JPS5811224B2 - Method for producing anti-blood coagulant polymer material - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an anti-blood coagulant polymer material, and more specifically, to an improved ethylene-vinyl acetate copolymer,
The present invention relates to a method for producing a polymeric material endowed with anticoagulant properties.

Ethylene-vinyl acetate copolymer (hereinafter referred to as EVA) is saponified to form ethylene-vinyl alcohol copolymer, which is used as a blood purification membrane for artificial kidneys.

Furthermore, as evidenced by the fact that EVA is used as an adhesive or a polymer modifier, it can be easily coated on other materials such as vinyl chloride resin, polyethylene, etc.
Or you can blend them.

Therefore, by coating other materials with EVA and then saponifying the surface, or conversely, coating saponified EVA and then introducing hydroxyl groups to the surface, it is also possible to obtain a coating material that has been imparted with hydrophilic properties.

As described above, ethylene-vinyl acetate copolymer is a material that can be used, or is expected to be used increasingly, as a medical polymer material that comes into contact with blood through modification.
At present, no effective method has been found to improve the anticoagulability of this material.

Therefore, an attempt could be made to make EVA saponified to make it hydrophilic, and to bind heparin to impart anticoagulant properties.

Several techniques have been attempted in the past to generally immobilize heparin on polymeric materials containing hydroxyl groups.

For example, in the Journal of Applied Physiology, Vol. 29, No. 5, pp. 723-730 (1,970), E.W. It has been shown that it is possible to combine by

Recently, in the Journal of Biomedical Materials Research, Vol. 13, pp. 347-364 (1979), M.B. Sefton et al. introduced a hydroxyl group into a styrene-butadiene-styrene block copolymer. It has been shown that polyvinyl alcohol and formaldehyde and glucurdehyde can be used to bind heparin.

It has been stated that all of these exhibit good anticoagulant properties.

However, since a highly toxic substance called aldehyde is used to fix heparin, it is extremely difficult to completely wash away the aldehyde.Furthermore, fixation with low molecular weight substances such as geltal aldehyde and formaldehyde does not allow heparin to co-exist. The amount of immobilization due to binding is extremely small, and the number of immobilization points per heparin molecule is large.
There was a problem in that the functional groups of heparin were blocked and the activity was likely to decrease.

The present invention has been developed as a result of intensive research aimed at overcoming the problems of the prior art and imparting even better anticoagulability to EVA. They found that if a graft polymer with a structure included in the graft chains of a base polymer is made, heparin can be easily immobilized on the graft chains, and the method of the present invention was completed.

That is, the present invention involves saponifying an ethylene-vinyl acetate copolymer with an alkali, and adding glycidyl acrylate (hereinafter referred to as GA) or glycidyl methacrylate (hereinafter referred to as GMA) to the saponified product in the presence of a polymerization initiator. This is a method for producing an anti-blood coagulant polymer material, which comprises graft polymerizing one or more vinyl monomers as one component, and binding heparin to the graft polymer.

In the method of the present invention, G
It is characterized in that A or GMA alone, and more preferably, other vinyl monomers are introduced together with it to form a polymer graft chain, and heparin is bonded to the epoxy group of GA or GMA present in this graft chain. .

Therefore, unlike conventional techniques in which heparin is directly bonded to a substrate or bonded with a low molecular weight compound such as geltal aldehyde, heparin can be immobilized in a surprisingly large amount and in a highly active state.

The EVA used in the present invention preferably has a vinyl acetate content of 15 parts or more.

The reason for this is that if the ethylene component is too large, the saponification reaction will be difficult, and if the proportion of saponified hydroxyl groups is low, it will be difficult for the graft polymerization reaction to occur sufficiently.
Due to its low hydrophilicity, it is difficult to bind heparin.

The EVA modification method of the present invention is preferably applied to molded articles.

