであり、使用した原液量は１００μｌであるから、コーティングチューブＡ接触前の菌数Ｎ_b は次式で示される。
Ｎ_b ＝１０⁴ Ｎ
浸漬原液４０ｍｌ中での４０ｃｍ分のコーティングチューブとの接触によるＮ_b →Ｎ_a の個数変化を表１に示した。接触によって菌数が減少するということはコーティングチューブの抗菌性が発揮されていることを示す。
【００２９】
コーティングチューブＡを使用しｉｎ ｖｉｖｏで抗血栓性を評価した。実験方法は次の通りである。
ペントバルビタール麻酔下でウサギ（日本白色種、オス、２．５〜３．０ｋｇ）の大腿静脈を剥離して、末梢側を糸で結紮し、糸から２〜３ｃｍのところを血管鉗子でクランプした。結紮部分の中枢側を眼下剪刀で血管径の１／４〜１／３切り、そこから試料であるコーティングチューブを１５ｃｍ、中枢側に向かって挿入した。挿入位置から１ｃｍほどのところで、血管外に出ているコーティングチューブの端部を縫いつけ、コーティングチューブが流されるのを防止した。切開部分を縫合し、抗生物質を投与して、以後試料を取り出すまで２週間にわたって飼育した。
２週間後、ヘパリン加ペントバルビタールで麻酔下、正中切開を施し、腹部大動脈より適当なチューブを用いて脱血してウサギを犠死させた後、コーティングチューブを挿入した部分の血管を切断した。血管を切開してコーティングチューブと血管内部を写真に撮るとともに、目視で観察し５段階評価を行った。結果は表１に示した。
【００３０】
【表１】

【００３１】
表１におけるｉｎ ｖｉｖｏ抗血栓性の５段階評価とは次の通りである。ａ：血小板凝集、血栓生成、フィブリン生成いずれも観察されない。ｂ：フィブリン生成または血小板凝集は見られるが血栓生成は観察されない。ｃ：フィブリン生成または血小板凝集が見られ血栓生成がわずかに観察される。ｄ：フィブリン生成または血小板凝集が見られ血栓生成がかなり観察される。ｅ：フィブリン生成または血小板凝集が見られ大量の血栓生成が観察される。
【００３２】
〈実施例２〉
実施例１で得たコーティングチューブＡをクエン酸加牛血漿に浸漬し、３７℃の振盪恒温槽で２週間にわたって溶出を行った。クエン酸加牛血漿は一日おきに交換した。以下、溶出後のコーティングチューブをコーティングチューブＡ’と呼ぶ。コーティングチューブＡと同様の方法でコーティングチューブＡ’での抗菌性について評価を行った。結果は表１に示した。
【００３３】
〈比較例１〉
Ｔｅｃｏ１４ｇ、実施例１で得た得たＴＢＬＰ−Ｈｅｐ２．８ｇにＴＨＦ３１９．２０ｇを加えて一様な溶液とし、コーティング溶液Ｂを得た。コーティング溶液Ｂは固形分濃度５重量％、良溶媒／貧溶媒重量比１／０、有機高分子材料に対する脂溶化ムコ多糖添加量２０ｐｈｒである。
【００３４】
外径１８Ｇのポリウレタン製チューブを上記で得たコーティング溶液Ｂに浸漬して引き揚げ、乾燥させることによってコーティングチューブＢを得た。コーティングチューブＢは乾燥することで全長の収縮が確認された。
【００３５】
実施例１のコーティングチューブＡと同様の方法でコーティングチューブＢでの抗菌性とｉｎ ｖｉｖｏ抗血栓性について評価を行った。結果は表１に示した。
【００３６】
〈比較例２〉
実施例２と同様の方法でコーティングチューブＢの血漿溶出を行った。溶出後のコーティングチューブをコーティングチューブＢ’と呼ぶ。コーティングチューブＡと同様の方法でコーティングチューブＢ’での抗菌性について評価を行った。結果は表１に示した。
【００３７】
〈比較例３〉
コーティングを行わない外径１８Ｇのポリウレタン製チューブをチューブＣとし、実施例１のコーティングチューブＡと同様の方法でチューブＣでの抗菌性とｉｎ ｖｉｖｏ抗血栓性について評価を行った。結果は表１に示した。
【００３８】
〈比較例４〉
実施例２と同様の方法でチューブＣの血漿溶出を行った。溶出後のチューブをチューブＣ’と呼ぶ。コーティングチューブＡと同様の方法でチューブＣ’での抗菌性について評価を行った。結果は表１に示した。
【００３９】
表１に示した結果からわかるように、本発明の抗菌性抗血栓性材料のコーティング方法で調製したコーティングカテーテルは優れた抗血栓性、抗菌性を示しており、溶出後も性能が維持されている。
コーティング溶媒を良溶媒であるＴＨＦのみにした場合、血漿溶出による抗菌性の低下が見られる。
【００４０】
【発明の効果】
本発明の抗菌性抗血栓性材料のコーティング方法は、優れた抗血栓性、抗菌性を付与することが可能であり、その性能は長期間の溶出操作後も維持される。また、本発明の抗菌性抗血栓性材料のコーティング方法より、既存の構造体に簡便に抗血栓性、抗菌性を付与することができ、医療用材料の抗血栓化、抗菌化を行う方法として優れた適性を有している。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for coating an antibacterial antithrombotic material on a substrate surface.
[0002]
[Prior art]
Artificial materials excellent in processability, elasticity, and flexibility have been widely used as medical materials in recent years, but artificial organs such as artificial kidneys, artificial lungs, auxiliary circulation devices, artificial blood vessels, It is expected that the use of disposable products such as syringes, blood bags, and cardiac catheters will continue to expand. In addition to sufficient mechanical strength and durability, these medical materials are required to have safety against living bodies, in particular, that blood does not clot when in contact with blood, that is, antithrombotic properties.
