JPWO1987006007A1 - Immobilized physiologically active substances - Google Patents

Abstract

(57) [Abstract] This publication contains application data prior to electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] Immobilized Physiologically Active Substances Technical Field The present invention relates to immobilized physiologically active substances and immobilization materials for immobilizing such physiologically active substances.

BACKGROUND ART A great deal of research has been conducted on the immobilization of physiologically active substances onto solid supports. Currently, the majority of this research focuses on the immobilization of enzymes, but other applications include medical materials immobilized with immunologically reactive substances such as antigens and antibodies, affinity chromatography separation substrates for adsorption of specific physiologically active substances, and even bioreactors and artificial organs immobilized with useful formed components such as bacteria and cells.

Here, immobilization refers to the process of rendering these naturally water-soluble physiologically active substances water-insoluble without impairing their functionality. Various methods are used for this, including carrier binding, cross-linking, and entrapment. However, carrier binding, which immobilizes the target physiologically active substance via a covalent bond to the carrier, is the most stable due to its strong binding strength and resistance to release. One commonly reported example is the diazonium compound formed by the reaction of a water-insoluble carrier (RNH2) with dilute sulfuric acid and sodium nitrite, which is then diazotically bonded to a protein (e.g., albumin) to immobilize it (Chamberl et al., Proc. Nat. Acad. Sci. 37:575 (1951)).

Recently, an improvement to this carrier-binding method has been made by introducing a direct-contact structure called a "spacer" or "arm" consisting of an n-alkyl chain between the carrier and the biologically active substance to be bound. This is because the introduction of such a structure alleviates steric hindrance when the biologically active substance immobilized at the tip reacts with the substrate, making it easier for its function to be expressed.

For example, Kim et al. immobilized the anticoagulant heparin on agarose beads containing n-alkyl chains of lengths 2, 4, 8, 10, and 12 as spacers, and found that its anticoagulant activity, measured by activated partial thromboplastin time (APTT), increased with increasing alkyl chain length, a phenomenon they termed the "spacer effect" (Kim et al., Thrombosis Research, 26:43 (1982)).

However, hydrophobic spacers such as n-alkyl chains have low affinity for physiologically active substances, which are inherently water-soluble. As a result, the immobilization rate decreases and the physiologically active substance may be denatured or inactivated during immobilization. Furthermore, long n-alkyl chains are water-insoluble regardless of the terminal functional group, and their incorporation into the support requires the use of organic solvents. However, depending on the support, organic solvents may cause dissolution, damage, or denaturation, significantly limiting the range of support options.

Furthermore, the hydrophobic surface of the support made hydrophobic by the introduction of such a hydrophobic spacer is prone to nonspecific adsorption and denaturation of other physiologically active substances, especially proteins, which may impair the original function of the immobilized physiologically active substances.

The present invention aims to provide a physiologically active substance immobilization material that can highly express the function of a physiologically active substance when immobilized, and to provide highly active immobilized physiologically active substances immobilized thereby.

DISCLOSURE OF THE INVENTION The above-mentioned objectives are achieved by introducing an alkylene oxide chain as a spacer. Specifically, the present invention relates to an immobilized physiologically active substance in which a carrier and a physiologically active substance are linked via an alkylene oxide chain, and to an immobilization material for the immobilized physiologically active substance.

BEST MODE FOR CARRYING OUT THE INVENTION The alkylene oxide chain preferably used as a spacer in this invention is a polyethylene oxide or ethylene oxide copolymer represented by the following formula: %Formula% (where m and n are integers between 2 and 100, m and n are 0 or positive integers, and □ is ≥ 0.5) 44g + 58m + 72n In the case of copolymers, they may be random copolymers, block copolymers, or mixtures thereof. However, block copolymers or homopolymers with a degree of polymerization between 2 and 100 and a content of ethylene oxide units (CH2CH20) of 50% by weight or more are particularly preferred.

The carrier used in the present invention is one whose surface is water-insoluble. That is, the entire carrier may be composed of a water-insoluble material, or the carrier may be composed of a water-soluble material whose surface is coated with a water-insoluble material. "Water-insoluble" here means that the carrier is substantially insoluble in water at 20°C. Such water-insoluble materials include natural or synthetic organic or inorganic polymers. Examples of suitable polymers include the following, which may be crosslinked as necessary to render them water-insoluble:

Cellulose-based materials such as cellulose acetate, polysaccharides such as dextran, collagens, polyolefins such as polyethylene and polypropylene, homo- or copolymers of vinyl compounds such as vinyl acetate, vinyl chloride, acrylonitrile, acrylic esters, methacrylic esters, and styrene, polyesters such as polyethylene terephthalate and polybutylene terephthalate, and aliphatic and aromatic polyamides.

