JP2007029716A - Porous substrate - Google Patents
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- JP2007029716A JP2007029716A JP2006169538A JP2006169538A JP2007029716A JP 2007029716 A JP2007029716 A JP 2007029716A JP 2006169538 A JP2006169538 A JP 2006169538A JP 2006169538 A JP2006169538 A JP 2006169538A JP 2007029716 A JP2007029716 A JP 2007029716A
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- human thrombomodulin
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Abstract
Description
本発明は、プロテインＣ活性化能を有した多孔質基材に関する。活性化プロテインＣは血栓形成を阻害するため、抗血栓性が必要な材料に幅広く用いることができる。具体的には、血液浄化用モジュール、人工腎臓、人工肺、体外循環医療カラム用補助用具などの医用材料に好適に用いられる。 The present invention relates to a porous substrate having protein C activation ability. Since activated protein C inhibits thrombus formation, it can be widely used for materials that require antithrombogenicity. Specifically, it is suitably used for medical materials such as a blood purification module, an artificial kidney, an artificial lung, and an auxiliary device for an extracorporeal circulation medical column.
医用材料において生体適合性は重要な問題であり、特に血栓形成を抑制する性質、即ち抗血栓性が要求される。 Biocompatibility is an important problem in medical materials, and in particular, a property of inhibiting thrombus formation, that is, antithrombogenicity is required.
そのため、材料に抗血栓性を付与する試みは、数多く行われている。例えば、血管内カテーテル・人工血管・心臓等は、血液凝固を防ぐためにヘパリンなどをその内面にコーティングする方法（特許文献１）や、アミノ硫酸基を材料表面に導入する方法（特許文献２）が開示されている。 For this reason, many attempts have been made to impart antithrombogenic properties to materials. For example, in an intravascular catheter, artificial blood vessel, heart, etc., a method of coating heparin or the like on its inner surface to prevent blood coagulation (Patent Document 1) or a method of introducing an aminosulfate group on the surface of a material (Patent Document 2). It is disclosed.
そのなかで、近年、材料にプロテインＣの活性化能を付与させることで、抗血栓性を達成させることが注目されてきている。活性化されたプロテインＣは、第Ｖ因子と第VIII因子の不活性化により血液凝固を阻害する。 Among them, in recent years, attention has been focused on achieving antithrombogenicity by imparting protein C activation ability to materials. Activated protein C inhibits blood clotting by inactivation of factor V and factor VIII.
プロテインＣの活性化はトロンボモジュリンにトロンビンが結合することによって数千倍に増幅される。トロンボモジュリンは、血管内皮細胞膜上の糖タンパク質であり、血管内および体外の血液凝固を制御している。すなわち、材料にトロンボモジュリンを固定化することにより、プロテインＣ活性化能を付与することができる。 Protein C activation is amplified several thousand times by binding of thrombin to thrombomodulin. Thrombomodulin is a glycoprotein on the vascular endothelial cell membrane and regulates blood clotting in and outside the blood vessels. That is, protein C activation ability can be imparted by immobilizing thrombomodulin on the material.
ヒト・トロンボモジュリンを材料に固定化する方法として、スペーサーを介してヒト・トロンボモジュリンを不溶性担体に共有結合により固定化する方法（特許文献３）や、ヒト・トロンボモジュリンを疎水化し、有機溶媒中で疎水性基材に被覆させる方法（特許文献４）が開示されている。また、人工腎臓用セルロース中空糸膜にヒト・トロンボモジュリンを固定化する方法が開示されている（ASAIO journal 1995 ; 41 : M369-M374）。しかしながら、これらの方法では、抗血栓性の効果は十分には得られていなかった。抗血栓性の効果を高めるために、グラフト鎖をフィルム表面に導入し、より多くのヒト・トロンボモジュリンを導入しようという試みも行われている（ASAIO journal 1994 ; 40 : M840-M845）が、このような方法でも、抗血栓性の効果には限界があること、また、操作が煩雑であるといった問題点があった。 As a method of immobilizing human thrombomodulin on a material, a method of covalently immobilizing human thrombomodulin to an insoluble carrier via a spacer (Patent Document 3), or hydrophobizing human thrombomodulin and making it hydrophobic in an organic solvent A method (Patent Document 4) for coating a substrate is disclosed. In addition, a method for immobilizing human thrombomodulin on a cellulose hollow fiber membrane for artificial kidney has been disclosed (ASAIO journal 1995; 41: M369-M374). However, these methods have not sufficiently obtained an antithrombotic effect. In order to increase the antithrombogenic effect, attempts have been made to introduce more human thrombomodulin (ASAIO journal 1994; 40: M840-M845) by introducing graft chains onto the film surface. However, there is a problem that the antithrombogenic effect is limited and the operation is complicated.
本発明の目的は、かかる従来技術の欠点を改良し、高いプロテインＣ活性化能をもつ抗血栓性の多孔質基材を提供することにある。 An object of the present invention is to provide an antithrombotic porous substrate having improved protein C activation ability by improving the drawbacks of the prior art.
本願発明者らは、鋭意研究の結果、担体として多孔質基材を用いると共に、担体の乾燥重量１ｇあたりプロテインＣを活性化させる生理活性物質を１μg以上保持しており、特に担体と血液とが直に接する表面だけではなく、担体の内孔部にもプロテインＣを活性化させる生理活性物質を導入することによって上記目的を達成することができることを見出し、本発明を完成した。 As a result of intensive studies, the inventors of the present invention use a porous substrate as a carrier and hold 1 μg or more of a physiologically active substance that activates protein C per gram of the dry weight of the carrier. It has been found that the above object can be achieved by introducing a physiologically active substance that activates protein C not only into the directly contacting surface but also into the inner pores of the carrier, thereby completing the present invention.
すなわち、本発明は、以下の構成を有する多孔質基材を提供する。
(1) 担体の乾燥重量１ｇあたりプロテインＣを活性化させる生理活性物質を１μg以上保持する多孔質基材。
(2) 前記の生理活性物質を担体表面および内孔部に保持していることを特徴とする(1)に記載の多孔質基材。
(3) 前記の生理活性物質がプロテインＣを２０％以上活性化させることを特徴とする(1)〜(2)のいずれかに記載の多孔質基材。
(4) 前記の生理活性物質がヒト・トロンボモジュリンもしくはその一部を有するペプチドであることを特徴とする(1)〜(3)のいずれかに記載の多孔質基材。
(5) 前記の担体が多孔質分離膜、多孔質ビーズ、多孔質繊維のいずれかであることを特徴とする請求項(1)〜(4)のいずれかに記載の多孔質基材。
(6) 前記の担体が多孔質分離膜であって、膜の両面を前記生理活性物質の水溶液に浸潤させ、またはさらに濾過をかけることによって得られた、(1)〜(5)のいずれかに記載の多孔質基材。
That is, the present invention provides a porous substrate having the following configuration.
(1) A porous substrate that holds 1 μg or more of a physiologically active substance that activates protein C per 1 g of dry weight of the carrier.
(2) The porous substrate according to (1), wherein the physiologically active substance is held on the surface of the carrier and the inner pore.
(3) The porous substrate according to any one of (1) to (2), wherein the physiologically active substance activates protein C by 20% or more.
(4) The porous substrate according to any one of (1) to (3), wherein the physiologically active substance is human thrombomodulin or a peptide having a part thereof.
(5) The porous substrate according to any one of (1) to (4), wherein the carrier is any one of a porous separation membrane, porous beads, and porous fibers.
(6) The carrier is a porous separation membrane, and is obtained by infiltrating both sides of the membrane into the aqueous solution of the physiologically active substance, or by further filtering, (1) to (5) A porous substrate as described in 1.
本発明によって、高いプロテインＣ活性化能をもつ抗血栓性の多孔質基材を提供することができる。 According to the present invention, an antithrombotic porous substrate having high protein C activation ability can be provided.