[0003]
Further, in a medical device that needs to be placed in the body for a long period of time, such as a highly nutrient transfusion catheter (hereinafter abbreviated as IVH catheter), infection from the bio-material interface has been a problem. Bacteria grow on thrombus produced by contact between blood and material, and enter the body to cause infection. Therefore, it is necessary for the material used for such a medical device to have antithrombogenicity and antibacterial properties at the same time. In order to meet the demand for such antibacterial and antithrombotic materials, we have already filed several patents such as JP-A-9-176379, JP-A-9-187501 and JP-A-9-187502.
[0004]
One of the methods conventionally tried to impart antithrombogenic properties to various medical devices is to coat a polymer having antithrombogenic properties. For example, Japanese Patent Publication No. 6-57247 discloses a method of using a good solvent and a non-solvent for a substrate as a coating solvent as a method for preventing exposure of the substrate on the coating surface and coating a thin layer.
[0005]
In the case of coating a polymer on a substrate, it is a conventional general method to use a good solvent for the substrate in order to improve the adhesion between the substrate and the coating polymer. The substrate may be exposed on the coating surface, and it was necessary to increase the thickness of the coating layer to avoid this. Japanese Patent Publication No. 6-57247 solves the above problem by adding a non-solvent of the base material to the coating solution.
[0006]
However, this method has been studied for a technique that avoids dissolution of the base material and the base material is not exposed on the surface even if the coating layer thickness is reduced, and is an organic polymer material that is one of the components of the coating material. The present invention, which improves antithrombogenicity and antibacterial properties by adding a non-solvent or poor solvent, is distinct from the present invention.
[0007]
[Problems to be solved by the invention]
We have already solved the above-mentioned drawbacks of the prior art and have already demonstrated antibacterial and antithrombotic materials that can exhibit antibacterial activity as well as long-term antithrombotic properties in addition to simplicity and versatility. Although several patents have been filed, the present invention has been accomplished as a result of intensive studies on a coating method that can be easily coated on a substrate and can exhibit excellent antibacterial and antithrombotic properties.