That is, after EvA is made into a molded article by some method, a saponification reaction is performed, or after saponification is made into a molded article, either one of GA or GMA is always included.
Graft polymerization of one or more vinyl monomers.

The reason for applying it to molded products is that the solvent in which saponified EVA can be dissolved changes depending on the ethylene content, so careful consideration must be given to the selection of the reaction solvent, and heparin is mainly bound only near the surface of the molded product. Choosing a homogeneous reaction system and binding heparin to all the saponified molecules of EVA when it would be sufficient is a waste of expensive heparin, and the fact that the heparin-soluble solvent is mainly aqueous (especially water) On the other hand, the solvent for EVA in saponification cannot necessarily be used as a solvent for heparin, which may make the heparin binding reaction difficult.

Molded products include, for example, membrane-like structures for blood purification, tube-shaped blood conduits, coatings on the inner surface of blood conduits such as vinyl chloride, or parts of headers in hollow fiber artificial kidney modules. There is a coating on the surface of the material that comes into contact with blood (the part surrounded by the opening of the hollow fiber solidified with a potting agent and the part into which blood is introduced or taken out from the part). .

EVA can be saponified using a commonly used aqueous caustic soda solution, but when the vinyl acetate content is low, it is preferable to use a caustic soda solution in a mixed solvent of acetone or ethanol and water.

The saponification rate may be determined experimentally in consideration of the physical properties of the target product and the amount of heparin bound, but it is usually preferably 70% or more.

GA or GMA used in the present invention is graft-polymerized to EvA in order to bind heparin to its epoxy group, but as described above, this GA or GMA
It may be used alone or in combination with other vinyl monomers.

However, when only GA or GMA is used, GA or G
Since the epoxy groups of MA are easy to ring open and bond to each other,
The amount of heparin binding may decrease due to gelation of the graft chain, and due to the presence of many epoxy groups in one graft chain, it bonds with the functional groups in the heparin chain at many points. Decreased heparin activity may also occur.

Therefore, it is more preferable to form a graft copolymer with another vinyl monomer such as acrylamide or N-vinylpyrrolidone to reduce the proportion of GA or GMA in the graft chain.

When using GA or GMA, either may be selected, but from an industrial standpoint, GMA is preferred because of its excellent stability.

The vinyl monomer other than GA or GMA used in the present invention is preferably hydrophilic so that the epoxy group in the formed graft chain reacts easily with water-soluble heparin.

However, it is also possible to incorporate somewhat hydrophobic vinyl monomers.

These vinyl monomers include, for example, acrylic acid or its salt, acrylamide, 2-hydroxyethyl methacrylate, acrylonitrile, N-vinylpyrrolidone, sodium p-styrenesulfonate, methyl acrylate, methyl methacrylate, vinyl acetate, allyl Examples include sodium sulfonate.

The polymerization initiator used in the present invention may be any one that can efficiently initiate graft polymerization of a saponified EVA, that is, a polymer having hydroxyl groups. Cerium ammonium,
cerium salts such as ceric ammonium sulfate,
Examples include hydrogen peroxide-ferrous sulfate system, hydrogen peroxide-ferric sulfate system, sodium periodate, and tributylboric acid.

However, preferred are cerium salts or hydrogen peroxide-ferrous sulfate systems.

As a solvent for carrying out the graft polymerization of the present invention, an aqueous system, particularly water, is most preferable from the viewpoint of economy and ease of handling. However, if there is a problem with the solubility of the vinyl monomer,
A mixed solvent of an organic solvent and water can also be used.

For example, methyl methacrylate, methyl acrylate, etc.
When trying to use MA or GA, it has extremely low solubility in water, so a mixed solvent such as water-acetone, water-methyl alcohol, water-ethyl alcohol, water-dimethylacetamide, water-dimethylformamide, etc. should be used. I can do it.

The mixing ratio is determined experimentally from the monomer solubility at the reaction temperature.