本発明は、担体の乾燥重量１ｇあたりプロテインＣを活性化させる生理活性物質を１μg以上保持していることを特徴とする多孔質基材であり、特に担体の表面だけではなく、内孔部にも保持していることが好ましい。プロテインＣを活性化させる生理活性物質（以下、単に生理活性物質という）を、多孔質の担体に付与させることで、担体重量あたりの生理活性物質の存在量を大幅に増やすことができる。 The present invention is a porous substrate characterized in that 1 μg or more of a physiologically active substance that activates protein C per 1 g of dry weight of the carrier is retained, and not only on the surface of the carrier but also in the inner pores. Is also preferably retained. By providing a porous carrier with a physiologically active substance that activates protein C (hereinafter simply referred to as a physiologically active substance), the amount of the physiologically active substance per carrier weight can be greatly increased.
プロテインＣを活性化させることで抗血栓性が得られるメカニズムは、以下の通りである。すなわち、活性化されたプロテインＣは第Ｖ因子と第VIII因子の不活性化により血液凝固を阻害する。プロテインＣの活性化はヒト・トロンボモジュリンにトロンビンが結合することによって数千倍に増幅される。ヒト・トロンボモジュリンは、血管内皮細胞膜上の糖タンパク質であり、血管内および体外の血液凝固を制御している。また、トロンビンはヒト・トロンボモジュリンと結合することで、凝固因子であるトロンビンのフィブリン形成作用および血小板凝集作用等が消失される。このため、生理活性物質としては、合成ペプチドやヒト・トロンボモジュリンなどのタンパク質もしくはその一部を有するペプチドなどが好適に用いられるが、トロンビンの凝固作用を消失できる機能を有していれば、抗血栓性にさらに有利であるため、ヒト・トロンボモジュリンもしくはその構造の一部分を有するペプチドが特に好適に用いられる。 The mechanism of obtaining antithrombogenicity by activating protein C is as follows. That is, activated protein C inhibits blood clotting by inactivating factor V and factor VIII. Protein C activation is amplified thousands of times by binding of thrombin to human thrombomodulin. Human thrombomodulin is a glycoprotein on the vascular endothelial cell membrane and regulates blood clotting both inside and outside the blood vessels. In addition, thrombin binds to human thrombomodulin, thereby eliminating the fibrin forming action and platelet aggregation action of thrombin, a coagulation factor. For this reason, as a physiologically active substance, a synthetic peptide, a protein such as human thrombomodulin or a peptide having a part thereof is preferably used. However, as long as it has a function capable of eliminating the coagulation action of thrombin, Since it is further advantageous for sex, human thrombomodulin or a peptide having a part of its structure is particularly preferably used.
つまり、ヒト・トロンボモジュリンがプロテインＣやトロンビンに接触できれば、抗血栓性が発揮される。したがって、ヒト・トロンボモジュリンが多孔質内孔部に存在していても、プロテインＣやトロンビンが多孔質内孔部に侵入できればよい。このような多孔質基材は、担体重量あたりのヒト・トロンボモジュリンの存在量が非常に多いので、良好な抗血栓性材料となる。 That is, if human thrombomodulin can contact protein C or thrombin, antithrombotic properties are exhibited. Therefore, even if human thrombomodulin is present in the porous inner pore portion, it is sufficient that protein C and thrombin can enter the porous inner pore portion. Such a porous substrate is a good antithrombogenic material because the amount of human thrombomodulin present per carrier weight is very large.
多孔質基材の特徴は、比表面積が大きいことである。比表面積が大きいほど、ヒト・トロンボモジュリンなどの生理活性物質をより多く保持できる。比表面積とは、重量あたりの表面積であり、多孔質体の場合は、内孔部を含めた表面積を指す。特に限定されるものではないが、比表面積は、１０００ｃｍ２／ｇ以上、好ましくは５０００ｃｍ２／ｇ以上、さらに好ましくは１００００ｃｍ２／ｇ以上である。 The feature of the porous substrate is that the specific surface area is large. The larger the specific surface area, the more biological active substances such as human thrombomodulin can be retained. The specific surface area is the surface area per weight, and in the case of a porous body, it refers to the surface area including the inner pores. Although not particularly limited, specific surface area, 1000 cm 2 / g or more, preferably 5000 cm 2 / g or more, more preferably 10000 cm 2 / g or more.
比表面積の値は、測定方法によって異なる場合があるので、注意が必要である。多孔質体の平均孔径が１００ｎｍ以下の場合は、気体吸着法が適しており、平均孔径が１００ｎｍ以上の場合は、水銀注入法が適している。平均孔径は、それぞれの測定方法において算出できる。 Since the value of the specific surface area may vary depending on the measurement method, it should be noted. When the average pore diameter of the porous body is 100 nm or less, the gas adsorption method is suitable, and when the average pore diameter is 100 nm or more, the mercury injection method is suitable. The average pore diameter can be calculated by each measurement method.
担体の乾燥重量１ｇあたり生理活性物質が１μg以上、好ましくは１０μg以上付与されていれば、十分な抗血栓性を得ることができる。なお、生理活性物質の付与量の上限は特にないが、コストなどの点から、通常、担体の乾燥重量１ｇあたり１００ｍｇ以下である。ここで乾燥重量とは、担体を乾燥させて、乾燥中の１時間での重量変化率が３％以内になった状態の重量をいう。 If the physiologically active substance is added in an amount of 1 μg or more, preferably 10 μg or more per 1 g of dry weight of the carrier, sufficient antithrombogenicity can be obtained. There is no particular upper limit on the amount of physiologically active substance applied, but it is usually 100 mg or less per gram of dry weight of the carrier from the viewpoint of cost. Here, the dry weight means the weight in a state where the carrier is dried and the weight change rate within 1 hour during drying is within 3%.
また、多孔質基材が分離膜の場合には、血液処理時に濾過作用が働くため、孔径が大きいと赤血球が分離膜中でトラップされてしまい、溶血を惹起するなどの懸念がある。また孔径が小さすぎると、プロテインＣが多孔質内部に入りにくくなるために、生理活性物質の存在量に比べて抗血栓性の効果が低くなる。このため、本発明において好適に用いられる分離膜は、デキストランの篩い係数が０．５の点のデキストラン分子量が５０００以上、好ましくは１万以上、さらに好ましくは１．５万以上であり、また、１０００万以下、好ましくは１００万以下、さらに好ましくは１０万以下である。 In addition, when the porous substrate is a separation membrane, a filtering action works during blood treatment. Therefore, if the pore size is large, there is a concern that red blood cells are trapped in the separation membrane and cause hemolysis. On the other hand, if the pore size is too small, protein C will not easily enter the inside of the porous body, so the antithrombotic effect will be lower than the amount of physiologically active substance present. For this reason, the separation membrane suitably used in the present invention has a dextran molecular weight of 5,000 or more, preferably 10,000 or more, more preferably 15,000 or more, with a dextran sieving coefficient of 0.5. It is 10 million or less, preferably 1 million or less, and more preferably 100,000 or less.
なお、デキストランの篩い係数測定において、分離膜の断面が非対称構造を有する場合、孔径が小さい方の面から、大きい方の面へデキストラン溶液を透過させる。 In the measurement of the dextran sieving coefficient, when the cross section of the separation membrane has an asymmetric structure, the dextran solution is permeated from the surface with the smaller pore diameter to the surface with the larger pore size.
なお、生理活性物質が保持されている担体は、多孔質であることは必要とされるが、分離膜のような貫通孔が必須なわけではない。 The carrier holding the physiologically active substance is required to be porous, but a through-hole such as a separation membrane is not essential.
