JP2004209423A - Polysulfone membrane having surface modified with dna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a polysulfone membrane which has improved hemocompatibility, and requires no injection of an antiflocculant by modifying the polysulfone membrane with DNA, and making the membrane hydrophilic. <P>SOLUTION: In the polysulfone membrane whose surface is made hydrophilic by fixing DNA to the surface and a method for producing the same, the hydrophilic DNA-fixed polysulfone membrane can be produced under simple and mild conditions where an untreated polysulfone membrane is dipped into a DNA solution, is thereafter taken out, is subjected to drying and ultraviolet radiation, and is sufficiently cleaned with water. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００１１】
ポリスルホン膜表面に不溶化ＤＮＡを固定するには、上述のように得られたポリスルホン膜を濃度として１０〜４０ｍｇ／ｍｌ程度、好ましくは２０ｍｇ／ｍｌの水性ＤＮＡ溶液に１２時間〜３０時間程度、好ましくは２４時間程度浸漬し、これを室温で乾燥後、例えば２５４ｎｍの紫外線で２時間程度照射後、水で洗浄することによって行われる。また、固定されたＤＮＡは中性ないし塩基性条件下においてはポリスルホン膜表面からの溶出量は少なく、安定である。得られたＤＮＡを固定したポリスルホン膜表面は十分な親水性をもち、血栓形成の原因となるタンパク質吸着量も減少するので、ポリスルホン膜の血液適合性を向上させる。
本発明の表面をＤＮＡで修飾したポリスルホン膜の形状は様々な形状が可能であり、例えば、通常の血液透析膜のような中空糸状の他、フィルム状、チューブ状などが挙げられる。さらにいえば、該ポリスルホン膜表面におけるＤＮＡの固定は膜が有する二つの面の両方でもよいが、いずれか一方についても行うことができる。
本発明はまた、これらの様々な形態および形状をもつ、本発明に係る膜表面にＤＮＡを固定したポリスルホン膜を使用した医療器具のための濾過膜、特に中空糸膜としての血液透析膜にも関する。
【００１２】
【実施例】
以下に示す実施例により、本発明をより詳細に説明する。これらの実施例は本発明の例示であり、本発明を限定することを目的とするものではない。
［主な使用試薬］
これらの材料は全て精製せずにそのまま使用した。
１．ポリスルホン膜製造に用いたポリスルホン（ＰＳｆ）：数平均分子量Ｍｎ約２６０００， Ａｌｄｒｉｃｈ Ｃｈｅｍｉｃａｌ Ｃｏｍｐａｎｙ Ｉｎｃ．製。
２．溶媒
１）Ｎ，Ｎ−ジメチルホルムアミド（ＤＭＦ）：化学試薬等級品，関東化学株式会社製
２）Ｎ−メチル−２−ピロリドン（ＮＭＰ）：ＨＰＬＣ用等級品、９９＋％， Ａｌｄｒｉｃｈ Ｃｈｅｍｉｃａｌ Ｃｏｍｐａｎｙ Ｉｎｃ．製
３．ＤＮＡ（ＤＮＡ−Ｎａ）：一本鎖ＤＮＡ、サルデ薬品株式会社製
４．牛血清アルブミン（ＢＳＡ）：ＲＩＡ等級品、Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．製
５．ドデシル硫酸ナトリウム（ＳＤＳ）：化学試薬等級品、和光純薬工業株式会社製
【００１３】
［例］ポリスルホン膜の製造
ポリスルホン膜は転相法（ｐｈａｓｅ ｉｎｖｅｒｓｉｏｎ）により製造した。ポリスルホンをＤＭＦに溶解し、１５重量％のポリスルホン溶液を得た。次いでこのポリマー溶液をガラスプレートにキャストしそして２５℃で均一な５００μｍ厚さ広げ、膜を製造した。
本実施例では、転相法を変更することにより４種類のポリスルホン膜を製造した。膜１（Ｍ１）は真空炉中５０℃で１日間ＤＭＦを蒸発させることにより製造した。膜２（Ｍ２）、膜３（Ｍ３）および膜４（Ｍ４）は全て１日間室温中で溶媒溶液の入った沈殿浴中に上記キャスティング溶液（２５℃で１５分蒸発後）を浸漬することにより製造した。膜Ｍ２、Ｍ３およびＭ４それぞれに対する沈殿溶媒溶液は水、アセトン、ならびにＤＭＦ溶液（ＤＭＦ／Ｈ_２Ｏ（体積／体積）＝１：２）を使用した。本実施例には脱イオン蒸留水を使用した。アセトンは化学試薬等級品のもので、さらに精製はしないで使用した。
蒸発（Ｍ１）および沈殿（Ｍ２〜Ｍ４）が完了した後、各膜をガラスプレートから取り外し、次いで８０℃で１時間、脱イオン水中でアニーリング及び抽出を行った。次に各膜を０．１Ｍ水酸化ナトリウム（ＮａＯＨ）溶液１００ｍｌに、次いで０．０１Ｍ塩酸（ＨＣｌ）１００ｍｌにそれぞれ３０分間浸漬し、そして次に蒸留水ですすぎ（１００ｍｌ×５回）そして使用するまで水中で保持した。
それぞれ得られた４種類の膜の厚みはミクロメーターで直接測定した。孔隙率はポリマー密度および乾燥前後の変化重量から下記式を使用して計算した。

Ｗ_Ｂ：乾燥前の膜重量（ｇ）
Ｗ_Ａ：乾燥後の膜重量（ｇ）
ρ_Ｗ：水の密度、ρ_Ｗ＝１．０ｇ／ｃｍ^３
ρ_Ｐ：ポリスルホンの密度、ρ_Ｐ＝１．２４ｇ／ｃｍ^３
【００１４】
さらに製法条件の異なるこれらの膜試料の形態を観察するため、走査型電子顕微鏡（ＳＥＭ）で観察した。おのおのの膜試料を試料液体窒素で破壊し試料支持体上に取り付けおよび金層でコートした。ＳＥＭ画像はＳ−２５００Ｃ顕微鏡（電圧＝６ｋＶ、倍率３００倍、日立製作所製）を使用して記録した。
【００１５】
異なる製造法で製造されたそれぞれの膜には顕著な違いが見られた。顕微鏡的には溶媒（ＤＭＦ）蒸発法により製造された膜Ｍ１は透明で薄く、そしてポリマー溶液を沈殿浴中で浸漬することによって製造したＭ２，Ｍ３およびＭ４は乳白色を呈した。膜の構造もまた異なる製造方法で変化した。Ｍ１はち密でなめらかであるが、Ｍ２，Ｍ３およびＭ４は多孔質であった。異なる方法により得られたポリスルホン膜の厚みおよび孔隙率を表１に示す。
【表１】

厚みおよび孔隙率の順番はＭ２，Ｍ４，Ｍ３，およびＭ１であった。Ｍ１は非常に薄く約０．０７４ｍｍであり、非常に低い孔隙率であり、すなわち膜中に殆ど孔はなかった。一方膜Ｍ２，Ｍ３およびＭ４は厚くそして孔隙率も大きかった。なかでもＭ２の厚みおよび孔隙率はこれらの膜中でもっとも大きく、それぞれ６８．４％および０．３１２ｍｍであった。
【００１６】
図１にポリスルホン膜の横断面（図１中、左列）と上面（図１中、右列）のＳＥＭ写真を示す。得られたポリスルホン膜の表面および本質的な構造も膜の形態に示されるように、膜Ｍ１，Ｍ２，Ｍ３およびＭ４間で異なるものであった。これらの写真から、各膜の表面および構造を理解しおよび、後述する固定化ＤＮＡおよびタンパク質吸着の量における膜の形態の効果を理解することは容易である。
Ｍ１の横断面および上面は密で、孔はみられなかった。Ｍ２およびＭ４ではスポンジ状構造が見いだされ、上面に多くの孔が見いだされた。Ｍ３の表面には窪みが見いだされた。
以上のとおり、異なる方法により製造された膜の形態は異なるものであった（表１および図１）。これらの違いは膜形成速度により説明しうる。初期の膜が水に浸漬される場合、表皮層は表面で速やかに形成され、ついで溶媒と非溶媒との交換が起こり、その結果孔隙率および厚みは非常に大きくなる。Ｍ４については、ＤＭＦを含む溶媒系の添加により、表皮層形成がＭ２よりもよりゆっくりである。Ｍ３およびＭ１については、表皮層の形成は非常に起こりにくく、その結果孔隙率および厚みの程度は非常に小さい。さらに、アセトンに浸漬することにより形成した膜Ｍ３は他とは異なり、初期の膜は特別なホルダーを使用しない場合には収縮した。