[0008]
[Means for Solving the Problems]
The present invention is as follows.
(1) Antibacterial antithrombotic material which is a composition comprising a fat-solubilized mucopolysaccharide comprising an ionic complex of at least one anticoagulant mucopolysaccharide and a quaternary onium, and an organic polymer material Characterized in that a mixed solvent of the fat-solubilized mucopolysaccharide and the good solvent of the organic polymer material and the non-solvent or the poor solvent of the organic polymer material is used as a coating solvent when coating the surface of the substrate. Antibacterial antithrombotic material coating method.
(2) The method for coating an antibacterial antithrombotic material according to the above (1), wherein the at least one mucopolysaccharide having anticoagulant property is heparin.
(3) The method for coating an antibacterial antithrombotic material as described in (1) or (2) above, wherein the quaternary onium is a quaternary phosphonium having a structure of the following formula 2.
[Chemical 2]

Wherein R ¹ , R ² and R ³ are alkyl groups having 1 to 12 carbon atoms, aryl groups having 6 to 12 carbon atoms, or aralkyl groups having 7 to 20 carbon atoms, and R ⁴ is 1 to 25 carbon atoms. And each may be the same or different.)
[0009]
The quaternary onium, which is an essential component of the antithrombotic antibacterial medical material of the present invention, is characterized by having the structure of the chemical formula 2. However, this quaternary phosphonium can be used even if only one kind is used. Different types may be used at the same time. Of the four hydrocarbon chains bonded to the phosphorus atom of the quaternary phosphonium, one is an alkyl group having 1 to 25 carbon atoms, preferably 3 to 20 carbon atoms, more preferably 6 to 20 carbon atoms. The other three hydrocarbon chains are alkyl groups having 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms, or aryl groups having 6 to 12 carbon atoms, preferably 6 to 10 carbon atoms, or 7 to 20 carbon atoms, preferably 7 carbon atoms. ˜12 aralkyl groups.
[0010]
Specific examples of the quaternary phosphonium include tributyl lauryl phosphonium, tributyl myristyl phosphonium, tributyl cetyl phosphonium, tributyl stearyl phosphonium, triphenyl lauryl phosphonium, triphenyl myristyl phosphonium, triphenyl cetyl phosphonium, triphenyl stearyl phosphonium, benzyl dimethyl lauryl phosphonium. , Benzyl dimethyl myristyl phosphonium, benzyl dimethyl cetyl phosphonium, benzyl dimethyl stearyl phosphonium, and the like are exemplified, but the compound is not limited to these as long as the compound has a structure represented by Chemical Formula 1.
[0011]
The antithrombotic antibacterial medical material of the present invention requires the use of a mucopolysaccharide having anticoagulant action, and examples of the mucopolysaccharide include heparin, chondroitin sulfate, hyaluronic acid, dermatan sulfate, and keratan sulfate. Among them, heparin is particularly preferable.
[0012]
A method for obtaining an ionic complex of mucopolysaccharide having anticoagulant action and quaternary phosphonium is not particularly limited. For example, a weakly acidic buffer solution or dispersion of mucopolysaccharide and a quaternary phosphonium salt are available. And a weakly acidic buffer solution or dispersion, and the resulting precipitate is recovered and freeze-dried. Solutes used in the buffer at this time include 2- (N-morpholino) ethanesulfonic acid, piperazine-1,4-bis (2-ethanesulfonic acid), N- (2-acetamido) -2-amino. Ethanesulfonic acid, N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid, 3- (N-morpholino) propanesulfonic acid, 3- (N-morpholino) -2-hydroxypropanesulfonic acid, 2 -[4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid is preferable, and 2- (N-morpholino) ethanesulfonic acid (hereinafter abbreviated as MES), piperazine-1,4-bis ( 2-ethanesulfonic acid) (hereinafter abbreviated as PIPES) and 3- (N-morpholino) propanesulfonic acid (hereinafter abbreviated as MOPS).