ここで、担体とは、プロテインＣを活性化させる生理活性物質を付与させる材料のことを指し、高分子材料が好ましい。高分子材料の例としては、ポリスルホンやポリスチレン、ポリウレタン、ポリエチレン、ポリプロピレン、ポリ塩化ビニル、セルロースや、これらの誘導体などが挙げられる。基材の形状としては、多孔質体であれば、特に問わないが、分離膜やビーズ、繊維が好適に用いられる。分離膜としては、中空糸膜でも、平膜であってもよい。これらは、具体的には、抗血栓性が必要な材料、例えば、血液浄化用モジュール、人工腎臓、人工肺、体外循環医療カラム用補助用具などの医用材料に好適に用いられる。 Here, the carrier refers to a material imparting a physiologically active substance that activates protein C, and a polymer material is preferable. Examples of the polymer material include polysulfone, polystyrene, polyurethane, polyethylene, polypropylene, polyvinyl chloride, cellulose, and derivatives thereof. The shape of the substrate is not particularly limited as long as it is a porous body, but a separation membrane, beads, and fibers are preferably used. The separation membrane may be a hollow fiber membrane or a flat membrane. Specifically, these are suitably used for materials requiring antithrombogenicity, for example, medical materials such as blood purification modules, artificial kidneys, artificial lungs, and extracorporeal circulation medical column auxiliary tools.
また、本発明でいうところの担体に生理活性物質を保持させるとは、担体と生理活性物質が相互作用している状態を指す。相互作用としては化学結合、吸着などが挙げられる。 In addition, holding the physiologically active substance in the carrier referred to in the present invention refers to a state in which the carrier and the physiologically active substance interact. Examples of the interaction include chemical bonding and adsorption.
例えば、担体を成型後、化学反応を用いて生理活性物質を架橋させても良い。このとき、生理活性物質と担体の間にスペーサーを介しても良い。化学反応を用いて架橋させる例としては、担体および生理活性物質にアミノ基やカルボキシル基がある場合には、１-エチル-３-（３-ジメチルアミノプロピル）カルボジイミド塩酸などの縮合剤を用いて生理活性物質と担体に化学結合を形成させることができる。 For example, after molding the carrier, the physiologically active substance may be cross-linked using a chemical reaction. At this time, a spacer may be interposed between the physiologically active substance and the carrier. As an example of crosslinking using a chemical reaction, when the carrier and the physiologically active substance have an amino group or a carboxyl group, a condensing agent such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is used. A chemical bond can be formed between the physiologically active substance and the carrier.
また、生理活性物質にアミノ基がある場合は、クロルアセトアミドメチル基をもつ担体と化学結合を形成させることができる。 In addition, when the physiologically active substance has an amino group, it can form a chemical bond with a carrier having a chloroacetamidomethyl group.
さらには、担体に生理活性物質を吸着させても良い。吸着は、間接的な相互作用であってもよい。例えば、生理活性物質が担体と水分子を介した水素結合によって吸着されていても良いし、担体上に電荷をもつ物質を導入し、静電相互作用によって吸着されていてもよい。具体的には、下記のいずれかよって、生理活性物質の担体への吸着が達成できるが、これに限定されるわけではない。１）担体を生理活性物質溶液に浸漬させる。２）担体を生理活性物質溶液に浸漬させた後、溶液からに抜き出し、湿潤状態で保持させる。３）担体を生理活性物質溶液に浸漬させた後、溶液から抜き出し、水に浸漬させる。 Furthermore, a physiologically active substance may be adsorbed on the carrier. Adsorption may be an indirect interaction. For example, a physiologically active substance may be adsorbed by hydrogen bonding via a carrier and water molecules, or a substance having a charge may be introduced onto the carrier and adsorbed by electrostatic interaction. Specifically, the adsorption of the physiologically active substance to the carrier can be achieved by any of the following, but is not limited thereto. 1) The carrier is immersed in the physiologically active substance solution. 2) After immersing the carrier in the physiologically active substance solution, the carrier is extracted from the solution and kept wet. 3) After immersing the carrier in the physiologically active substance solution, the carrier is extracted from the solution and immersed in water.
ここで、湿潤状態とは、担体を浸漬していた溶液を除去して乾燥させない状態のことを言う。特に限定されるものではないが、担体の乾燥重量に対して３重量％以上の水分を含んでいることが好ましい。 Here, the wet state means a state where the solution in which the carrier is immersed is removed and not dried. Although not particularly limited, it preferably contains 3% by weight or more of moisture with respect to the dry weight of the carrier.
また、生理活性物質溶液の溶媒としては、水が好適に用いられるが、緩衝液などのように塩を含んでいても、アルコールなどの有機溶媒を含んでいてもよい。 In addition, water is preferably used as a solvent for the physiologically active substance solution, but it may contain a salt such as a buffer solution or an organic solvent such as alcohol.
成型後の担体に生理活性物質を多孔質内孔部に保持させるためには、担体を反応溶液や吸着させるための生理活性物質溶液にすべて浸るようにしたほうが良い。例えば、人工腎臓などに使用される多孔質中空糸膜の場合は、中空糸膜の内側と外側に生理活性物質溶液を満たすことによって、簡便に多孔質内孔部に保持させることができる。さらには、これらの溶液を濾過をかけて導入することで、効率的に多孔質内孔部に導入することができる。 In order to retain the physiologically active substance in the porous inner pores in the carrier after molding, it is better to immerse the carrier in the reaction solution or the physiologically active substance solution for adsorption. For example, in the case of a porous hollow fiber membrane used for an artificial kidney or the like, it can be easily held in the porous inner hole portion by filling the inside and outside of the hollow fiber membrane with a physiologically active substance solution. Furthermore, these solutions can be efficiently introduced into the porous inner pores by introducing them through filtration.
ここでいう濾過とは、分離膜の場合は、分離膜を通して溶液が濾されることをいう。濾過がかかった状態とは、溶液の通液流量の１％以上好ましくは１０％以上、さらに好ましくは５０％以上が濾液として出てきている状態をいう。分離膜が平膜で、濾液流量が分離膜の場所によって不均一である場合は、分離膜全体の平均値を取ればよい。多孔質ビーズにおける濾過とは、カラムに内蔵された際、上記の溶液を導入する際に、カラムの入口と出口で、１０ｍｍＨｇ以上、好ましくは２０ｍｍＨｇ以上、さらに好ましくは３０ｍｍＨｇ以上の圧力差を生じるように圧力をかけた状態で導入することをいう。 In the case of a separation membrane, the filtration here means that the solution is filtered through the separation membrane. The filtered state refers to a state in which 1% or more, preferably 10% or more, more preferably 50% or more of the flow rate of the solution has come out as a filtrate. When the separation membrane is a flat membrane and the filtrate flow rate is uneven depending on the location of the separation membrane, an average value of the entire separation membrane may be taken. The filtration in the porous beads means that when the above solution is introduced into the column, a pressure difference of 10 mmHg or more, preferably 20 mmHg or more, more preferably 30 mmHg or more is generated at the inlet and outlet of the column. Introducing under pressure.
また、担体成型原液に生理活性物質を混練させ、物理的に生理活性物質を担体に内包させる形にしても良い。混練させることで、生理活性物質を多孔質の内孔部に保持させることができるので好ましいが、成型原液中で生理活性物質が変性しないようにすることが必要である。 Alternatively, a physiologically active substance may be kneaded in the carrier molding stock solution, and the physiologically active substance may be physically encapsulated in the carrier. Kneading is preferable because the physiologically active substance can be held in the porous inner pore, but it is necessary to prevent the physiologically active substance from being denatured in the molding stock solution.