Because the carrier of the present invention is bonded with an alkylene oxide chain as a spacer, it is necessary for the carrier to contain a functional group that can bond to the spacer. Therefore, if such a functional group does not exist, an activation treatment is performed to introduce the functional group. Typical examples of functional groups are amino and epoxy groups. Furthermore, as described below, alkylene oxide chains can also be introduced into the carrier by using a monomer having an alkylene oxide chain as a side chain as a copolymerization component of the polymer that constitutes the carrier.

The form of the carrier is determined appropriately depending on the purpose. Examples include spheres, cylinders, disks, test tubes, cylinders, fibers, membranes, particles, meshes, and microtrays. A particularly preferred embodiment uses fine fibers of 1.0 denier or less as the carrier. The polymer constituting the ultrafine fibers is appropriately selected from polyester, polyamide, polytetrafluoroethylene, polystyrene, polyolefin, cellulose, polyamino acid, collagen, etc., with polyester being particularly preferred. When multicomponent fibers are used, the final remaining polymer is the above-mentioned polymer, but other components may include polystyrene, polyethylene, water-soluble polyamide, aqueous alkali-soluble polyester, water-soluble polyvinyl alcohol, etc. Ultrafine fibers made of the above-mentioned polymers are used in the carrier of the present invention in the form of woven, knitted, nonwoven, or other structures.

Methods for bonding alkylene oxide chains to the support surface include, for example, (1) a method utilizing a coupling reaction, or (2) a method utilizing a polymer or copolymer composed of a monomer having an alkylene oxide chain in its side chain and a polymerizable carbon-carbon double bond. Method (1) is preferred, and it is particularly preferred to use an alkylene oxide chain having an amino or epoxy group as a functional group at its terminal.

Specifically, this can be achieved by reacting an excess of alkylene oxide chains having amino groups at both ends with a polymeric support having functional groups capable of bonding with amino groups. Such functional groups include, for example, epoxy groups. Examples of such polymers include those containing glycidyl methacrylate as a copolymerization component, uncured epoxy resins, and the like.

On the other hand, method (2) can utilize a support made of a water-insoluble copolymer containing a monomer unit represented by the formula (where p is an integer of 2 or greater, and R is H or CH3), or a support coated with this monomer. The copolymerization component may be any component capable of copolymerizing with the monomer to form a water-insoluble polymer, such as methyl methacrylate, acrylonitrile, styrene, or vinyl chloride. Copolymerization with a crosslinkable monomer, such as diethylene glycol dimethacrylate, is also a preferred method.

When this copolymer is used as a coating agent, the thickness of the coating layer is not particularly limited, but is usually preferably 100 Å or more. In this case, the material of the interior of the carrier is not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include organic polymers, glass, and metals.

The amount of alkylene oxide chains bound to the support surface can be adjusted by changing the chemical composition of the support, reaction conditions, etc., but to achieve an effective immobilization amount of a physiologically active substance, it is recommended that at least 10-8 mol of alkylene oxide chains be bound per support.

To immobilize a physiologically active substance on an alkylene oxide chain attached to a support, a functional group capable of forming a covalent bond with the physiologically active substance is introduced at the end of the alkylene oxide chain, and the physiologically active substance is covalently bonded to the functional group, thereby easily achieving immobilization.

Functional groups capable of reacting with physiologically active substances to form covalent bonds include, for example, amino, carboxyl, epoxy, hydroxyl, carbodiimide, aldehyde, isocyanate, and imidazole groups. However, hydroxyl, amino, and epoxy groups are particularly preferred due to their ease of introduction to the termini of polyethylene oxide compounds. Covalent bonds between these functional groups and physiologically active substances can be formed using well-known techniques, such as those described in "Fixed Enzymes" (edited by Senhata, pp. 11-40, Kodansha, 1975).

For example, when a polyethylene oxide compound bound to the surface of a water-insoluble support has a free amino terminal, glutaraldehyde can be used as a binder to form a Schiff base between the amino group of the polyethylene oxide compound and an amino group in a physiologically active substance, such as a protein, thereby allowing immobilization.