[0013]
The present invention is characterized by coating a composition comprising an ionic complex of mucopolysaccharide having anticoagulant action and quaternary onium, and an organic polymer material. The surface of the material is inactivated by the blend of the fat-solubilized mucopolysaccharide, and at the same time, it is considered that a part of the material is gradually released from the organic polymer material to exhibit antithrombotic and antibacterial properties. In the antithrombotic antibacterial medical material of the present invention, the sustained release of the fat-solubilized mucopolysaccharide is controlled by the affinity between the organic polymer material and the fat-solubilized mucopolysaccharide. It is possible to maintain. Furthermore, due to the effect of the quaternary onium functioning as a fat solubilizing agent, it is possible to introduce antithrombotic and antimicrobial properties into the material.
[0014]
The amount of addition of the fat-solubilized mucopolysaccharide to the organic polymer material is not particularly limited, but the fat-solubilized mucopolysaccharide is preferably 0.1 parts by weight to 100 parts by weight of the organic polymer material. It is recommended to add in an amount of about 50 parts by weight, more preferably about 1 part by weight to 30 parts by weight (hereinafter, when 1 part by weight of the additive is added to 100 parts by weight of the organic polymer material, the additive is added). The amount added is expressed as 1 phr).
[0015]
The composition of the organic polymer material and the synthesis method are not particularly limited, and for example, polyvinyl chloride (hereinafter abbreviated as PVC), polyurethane, and polyurethane urea can be used. In particular, a segmented polyether urethane (urea) having a moderately stretched hydrophilic segment and a supple structure is preferred. This is because the mechanical properties such as processability, elasticity and flexibility required as a medical material are satisfied first, and secondly, it is advantageous for exhibiting antithrombogenicity and antibacterial properties. Although the detailed mechanism is not clear, when the chemical structure of the polyaddition reactant is flexible, the lipid-solubilized mucopolysaccharide has improved motility, and the material-biological component has a conformation that is more active. This is thought to be due to exudation at the interface.
[0016]
The present invention relates to a method of coating a substrate surface with an antibacterial antithrombotic material which is a composition comprising a fat-solubilized mucopolysaccharide and an organic polymer material. There are no particular limitations on the material of the other structural bodies that serve as the base material, and polyether urethane, polyurethane, polyurethane urea, PVC, polyvinylidene chloride, polyester, polypropylene, polyethylene, polycarbonate, etc. have been used conventionally. The materials that are used and the materials that will be used in the future are widely available. Further, it can be introduced for the purpose of imparting antithrombogenicity to blood treatment agents such as hemodialysis membranes, plasma separation membranes, adsorbents and the like made of existing and novel materials.
[0017]
The coating method of the antibacterial antithrombotic material of the present invention is carried out by coating the antibacterial antithrombotic material, which is a composition comprising a fat solubilized mucopolysaccharide and an organic polymer material, on the substrate surface. A mixed solvent of a polysaccharide and a good solvent for the organic polymer material (hereinafter simply referred to as a good solvent) and a non-solvent or a poor solvent for the organic polymer material (hereinafter simply referred to as a poor solvent) is used as a coating solvent. It is a feature.
[0018]
The good solvent used in the present invention needs to be appropriately selected depending on the quaternary phosphonium or organic polymer material used as a raw material for the fat-solubilized mucopolysaccharide, but when the organic polymer material is polyurethane, tetrahydrofuran (hereinafter abbreviated as THF). In the case where the polymer material is PVC, dioxane, THF and the like are preferable.
[0019]
Any poor solvent may be used as long as it does not dissolve the organic polymer material, but a good solvent for the fat-solubilized mucopolysaccharide is preferable, and methanol, ethanol, acetone, and the like are preferable.