担体に保持した生理活性物質の溶出は少ないことが望まれる。溶出量が多いと、患者によっては出血傾向を助長するなど、安全性に問題が生じる可能性がある。特に限定されるわけではないが、担体の乾燥重量１ｇあたり２７３ｍｌの水で洗浄した後、室温で４５ｍｌの水を用いて２時間抽出した水中の生理活性物質濃度が、１０ｎｇ／ｍｌ以下であることが好ましい。ここで、水の代わりに生理食塩水もしくは緩衝液を用いてもよい。 Less elution of the physiologically active substance held on the carrier is desired. If the amount of elution is large, there may be a problem in safety, such as promoting a bleeding tendency in some patients. Although not particularly limited, the concentration of the physiologically active substance in water extracted with 45 ml of water at room temperature for 2 hours after washing with 273 ml of water per gram of dry weight of the carrier is 10 ng / ml or less. Is preferred. Here, physiological saline or a buffer may be used instead of water.
なお、本発明においては、プロテインＣの活性が２０％以上増加する生理活性物質を用いることが好ましい。プロテインＣの活性化度の測定方法についての詳細は後述するが、４０５ｎｍの吸光度で比較する。プロテインＣが活性化されているとは、吸光度の値がブランクよりも高いことをいう。プロテインＣの活性が２０％以上増加しているとは、ブランクに対して、吸光度の値が２０％以上増加していることを意味する。プロテインＣの活性化が２０％より少ない場合、抗血栓性の効果が充分でない場合がある。 In the present invention, it is preferable to use a physiologically active substance that increases the activity of protein C by 20% or more. Details of the method for measuring the degree of activation of protein C will be described later. Protein C being activated means that the absorbance value is higher than that of the blank. The fact that the activity of protein C is increased by 20% or more means that the absorbance value is increased by 20% or more with respect to the blank. If the activation of protein C is less than 20%, the antithrombotic effect may not be sufficient.
以下実施例を挙げて本発明を説明するが、本発明はこれらの例によって限定されるものではない。 EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.
実施例１
１．ポリスルホン／ポリビニルピロリドン混合中空糸膜ミニカラム
抗血栓性を付与する担体にポリスルホン／ポリビニルピロリドン混合中空糸膜を用いた。人工腎臓のモデルとして、該中空糸膜のミニカラムを作成した。ポリスルホン（ソルベイ社製ユーデルポリスルホン（登録商標）Ｐ-３５００）１８重量部およびポリビニルピロリドン（ＢＡＳＦ社製Ｋ３０）９重量部をＮ，Ｎ'-ジメチルアセトアミド７２重量部および水１重量部の混合溶媒に加え、９０℃で１４時間加熱して溶解し、製膜原液を得た。この製膜原液を外側の内径０．３ｍｍ、内側の内径０．２ｍｍのオリフィス型二重円筒型口金の外側の管より吐出した。芯液としてＮ，Ｎ'-ジメチルアセトアミド５８重量部および水４２重量部からなる溶液を内側の管より吐出した。吐出された製膜原液は、乾式長３５０ｍｍを通過した後、水１００％の凝固浴に導かれ、中空糸が得られた。
Example 1
1. Polysulfone / polyvinylpyrrolidone mixed hollow fiber membrane minicolumn A polysulfone / polyvinylpyrrolidone mixed hollow fiber membrane was used as a carrier for imparting antithrombogenicity. As a model of an artificial kidney, a minicolumn of the hollow fiber membrane was prepared. A mixed solvent of 18 parts by weight of polysulfone (Udelpolysulfone (registered trademark) P-3500 manufactured by Solvay) and 9 parts by weight of polyvinylpyrrolidone (K30 manufactured by BASF) and 72 parts by weight of N, N′-dimethylacetamide and 1 part by weight of water In addition, the film was dissolved by heating at 90 ° C. for 14 hours to obtain a film forming stock solution. This film-forming stock solution was discharged from an outer tube of an orifice type double cylindrical die having an inner diameter of 0.3 mm and an inner diameter of 0.2 mm. A solution consisting of 58 parts by weight of N, N′-dimethylacetamide and 42 parts by weight of water was discharged from the inner tube as the core liquid. The discharged film-forming stock solution passed through a dry length of 350 mm, and was then introduced into a 100% water coagulation bath to obtain a hollow fiber.
中空糸膜を１００本束ね、直径約７ｍｍ、長さは１２ｃｍのプラスチック管ミニカラムケースに挿入した。中空糸膜の両末端を、中空糸膜中空部を閉塞しないようにウレタン系ポッティング剤で固定し、図１に示すようなミニカラムを作成した。ミニカラムの中空糸膜内側および外側を３７℃の超純水を１ｍｌ／分の流速で３０分間洗浄した。 100 hollow fiber membranes were bundled and inserted into a plastic tube mini-column case having a diameter of about 7 mm and a length of 12 cm. Both ends of the hollow fiber membrane were fixed with a urethane potting agent so as not to block the hollow portion of the hollow fiber membrane, and a minicolumn as shown in FIG. 1 was prepared. The inside and outside of the hollow fiber membrane of the mini column were washed with ultrapure water at 37 ° C. for 30 minutes at a flow rate of 1 ml / min.
２．比表面積の測定
日本ベル（株）製高精度全自動ガス吸着装置（ＢＥＬＳＯＲＰ ３６）を用いて、中空糸膜を１００℃で減圧脱気の後、液体窒素温度（７７Ｋ）で窒素の吸着等温線を測定した。この等温線をＢＥＴ法で解析し、比表面積を求めた。比表面積は、１８６ｍ２／ｇであった。
2. Measurement of specific surface area Using a high-precision fully automatic gas adsorption device (BELSORP 36) manufactured by Nippon Bell Co., Ltd., degassing the hollow fiber membrane at 100 ° C, followed by adsorption isotherm of nitrogen at liquid nitrogen temperature (77K) Was measured. This isotherm was analyzed by the BET method to determine the specific surface area. The specific surface area was 186 m 2 / g.
３．デキストランの篩い係数測定
分子量１５００，６０００，２万，４万，６万，２０万の６種類のデキストランが各０．５重量％水溶液になるように調整した。
3. Measurement of dextran sieving coefficient Six kinds of dextran having molecular weights of 1500, 6000, 20,000, 40,000, 60,000 and 200,000 were adjusted to be 0.5 wt% aqueous solutions.
中空糸膜ミニカラムの図１の１に、内径２ｍｍ、外径４ｍｍ、長さ５０ｃｍのシリコーンチューブ（製品名ＡＲＡＭ（登録商標））を、図１の２に、内径２ｍｍ、外径４ｍｍ、長さ１００ｃｍのシリコーンチューブを接続した。中空糸膜ミニカラムの図１の４はキャップをつけ、図１の３は、開放した。 A hollow fiber membrane mini-column 1 in FIG. 1 has a silicone tube (product name: ARAM (registered trademark)) having an inner diameter of 2 mm, an outer diameter of 4 mm, and a length of 50 cm, and FIG. 1 has an inner diameter of 2 mm, an outer diameter of 4 mm, and a length. A 100 cm silicone tube was connected. The hollow fiber membrane mini-column 4 in FIG. 1 was capped and 3 in FIG. 1 was opened.
中空糸膜ミニカラムを垂直に立てて、デキストラン水溶液を下から上（図１の２から１）へワンパスで通液した。このとき、入口側（図１の２）の流速を５ｍｌ／ｍｉｎで、出口側（図１の１）の流速を４ｍｌ／ｍｉｎとすることで、中空糸膜ミニカラムの図１の３から１ｍｌ／ｍｉｎの濾液が出てくるようにした。 The hollow fiber membrane mini-column was set up vertically, and the aqueous dextran solution was passed from the bottom to the top (2 to 1 in FIG. 1) in one pass. At this time, by setting the flow rate on the inlet side (2 in FIG. 1) to 5 ml / min and the flow rate on the outlet side (1 in FIG. 1) to 4 ml / min, 3 to 1 ml / min of the hollow fiber membrane minicolumn in FIG. The filtrate of min was made to come out.