Furthermore, for example, if the physiologically active substance to be immobilized has a carboxyl group, the physiologically active substance can be easily immobilized on the aforementioned support by first treating the substance with a condensing agent such as a carbodiimide reagent or Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3-sulfonate).

Examples of physiologically active substances that can be immobilized include enzymes such as asparaginase, urease, and urokinase; hormones such as human chorionic gonadotropin and thyroid-stimulating hormone; albumin; various complement-related substances; plasma proteins such as coagulation factors such as thrombin and coagulation inhibitors such as antithrombin; immunoreactive substances such as mycoplasma antigens and anti-estrogen antibodies; mucopolysaccharides such as heparin, heparan sulfate, chondroitin sulfate, and hyaluronic acid; alanine; These include amino acids such as glutamic acid, glutamic acid, and aspartic acid, various prostaglandin derivatives, thimble derivatives such as phosphatidylcholine, glycolipids such as lipopolysaccharide, antibiotics such as polymyxin B and chloramphenicol, blood cells such as red blood cells, white blood cells (granulocytes and macrophages), and lymphocytes, epithelial or endothelial cells such as vascular endothelial cells, hepatocytes, and pancreatic beta cells, various differentiation factors related to the differentiation and growth of these cells such as ECGF and C3F, growth factors, and gene-related factors such as DNA and RNA. Selection of appropriate agents is possible depending on the purpose.

The present invention will be specifically described below with reference to examples.

Example 1 and Comparative Example 1.2 (Spacer Synthesis) A mixture of polyethylene glycol with a degree of polymerization of 23 (number-average molecular weight of 1000) and acrylonitrile was subjected to a cyanoethylation reaction in the presence of a fine particle of strong basic anion exchange resin, "Amberlite IR A-400" (Rohm & Haas), to obtain a polyethylene oxide compound bearing β-cyanoethoxy groups at both ends. 100 g of this compound was dissolved in 1.1 L of water and hydrogenated at 60°C for 6 hours to synthesize 91 g of a bisaminopolyethylene oxide compound (PGD-1000) with a degree of polymerization of 23 and amino groups at both ends.

(Synthesis of Carrier) A carrier was synthesized by incorporating the above-mentioned PGD-1000 into an epoxy group-containing polyvinyl chloride copolymer film using the following procedure.

First, 4 g of commercially available polyvinyl chloride (PVC) was mixed with N.

The resulting solution was dissolved in N-dimethylformamide (DMF), 11 g of sodium diethyldithiocarbamate (DTC) was added, and the mixture was reacted at 50°C for 3 hours in the dark to synthesize photofunctional DTC-modified PVC.

50 g of this DTC-modified PVC was dissolved in 0 g of tetrahydrofuran (THF), 80 g of glycidyl methacrylate (GMA) was added, and a photografting reaction was carried out for 6 hours at 40°C using a 100 W high-pressure mercury lamp, synthesizing epoxy-containing poly(vinyl chloride)-glycidyl methacrylate graft copolymer (PVC-g-GMA). The graft ratio of this graft copolymer, as determined by elemental analysis, was 86%.

A film with a thickness of approximately 100 μm was prepared from a 5% THF solution of this polymer by solvent casting using a glass plate. This film was then immersed in a 50% aqueous solution of PGD-1000 and reacted at 60°C for 24 hours to introduce spacers into the film surface.

Quantitation of introduced amino groups using adduct formation between benzylamine and 2-hydroxy-1-naphthylamide (HNA) (C, D, Ebert et al., J. Biomed. Hater. Res., 16:629 (1982)) revealed that the amount of PGD-1000 introduced onto the film surface was 0.7 μmO1/co?. Furthermore, the introduction of PGD-1000 reduced the water contact angle on the film surface (measured using a Kyowa Kagaku Co., Ltd. CA-P contact angle measuring device) from 63° to 43°, clearly demonstrating hydrophilicity of the film surface.

(Heparin Immobilization) 0.7 g of heparin sodium 800 μg, Woodward's reagent KO, was placed in a glass container and dissolved in 100 ml of phosphate-buffered saline (PBS). The solution was stirred slowly at 4°C for 8 hours to activate the heparin. The PGD-1000-incorporated film described above was immersed in this activated heparin solution and allowed to react at 4°C for 8 hours to immobilize the heparin on the film surface.