【００１７】
実施例１：ポリスルホン膜表面への不溶化ＤＮＡの固定
上記例により得られたＭ１〜Ｍ４の未処理のポリスルホン膜（２×２ｃｍ^２）を水性ＤＮＡ溶液（水中、２０ｍｇ／ｍｌＤＮＡ）中に２４時間浸漬した。室温で乾燥後、膜を紫外線（Ｒ−５２Ｇ、米国、Ｕｌｔｒａ ｖｉｏｌｅｔ Ｉｎｃ． 社製）で各々の表面につき２５４ｎｍにて２時間照射した。紫外線強度は試料の位置において５６００μＷ／ｃｍ^２とした。紫外線処理膜を水中に浸漬しそしてヤマトウォーターバスインキュベータモデル１３７−２５により４８時間１２０回／分で振とうし、水を数回交換し、次いで蒸留水（１００ｍｌ×５回）で洗浄した。これを膜の使用時まで、水中で保存した。
【００１８】
以下、ＤＮＡを固定したポリスルホン膜の性能を評価する。
Ｉ．測定法
（１）固定されたＤＮＡの濃度の測定
ポリスルホン膜表面に適用され、固定されたＤＮＡの量は以下の方法により測定した：ＤＮＡを固定した膜を１Ｍ ＨＣｌ溶液で１００℃で１時間加水分解した。溶液中のＤＮＡの量はＵＶ−ＶＩＳ分光光度計Ｕ−２００Ａ（（株）日立製作所製）２６０ｎｍの吸光度により定量した。
（２）接触角の測定
上述のＭ１〜Ｍ４よりなる異なる転相法で製造されたポリスルホン膜の表面およびＤＮＡを固定したポリスルホン膜の表面の水接触角測定を行った。
表面の湿潤性、即ち親水性の指標となる水接触角は２５℃で、接触角計ＣＡ−Ｄ（協和界面科学（株）製）を使用してセサイルドロップ（ｓｅｓｓｉｌｅ ｄｒｏｐ）法により測定した。蒸留水の液滴を膜表面に置き、接触角の直接顕微鏡測定をゴニオメーターにて行った。
角度は、膜の２表面における接触角を測定し、２表面における８−１０滴の測定値の平均をデータとした。
（３）タンパク質の吸着量の測定
おのおのの膜へのタンパク質吸着性をリン酸緩衝溶液（ＰＢＳ、ｐＨ＝７．４）中１０ｇ／ｌ濃度の牛血清アルブミン（ＢＳＡ）を用いて測定した。
ＤＮＡを固定していないポリスルホン膜をＢＳＡ溶液に２４時間、浸漬した。タンパク質を吸着させた膜をＰＢＳで注意して洗浄し、および蒸留水ですすいだ。吸着されたタンパク質を１６時間４．０ｍｌＳＤＳ溶液（２重量％）で定量的に溶出する。ＳＤＳ溶液中のＢＳＡの量をＵＶ−ＶＩＳ分光光度計Ｕ−２００Ａ（（株）日立製作所製）を使用して２７９ｎｍにおける吸光度により定量し、膜表面に吸着されたＢＳＡを計算する。
ＤＮＡを表面に固定した膜もまた２４時間ＢＳＡ溶液（１０ｇ／ｌ）に浸漬した。ここでＳＤＳ溶液が吸着されたタンパク質を膜から除去するために使用された場合、ＤＮＡの一部はＳＤＳ溶液に溶出する。そのためＢＳＡの量を測定する場合、波長２６０ｎｍ、２７０ｎｍおよび２８０ｎｍを使用することによるＵＶ−ＶＩＳ分光光度計Ｕ−２００Ａの多成分決定機能が使用された。
（４） ＢＳＡ吸着膜の赤外分光分析
ＢＳＡの立体配座変化を調べるため、フーリエ変換赤外分光装置ＦＴ−２１０（４ｃｍ^−１分解能で１２０スキャン、２５±２℃、（株）堀場製作所製）によってＢＳＡ吸着膜の赤外分光スペクトルを測定した。ポリマー溶液をキャスティングすることによって、ポリスルホンの薄い高密度のフィルムをセレン化亜鉛（ＺｎＳｅ）プレート（１０ｍｍ幅、５０ｍｍ長）上にコートした。このポリマー溶液濃度はＮＭＰ溶液中１％（重量／体積）であった。使用前にコーティングを真空炉中４０℃で１２時間乾燥させ、室温にてＰＢＳですすいだ。ＤＮＡによる修飾のために、２％ＤＮＡ溶液２滴をコーティング上に加え、室温で乾燥し、紫外線で照射し、ついで水とＰＢＳ溶液ですすいだ。バックグラウンドスペクトルは結晶とポリマーから得た。吸着性の試験のために、ＢＳＡ溶液（ＰＢＳ溶液中１０ｇ／ｌ）１滴をコーティング上に加え、室温にて３０分チャンバー中に貯蔵し、次にＢＳＡ溶液を注意深く取り除き、そしてＰＢＳ溶液で注意深くすすぎ、室温で乾燥後にスペクトルを測定した。
【００１９】
ＩＩ 測定結果
Ａ． 固定されたＤＮＡの量に関するＤＮＡ濃度の効果
膜表面に固定されたＤＮＡの量と空気−水（ａｉｒ−ｗａｔｅｒ）接触角におけるＤＮＡ濃度の効果をＭ２について試験した。図２はポリスルホン膜表面に固定されたＤＮＡの量および接触角と、ＤＮＡ処理溶液濃度との関係を示すグラフである。図２に示すように膜（１６時間浸漬）を処理するのに使用したＤＮＡ溶液の濃度が増加するにつれ、膜表面に固定されるＤＮＡの量は増加し、そしてＤＮＡ固定化ポリスルホン膜表面に対する接触角は減少した。固定されるＤＮＡの量はＤＮＡ濃度に非常に影響を受け、そして２０ｍｇ／ｍｌまではＤＮＡ濃度の増加とともに増加がみられた。接触角の減少は、ＤＮＡ溶液の濃度が１５ｍｇ／ｍｌを越えるとき、顕著な違いは示さなかった。
空気−水接触角およびポリスルホン膜表面に固定されたＤＮＡ量に関するＤＮＡ溶液（２０ｍｇ／ｍｌ）中に浸漬されるポリスルホン膜に対する浸漬時間の効果はＤＮＡ濃度の効果と同様の傾向を示した（データは示さず）。開始時には速やかに接触角は減少しおよび膜表面に固定されるＤＮＡの量は増加したが、１２時間後には変化は見られなかった。
【００２０】
Ｂ． 膜表面に固定されたＤＮＡの安定性
膜表面に固定されたＤＮＡの安定性をＨ_２Ｏ、ＮａＣｌ溶液、ＳＤＳ溶液およびＨＣｌ溶液を含む種々の溶液を使用して試験し、ＤＮＡを固定した膜表面から溶出したＤＮＡの量を測定した（図３ａは膜Ｍ１についてのデータ；図３ｂはＭ２についてのデータに基づく）。Ｍ１およびＭ２についてはＤＮＡを固定した膜は水およびＮａＣｌ溶液（０．９重量％）中では安定であり、９６時間インキュベート後も層から溶出したのは当初固定化されたＤＮＡの１０％以下であった。しかし酸性条件下でインキュベートした場合には大量のＤＮＡが溶液に溶出し、そして当初固定化されたＤＮＡの約８０％が溶出した。
図３ａおよび図３ｂを比較すると、膜表面に固定されたＤＮＡの安定性は、特にＳＤＳ溶液において、Ｍ１とＭ２で異なることが見いだされた。当初固定されたＤＮＡの約２０％がＭ１からＳＤＳ溶液に溶出し、一方４５％以上が、Ｍ２からＳＤＳ溶液に溶出した。２つの膜表面から溶出したＤＮＡの量は４８時間インキュベート後、徐々に平衡値に到達した。平衡値はインキュベートした溶液および膜に依存して異なる。
また、殆ど全ての固定されたＤＮＡがＨＣｌ溶液（１Ｍ）中に溶出したが、これはＤＮＡのホスホジエステルが加水分解されたと考えられうる。ＤＮＡの一部はまたＳＤＳの表面活性のため、２重量％ＳＤＳ溶液にも溶出した。これらの結果は表面にＤＮＡを固定したポリスルホン膜が中性および塩基性条件下で生体材料として使用に利用できるであろうことおよび血液精製用のような生医学的材料として使用可能であることを示す。
【００２１】
Ｃ． 異なる膜表面におけるＤＮＡ固定化量
異なる製造方法により得られた異なる形態および構造をもつ膜表面における、紫外線によるＤＮＡ固定化量は図４に示される。表１に示したように孔隙率は膜ごとに著しく異なりおよびＤＮＡは膜の孔に存在するであろうことから、膜表面に固定されたＤＮＡ量（および後述するＢＳＡ吸着量）を評価するのに２つの単位μｇ／ｃｍ^２およびｍｇ／ｇを使用した。ポリスルホン膜表面の固定化ＤＮＡの量は膜構造に依存して顕著に異なることが示された。その密でない構造および大きい孔隙率により、Ｍ２表面には約２．８６ｍｇ／ｇのＤＮＡが固定されたが、Ｍ１表面には単に０．３９ｍｇ／ｇ（１．２５μｇ／ｃｍ^２）のＤＮＡが固定された。
【００２２】
なお、本発明によるＤＮＡのポリスルホン膜表面への固定化の機構は以下のように考えられる。
紫外線照射後にＤＮＡは水不溶性になり、および上記結果からポリスルホン膜表面に固定されたと結論づけられる。ポリスルホン膜表面に固定されたＤＮＡは２つの部分からなる：膜表面へ物理的に吸着されたＤＮＡおよび膜表面における電子共有化により固定されたＤＮＡである。紫外線照射後に水不溶性となるであろうとされるにもかかわらず、物理的に吸着されたＤＮＡはＳＤＳ溶液により溶出できる。