通流開始４分後から３分間、中空糸膜ミニカラムの図１の３から出てくる濾液をサンプリングした。この液を、ｆ液と呼ぶ。 The filtrate from 3 of FIG. 1 of the hollow fiber membrane mini-column was sampled for 3 minutes from 4 minutes after the start of flow. This liquid is called liquid f.
通液開始から７分後に、通液を停止し、図１の１に接続した１００ｃｍのチューブ内に残っているデキストラン水溶液をサンプリングした。この液を、Ｂｏ液と呼ぶ。また、中空糸膜ミニカラムに通過する前のデキストラン水溶液をＢｉ液と呼ぶ。 Seven minutes after the start of the flow, the flow was stopped, and the dextran aqueous solution remaining in the 100 cm tube connected to 1 in FIG. 1 was sampled. This liquid is called Bo liquid. The aqueous dextran solution before passing through the hollow fiber membrane minicolumn is referred to as Bi solution.
これらのサンプル液をゲルパーミエーションクロマトグラフィにて、デキストランの分子量と濃度を測定し、デキストランの各分子量における篩い係数を算出した。その結果、篩い係数が０．５のときのデキストラン分子量は１．８万であった。 These sample solutions were subjected to gel permeation chromatography to measure the molecular weight and concentration of dextran, and the sieving coefficient at each molecular weight of dextran was calculated. As a result, the dextran molecular weight when the sieving coefficient was 0.5 was 18,000.
４．ヒト・トロンボモジュリン
プロテインＣを活性化させる生理活性物質として、ヒト・トロンボモジュリンを使用した。ヒト・トロンボモジュリンとしては、特開平１-６２１９号公報の記載に従い、遺伝子工学的手法によって得たヒト・トロンボモジュリンを用いた。
4). Human thrombomodulin Human thrombomodulin was used as a physiologically active substance that activates protein C. As human thrombomodulin, human thrombomodulin obtained by a genetic engineering technique was used in accordance with the description in JP-A-1-6219.
このヒト・トロンボモジュリン１ｎｇを５ｕｎｉｔ／ｍｌのトロンビンおよび１００ｎＭのプロテインＣを含む溶液（５０ｍＭトリス-塩酸緩衝液（ｐＨ８．０）、０．１重量％ＮａＣｌ、０．１重量％ＢＳＡ、２ｍＭ ＣａＣｌ２）０．５ｍｌ中３７℃で１時間インキュベートした。１０ｕｎｉｔ／ｍｌのアンチトロンビンIIIおよび８ｕｎｉｔ／ｍｌのヘパリンを含む溶液（５０ｍＭトリス-塩酸緩衝液（ｐＨ８．０）、０．１重量％ＮａＣｌ、０．１重量％ＢＳＡ、２ｍＭ ＣａＣｌ２）０．５ｍｌを加え、さらに３７℃で３０分間インキュベートし、ヒト・トロンボモジュリンとプロテインＣとの反応を停止させた。 A solution containing 1 ng of this human thrombomodulin containing 5 units / ml thrombin and 100 nM protein C (50 mM Tris-HCl buffer (pH 8.0), 0.1 wt% NaCl, 0.1 wt% BSA, 2 mM CaCl 2 ) Incubated for 1 hour at 37 ° C. in 0.5 ml. 0.5 ml of a solution containing 10 units / ml antithrombin III and 8 units / ml heparin (50 mM Tris-HCl buffer (pH 8.0), 0.1 wt% NaCl, 0.1 wt% BSA, 2 mM CaCl 2 ) And further incubated at 37 ° C. for 30 minutes to stop the reaction between human thrombomodulin and protein C.
この上清５０μｌに１ｍＭのＳ-２２３８（２０ｍＭトリス-塩酸緩衝液（ｐＨ７．４）、０．１重量％ＮａＣｌ、０．１重量％ＢＳＡの混合溶液）１００μｌを加えて３７℃で５分間インキュベートした。酢酸５μlを加えて発色を停止させた後、４０５ｎｍの吸光度を測定した。その結果、吸光度は０．１５８であった。一方で、トロンボモジュリンを添加しなかったブランク溶液の吸光度は０．０６１であった。ブランクに対して吸光度は２５０％増加していることから、このヒト・トロンボモジュリンがプロテインＣ活性化能を有していることが確認できた。 To 50 μl of this supernatant, 100 μl of 1 mM S-2238 (20 mM Tris-HCl buffer (pH 7.4), 0.1 wt% NaCl, 0.1 wt% BSA mixed solution) was added and incubated at 37 ° C. for 5 minutes. did. The color development was stopped by adding 5 μl of acetic acid, and then the absorbance at 405 nm was measured. As a result, the absorbance was 0.158. On the other hand, the absorbance of the blank solution to which thrombomodulin was not added was 0.061. Since the absorbance increased by 250% with respect to the blank, it was confirmed that this human thrombomodulin has the ability to activate protein C.
５．ヒト・トロンボモジュリンの導入
ヒト・トロンボモジュリンのリン酸緩衝生理食塩水（以下、ＰＢＳ）溶液（ヒト・トロンボモジュリン濃度は２５μｇ／ｍｌ）２．５ｍｌを、前記１.で得られた中空糸膜ミニカラム（図１）の１から２へ、続いて３から４へ通液し（図１中の矢印参照）、１０分間、灌流させた。灌流は、室温で、流速は１ｍｌ／分で行なった。中空糸膜内側のみならず外側にも灌流させることで、ヒト・トロンボモジュリンを中空糸内側、外側両方に満たし、中空糸膜の内孔部にもヒト・トロンボモジュリンを保持させた。
5. Introduction of human thrombomodulin 2.5 ml of a phosphate buffered saline (hereinafter referred to as PBS) solution of human thrombomodulin (human thrombomodulin concentration of 25 μg / ml) was added to the hollow fiber membrane mini-column obtained in 1 above (FIG. 1). ) From 1 to 2 and then from 3 to 4 (see arrows in FIG. 1), and allowed to perfuse for 10 minutes. Perfusion was performed at room temperature and a flow rate of 1 ml / min. By irrigating not only the inside of the hollow fiber membrane but also the outside, the human thrombomodulin was filled both inside and outside the hollow fiber, and the human thrombomodulin was also retained in the inner pores of the hollow fiber membrane.
６．ヒト・トロンボモジュリンの溶出実験
中空糸膜ミニカラムについて、ヒト・トロンボモジュリン溶出試験を始める直前に、ＰＢＳ３０ｍｌ（中空糸内表面１ｍ２換算でＰＢＳ４Ｌ、中空糸膜乾燥重量１ｇ換算で約２７３ｍｌに相当する）を室温にて１ｍｌ／分で３０分循環し、洗浄した。ＰＢＳ５ｍｌ（中空糸内表面１ｍ２換算で６６７ｍＬ、中空糸膜乾燥重量１ｇ換算で約４５ｍｌに相当する）を０．５ｍｌ／分の流速で、室温で２時間循環させた。なお、後述する血液循環試験と対応させるために、循環開始後の最初の１ｍｌは廃棄した後、循環した。２時間循環させた液に溶出したヒト・トロンボモジュリンの濃度は、ＥＬＩＳＡ法にて測定した。
6). Human thrombomodulin elution experiment Immediately before starting the human thrombomodulin elution test, 30 ml of PBS (corresponding to PBS 4L in terms of 1 m 2 of hollow fiber inner surface and approximately 273 ml in terms of 1 g of dry weight of hollow fiber membrane) was measured at room temperature. Was circulated at 1 ml / min for 30 minutes and washed. 5 ml of PBS (equivalent to 667 mL in terms of 1 m 2 of hollow fiber inner surface and approximately 45 ml in terms of dry fiber membrane dry weight of 1 g) was circulated at room temperature for 2 hours at a flow rate of 0.5 ml / min. In order to correspond to the blood circulation test described later, the first 1 ml after the start of circulation was discarded and then circulated. The concentration of human thrombomodulin eluted in the solution circulated for 2 hours was measured by ELISA.