After the reaction was complete, the film was washed three times with PBS to remove unimmobilized heparin. A 2 M ethanolamine PBS solution adjusted to pH 17.4 with hydrochloric acid was added, and the film was immersed at 4°C for 24 hours to block residual active groups in the heparin. ESCA (Electron Spectroscopy for Chemical Analysis, Shimabara X-ray Electron Spectrometer ESCA 750) analysis revealed that 0°2/day of heparin was immobilized on the surface of the resulting heparin-immobilized film.

(Heparin Activity Measurement) The activated partial thromboplastin time (APTT) was used to measure the anticoagulant activity of immobilized heparin.

Specifically, bovine plasma, which had been centrifuged to remove cellular components, was used as the test plasma, using 3.8% sodium citrate as an anticoagulant. A heparin-immobilized film with a surface area of 20 Å was placed in 1 ml of this plasma and stirred at 37°C for 30 minutes, after which the plasma and film were separated. This plasma was placed in a siliconized glass test tube, and 0.1 ml of an activated phospholipid preparation was added. The mixture was then heated at 37°C for 5 minutes, followed by the addition of 0.1 ml of a 1/40 M CaCl2 solution. The mixture was then left to stand at 37°C for 30 seconds. The test tube was then gently tilted, and the time until fibrin precipitation was measured as ATP.

As a comparative example, a carrier having hexamethylenediamine and diaminododecane as spacers was synthesized under the same conditions, and heparin was immobilized on the carrier, and its activity was measured. The results are shown in Table 1.

As can be seen from this table, the anticoagulant activity of heparin immobilized using the spacer of the present invention was significantly higher than that of heparin immobilized using other known hydrophobic n-alkyl spacers.

Example 2 and Comparative Example 3: 120 g of methyl methacrylate (MMA), 80 g of polyethylene glycol monomethacrylate (number-average degree of polymerization of the polyethylene glycol moiety: 9, manufactured by Nippon Oil & Fats Corporation, "Blenmer PE-350"), and 1.4 g of α,α'-azobis(α,α'-dimethylvaleronitrile) were added to 100 g of isopropanol and reacted at 50°C under a nitrogen stream. After 5 hours, the resulting polymer solution was transferred to a vessel with a 2-hole bottom and re-precipitated in stirred cold water from a height of approximately 1 m, yielding polymer particles with diameters of 50 to 100 μm. ESC analysis revealed that approximately 20 mmol/g of polyethylene glycol chains were present on the surface of the polymer particles.

10 g of these polymer particles were thoroughly washed with water and suspended in 880 ml of LM Na O. 10 ml of epichlorohydrin was added and the mixture was allowed to react with vigorous stirring at room temperature for 24 hours.

After the reaction is complete, the support bearing the epichlorohydrin-activated polyethylene oxide spacer is collected and the excess epichlorohydrin is removed by washing with water. The support is then suspended in a 2 M potassium carbonate solution (pH 10) containing 3 g of sulfanilic acid.

The pH was then adjusted to 10 and the mixture was allowed to stand for 6 days. The carrier obtained by the above method was then treated with phloroglucitur. 5% divinyl sulfone was added to the phloroglucitur derivative, and the mixture was allowed to react at pH 11 for 30 minutes. After that, soybean trypsin inhibitor was added as a ligand at pH 9.

This method enabled us to obtain a support for immobilizing soybean trypsin inhibitors using polyethylene oxide with a degree of polymerization of 9 as a spacer. For comparison, polymer particles were prepared under the same conditions using hydroxyethyl methacrylate as the copolymer component of MMA, and a support for immobilizing soybean trypsin inhibitors was prepared. The results of the comparison of trypsin adsorption capacities are shown in Table 2.

As described above, the adsorbent obtained by this method showed a high adsorption capacity.

Example 3 and Comparative Example 4 400 g of islands-in-sea composite fibers, consisting of 50 parts of polypropylene (Mitsui "Noblen" J3HG) as the island component and a mixture of 46 parts of polystyrene ("Styron" 679) and 4 parts of polypropylene (Mitsui "Lebran" WF-727-F) as the sea component, were immersed in a mixed solution consisting of 560 g of N-methylol-α-chloroacetamide, 3100 ml of nitrobenzene, 2020 ml of concentrated sulfuric acid, and 8 g of paraformaldehyde, and reacted at 20°C for 1 hour. The fibers were removed from the reaction solution and placed in 5.2 ml of ice water at 0°C to terminate the reaction. They were then washed with water, and the nitrobenzene remaining on the fibers was extracted with methanol and removed. The fibers were then vacuum-dried at 40°C to obtain chloroacetamidomethylated fibers (fiber A).