ＤＮＡと膜表面の電子共有化による固定はＤＮＡとポリスルホン間の化学反応によるものであり、ＤＮＡとポリスルホンの光反応性によるものである。ポリスルホン膜表面に電子共有化により固定されたＤＮＡの存在は以下の手順により、証明される。ＤＮＡ固定化膜を４８時間ＳＤＳ溶液中でインキュベートし、およびＳＤＳ溶液を数回交換し、ついで蒸留水で数回洗浄し、水で２４時間インキュベートし、水で洗浄し、固定されたＤＮＡの量が測定された。結果（データ示さず）はＳＤＳ溶液により一部分が溶出したが、ＤＮＡの殆どがポリスルホン膜表面に存在した。
ポリスルホンは光反応性を有し、ポリスルホン膜材料のそして紫外線スペクトルは、ポリスルホンが最大吸光度２６８ｎｍを持つことを示した。ポリスルホンフィルムおよび膜における紫外線照射の効果は紫外線吸光光度計により測定でき、全体の崩壊過程は試料の黄変（黄変の度合いは明らかに観察される、データは示さない）を伴うが、従来の研究によれば特定の構造情報は得られていない（非特許文献９）。
紫外線照射によるポリスルホン膜の修飾は例えば文献（非特許文献５−６，９）に報告されている。ビニルモノマーが紫外線照射下、直接的な化学結合により直接的にポリスルホン鎖に結合できることが見出されている。ポリスルホン膜の開裂に対する機構は従来以下のように提案されている。第一ステップとしてポリマー鎖のバックボーンにあるフェノキシフェニルスルホン発色団による光の吸収を含む。光励起はスルホン結合において炭素−硫黄結合のホモリティック開裂を発生させる。このステップは各々のポリマー鎖のそれぞれの末端に２つのラジカル部位を発生するポリマーバックボーンのランダム開裂を生じる。開裂において発生したアリールラジカルおよびスルホニルラジカルの両方は反応性であり、そして化学反応がそれぞれの部位に生じる。
ＤＮＡ鎖におけるピリミジン塩基中の二重共有結合が励起する。弱いπ結合が紫外線によって崩壊する。単にＤＮＡのみが励起する場合、ＤＮＡ鎖における隣接するピリミジン塩基のサイトは紫外線により損傷をうけて、これらの塩基の間に二量体を形成する：シクロブタンピリミジン二量体（ＣＰＤ）、６−４光生成物（６−４ＰＰ）またはそのデュワー（Ｄｅｗｅｒ）体のいずれかである。ピリミジン二量体の主要部は特定の短波紫外線に対し特異性をもつと考えられている。上述したアリールラジカルおよびスルホニルラジカルの存在下で、これらのラジカルとピリミジン塩基との反応が起こりうる。
これらの反応はＤＮＡの水不溶への変換を誘導しそしてポリスルホン膜へ結合し得ると考えられる。
【００２３】
Ｄ． 異なる製造方法における、接触角への影響
膜表面の湿潤性の指標となる水接触角は異なるポリスルホン膜表面に対するＤＮＡの固定化の前後に測定され、図５にまとめられた。データは８−１２滴に対する膜の２表面の平均である。いずれの膜においても、ＤＮＡ固定化後の接触角は固定化前の接触角よりも減少し、ＤＮＡ固定化ポリスルホン膜は親水性になったことが判る。
ＤＮＡ固定化前では膜Ｍ１表面の接触角は大きく、同時に膜Ｍ２、Ｍ３およびＭ４表面の接触角はわずかに小さかった。相転法により形成された膜の間に顕著な違いはみられなかった。これは沈殿法により製造された膜は溶媒蒸発法により製造された膜（Ｍ１）に比べてわずかにより親水性であること示す。それはＭ１表面が非常に滑らかである一方Ｍ３は非常に粗く、Ｍ２およびＭ４の表面には多くの孔があることによるものであろう（図２）
膜表面にＤＮＡが固定されると接触角は約８０度から約５５度に減少し、親水性が増加する。しかし、ＤＮＡを固定した後に減少した接触角の程度は、Ｍ１，Ｍ２、Ｍ３およびＭ４間に違いはみられなかった。これは接触角が表面特性によるためと考えられる。さらに、接触角は同じ膜の２表面間にわずかな違いが見られることは注目するべきである。これは空気への曝露時間が異なることよるものであろう。
【００２４】
Ｅ．タンパク質吸着量および立体配座変化
血漿タンパク質吸着は生体材料のトロボゲン生成性（ｔｈｒｏｂｏｇｅｎｉｃｉｔｙ）を決定する１つの重要な事象である。そして物質の表面において吸収されたタンパク質の量は物質の血液適合性を評価する主要な因子の１つであることがよく知られている。さらに表面で吸着されたタンパク質の立体配座変化もまた血液適合性を評価するのに重要である。
図６に示すようにＤＮＡを固定する前後における上記４種類のポリスルホン膜に関し、膜表面に吸着されたＢＳＡの量を測定した。その結果、いずれの製造方法の膜においても、ＤＮＡの固定後の膜表面に吸着されるタンパク質の量は固定化前の量よりも減少することが見出された。
なお、膜の種類によって吸着量は、固定化ＤＮＡの量における膜形態の効果と同様に変化することが見出された。膜の表面構造および多孔性の違いにより、膜Ｍ２、次にＭ４、Ｍ３およびＭ１の順にかなりのＢＳＡが吸着された。これらの結果はＢＳＡが膜表面だけでなく膜に存在する孔の表面にも吸着されることが推定される。
吸着されたタンパク質の量は親水性に相関性があり、一般的にはより親水性であれば、反対により少ない量のタンパク質が吸着されるといわれる。しかし、図６からは、ＤＮＡの固定による修飾後、膜表面に吸着されたＢＳＡの量の減少割合に比べて、親水性の増加割合の方が大きい（図６）。この理由は接触角は表面特性であり、そしてＤＮＡとタンパク質間の相互作用がこのような結果を招いたと推定される。タンパク質はＤＮＡと、部位または非部位にて結合し、それらの相互作用は共有結合ではないが、例えば静電結合、水素結合、ファンデルワールス力および疎水力のような他の弱い力である。修飾前のＢＳＡとポリスルホン膜との相互作用に比べ、ＤＮＡ固定化膜にはより多くの水素結合が存在するであろうし、他方疎水力は弱くなっている。
【００２５】
ポリスルホン膜上に吸着されたＢＳＡの立体配座の変更はＦＴ−ＩＲによって検討した。ペプチド基の振動から生じるアミドバンドはタンパク質の二次構造における情報を与える。タンパク質の構造研究に最も広く使用される様態は中赤外領域にあり、アミドＩ（１７００−１６００ｃｍ^−１）、アミドＩＩ（１６００−１５００ｃｍ^−１）およびアミドＩＩＩ（１３２０−１２３０ｃｍ^−１）である。ペプチド基のアミドＩバンドは主にＣ＝Ｏ伸縮振動から起こる。アミドＩＩバンドは主にＣ−Ｎ伸縮振動が関与するＮ−Ｈベンディングであり、吸光強度は吸着されたタンパク質の量に比例する。赤外において主にＮ−ＨベンディングおよびＣ−Ｎ伸縮振動から生じるアミドＩＩＩ吸収は通常非常に弱い。
図７に示すのは、ＺｎＳｅプレート、ならびにポリスルホン膜を吸着したＺｎＳｅプレートおよびＤＮＡを表面に固定したポリスルホン膜を吸着したＺｎＳｅプレートにおけるアミドＩおよびアミドＩＩ領域におけるＢＳＡの赤外スペクトルである。スペクトルを比較すると違いは明らかである。約１６５４ｃｍ^−１および１５４２ｃｍ^−１に中心をおくアミドＩおよびアミドＩＩバンドの関連する吸収強度はそれぞれ変化した。ポリスルホン膜上のＢＳＡのアミドＩおよびアミドＩＩバンドの吸収強度の比は２．０１±０．２８であるが、ＤＮＡ固定化ポリスルホン膜およびＺｎＳｅプレート上のＢＳＡの前記比はそれぞれ１．５４±０．０８と、１．４６±０．０６である。アミドＩおよびアミドＩＩピークの強度比の違いは第二次および第三次構造の不特定の変化による。ポリスルホン膜における吸着されたＢＳＡに対するバンド高さの７０％におけるアミドＩのバンド幅（３４±２ｃｍ^−１）は、ＤＮＡ固定化膜およびＺｎＳｅプレート（それぞれ２９±２ｃｍ^−１および２９±１ｃｍ^−１）におけるタンパク質に対するものと比較して、より大きい。吸着におけるバンドの拡張はある種の構造的変更を示唆するより広い領域の振動度数を示した。
強度比が増大しおよびバンド幅が拡張したこれらの結果は、ポリスルホン膜におけるＢＳＡ吸着の立体配座において、ＤＮＡ固定化ポリスルホン膜の場合よりもより大きい変化があったことが示される。
以上のとおり、未処理のポリスルホン膜に比べて、ＤＮＡ−固定化ポリスルホン膜に表面に吸着されるタンパク質の量はより少ないことは、ＤＮＡ修飾ポリスルホン膜がより血栓形成性が低いことを示し、また、未処理のポリスルホン膜に吸着したタンパク質よりもＤＮＡ−固定化ポリスルホン膜に吸着したタンパク質が小さい立体配座変化を示すことは、より血漿性タンパク質の変性が小さいことを示すので、ＤＮＡを固定化したポリスルホン膜は血液適合性が改善されたことがわかる。