７．ヒト・トロンボモジュリン濃度測定
ヒト・トロンボモジュリンの濃度は、ヒトＣＤ１４１ ＥＬＩＳＡキット（ＤＩＡＣＬＯＮＥ社製造）を使用した。実験手順は、以下の通りである。ＥＬＩＳＡプレートのウェルにサンプルを１００μｌずつ加えた。ビオチン化ａｎｔｉ-ＣＤ１４１を調整し、５０μｌずつウェルに加えた。プレートカバーでカバーし、１時間室温下で定温放置した。カバーを外し、プレートをマイクロプレートウオッシャー（ＢＩＯ-ＲＡＤ ＩｍｍｕｎｏＷａｓｈ Ｍｏｄｅｌ １５７５）にて、以下のように洗浄した。
１） 全てのウェルから溶液を吸い出した。
２） 全てのウェルに洗浄液を３００μｌずつ満たした。
３） 再度全てのウェルから溶液を吸い出した。
４） 手順２）と３）を二度繰り返した。
7). Measurement of Human Thrombomodulin Concentration The human CD141 ELISA kit (manufactured by DIACLONE) was used for the concentration of human thrombomodulin. The experimental procedure is as follows. 100 μl of sample was added to each well of the ELISA plate. Biotinylated anti-CD141 was prepared and 50 μl was added to each well. Covered with a plate cover and allowed to stand at room temperature for 1 hour. The cover was removed, and the plate was washed with a microplate washer (BIO-RAD ImmunoWash Model 1575) as follows.
1) Solution was aspirated from all wells.
2) All wells were filled with 300 μl of washing solution.
3) The solution was aspirated again from all wells.
4) Procedures 2) and 3) were repeated twice.
洗浄操作後、ストレプトアビジン-ＨＲＰ溶液を調整し、ウェルに１００μｌ加えた。プレートカバーでカバーし、３０分間室温下で定温放置した。カバーを外してウェルを、上記洗浄操作１）〜４）と同様に洗浄を行った。ｒｅａｄｙ-ｕｓｅ（レディ・ユース）ＴＭＢ基質溶液をウェルに１００μｌ加えた。プレートをアルミホイルで包んで光を遮断し１５分間室温で定温放置した。ウェルに１００μｌの停止試薬Ｈ２ＳＯ４を加えて、酵素基質反応を停止した。停止試薬Ｈ２ＳＯ４を加えた後に、速やかにマイクロプレートリーダー（ＢＩＯ-ＲＡＤ マイクロプレートリーダー モデル６８０）を用いて４５０ｎｍの吸光度を読み取った。既知濃度の吸光度の値から検量線を引き、サンプルのヒト・トロンボモジュリン濃度を算出した。 After the washing operation, a streptavidin-HRP solution was prepared, and 100 μl was added to the well. The plate was covered with a plate cover and allowed to stand at room temperature for 30 minutes. After removing the cover, the wells were washed in the same manner as in the washing operations 1) to 4). 100 μl of ready-use TMB substrate solution was added to the wells. The plate was wrapped with aluminum foil to block the light and left at room temperature for 15 minutes. The enzyme substrate reaction was stopped by adding 100 μl of stop reagent H 2 SO 4 to the wells. After adding the stop reagent H 2 SO 4 , the absorbance at 450 nm was immediately read using a microplate reader (BIO-RAD microplate reader model 680). A calibration curve was drawn from the absorbance value at a known concentration to calculate the human thrombomodulin concentration of the sample.
８．抗血栓性の評価（血液循環試験）
中空糸膜ミニカラムの片側（図１の１）に、内径１ｍｍ、外径２ｍｍ、長さ５０ｃｍのシリコーンチューブ（製品名ＡＲＡＭ（登録商標））を接続した。中空糸膜ミニカラムについて、血液循環試験を始める直前に、ＰＢＳ３０ｍｌを室温にて１ｍｌ／分で３０分、図１の１から２へ循環し、洗浄した。
8). Antithrombogenicity evaluation (blood circulation test)
A silicone tube (product name: ARAM (registered trademark)) having an inner diameter of 1 mm, an outer diameter of 2 mm, and a length of 50 cm was connected to one side (1 in FIG. 1) of the hollow fiber membrane minicolumn. With respect to the hollow fiber membrane mini-column, immediately before starting the blood circulation test, 30 ml of PBS was circulated from 1 to 2 in FIG.
健常者ボランティアから静脈血を採血後、十秒内にヘパリンを０．５Ｕ／ｍｌになるように添加した。採血後、実験開始までにある程度の時間が経過してしまうことなど、循環実験以外の要因で血液が活性化される。ヘパリンを０．５Ｕ／ｍｌ程度添加することで、そのような活性化を抑制できると考えられる。ヘパリン添加後、数分以内に、該血液をカラムで循環させた。循環は５ｍｌの血液を、垂直に立てた中空糸膜ミニカラムの上から下に（図１の１から２に）、０．５ｍｌ／分の流速で、室温で行った。なお、ミニカラム内および回路内には、はじめはＰＢＳが充填してあるので、循環開始後の最初の１ｍｌは廃棄した後、循環した。該条件での、循環可能時間を測定した。なお、ここでいう循環可能時間とは、回路中もしくはミニカラム中に血栓などが生じて血液が流れなくなるまでの時間、もしくは血液が流れにくくなったため、回路接続部などから血液が漏れだしてくるまでの時間をいう。 After collecting venous blood from healthy volunteers, heparin was added to 0.5 U / ml within 10 seconds. After blood collection, blood is activated by factors other than the circulation experiment, such as a certain amount of time elapses before the start of the experiment. It is thought that such activation can be suppressed by adding about 0.5 U / ml of heparin. The blood was circulated through the column within minutes after the addition of heparin. Circulation was carried out at room temperature at a flow rate of 0.5 ml / min, 5 ml of blood from the top to the bottom of a vertically standing hollow fiber membrane minicolumn (from 1 to 2 in FIG. 1). Since the minicolumn and the circuit were initially filled with PBS, the first 1 ml after the start of circulation was discarded and then circulated. Under this condition, the circulation possible time was measured. Note that the circulatorable time here is the time until blood clots occur in the circuit or mini-column and blood does not flow, or until blood leaks from the circuit connection etc. Of time.
前記３において、ミニカラムに循環する前後のヒト・トロンボモジュリン濃度および、前記４の溶出試験でのヒト・トロンボモジュリン濃度を測定することで、中空糸膜に保持させたヒト・トロンボモジュリン量を求めた。中空糸膜の乾燥重量は０．１１ｇであり、ヒト・トロンボモジュリン保持量は１７μｇ／ｇであった。前記３で得られた中空糸膜ミニカラムについて、ヒト・トロンボモジュリンの溶出実験の結果、ヒト・トロンボモジュリン濃度は１ｎｇ／ｍｌ以下であった。該中空糸膜ミニカラムの抗血栓性を評価した結果、循環可能時間は２時間３０分であった。 In 3 above, the concentration of human thrombomodulin retained in the hollow fiber membrane was determined by measuring the concentration of human thrombomodulin before and after circulating in the minicolumn and the concentration of human thrombomodulin in the elution test of 4 above. The dry weight of the hollow fiber membrane was 0.11 g, and the retained amount of human thrombomodulin was 17 μg / g. As a result of the elution experiment of human thrombomodulin, the human thrombomodulin concentration of the hollow fiber membrane minicolumn obtained in 3 was 1 ng / ml or less. As a result of evaluating the antithrombogenicity of the hollow fiber membrane minicolumn, the circulation possible time was 2 hours 30 minutes.
