JPS61114734A - Method for immobilizing physiologically active substance and fiber therefor - Google Patents

Abstract

PURPOSE:To immoilize a physiologically active substance at high density by an insoluble vinyl polymer molded product, by immobilizing the physiologically active substance by the insoluble vinyl polymer molded product in the presence of an org. solvent capable of swelling said molded product. CONSTITUTION:An insoluble vinyl polymer molded product is immersed in a solvent such as a polar aprotic solvent with dielectric constant of 15 or more, for example, dimethylsulfoxide or N,N-dimethylformamide to be swollen. In this state, a physiologically active substance is immobilized by the insoluble vinyl polymer molded product according to a covalent method but, concretely, a functional group such as an alpha-halogenated acyl group, a halogenated alkyl group, an isocyanic acid group, a carboxyl group or a carboxylate group is bonded to the aforementioned polymer molded product. As the physiologically active substance, polymixine, colistin or hystamine can be designated.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for immobilizing a physiologically active substance on an insoluble vinyl polymer molded article and to the immobilized fiber.

(Prior art) Materials in which enzymes, microorganisms, and other physiologically active substances are chemically bonded to insoluble carriers have been applied or researched in the fields of engineering, science, and medicine as bioreactors, electrodes, and affinity adsorbents for blood purification. Examples of insoluble carriers that can be used include hydrophilic supports such as agarose-based, cross-linked dextran-based, and cross-linked polyacrylamide-based supports, granular supports such as porous silica beads, and granular supports such as those already proposed by the present inventors (particularly Examples include miscellaneous carriers.

In the method of chemically immobilizing, for example, a physiologically active substance on such an insoluble carrier, the reaction rate is very slow and the immobilization density cannot be easily increased because the reaction is in a solid-liquid heterogeneous system. There were drawbacks. Furthermore, in affinity adsorbents, etc., it is desirable that Gantts that selectively or specifically interact with the target substance be immobilized at high density, which has caused problems in the development of such materials.

[Problem to be interpreted by the invention]

Immobilizing physiologically active substances in insoluble vinyl polymer molded articles at high density.

(Means for Solving the Problems) The present invention provides (1) a method for immobilizing a physiologically active substance on an insoluble vinyl polymer molded article, in which the molded article is immobilized in the presence of an organic solvent capable of swelling; A method for immobilizing a physiologically active substance, characterized by:

(2) The present invention relates to a bioactive substance-immobilized fiber, characterized in that the bioactive substance is polymyxin, and the immobilization density is 10IIIg or more per gram of fiber.

Examples of insoluble vinyl polymers include homopolymers or copolymers of monovinyl compounds such as styrene, vinyl chloride, acrylic amide, acrylonitrile, maleic anhydride, and methacrylic esters, or divinylbenzene or methylene bis. In addition to copolymers with polyvinyl compounds typified by acrylamide and the like, examples include those obtained by crosslinking the monovinyl compound polymer molded article with formaldehyde, chlorosulfonic acid, etc. Crosslinked polymers have no fluidity and are difficult to mold, so
It is preferable to crosslink the material after forming it into particles, membranes, fibers, etc.

In order to immobilize a physiologically active substance on a vinyl polymer molded article by a covalent bonding method, an α-halogenated acyl group,
It is necessary that a functional group such as a halogenated alkyl group, an isocyanate group, a carboxyl group, or a carboxylic acid ester group is bonded to the polymer directly or via a spacer.

The solvent that imparts swelling properties to the molded article must also be able to dissolve the physiologically active substance to be immobilized. Examples include dimethyl sulfoxide, N,N-dimethylformamide, dimethylacetamide, and hexamethylphosphorol. Ruamide, tetramethyl l;i! The dielectric constant of element is 1
Polar aprotic solvents of 5 or more are preferred. However, it is not limited to the example shown here. The reaction may be carried out at room temperature or may be heated. If the temperature is too high, powder may fall off from the vinyl polymer molded product, so a temperature of 100° C. or lower is preferable. In the present invention, the reaction solvent may be a mixed solvent with water as long as the organic solvent is present in an amount sufficient to swell the molded article. Even if the solvent is a small component, the swelling effect may be sufficiently exerted.

By this immobilization method, the immobilization density of the fibers immobilized with polymyxin, which is an antibiotic that has a bactericidal effect on Durham-negative bacteria, has been improved. -112695> Not only does it exhibit excellent endotoxin absorption ability as an endotoxin neutralizer, but it also unexpectedly has improved antibacterial properties against Durham-negative bacteria such as Salmonella typhimurium and Escherichia coli. .