【００２６】
【発明の効果】
本発明によって、ポリスルホン（ＰＳｆ）膜の表面ないし孔隙部分に生体成分であるＤＮＡを固定することにより、該表面ないし孔隙部分を親水性にしたポリスルホン膜であって、かつ、膜表面へのタンパク質吸着量もより少なく、立体配座変化もより少ない血液適合性の改善されたポリスルホン膜が提供できる。従って、本発明の表面にＤＮＡを固定したポリスルホン膜を、特に中空糸状の血液透析膜として利用すれば、抗−凝集剤を使用しなくとも、目詰まりや、血栓形成反応を起こしにくい血液透析器が提供できる。
さらに本発明は人工器官および医療器具、例えば血液透析濾過、血漿交換、血漿採取および手術中の血液酸素付加のための装置への濾過膜としても使用することができる。またその他の工学的、医学的、バイオテクノロジー分野における、分離技術分野、例えば限外濾過膜、精密濾過膜、ならびに逆浸透膜の支持膜等への適用も可能である。
また、本発明の膜表面を親水性にしたポリスルホン膜は、ポリスルホン膜を、ＤＮＡ溶液中に浸漬し、室温において乾燥し、紫外線照射しおよび次いで蒸留水により洗浄しおよび水に貯蔵するという、極めて簡素な工程と、穏やかな条件下で製造できるので、製造に大規模な装置などを必要とせずに親水性ポリスルホン膜を効率よく生産することができる。
【図面の簡単な説明】
【図１】図１は異なる製法で製造したポリスルホン膜の横断面と上面の形態を示す、ＳＥＭの写真（電圧６．０ｋＶ，倍率３００倍）である。
【図２】図２はポリスルホン膜表面に固定されたＤＮＡの量と接触角における、ＤＮＡ処理溶液濃度の効果を示すグラフである。
【図３】図３（ａ）は膜Ｍ１における種々の溶液中のＤＮＡ−固定化ポリスルホン膜の安定度を示すグラフである。図３（ｂ）は膜Ｍ２における種々の溶液中のＤＮＡ−固定化ポリスルホン膜の安定度を示すグラフをである。
【図４】図４は異なる方法で製造されたポリスルホン膜表面に固定化されたＤＮＡの量を示すグラフである。
【図５】図５はＤＮＡを固定する前後の、異なるポリスルホン膜表面における接触角を示すグラフである。
【図６】図６はＤＮＡを固定する前後における異なるポリスルホン膜に吸着されたタンパク質の量を示すグラフであり、Ａはμｇ／ｃｍ^２単位で、Ｂはμｇ／ｍｇ単位で示すものである。
【図７】図７はＳｅＺｎプレート、その上にポリスルホン膜を吸着させたプレート、およびその上にＤＮＡを固定したプレートそれぞれに吸着された牛血清アルブミン（ＢＳＡ）のアミドＩおよびアミドＩＩ領域における赤外線吸収スペクトルを示すグラフである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a polysulfone membrane which has been made hydrophilic by fixing the surface of the polysulfone membrane with DNA. Since the polysulfone membrane having the hydrophilic surface has good blood compatibility, it can be used for a hemodialysis membrane or the like.
[0002]
[Prior art]
Polymeric materials are biomedical for advanced separation technology and prostheses and medical devices such as, for example, hemodialysis, hemodiafiltration, hemofiltration, plasma exchange, plasma collection and oxygenation of blood during cardiac surgery. Widely used in the field. Currently used polymers are cellulose acetate (CA), polymethyl methacrylate (PMMA), polyethylene (PE), polypropylene (PP), polyacrylonitrile (PAN), ethylene vinyl alcohol copolymer (EVAL), polyvinyl alcohol (PVA) ) And polysulfone (PSf).
Of these materials, polysulfone (PSf) is one of the most important materials, and polysulfone and polysulfone-based membranes have remarkable oxidative, thermal and hydrolytic stability and good mechanical properties And film forming properties. And when this membrane is used as a hemodialysis hollow fiber, it shows high permeability to low molecular weight proteins (Non-Patent Documents 1 and 2).
[0003]
However, polysulfone membranes are hydrophobic and have poor blood compatibility, i.e., antithrombotic properties. Therefore, the biggest problem is that the blood coagulation reaction easily occurs, and the hollow fiber membrane used for dialysis, that is, the circulation of blood becomes impossible. For this reason, the use of a polysulfone membrane requires the injection of an anti-aggregant during hemodialysis.
Numerous studies have been reported on modifying the surface of biomedical devices with other polymers or monomers to improve blood compatibility. For example, these methods can be divided into six types.
(1) A blend with a hydrophilic polymer, for example, a blend with 2-methacryloyloxyethyl phosphorylcholine (MPC) (Patent Document 1), a blend with polyvinylpyrrolidone (PVP), or a block containing, for example, polyethylene oxide (PEO) and PEO Blends with other amphiphilic polymers, including copolymers (Patent Document 2),
(2) Grafting with other polymers, such as grafting of polyethylene glycol (PEG), by radiation including UV irradiation (Non-patent Document 3, Patent Document 3) or by a low-temperature plasma method (Non-patent Document 4).