すなわち、ヒト・トロンボモジュリンの溶出は、ほとんどないうえに、プロテインＣを活性化することで抗血栓性を有する多孔質な中空糸膜ミニカラムを得ることができた。 That is, there was almost no elution of human thrombomodulin, and by activating protein C, a porous hollow fiber membrane minicolumn having antithrombotic properties could be obtained.
実施例２
アイソタクチック−ポリメタクリル酸メチル５重量部とシンジオタクチック−ポリメタクリル酸メチル２０重量部を、ジメチルスルホキシド７５重量部に加え、加熱溶解し製膜原液を得た。この製膜原液をオリフィス型二重円筒型口金から吐出し、空気中を３００ｍｍ通過させた後、水１００％の凝固浴中に導き中空糸膜を得た。この際、内部注入気体として乾燥窒素を用いた。得られた中空糸分離膜の内径は０．２ｍｍであり 、膜厚は０．０３ｍｍであった。
Example 2
5 parts by weight of isotactic-polymethyl methacrylate and 20 parts by weight of syndiotactic-polymethyl methacrylate were added to 75 parts by weight of dimethyl sulfoxide and dissolved by heating to obtain a film forming stock solution. This membrane-forming stock solution was discharged from an orifice-type double cylindrical die, allowed to pass through 300 mm in air, and then led into a 100% water coagulation bath to obtain a hollow fiber membrane. At this time, dry nitrogen was used as the internal injection gas. The resulting hollow fiber separation membrane had an inner diameter of 0.2 mm and a film thickness of 0.03 mm.
中空糸膜を１００本束ね、直径約７ｍｍ、長さは１２ｃｍのプラスチック管ミニカラムケースに挿入した。中空糸膜の両末端を、中空糸膜中空部を閉塞しないようにウレタン系ポッティング剤で固定し、図１に示すようなミニカラムを作成した。ミニカラムの中空糸膜内側および外側を３７℃の超純水を１ｍｌ／分の流速で３０分間洗浄した。 100 hollow fiber membranes were bundled and inserted into a plastic tube mini-column case having a diameter of about 7 mm and a length of 12 cm. Both ends of the hollow fiber membrane were fixed with a urethane potting agent so as not to block the hollow portion of the hollow fiber membrane, and a minicolumn as shown in FIG. 1 was prepared. The inside and outside of the hollow fiber membrane of the mini column were washed with ultrapure water at 37 ° C. for 30 minutes at a flow rate of 1 ml / min.
実施例１と同様に比表面積を測定した結果、２４２ｍ２／ｇであった。また、実施例１と同様にデキストラン篩い係数を測定した結果、篩い係数が０．５のときのデキストラン分子量は１．４万であった。 It was 242 m < 2 > / g as a result of measuring a specific surface area like Example 1. FIG. Moreover, as a result of measuring the dextran sieving coefficient in the same manner as in Example 1, the dextran molecular weight when the sieving coefficient was 0.5 was 14,000.
実施例１と同様にヒト・トロンボモジュリンを保持させた。ヒト・トロンボモジュリン量は、５μｇ／ｇであった。また、ヒト・トロンボモジュリンの溶出実験の結果、ヒト・トロンボモジュリン濃度は１ｎｇ／ｍｌ以下であった。 Human thrombomodulin was retained in the same manner as in Example 1. The amount of human thrombomodulin was 5 μg / g. As a result of the elution experiment of human thrombomodulin, the human thrombomodulin concentration was 1 ng / ml or less.
該中空糸膜ミニカラムの抗血栓性を評価した結果、循環可能時間は２時間２０分であった。すなわち、ヒト・トロンボモジュリンの溶出は、ほとんどないうえに、プロテインＣを活性化することで抗血栓性を有する多孔質な中空糸膜ミニカラムを得ることができた。 As a result of evaluating the antithrombogenicity of the hollow fiber membrane minicolumn, the circulation possible time was 2 hours and 20 minutes. That is, there was almost no elution of human thrombomodulin, and by activating protein C, a porous hollow fiber membrane minicolumn having antithrombotic properties could be obtained.
実施例３
１．海島型複合繊維の多孔質化およびクロルアセトアミドメチル化
ポリプロピレン５０重量部を島成分とし、ポリスチレン４６重量部、ポリプロピレン４重量部の混合物を海成分とする海島型複合繊維（島数１６、単糸繊度２６デニール、引張強度２９ｇ／ｄ、伸度５０％、フィラメント数４２） ５ｇを、Ｎ−メチロール−α−クロルアセトアミド５ｇ、ニトロベンゼン４０ｇ、９８重量％硫酸４０ｇおよびパラホルムアルデヒド０．０８５ｇからなる混合溶液中に浸し、２０℃で１時間反応させた。繊維を反応液から取り出し、０℃の氷水中に投じて反応停止させたのち、水で洗浄し、次に、繊維に付着しているニトロベンゼンを抽出除去した。この繊維を５０℃で真空乾燥し、クロルアセトアミドメチル化多孔質繊維を得た。なお、パラホルムアルデヒドは架橋構造を形成し、ニトロベンゼンは海成分であるポリスチレンを溶解するために、多孔質な繊維を得ることができる。
Example 3
1. Porous and chloracetamidomethylation of sea-island type composite fiber Sea-island type composite fiber (16 islands, single yarn fineness) containing 50 parts by weight of polypropylene as an island component and a mixture of 46 parts by weight of polystyrene and 4 parts by weight of polypropylene as sea components 26 denier, tensile strength 29 g / d, elongation 50%, filament number 42) 5 g in a mixed solution consisting of 5 g of N-methylol-α-chloroacetamide, 40 g of nitrobenzene, 40 g of 98 wt% sulfuric acid and 0.085 g of paraformaldehyde And allowed to react at 20 ° C. for 1 hour. The fiber was taken out from the reaction solution, poured into ice water at 0 ° C. to stop the reaction, washed with water, and then nitrobenzene adhering to the fiber was extracted and removed. This fiber was vacuum-dried at 50 ° C. to obtain chloracetamidomethylated porous fiber. In addition, since paraformaldehyde forms a crosslinked structure and nitrobenzene dissolves polystyrene, which is a sea component, porous fibers can be obtained.
２．比表面積の測定
実施例１の２と同様にして行った。比表面積は、２．３ｍ２／ｇであった。
2. Measurement of specific surface area The measurement was performed in the same manner as in Example 1-2. The specific surface area was 2.3 m 2 / g.
３．ヒト・トロンボモジュリンの導入
前記１で得られた繊維０．１ｇ（乾燥重量）を、実施例１の２のヒト・トロンボモジュリンのＰＢＳ溶液（ヒト・トロンボモジュリン濃度は２５μｇ／ｍｌ）２．５ｍｌに４０℃で２時間浸漬、振盪させ、クロルアセトアミドメチル基とヒト・トロンボモジュリンを反応させた。ヒト・トロンボモジュリン固定化多孔質繊維を、ＰＢＳ溶液２．５ｍｌに、４０℃で１０分間浸漬、振盪させることで吸着したヒト・トロンボモジュリンを洗浄した。この洗浄操作を３回繰り返し、３回目のＰＢＳ溶液中のヒト・トロンボモジュリン濃度が１ｎｇ／ｍｌ以下になったことを確認した。ヒト・トロンボモジュリンの反応前後の濃度変化および、洗浄液中のヒト・トロンボモジュリン濃度から、固定化した量を算出したところ、１．６μｇ（１６μｇ／ｇ）であった。
3. Introduction of human thrombomodulin 0.1 g (dry weight) of the fiber obtained in 1 above was added to 2.5 ml of the PBS solution of human thrombomodulin of Example 1 (concentration of human thrombomodulin of 25 μg / ml) at 40 ° C. It was immersed for 2 hours and shaken to react chloroacetamidomethyl group with human thrombomodulin. The adsorbed human thrombomodulin was washed by immersing the human thrombomodulin-immobilized porous fiber in 2.5 ml of PBS solution at 40 ° C. for 10 minutes and shaking. This washing operation was repeated three times, and it was confirmed that the human thrombomodulin concentration in the third PBS solution was 1 ng / ml or less. The amount immobilized was calculated from the change in the concentration of human thrombomodulin before and after the reaction and the concentration of human thrombomodulin in the washing solution, and it was 1.6 μg (16 μg / g).