Examples of physiologically active substances in the present invention include polymyxin, colistin, histamine, gentamicin, and serotonin. Among these, polymyxin has a bactericidal property against Durham-negative bacteria, and its mechanism is It has the characteristic of selectively binding to and destroying endotoxins in the cell walls of Durham-negative bacteria, so fibers with immobilized endotoxins have excellent performance as materials for removing endotoxins from liquids to be treated such as water and blood. Demonstrate.

[Mechanism of action of the invention]

By immobilizing with a solvent system that imparts swelling properties, the vinyl polymer molded product swells, and the reaction can take place more deeply inside the molded product than when immobilized with an aqueous system. This is considered to be the reason why the immobilization density has improved.

Furthermore, in the fibers in which polymyxin was immobilized by the method of the present invention, the peak positions of amide-I and amide-■ in the infrared rays shifted compared to those in the case of immobilization in a conventional aqueous system, and the peak positions of amide-I and amide-■ in the infrared rays were shifted, resulting in a rough difference. From the appearance of a peak, it can be inferred that the immobilization structure has changed. It is inferred that the improvement in immobilization density and the change in immobilization mode result in excellent endotoxin adsorption ability and antibacterial activity.

〔Example〕

Example 1 Polypropylene (Mizuki"'Noblen"J3HCI>5
0 part is the island component, polystyrene (“Styron” 67
9) 46 parts, polypropylene (Sumitomo'')-Brene''W
F-727-F) 400 g of sea-island composite fibers containing 4 parts of warm mixture as sea component, 560 g of N-methylol-α-chloraledamide, 3100 g of nitrobenzene, 202 O20 of Im sulfuric acid, and 89 to The sample was immersed in a mixed solution of 20° C. and reacted at 20° C. for 1 hour. The fibers were taken out from the reaction solution and poured into ice water 5α at 0° C. to stop the reaction, washed with water, and then nitrobenzene adhering to the fibers was extracted with methanol and removed. This fiber was vacuum dried at 40°C to yield 1 q of chloroacetamide methylated fiber (41 fiber A).

10 g of polymyxin B sulfate (contains sugar, manufactured by Pfizer) was dissolved in 10100O dimethyl sulfoxide. To this, 359% of fiber 4'lIA was added, and 5% of magnesium oxide was added.
g was added thereto, and the mixture was reacted in a 90°C oil bath for 8 hours with thorough stirring. Reaction completed (1, remove ffl?l, M411 0.1NJ3afi95α, hot water <90
Thorough washing was carried out in the order of ℃>1111.

A portion of the fibers thus obtained was taken out and vacuum dried, followed by 6N! ! Hydrolysis was carried out in acid at 110°C for 24 hours, and the amount of immobilization was calculated by amino acid analysis.
157.9 mO of polymyxin B was immobilized per g.

Comparative Example 10 g of polymyxin B sulfate (white sugar, manufactured by Iser) was dissolved in 10,100 O of distilled water.

Add 35g of fiber A to this, and 5g of magnesium oxide.
q was added thereto, and the mixture was allowed to react in a 90° C. oil bath for 8 hours with sufficient stirring. Immobilized fibers were prepared by washing in the same manner as in the Example.

The amount of immobilization determined by amino acid analysis was 1, O per milligram.
mq, which was an extremely small value.

Example 2 I! obtained in Example 1! 0.5Q of fiber (dried product) was weighed and placed into sterilized tubes △ and △, respectively. Next, add 5 ml each of Escherichia coli bacteria solution (28 x 105 cells) and Murine typhoid bacteria solution (7 x 107 cells) to this and incubate at narrow temperature for 12 hours.
Shake for an hour. As a control, a test tube containing only the bacterial solution was prepared and shaken in the same manner.

After shaking, the bacterial solution was quantitatively cultured, and the number of colonies was compared with that of the control to determine antibacterial properties.

As a result, in the case of murine Salmonella typhimurium, the bacterial reduction rate was 9.
It was found that it exhibited significant antibacterial activity of 9.7%. Furthermore, in the case of Escherichia coli, the number of bacteria was reduced by 97.9%, indicating that it has an antibacterial effect.

〔Effect of the invention〕

According to the present invention, physiologically active substances can be immobilized in an insoluble vinyl polymer molded article at high density. Furthermore, it becomes possible to have an immobilization structure different from that in the case of immobilization in a conventional aqueous system. Furthermore, the fibers produced by the method of the present invention are well spread between single filaments, and when used as an absorbent for endotoxins, etc., the contact area with the liquid is large, so the adsorption rate becomes high. It has the advantage of increasing the amount of suction.

Claims (2)

[Claims] (1) A method for immobilizing a physiologically active substance on an insoluble vinyl polymer molded article, the method comprising immobilizing the biologically active substance in the presence of an organic solvent that can swell the molded article. (2) A bioactive substance-immobilized fiber, characterized in that the bioactive substance is polymyxin, and the immobilization density is 10 mg or more per 1 g of fiber.