(3) graft copolymerization of monomers (Non-Patent Documents 5-7, Patent Document 4),
(4) grafting of low molecular weight substances (Non-Patent Document 8),
(5) Grafting reactive groups as model monomers and carriers, followed by electron-pair covalent immobilization of large molecules (Non-Patent Document 9).
(6) Coating with a hydrophilic copolymer (Non-Patent Document 10).
[0004]
Further, as other patent documents, for example, a water-soluble polymer substance such as a water-soluble cellulose derivative or polyethylene glycol is attached to the surface of a polysulfone membrane, and the water-soluble polymer substance is a hydrophilic polyphenol such as tannin. There is also one that describes a hydrophilic polysulfone membrane that has been insolubilized by the presence of (Patent Document 5).
[0005]
All of the membrane surface modifications described above assume that the material used for the modification is inherently more hydrophilic and provides less protein adsorption than the underlying substrate. Of these methods, the photochemical surface modification method is attractive and has a number of advantages. Applicable under mild reaction conditions and low temperatures; high selectivity is possible by selecting reactive groups or monomers and their respective excitation wavelengths; they can be easily incorporated into the final stages of the manufacturing process. Polysulfone is inherently photoreactive, and UV-induced modification of polysulfone membranes has been reported in previous literature (Non-Patent Documents 5-7 and 9, Patent Document 4).
[0006]
On the other hand, DNA, one of the most important materials for the genetic process of living organisms, can also be considered as a natural and highly specific functional biopolymer. And DNA also has a biomembrane-like structure and carries phosphate groups that will help improve blood compatibility when used as a modifier. Recent studies have produced water-insoluble DNA-films and gels conjugated to alginic acid or collagen, as well as cellulosic nonwovens or glass beads on which DNA has been immobilized by ultraviolet irradiation, which is composed of ethidium bromide, dioxin derivatives, [A] It has been found to adsorb pyrene and DNA binding inserts such as metallic iron (eg, Non-Patent Documents 11 and 12). These results suggest that water-insoluble DNA can be produced and immobilized by stably binding to other material substrates.
[0007]
[Non-patent document 1]
T. Nishimura, Polymer material for purification, In: Tsuruta et al. , Editors, Biomedical applications of polymeric materials, CRC Press, BocaRaton, PA (1993), pp. 147-64. 191-218
[Non-patent document 2]
B. Schmidt, Membranes in Artificial Organs, Artificial Organs, 20 (1996) 375
[Non-Patent Document 3]
Fu Zhang, et al. , Surface modification of stainless steel by grafting of poly (ethylene glycol) for reduction in protein advertisement, Biomaterials, 22.
[Non-patent document 4]
Y. Q. Song, et al. , Surface modification of polysulfone membranes by low temperature plasma-graft poly (ethylene glycol) onto polysulfone membranes, J. Appl. Polym. Sci. , 78 (2000) 979
[Non-Patent Document 5]
M. See Ulbricht, et al. , Ultrafiltration membrane surfaces with grafted polymer 'tentacles'; preparation, characterization and application for covalent protein binding, 19th year, 19th year, 1998
[Non-Patent Document 6]
H. Yamagishi, et al. , Development of a novel photochemical technique for modifying poly (arylsulfone) ultrafiltration membranes, J. Mol. Membrane Sci. , 105 (1995) 237
[Non-Patent Document 7]
B. Kaeselv, et al. J., Photoinduced grating of ultrafiltration membranes: comparison of poly (ether sulphone) and poly (sulfone); Membrane Sci. , 194 (2001), 245.
[Non-Patent Document 8]
Y. J. Kima, et al. , Surface Characterization and In Vitro Blood Compatibility of Poly (Ethylene Terephthalate) Immobilized with Insulin and a Gloss in the United States
[Non-Patent Document 9]
M. Ubricht, et al. , Novel photochemical surface functionalization of polysulfone ultrafiltration membranes for covalent immobilization of biomolecules, J. et al. Membrane Sci. , 120 (1996), 239
[Non-Patent Document 10]
A. Lewis, et al. , Synthesis and Characterization of Phosphorylcholine-Based Polymer Useful For Coating Blood Filtration Devices, Biomaterials, 21 (2000) 1847.
[Non-Patent Document 11]
K. Iwata, et al. Utilization of DNA as functional materials: preparation of filters containing DNA insolubilized with organic acid gel Int. J. Biol. Macromol. , 18 (1996) 149.
[Non-Patent Document 12]
M. Yamada, et al. , UV-irradiation-induced immobilization and functionalization of DNA on non-woven cellulosic fabric, Biomaterials, 22 (2001) 3121.
[Patent Document 1]
JP-A-10-296063 (pages 2 to 8)
[Patent Document 2]
JP-A-9-234244 (page 2, page 4 to page 11)
[Patent Document 3]
JP-A-6-238139 (pages 2 to 5)
[Patent Document 4]
JP-A-10-60142 (page 2, page 4 to page 8)
[Patent Document 5]
JP-A-7-185280 (pages 2 to 5)
[0008]
[Problems to be solved by the invention]
Based on these prior arts, an object of the present invention is to improve the blood compatibility by making the surface of the polysulfone membrane hydrophilic, and particularly when the polysulfone membrane is used as a hollow fiber-shaped hemodialysis membrane, it has an anti-dialysis effect on the hemodialyzer. Clogging in the absence of a coagulant is to provide a polysulfone membrane that is less prone to thrombogenic reactions.
[0009]
[Means for Solving the Problems]
Therefore, the present invention relates to a polysulfone membrane whose surface is made hydrophilic by immobilizing DNA on at least one surface of the polysulfone membrane. The surface modification of the polysulfone membrane with DNA of the present invention includes those in which DNA is fixed also in the pores on the surface of the porous polysulfone membrane.
The DNA particularly used in the present invention may be either a single helix or a double helix, but is preferably a single helix DNA having reactivity, and a DNA analog having a phosphate ester group and having reactivity is also available. Can be used.
The polysulfone membrane whose surface is modified with DNA, that is, the DNA-immobilized polysulfone membrane of the present invention, is manufactured by utilizing the fact that DNA is insolubilized and immobilized by a photoreaction by ultraviolet irradiation.
The polysulfone membrane whose surface is made hydrophilic according to the present invention is, specifically, first, an untreated polysulfone membrane produced is immersed in a DNA solution, taken out and dried, and then irradiated with ultraviolet light, Thereafter, the membrane is obtained by sufficiently washing the membrane with water.
Therefore, the polysulfone membrane having a hydrophilic surface according to the present invention can be produced under extremely mild conditions.
However, DNA can be immobilized on the polysulfone membrane by a physical method by irradiating plasma or radiation, or other chemical methods, in addition to the above-described ultraviolet light.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
The polysulfone membrane in the present invention is typically, for example,
It is composed of a polysulfone composed of a repeating unit represented by the following formula (I) or (II), but is not limited to such examples.
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[0011]
In order to immobilize the insolubilized DNA on the surface of the polysulfone membrane, the concentration of the polysulfone membrane obtained as described above is about 10 to 40 mg / ml, preferably about 20 to 30 mg / ml in an aqueous DNA solution for about 12 to 30 hours, preferably about 20 to 30 hours. This is performed by immersing for about 24 hours, drying this at room temperature, irradiating with ultraviolet light of, for example, 254 nm for about 2 hours, and washing with water. The immobilized DNA is stable under neutral or basic conditions with a small amount of elution from the surface of the polysulfone membrane. Since the surface of the polysulfone membrane on which the obtained DNA is immobilized has sufficient hydrophilicity and the amount of protein adsorbed to cause thrombus formation is reduced, blood compatibility of the polysulfone membrane is improved.
The shape of the polysulfone membrane whose surface is modified with DNA according to the present invention can be various shapes, and examples thereof include a film shape, a tube shape, and the like, in addition to a hollow fiber shape such as an ordinary hemodialysis membrane. Furthermore, the immobilization of DNA on the surface of the polysulfone membrane may be performed on both surfaces of the membrane, but may be performed on either one of the surfaces.