[0020]
A preferable mixing ratio of the good solvent and the poor solvent varies depending on the types of the good solvent, the poor solvent, the organic polymer material, and the substrate. For example, when the organic polymer material is polyurethane and the substrate is polyurethane, THF as a good solvent and methanol or acetone as a poor solvent in a weight ratio of 10: 1 to 1: 3, preferably 5: 1 to 1: 2. It is preferred to use a mixed solvent. When the addition amount of the poor solvent is larger than this, the solubility of the organic polymer material in the good solvent-poor solvent mixed solvent is not sufficient, and coating becomes difficult, which is not preferable. In addition, when the amount of the poor solvent added is less than this, it is difficult to obtain antibacterial and antithrombogenic properties over a long period of time.
[0021]
It is not clear about the detailed mechanism by which antibacterial and antithrombotic properties are realized for a long time by using a mixed solvent of good solvent and poor solvent as a coating solvent, but in the process of coating and drying, the fat-solubilized mucopolysaccharide It is presumed that the distribution in the organic polymer material is optimized and an appropriate amount is easily released over a long period of time.
[0022]
The coating solution is preferably prepared by first dissolving the fat-solubilized mucopolysaccharide and the organic polymer material in a good solvent and adding a poor solvent, but the fat-solubilized mucopolysaccharide and the organic polymer are mixed in a mixed solvent of the good solvent and the poor solvent. The material may be dissolved to form a coating solution. The solid content concentration in the coating solution is 0.01 to 30% by weight, preferably 0.1 to 15% by weight.
[0023]
Although it does not specifically limit about the coating method to a base material, The application | coating method, the spray method, the dip method etc. are illustrated.
[0024]
DETAILED DESCRIPTION OF THE INVENTION
In this manner, the antibacterial antithrombotic material coating method of the present invention is carried out. Although the detailed mechanism is not clear, the antibacterial antithrombotic material coated according to the present invention can maintain a good anticoagulant property not only at the initial stage of contact with a biological component but also after prolonged contact. In addition, anti-thrombotic and antibacterial properties can be introduced by the effect of quaternary phosphonium.
Taking advantage of such advantages, the coating method of the antibacterial antithrombotic material of the present invention can be widely applied to various medical instruments or devices. Specifically, antibacterial and antithrombotic properties can be imparted to hemodialysis membranes, plasma separation membranes, and coating agents for adsorbing blood waste products. Further, it can be used in a wide range of fields such as artificial lung membrane materials (blood and oxygen partition walls), artificial lung lung sheet material, aortic balloons, blood bags, catheters, cannulas, shunts, blood circuits, and the like. In addition, it is particularly preferable to apply to IVH or the like, in which infection from the bio-material interface has been a problem, utilizing the feature that can impart antibacterial properties at the same time.
[0025]
Hereinafter, the present invention will be described using examples.
<Example 1>
Heparin sodium salt (10.00 g) was dissolved in ion exchange water to make a total volume of 100 ml. 16.76 g of tri-n-butyllaurylphosphonium chloride (hereinafter abbreviated as TBLP · Cl) was dissolved in ion-exchanged water to make a total amount of 168 ml. Both solutions were mixed under ice cooling and allowed to stand at 4 ° C. for 15 hours to obtain a suspension. The suspension was centrifuged at 3300 rpm to recover the precipitate. Further, the operation of suspending by adding distilled water and washing the precipitate by centrifugation was repeated three times. Thereafter, the precipitate was dried, and TBLP · Cl and heparin were dried. To obtain a composite (hereinafter abbreviated as TBLP-Hep). This TBLP-Hep was soluble in organic solvents such as benzene, DMF, THF, chloroform and the like.
[0026]
147.6 g of commercially available aliphatic polyurethane (Tecoflex (trade name) EG80A, hereinafter abbreviated as Teco) and 2.8 g of TBLP-Hep obtained above were added with 127.68 g of THF to obtain a uniform solution. 191.52 g of acetone was added to this solution to obtain a coating solution A. The coating solution A has a solid content concentration of 5% by weight, a good solvent / poor solvent weight ratio of 2/3, and an addition amount of fat-solubilized mucopolysaccharide to the organic polymer material of 20 phr.