The fiber had a diameter of 0.5 denier. Next, 3000 ml of a 20 wt % dimethyl sulfoxide solution of the same bisaminopolyethylene oxide compound (PGD-1000) used in Example 1 was added to 80 g of the fiber A. The mixture was allowed to react at room temperature for 48 hours, then washed with 20 ml of water and then with 1N HCl. The resulting mixture was washed again with 20 ml of water to obtain fibers with polyethylene oxide chains terminated in amine groups.

163 g of this fiber was placed in a flask, and a 3% solution of glutaraldehyde in phosphate-buffered saline (PBS) was added and the mixture was immersed at room temperature for 4.5 hours.

After thorough washing with PBS, a 2.0 wt% aqueous solution of the polypeptide antibiotic polymyxin B sulfate (Pfizer) and 7 ml of PBS were added and immobilized at 4°C for 24 hours with gentle stirring. The film was then immersed in Tris buffer at 4°C for 6 hours to block any remaining free amino groups. The film was then transferred to a 5 ml + 1 solution of 0.1 wt% sodium borohydride in PBS and stirred at room temperature for 24 hours to reduce the formed Schiff base, yielding polymyxin B-immobilized fibers (PMX-PEO).

The amount of polymyxin B immobilized on the fibers was determined by amino acid analysis to be 3.5 μg/g.

As a comparative example, fibers (P-1X-Cl2) were obtained in which polymyxin B was immobilized using diaminododecane as a spacer in a similar manner. 3.8 mg/g of polymyxin B was immobilized on these fibers.

The antibacterial activity of these fibers was measured using Escherichia coli (ATCC 25922), and the results are shown in Table 3.

As described above, polymyxin B immobilized using the spacer of the present invention exhibited higher antibacterial activity than polymyxin B immobilized using an alkyl spacer.

INDUSTRIAL APPLICABILITY The physiologically active substance immobilization material of the present invention can immobilize physiologically active substances while maintaining their functionality to a high degree. Therefore, physiologically active substances immobilized in this manner can be used as substrates for affinity chromatography for substance separation and purification, as components of kits for disease diagnosis and treatment, and as in vivo implant materials.

Claims (11)

[Claims] 1. An immobilized physiologically active substance in which a carrier and a physiologically active substance are bound via an alkylene oxide chain. 2. The immobilized biologically active substance according to claim 1, wherein the alkylene oxide chain is a polymer represented by the formula: ▲Mathematical formula, chemical formula, table, etc.▼ (where l is an integer between 2 and 100, m and n are 0 or positive integers, and 44l/44l + 58m + 72n ≥ 0.5). 3. The immobilized physiologically active substance according to claim 2, wherein m and n are 0. 4. The immobilized physiologically active substance according to claim 1, wherein the carrier is made of a water-insoluble material. 5. The immobilized physiologically active substance according to claim 1, wherein the carrier is an ultrafine fiber having a denier of 1.0 or less. 6. A physiologically active substance immobilization material in which one end of an alkylene oxide chain is bound to a support and the other end has a functional group reactive with a physiologically active substance introduced therein. 7. A physiologically active substance immobilization material according to claim 6, wherein the alkylene oxide chain is a polymer represented by the formula ▲Mathematical formula, chemical formula, table, etc.▼ (where l is an integer between 2 and 100, m and n are 0 or positive integers, and 44l/44l + 58m ÷ 72n ≧ 0.5). 8. The physiologically active substance immobilizing material according to claim 6, wherein m and n are 0. 9. The physiologically active substance immobilization material according to claim 6, wherein the carrier is made of a water-insoluble material. 10. The physiologically active substance immobilization material according to claim 6, wherein the carrier is an ultrafine fiber of 1.0 denier or less. 11. The physiologically active substance immobilizing material according to claim 6, wherein the functional group is one or more selected from the group consisting of an amino group, a carboxyl group, an epoxy group, a hydroxyl group, a carbodiimide group, an aldehyde group, an isocyanate group, and an imidazole group.