The present invention also provides a filtration membrane for a medical device using a polysulfone membrane having DNA immobilized on the membrane surface according to the present invention having these various forms and shapes, particularly a hemodialysis membrane as a hollow fiber membrane. Related.
[0012]
【Example】
The following examples illustrate the invention in more detail. These examples are illustrative of the present invention and are not intended to limit the present invention.
[Main reagents used]
All of these materials were used without purification.
1. Polysulfone (PSf) used for the production of the polysulfone membrane: number average molecular weight Mn: about 26,000, Aldrich Chemical Company Inc. Made.
2. solvent
1) N, N-dimethylformamide (DMF): Chemical reagent grade, manufactured by Kanto Chemical Co., Ltd.
2) N-methyl-2-pyrrolidone (NMP): grade for HPLC, 99 +%, Aldrich Chemical Company Inc. Made
3. DNA (DNA-Na): single-stranded DNA, manufactured by Sarde Pharmaceutical Co., Ltd.
4. Bovine serum albumin (BSA): RIA grade, Sigma Chemical Co. Made
5. Sodium dodecyl sulfate (SDS): Chemical reagent grade product, manufactured by Wako Pure Chemical Industries, Ltd.
[0013]
[Example] Production of polysulfone membrane
The polysulfone membrane was manufactured by a phase inversion method. Polysulfone was dissolved in DMF to obtain a 15% by weight polysulfone solution. The polymer solution was then cast on a glass plate and spread at 25 ° C. to a uniform thickness of 500 μm to produce a membrane.
In this example, four types of polysulfone membranes were produced by changing the phase inversion method. Membrane 1 (M1) was prepared by evaporating DMF in a vacuum furnace at 50 ° C. for 1 day. Membrane 2 (M2), Membrane 3 (M3) and Membrane 4 (M4) were all immersed in a precipitation bath containing the solvent solution at room temperature for 1 day by immersing the casting solution (after evaporation at 25 ° C. for 15 minutes). Manufactured. The precipitation solvent solution for each of the membranes M2, M3 and M4 was water, acetone, and a DMF solution (DMF / H₂O (vol / vol) = 1: 2) was used. In this example, deionized distilled water was used. Acetone was of chemical reagent grade and used without further purification.
After evaporation (M1) and precipitation (M2-M4) were completed, each membrane was removed from the glass plate and then annealed and extracted in deionized water at 80 ° C. for 1 hour. Each membrane is then immersed in 100 ml of 0.1 M sodium hydroxide (NaOH) solution and then in 100 ml of 0.01 M hydrochloric acid (HCl) for 30 minutes each, and then rinsed with distilled water (100 ml × 5 times) and used. It was kept in water until.
The thickness of each of the four types of films obtained was directly measured with a micrometer. The porosity was calculated from the polymer density and the weight changed before and after drying using the following formula.

W_B: Membrane weight before drying (g)
W_A: Weight of membrane after drying (g)
ρ_W: Water density, ρ_W= 1.0 g / cm³
ρ_P: Density of polysulfone, ρ_P= 1.24 g / cm³
[0014]
Further, in order to observe the morphology of these film samples having different manufacturing conditions, they were observed with a scanning electron microscope (SEM). Each membrane sample was broken with sample liquid nitrogen, mounted on a sample support and coated with a gold layer. The SEM image was recorded using an S-2500C microscope (voltage = 6 kV, magnification 300 times, manufactured by Hitachi, Ltd.).
[0015]
Significant differences were observed between the films manufactured by different manufacturing methods. Microscopically, the membrane M1 produced by the solvent (DMF) evaporation method was transparent and thin, and M2, M3 and M4 produced by immersing the polymer solution in a precipitation bath exhibited a milky white color. The structure of the membrane also changed with different manufacturing methods. M1 was dense and smooth, while M2, M3 and M4 were porous. Table 1 shows the thickness and porosity of the polysulfone membrane obtained by different methods.
[Table 1]

The order of thickness and porosity was M2, M4, M3, and M1. M1 was very thin, about 0.074 mm, and had very low porosity, ie, there were few pores in the membrane. On the other hand, films M2, M3 and M4 were thick and had high porosity. Among them, the thickness and porosity of M2 were the largest among these films, being 68.4% and 0.312 mm, respectively.
[0016]
FIG. 1 shows SEM photographs of the cross section (left column in FIG. 1) and the top surface (right column in FIG. 1) of the polysulfone membrane. The surface and essential structure of the obtained polysulfone membrane also differed among the membranes M1, M2, M3 and M4 as shown in the morphology of the membrane. From these photographs, it is easy to understand the surface and structure of each membrane and to understand the effect of membrane morphology on the amount of immobilized DNA and protein adsorbed, as described below.
The cross section and upper surface of M1 were dense and no holes were found. In M2 and M4, a sponge-like structure was found, and many holes were found in the upper surface. A depression was found on the surface of M3.
As described above, the morphologies of the films manufactured by different methods were different (Table 1 and FIG. 1). These differences can be explained by the film formation speed. If the initial membrane is immersed in water, the skin layer forms quickly on the surface, and then exchanges between solvent and non-solvent, resulting in very high porosity and thickness. For M4, the formation of the skin layer is slower than M2 due to the addition of the solvent system containing DMF. For M3 and M1, skin layer formation is very unlikely, resulting in very low porosity and thickness. Further, the film M3 formed by immersion in acetone was different from the others, and the initial film shrank when no special holder was used.
[0017]
Example 1Immobilization of immobilized DNA on polysulfone membrane surface
The untreated polysulfone membrane of M1 to M4 obtained by the above example (2 × 2 cm²) Was immersed in an aqueous DNA solution (20 mg / ml DNA in water) for 24 hours. After drying at room temperature, the membrane was irradiated with ultraviolet light (R-52G, Ultra Violet Inc., USA) at 254 nm on each surface for 2 hours. UV intensity is 5600 μW / cm at sample position²And The UV treated membrane was immersed in water and shaken at 120 times / min for 48 hours using a Yamato Water Bath Incubator Model 137-25, the water was changed several times, and then washed with distilled water (100 ml × 5 times). This was stored in water until use of the membrane.
[0018]
Hereinafter, the performance of the polysulfone membrane on which DNA is immobilized will be evaluated.
I. Measurement method
(1) Measurement of the concentration of immobilized DNA
The amount of DNA applied and immobilized on the polysulfone membrane surface was determined by the following method: The DNA-immobilized membrane was hydrolyzed with a 1M HCl solution at 100 ° C. for 1 hour. The amount of DNA in the solution was quantified by UV-VIS spectrophotometer U-200A (manufactured by Hitachi, Ltd.) at 260 nm absorbance.
(2) Measurement of contact angle
The water contact angles of the surface of the polysulfone membrane produced by the different phase inversion method consisting of M1 to M4 and the surface of the polysulfone membrane on which DNA was immobilized were measured.
The wettability of the surface, that is, the water contact angle as an index of hydrophilicity, was measured at 25 ° C. by a sesile drop method using a contact angle meter CA-D (manufactured by Kyowa Interface Science Co., Ltd.). . A droplet of distilled water was placed on the surface of the membrane, and the contact angle was directly measured with a goniometer by a microscope.
As for the angle, the contact angle on the two surfaces of the film was measured, and the average of the measured values of 8-10 drops on the two surfaces was used as data.
(3) Measurement of protein adsorption amount
Protein adsorption to each membrane was measured using bovine serum albumin (BSA) at a concentration of 10 g / l in phosphate buffered saline (PBS, pH = 7.4).
The polysulfone membrane on which DNA was not immobilized was immersed in a BSA solution for 24 hours. The protein-adsorbed membrane was carefully washed with PBS and rinsed with distilled water. The adsorbed protein is quantitatively eluted with a 4.0 ml SDS solution (2% by weight) for 16 hours. The amount of BSA in the SDS solution is quantified by the absorbance at 279 nm using a UV-VIS spectrophotometer U-200A (manufactured by Hitachi, Ltd.), and the BSA adsorbed on the membrane surface is calculated.
The membrane with the DNA immobilized on the surface was also immersed in a BSA solution (10 g / l) for 24 hours. Here, when the SDS solution is used to remove the adsorbed protein from the membrane, a part of the DNA elutes into the SDS solution. Therefore, when measuring the amount of BSA, the multi-component determination function of the UV-VIS spectrophotometer U-200A by using wavelengths 260 nm, 270 nm and 280 nm was used.