[0027]
A polyurethane tube having an outer diameter of 18G was dipped in the coating solution A obtained above, pulled up, and dried to obtain a coating tube A.
[0028]
The antibacterial properties of the coated tube A were evaluated by the following methods. All the series of operations were performed aseptically.
A staphylococcus aureus solution (hereinafter referred to as a bacterial stock solution) having a concentration of about 1 × 10 ⁷ cells / ml was prepared with a broth solution (diluted 50 times with sterile physiological saline). The concentration of this bacterial stock solution was measured as follows. After diluting the bacterial stock solution 10 ⁴ times, 100 μl was spread on a normal agar plate, and the number of colonies of Pseudomonas aeruginosa formed 24 hours later was counted. When the number of colonies is N, the concentration C of the bacterial stock solution is C = 10 ⁴ × N / 0.1 = 10 ⁵ × N [cells / ml]
It is indicated.
100 μl of this bacterial stock solution was diluted with a broth solution (diluted 40-fold with sterilized physiological saline) to prepare a total volume of 40 ml (hereinafter, this solution is referred to as a soaking stock solution). A 40 cm portion of EOG-sterilized coating tube A was cut into an appropriate length and immersed in the soaking stock solution, and cultured at 37 ° C. for 24 hours. After culturing, the stock solution was diluted to 10 ⁴ times in a 10-fold series with sterile physiological saline (hereinafter abbreviated as 10 ^n- fold diluted solution). 100 μl of each diluted solution was put on a normal agar medium, and after 24 hours, the number of S. aureus colonies formed on the normal agar plate was counted from 30 to 300. When the number of colonies obtained by measuring the N _n pieces, the number of bacteria N _a after contact with the coating tube A is given by the following equation.
Na = 40 × 10 ⁿ × N _n /0.1
The concentration of the bacterial stock solution before contacting the coating tube A is as described in C above, and the amount of the stock solution used is 100 μl. Therefore, the bacterial count N _b before contacting the coating tube A is expressed by the following equation.
N _b = 10 ⁴ N
Table 1 shows the change in the number of N _b → N _a due to contact with the coating tube for 40 cm in the immersion stock solution. The decrease in the number of bacteria by contact indicates that the antibacterial property of the coating tube is exerted.
[0029]
Coated tube A was used to evaluate antithrombogenicity in vivo. The experimental method is as follows.
Under anesthesia with pentobarbital, the femoral vein of a rabbit (Japanese white species, male, 2.5 to 3.0 kg) was peeled off, the distal side was ligated with a thread, and the place 2 to 3 cm from the thread was clamped with vascular forceps . The central side of the ligated part was cut by 1/4 to 1/3 of the blood vessel diameter with a lower eye knife, and a coating tube as a sample was inserted 15 cm toward the central side. At about 1 cm from the insertion position, the end of the coating tube that had come out of the blood vessel was sewed to prevent the coating tube from flowing away. The incision was sutured, antibiotics were administered, and the animals were raised for 2 weeks until the samples were removed.
Two weeks later, a midline incision was made under anesthesia with heparin-added pentobarbital, blood was removed from the abdominal aorta using a suitable tube, and the rabbit was sacrificed, and then the blood vessel in the portion where the coating tube was inserted was cut. The blood vessel was incised, and the coating tube and the inside of the blood vessel were photographed and visually observed to make a five-step evaluation. The results are shown in Table 1.
[0030]
[Table 1]

[0031]
The in vivo antithrombogenicity 5-grade evaluation in Table 1 is as follows. a: Platelet aggregation, thrombus formation, and fibrin formation are not observed. b: Fibrin formation or platelet aggregation is observed, but thrombus formation is not observed. c: Fibrin formation or platelet aggregation is observed, and thrombus formation is slightly observed. d: Fibrin formation or platelet aggregation is observed, and thrombus formation is considerably observed. e: Fibrin formation or platelet aggregation is observed, and massive thrombus formation is observed.