The temperature during graft polymerization in the present invention is preferably as low as possible.

The reason for this is that the lower the graft polymerization temperature, the greater the amount of heparin bound at the same grafting rate (the percentage of the weight of the graft polymer reacted and bonded to the saponified EVA to the weight of the base saponified EVA).

However, the lower the temperature, the longer the reaction temperature.

Therefore, the preferred reaction temperature range is above the substantial freezing point of the reaction solution, up to about 70°C where the ring-opening reaction of the epoxy group of GMA or GA can be ignored, and more preferably about 50°C.
~40°C.

In the case of a molded article, the amount of graft polymer per unit surface area can be determined using the amount of bound heparin as the main indicator.

Generally, when graft polymerizing only GA or GMA, 1.5 x 10-' to 3.5 x 1010
The amount of heparin binding reaches the maximum at a surface grafting amount of 101O-4 (7), but when graft copolymerizing with other vinyl monomers, the maximum amount of heparin binding generally shifts toward a higher grafting amount.

Although it varies depending on the type of vinyl monomer to be cograft-polymerized, generally 3 x 10-4 to 10 x 10-'
(gr/cm2) is often the maximum value.

Further, factors affecting the amount of graft polymerization include reaction temperature, monomer concentration, polymerization initiator concentration, polymerization time, etc.

An example of the reaction charge ratio in the graft copolymerization of the present invention is as follows, based on 100 parts by weight of water as a reaction solvent.

In other words, GA or GMA is in the range of 0.01 to 1.2 parts by weight, the lower limit is determined by the value at which heparin can be substantially fixed even when reacted with other vinyl monomers, and the upper limit is determined by the value that allows heparin to be substantially fixed even when reacted simultaneously with other vinyl monomers. It is determined from the solubility in

The polymerizable vinyl monomer other than GA or GMA is GA
The amount of heparin to be charged differs depending on the reaction rate ratio with GMA, but in general, heparin binding occurs at about 500 times the amount of GA or GMA, so 0.01
~600 parts by weight.

However, this upper limit is limited by the solubility in the solvent.

The polymerization initiator varies depending on its type, but in the case of ceric ammonium nitrate and hydrogen peroxide, it is typically in the range of about 1 mmol to 5 mmol of O,OO, and nitric acid used together with ceric ammonium nitrate, or The amount of ferrous sulfate used together with hydrogen peroxide is about % to 5 times the amount of the main initiator.

Since the amount of saponified EVA is often reacted in the form of a molded product, it is convenient to express it in terms of the reaction area relative to the weight of the monomers charged.

The lower limit of the reaction surface area is arbitrary, but the upper limit is empirically determined to be approximately 6X10-3gr (monomer)7c
m2 (reaction surface area).

The graft polymerization reaction of the present invention is stopped by taking out the reactants from the reaction vessel and washing them with a large amount of water.
After stopping the reaction, it is preferable to wash the sample with a large amount of water and use it for the next heparin binding reaction.

Since the epoxy groups of GA or GMA ring open when left in water for a long time even at room temperature, it is necessary to carry out the heparin binding reaction as quickly as possible.

The heparin used in the heparin binding reaction may be commercially available from pig intestinal mucosa, fresh heparin, or the like.

Usually, these are used in the form of powdered heparinum salt of about 100 to 200 (units/m9), and the EVA modified product that has undergone graft polymerization is immersed in this dissolved in water, and the temperature is raised to about 30°C. ~80°C, preferably 50-7
The mixture is kept at 0°C and allowed to react for about 1 to 48 hours.

The lower the reaction temperature, the better in order to suppress the decomposition and decrease in activity of heparin, and the higher the reaction temperature, the better in order to increase the reaction rate and amount of binding.

In addition, the concentration of the heparin aqueous solution during the reaction is high, and the amount of layer bonding is high, but it is economical and the viscosity becomes too high at high concentrations, making it difficult to immerse the molded product.
% (W/V), preferably about 5 to 30%.