(4) Infrared spectroscopy analysis of BSA adsorption film
To examine the conformational change of BSA, a Fourier transform infrared spectrometer FT-210 (4 cm^-1The infrared spectrum of the BSA adsorption film was measured by a resolution of 120 scans, 25 ± 2 ° C., manufactured by Horiba, Ltd.). A thin high density film of polysulfone was coated on a zinc selenide (ZnSe) plate (10 mm wide, 50 mm long) by casting the polymer solution. The polymer solution concentration was 1% (weight / volume) in the NMP solution. Prior to use, the coating was dried in a vacuum oven at 40 ° C. for 12 hours and rinsed with PBS at room temperature. For modification with DNA, two drops of a 2% DNA solution were added on the coating, dried at room temperature, irradiated with UV light, and then rinsed with water and a PBS solution. Background spectra were obtained from crystals and polymers. For the adsorption test, one drop of BSA solution (10 g / l in PBS solution) is added onto the coating, stored in the chamber for 30 minutes at room temperature, then the BSA solution is carefully removed and carefully washed with PBS solution. The spectrum was measured after rinsing and drying at room temperature.
[0019]
II Measurement result
A. Effect of DNA concentration on the amount of immobilized DNA
The effect of DNA concentration on the amount of DNA immobilized on the membrane surface and the air-water contact angle was tested for M2. FIG. 2 is a graph showing the relationship between the amount and contact angle of DNA immobilized on the polysulfone membrane surface and the concentration of the DNA treatment solution. As shown in FIG. 2, as the concentration of the DNA solution used to treat the membrane (16 hours soak) increases, the amount of DNA immobilized on the membrane surface increases, and the contact with the DNA-immobilized polysulfone membrane surface increases. The corner has decreased. The amount of DNA immobilized was greatly affected by the DNA concentration, and increased with increasing DNA concentration up to 20 mg / ml. The decrease in contact angle did not show a significant difference when the concentration of the DNA solution exceeded 15 mg / ml.
The effect of immersion time on the polysulfone membrane immersed in a DNA solution (20 mg / ml) on the air-water contact angle and the amount of DNA immobilized on the polysulfone membrane surface showed a similar trend as the effect of DNA concentration (data shown). Not shown). At the start, the contact angle decreased rapidly and the amount of DNA immobilized on the membrane surface increased, but did not change after 12 hours.
[0020]
B. Stability of DNA immobilized on membrane surface
The stability of the DNA immobilized on the membrane surface₂The test was carried out using various solutions including O, NaCl solution, SDS solution and HCl solution, and the amount of DNA eluted from the surface of the membrane on which DNA was immobilized was measured (FIG. 3a is data for the membrane M1; FIG. 3b is M2). For M1 and M2, the membrane on which the DNA was immobilized was stable in water and NaCl solution (0.9% by weight), and after 96 h incubation less than 10% of the initially immobilized DNA eluted from the layer. there were. However, when incubated under acidic conditions, large amounts of DNA eluted into solution and about 80% of the initially immobilized DNA eluted.
3a and 3b, it was found that the stability of the DNA immobilized on the membrane surface was different between M1 and M2, especially in the SDS solution. About 20% of the initially immobilized DNA eluted from M1 into the SDS solution, while over 45% eluted from M2 into the SDS solution. The amount of DNA eluted from the two membrane surfaces gradually reached an equilibrium value after 48 hours of incubation. Equilibrium values differ depending on the solution and the membrane incubated.
Also, almost all of the immobilized DNA eluted in the HCl solution (1M), which may be attributed to hydrolysis of the phosphodiester of DNA. Some of the DNA also eluted in the 2% by weight SDS solution due to SDS surface activity. These results indicate that a polysulfone membrane with DNA immobilized on its surface could be used as a biomaterial under neutral and basic conditions and that it could be used as a biomedical material such as for blood purification. Show.
[0021]
C. DNA immobilization on different membrane surfaces
FIG. 4 shows the amount of DNA immobilized by ultraviolet light on the surface of the membrane having different morphologies and structures obtained by different manufacturing methods. As shown in Table 1, the porosity varies significantly from membrane to membrane and DNA will be present in the pores of the membrane, so the amount of DNA immobilized on the membrane surface (and the amount of BSA adsorbed as described below) is evaluated. 2 units μg / cm²And mg / g were used. It was shown that the amount of immobilized DNA on the surface of the polysulfone membrane was significantly different depending on the membrane structure. Due to its loose structure and high porosity, about 2.86 mg / g of DNA was immobilized on the M2 surface, while only 0.39 mg / g (1.25 μg / cm) was immobilized on the M1 surface.²DNA) was fixed.
[0022]
The mechanism of immobilization of DNA on the surface of the polysulfone membrane according to the present invention is considered as follows.
It is concluded that the DNA became water-insoluble after UV irradiation and was fixed on the polysulfone membrane surface from the above results. DNA immobilized on a polysulfone membrane surface consists of two parts: DNA physically adsorbed to the membrane surface and DNA immobilized by electron sharing on the membrane surface. Physically adsorbed DNA can be eluted with the SDS solution, even though it will become water-insoluble after UV irradiation.
The immobilization of the DNA and the membrane surface by electron sharing is based on a chemical reaction between the DNA and the polysulfone, and is based on the photoreactivity of the DNA and the polysulfone. The presence of DNA immobilized on the polysulfone membrane surface by electron sharing is proved by the following procedure. The DNA-immobilized membrane was incubated in the SDS solution for 48 hours and the SDS solution was changed several times, then washed several times with distilled water, incubated for 24 hours with water, washed with water, and the amount of immobilized DNA Was measured. The results (data not shown) were partially eluted by the SDS solution, but most of the DNA was present on the polysulfone membrane surface.
The polysulfone was photoreactive and the UV spectrum of the polysulfone membrane material showed that the polysulfone had a maximum absorbance of 268 nm. The effect of UV irradiation on polysulfone films and membranes can be measured by UV absorption spectroscopy, and the overall disintegration process is accompanied by yellowing of the sample (the degree of yellowing is clearly observed, data not shown), but According to research, no specific structural information has been obtained (Non-Patent Document 9).
Modification of the polysulfone membrane by ultraviolet irradiation has been reported, for example, in the literature (Non-Patent Documents 5-6, 9). It has been found that vinyl monomers can be directly bonded to polysulfone chains by direct chemical bonding under ultraviolet irradiation. The mechanism for cleavage of the polysulfone membrane has been conventionally proposed as follows. The first step involves the absorption of light by the phenoxyphenyl sulfone chromophore in the backbone of the polymer chain. Photoexcitation causes homolytic cleavage of the carbon-sulfur bond at the sulfone bond. This step results in random cleavage of the polymer backbone, generating two radical sites at each end of each polymer chain. Both the aryl and sulfonyl radicals generated in the cleavage are reactive, and a chemical reaction occurs at each site.
The double covalent bond in the pyrimidine base in the DNA strand is excited. Weak π bonds are broken by ultraviolet light. If only DNA is excited, the sites of adjacent pyrimidine bases in the DNA strand are damaged by ultraviolet light and form dimers between these bases: cyclobutane pyrimidine dimer (CPD), 6-4 Either the photoproduct (6-4PP) or its Dewar form. The major part of the pyrimidine dimer is believed to have specificity for certain short-wave ultraviolet radiation. In the presence of the above-mentioned aryl radicals and sulfonyl radicals, a reaction of these radicals with a pyrimidine base can take place.
It is believed that these reactions induce the conversion of DNA to water insoluble and can bind to the polysulfone membrane.
[0023]
D. Effect of contact angle on different manufacturing methods
Water contact angles, which are indicators of membrane wettability, were measured before and after immobilization of DNA on different polysulfone membrane surfaces and are summarized in FIG. Data is the average of the two surfaces of the membrane for 8-12 drops. In any of the membranes, the contact angle after immobilization of DNA was smaller than that before immobilization, indicating that the polysulfone membrane on which DNA was immobilized became hydrophilic.