[0032]
<Example 2>
The coated tube A obtained in Example 1 was immersed in citrated cow plasma and eluted for 2 weeks in a 37 ° C. shaking thermostat. The citrated cow plasma was changed every other day. Hereinafter, the coating tube after elution is referred to as coating tube A ′. The antibacterial properties of the coating tube A ′ were evaluated in the same manner as the coating tube A. The results are shown in Table 1.
[0033]
<Comparative example 1>
A coating solution B was obtained by adding 319.20 g of THF to 14 g of Teco and 2.8 g of TBLP-Hep obtained in Example 1 to obtain a uniform solution. The coating solution B has a solid content concentration of 5% by weight, a good solvent / poor solvent weight ratio of 1/0, and an addition amount of fat-solubilized mucopolysaccharide to the organic polymer material of 20 phr.
[0034]
A polyurethane tube having an outer diameter of 18G was dipped in the coating solution B obtained above, pulled up, and dried to obtain a coating tube B. The coating tube B was confirmed to be fully contracted by drying.
[0035]
The antibacterial properties and in vivo antithrombotic properties of the coating tube B were evaluated in the same manner as the coating tube A of Example 1. The results are shown in Table 1.
[0036]
<Comparative example 2>
Plasma elution of the coating tube B was performed in the same manner as in Example 2. The coating tube after elution is called coating tube B ′. The antibacterial properties of the coating tube B ′ were evaluated in the same manner as the coating tube A. The results are shown in Table 1.
[0037]
<Comparative Example 3>
The tube made of polyurethane having an outer diameter of 18G without coating was designated as tube C, and the antibacterial activity and in vivo antithrombogenicity of tube C were evaluated in the same manner as the coating tube A of Example 1. The results are shown in Table 1.
[0038]
<Comparative example 4>
Plasma elution of tube C was performed in the same manner as in Example 2. The tube after elution is called tube C ′. The antibacterial properties of the tube C ′ were evaluated in the same manner as the coating tube A. The results are shown in Table 1.
[0039]
As can be seen from the results shown in Table 1, the coating catheter prepared by the coating method of the antibacterial and antithrombotic material of the present invention exhibits excellent antithrombotic and antibacterial properties, and the performance is maintained after elution. Yes.
When only the good solvent, THF, is used as the coating solvent, a decrease in antibacterial properties due to plasma elution is observed.
[0040]
【The invention's effect】
The antibacterial antithrombotic material coating method of the present invention can provide excellent antithrombotic and antibacterial properties, and the performance is maintained even after a long-term elution operation. In addition, according to the coating method of the antibacterial antithrombotic material of the present invention, an antithrombotic and antibacterial property can be easily imparted to an existing structure. Has excellent aptitude.

Claims (3)

Coating the surface of the substrate with an antibacterial antithrombotic material, which is a composition comprising a fat-solubilized mucopolysaccharide comprising an ionic complex of at least one kind of anticoagulant mucopolysaccharide and quaternary onium, and polyurethane when the coating method of the antibacterial antithrombogenic material characterized by using a mixed solvent weight ratio of Te tetrahydrofuran / acetone consisting of 5 / 1-1 / 2 as the coating solvent. The method for coating an antibacterial antithrombotic material according to claim 1, wherein the at least one anticoagulant mucopolysaccharide is heparin. The method for coating an antibacterial and antithrombotic material according to claim 1 or 2, wherein the quaternary onium is a quaternary phosphonium having a structure of Chemical Formula 1 below.

Wherein R ¹ , R ² and R ³ are alkyl groups having 1 to 12 carbon atoms, aryl groups having 6 to 12 carbon atoms, or aralkyl groups having 7 to 20 carbon atoms, and R ⁴ is 1 to 25 carbon atoms. And each may be the same or different.)