この後、該繊維を４．５ｍｌのＰＢＳ溶液に浸漬させ、２時間、室温にて振盪させることでヒト・トロンボモジュリンの溶出の有無を確認した。振盪後のＰＢＳ溶液中ヒト・トロンボモジュリン濃度は１ｎｇ／ｍｌ以下であり、溶出は確認されなかった。 Then, the presence or absence of elution of human thrombomodulin was confirmed by immersing the fiber in a 4.5 ml PBS solution and shaking for 2 hours at room temperature. The concentration of human thrombomodulin in the PBS solution after shaking was 1 ng / ml or less, and elution was not confirmed.
なお、ヒト・トロンボモジュリン濃度の測定は、実施例１の５と同様の操作で行った。 The measurement of the human thrombomodulin concentration was carried out in the same manner as in Example 1-5.
４．抗血栓性の評価（血液振盪試験）
健常者ボランティアから静脈血を採血した全血１ｍｌに前記２で得られたヒト・トロンボモジュリン固定化多孔質繊維０．１ｇを浸漬させた。採血後、繊維を浸漬させるまでの時間は数十秒以内に行った。室温で３０分間振盪させても、ヒト・トロンボモジュリン固定化多孔質繊維に血栓が付着することはなかった。
4). Evaluation of antithrombogenicity (blood shaking test)
0.1 g of human thrombomodulin-immobilized porous fiber obtained in 2 above was immersed in 1 ml of whole blood obtained by collecting venous blood from healthy volunteers. After blood collection, the time until the fibers were immersed was within tens of seconds. Even after 30 minutes of shaking at room temperature, no thrombus adhered to the human thrombomodulin-immobilized porous fiber.
すなわち、ヒト・トロンボモジュリンの溶出は、ほとんどないうえに、プロテインＣを活性化することで抗血栓性を有する多孔質繊維を得ることができた。 That is, there was almost no elution of human thrombomodulin, and a porous fiber having antithrombogenicity could be obtained by activating protein C.
比較例１
実施例１の３の中空糸膜ミニカラムにおいてヒト・トロンボモジュリン溶液の代わりに、水を用いた。該中空糸膜ミニカラムについてヒト・トロンボモジュリンの溶出実験の結果、ヒト・トロンボモジュリン濃度は１ｎｇ／ｍｌ以下であった。該中空糸膜ミニカラムの抗血栓性を評価した結果、循環可能時間は１時間２０分であった。抗血栓性の低い中空糸膜ミニカラムであった。
Comparative Example 1
Instead of the human thrombomodulin solution in the three hollow fiber membrane minicolumns of Example 1, water was used. As a result of the human thrombomodulin elution experiment on the hollow fiber membrane minicolumn, the human thrombomodulin concentration was 1 ng / ml or less. As a result of evaluating the antithrombogenicity of the hollow fiber membrane minicolumn, the circulation possible time was 1 hour 20 minutes. It was a hollow fiber membrane minicolumn with low antithrombotic properties.
比較例２
実施例１の３の中空糸膜ミニカラムにおいてヒト・トロンボモジュリン溶液を中空糸内側だけに通し、外側には水を充填した。すなわち、図１の１から２にヒト・トロンボモジュリン溶液を、図１の３から４に水を通した。該中空糸膜ミニカラムについて、実施例１と同様にヒト・トロンボモジュリンの保持量を求めた結果、０．７７μｇ／ｇであった。ヒト・トロンボモジュリンの溶出実験の結果、ヒト・トロンボモジュリン濃度は１ｎｇ／ｍｌ以下であった。さらに該中空糸膜ミニカラムの抗血栓性を評価した結果、循環可能時間は１時間５０分であった。比較例１に比べて抗血栓性を有するが、実施例１には及ばない結果となった。
Comparative Example 2
In the hollow fiber membrane mini-column of Example 1, the human thrombomodulin solution was passed only inside the hollow fiber, and the outside was filled with water. That is, the human thrombomodulin solution was passed from 1 to 2 in FIG. 1, and water was passed from 3 to 4 in FIG. With respect to the hollow fiber membrane minicolumn, the retained amount of human thrombomodulin was determined in the same manner as in Example 1. As a result, it was 0.77 μg / g. As a result of the elution experiment of human thrombomodulin, the concentration of human thrombomodulin was 1 ng / ml or less. Furthermore, as a result of evaluating the antithrombogenicity of the hollow fiber membrane minicolumn, the circulation possible time was 1 hour 50 minutes. Although it has antithrombogenicity as compared with Comparative Example 1, the results were not as good as Example 1.
比較例３
実施例３の１におけるニトロベンゼンとパラホルムアルデヒドを添加しない以外は、すべて同一の操作を行った。実施例１の２と同様に比表面積の測定を行ったところ、８０ｃｍ２／ｇであった。該繊維０．１ｇ（乾燥重量）を実施例３の３と同様の操作によって、ヒト・トロンボモジュリン固定化繊維を得た。ヒト・トロンボモジュリンの固定化量は、０．０８μｇ（０．８μｇ／ｇ）であった。また、実施例３の３と同様にヒト・トロンボモジュリンの溶出の有無を確認したところ、振盪後のＰＢＳ溶液中ヒト・トロンボモジュリン濃度は１ｎｇ／ｍｌ以下であり、溶出は確認されなかった。
Comparative Example 3
All operations were the same except that nitrobenzene and paraformaldehyde in Example 3 were not added. It was 80 cm < 2 > / g when the specific surface area was measured similarly to 2 of Example 1. FIG. Human thrombomodulin-immobilized fiber was obtained in the same manner as in Example 3 3 by using 0.1 g (dry weight) of the fiber. The amount of human thrombomodulin immobilized was 0.08 μg (0.8 μg / g). Further, as in Example 3-3, the presence or absence of elution of human thrombomodulin was confirmed. As a result, the concentration of human thrombomodulin in the PBS solution after shaking was 1 ng / ml or less, and elution was not confirmed.
実施例３の３と同様に抗血栓性を評価した結果、３０分間振盪時点で、繊維に血栓が付着していた。 As a result of evaluating the antithrombogenicity in the same manner as 3 in Example 3, the thrombus was attached to the fiber at the time of shaking for 30 minutes.
１ 中空糸膜内側への連結口（血液流入口）
２ 中空糸膜内側への連結口（血液流出口）
３ 中空糸膜外側への連結口（入口）
４ 中空糸膜外側への連結口（出口）
1 Connection port to the inside of the hollow fiber membrane (blood inlet)
2 Connection port to the inside of the hollow fiber membrane (blood outlet)
3 Connection port (inlet) to the outside of the hollow fiber membrane
4 Connection port (outlet) to the outside of the hollow fiber membrane
Claims (6)
The said support | carrier is a porous separation membrane, Comprising: Both sides of the membrane were made to infiltrate the aqueous solution of the said bioactive substance, or obtained by further filtering, The Claim 1 any one of Claims 1-5 obtained. Porous substrate.
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| JPH0415063A (en) * | 1990-05-09 | 1992-01-20 | Mitsuru Akashi | Antithrombotic material having human thrombomoduline fixed |
| JPH07148243A (en) * | 1993-09-24 | 1995-06-13 | Takiron Co Ltd | Implant material |
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