Before DNA immobilization, the contact angle on the surface of the membrane M1 was large, and at the same time, the contact angle on the surface of the membranes M2, M3 and M4 was slightly small. No significant difference was observed between the films formed by the phase inversion method. This indicates that the membrane produced by the precipitation method is slightly more hydrophilic than the membrane (M1) produced by the solvent evaporation method. This may be due to the fact that the M1 surface is very smooth while the M3 is very rough, and the M2 and M4 surfaces have many holes (FIG. 2).
When the DNA is immobilized on the membrane surface, the contact angle decreases from about 80 degrees to about 55 degrees, and the hydrophilicity increases. However, there was no difference between M1, M2, M3 and M4 in the extent of the reduced contact angle after immobilization of DNA. This is probably because the contact angle depends on the surface characteristics. Furthermore, it should be noted that the contact angle shows a slight difference between the two surfaces of the same film. This may be due to different air exposure times.
[0024]
E. FIG. Protein adsorption and conformational change
Plasma protein adsorption is one important event that determines the trobogenicity of a biomaterial. And it is well known that the amount of protein absorbed on the surface of a substance is one of the major factors in assessing the blood compatibility of the substance. In addition, conformational changes of proteins adsorbed on the surface are also important in assessing blood compatibility.
As shown in FIG. 6, the amount of BSA adsorbed on the surface of the four types of polysulfone membranes before and after immobilizing DNA was measured. As a result, it was found that the amount of protein adsorbed on the surface of the membrane after immobilization of DNA was smaller than that before immobilization in the membranes of any of the production methods.
It has been found that the amount of adsorption varies depending on the type of the membrane, similarly to the effect of the membrane morphology on the amount of immobilized DNA. Due to differences in the surface structure and porosity of the membrane, significant BSA was adsorbed in the order of membrane M2, then M4, M3 and M1. These results suggest that BSA is adsorbed not only on the membrane surface but also on the surface of pores present in the membrane.
The amount of protein adsorbed is correlated with hydrophilicity, and generally it is said that a more hydrophilic protein will adsorb a smaller amount of protein. However, from FIG. 6, the rate of increase in hydrophilicity is greater than the rate of decrease in the amount of BSA adsorbed on the membrane surface after modification by immobilization of DNA (FIG. 6). The reason for this is that contact angle is a surface property and it is presumed that the interaction between DNA and protein has led to such a result. Proteins bind DNA at sites or non-sites, and their interactions are not covalent, but are other weak forces such as, for example, electrostatic, hydrogen, van der Waals and hydrophobic forces. Compared to the interaction of BSA with the polysulfone membrane before modification, the DNA immobilized membrane will have more hydrogen bonds, while the hydrophobicity is weaker.
[0025]
The change of the conformation of BSA adsorbed on the polysulfone membrane was examined by FT-IR. Amide bands resulting from vibrations of the peptide group provide information on the secondary structure of the protein. The most widely used mode for studying protein structure is in the mid-infrared region, where amide I (1700-1600 cm^-1), Amide II (1600-1500 cm^-1) And amide III (1320-1230 cm)^-1). The amide I band of the peptide group mainly arises from C = O stretching vibration. The amide II band is NH bending mainly involving CN stretching vibration, and the absorption intensity is proportional to the amount of the adsorbed protein. In the infrared, the amide III absorption resulting mainly from NH bending and CN stretching vibrations is usually very weak.
FIG. 7 shows the infrared spectra of BSA in the amide I and amide II regions of the ZnSe plate, the ZnSe plate on which the polysulfone membrane was adsorbed, and the ZnSe plate on which the polysulfone membrane having DNA immobilized on the surface was adsorbed. The difference is clear when comparing the spectra. About 1654cm^-1And 1542 cm^-1The associated absorption intensities of the amide I and amide II bands centered on, respectively, changed. The ratio of the absorption intensities of the amide I and amide II bands of BSA on the polysulfone membrane is 2.01 ± 0.28, whereas the ratio of BSA on the DNA-immobilized polysulfone membrane and the ZnSe plate is 1.54 ± 0, respectively. .08 and 1.46 ± 0.06. The difference in the intensity ratio of the amide I and amide II peaks is due to unspecified changes in the secondary and tertiary structures. Amide I bandwidth (34 ± 2 cm) at 70% of band height for adsorbed BSA on polysulfone membrane^-1) Are DNA immobilized membrane and ZnSe plate (29 ± 2 cm each)^-1And 29 ± 1cm^-1) Is greater than for the protein in). The broadening of the band on adsorption showed a broader frequency range suggesting certain structural changes.
These results, with increased intensity ratio and broadened bandwidth, indicate that there was a greater change in the conformation of BSA adsorption on the polysulfone membrane than on the DNA-immobilized polysulfone membrane.
As described above, the lower amount of protein adsorbed on the surface of the DNA-immobilized polysulfone membrane as compared to the untreated polysulfone membrane indicates that the DNA-modified polysulfone membrane has lower thrombus-forming properties, and The fact that the protein adsorbed on the DNA-immobilized polysulfone membrane shows a smaller conformational change than the protein adsorbed on the untreated polysulfone membrane indicates that the denaturation of the plasma protein is smaller, and thus the DNA is immobilized. It can be seen that the obtained polysulfone membrane has improved blood compatibility.
[0026]
【The invention's effect】
According to the present invention, a polysulfone membrane whose surface or pores are made hydrophilic by immobilizing DNA as a biological component on the surface or pores of the polysulfone (PSf) membrane, and protein adsorption on the membrane surface A polysulfone membrane having an improved blood compatibility with a smaller amount and a smaller conformational change can be provided. Therefore, if the polysulfone membrane having DNA immobilized on the surface of the present invention is used as a hollow fiber hemodialysis membrane, a clogging or thrombus formation reaction is less likely to occur without using an anti-aggregating agent. Can be provided.
Furthermore, the present invention can be used as a filtration membrane for devices for prostheses and medical devices such as hemodiafiltration, plasma exchange, plasma collection and blood oxygenation during surgery. Further, the present invention can be applied to other technical fields such as engineering, medical, and biotechnology, such as separation membranes such as ultrafiltration membranes, microfiltration membranes, and reverse osmosis membranes.
Further, the polysulfone membrane of the present invention having a hydrophilic membrane surface can be obtained by immersing the polysulfone membrane in a DNA solution, drying at room temperature, irradiating with ultraviolet rays, and then washing with distilled water and storing in water. Since the production can be performed under a simple process and under mild conditions, a hydrophilic polysulfone membrane can be efficiently produced without requiring a large-scale apparatus for the production.
[Brief description of the drawings]
FIG. 1 is an SEM photograph (voltage: 6.0 kV, magnification: 300 ×) showing a cross section and a top view of a polysulfone membrane manufactured by a different manufacturing method.
FIG. 2 is a graph showing the effect of DNA treatment solution concentration on the amount of DNA immobilized on a polysulfone membrane surface and the contact angle.
FIG. 3 (a) is a graph showing the stability of the DNA-immobilized polysulfone membrane in various solutions in the membrane M1. FIG. 3 (b) is a graph showing the stability of the DNA-immobilized polysulfone membrane in various solutions in the membrane M2.
FIG. 4 is a graph showing the amount of DNA immobilized on the surface of a polysulfone membrane produced by different methods.
FIG. 5 is a graph showing contact angles on different polysulfone membrane surfaces before and after immobilizing DNA.
FIG. 6 is a graph showing the amounts of proteins adsorbed on different polysulfone membranes before and after immobilizing DNA, where A is μg / cm.²In units, B is in μg / mg.
FIG. 7 shows infrared rays in the amide I and amide II regions of bovine serum albumin (BSA) adsorbed on a SeZn plate, a plate on which a polysulfone membrane is adsorbed, and a plate on which DNA is immobilized, respectively. It is a graph which shows an absorption spectrum.

Claims (5)

A polysulfone membrane whose surface is made hydrophilic by immobilizing DNA on at least one surface of the polysulfone membrane. 2. The polysulfone membrane according to claim 1, wherein DNA is fixed also inside the pores on the surface of the porous polysulfone membrane. The polysulfone membrane according to claim 1, wherein the DNA is a single helix DNA. The method for producing a polysulfone membrane according to claim 1, comprising immersing the polysulfone membrane in a DNA solution, drying the polysulfone membrane, and then irradiating the membrane with ultraviolet rays to fix the DNA to the polysulfone membrane. A hemodialysis membrane using the polysulfone membrane according to any one of claims 1 to 3.

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