Tertiary amines, acid catalysts, and the like can be used as catalysts for the heparin binding reaction, but this is not necessarily advantageous in view of the time and effort required for washing.

The EVA-modified heparin conjugate produced in this way has extremely high heparin activity because it binds a large amount of heparin, and because the reaction is conducted under mild conditions and heparin is bound to the polymer graft chain. It is useful for medical purposes, especially as a material that comes into contact with blood.

For example, it can be applied to blood purification membranes in the form of flat membranes or hollow fiber membranes made of saponified EVA, medical tubes and catheters made of EVA, blood tubes for extracorporeal circulation whose inner surfaces are coated with EVA, and the like.

Next, the present invention will be explained in more detail with reference to Examples.

Example I EVA (vinyl acetate content 70%, melt index 25 (9)
710 minutes) manufactured by Nippon Gosei Kagaku Kogyo ■) was dissolved in ethyl acetate to make a 10% (W/V) solution, and a 50 μm thick film was prepared by the desolvent evaporation method.

This was saponified by immersing it in a 1N caustic soda solution using a 60% ethanol aqueous solution as a solvent at 30°C for 2 hours.

The degree of saponification was 95 as measured by an infrared method, which was determined by the disappearance of acetyl groups in the film.

This saponified EVA film was thoroughly washed with water, and a portion thereof was used for calculating the weight per area.

Pour 300 ml of distilled water into a four-bottle flask with an internal volume of 500 mA kept at 25°C, add 2 g of GMAo, 5 g of N,
- Added 2.5 grams of vinylpyrrolidone and completely dissolved it, added 1 gram of the above-mentioned saponified EVA film, 3 ml of nitric acid (1/1 ON), and 0.3 grams of ceric ammonium nitrate as a polymerization initiator. Thereafter, the reactor was immediately sealed tightly and the operations of reducing the pressure and introducing nitrogen and fluorine gas were repeated to perform nitrogen substitution and to start the reaction.

After 1.5 hours, the film was taken out from inside the reaction flask, washed with a large amount of water, and then added to the heperin 2 film kept at 60°C.
It was immersed in a 0% aqueous solution (W/V) and kept under sealed conditions for 24 hours.

After 5 days, the samples were removed, washed with copious amounts of water, and stored in physiological saline at 4°C.

This film was tested for anticoagulant properties in the following manner.

That is, the film to be tested is immersed in 500 ml of 25% saline at a rate of about 1 gr for 10 hours, repeated twice with the saline solution being changed to zero, and then thoroughly washed with physiological saline. Place it on a glass plate 1.9cm in the center
It is held down with a 5 mm thick silicone gasket with a hole of φ, and is further pressed down and tightened with a glass plate having a hole of the same size.

Next, after pouring out the first 0.5 ml from the dog's jugular vein, replace only the syringe and collect 5 ml of blood. Drop 0.5 ml per sample film as described above and spread it evenly on the film. , every 5 minutes for the first 20 minutes, 2
After 0 minutes, the device was tilted at 45 degrees every 2 minutes to measure the time until the blood coagulated and stopped moving.

The clotting time was measured once for each of the three dogs, and the average value was used to show the clotting time.

Illegal is the Lindholm test applied to dog blood.

The coagulation temperature was room temperature, and the results are shown in comparison with a control film measured at the same time.

The coagulation times measured in this manner are shown below in comparison with other materials.

Illegal heparin fixed EVA film EVA film that has been saponified for more than 120 minutes 22 minute glass
It can be seen that the blood coagulation time is dramatically extended by 15 to 20 minutes, that is, by the anticoagulation method of the present invention.

The amount of bonding in the graft polymerization and heparin bonding of this film was determined by the gravimetric method, and the amount per film area was as follows.

Graft polymerization amount 3.8 x 10-4 (gr/cr
1. ) Heparin binding amount 1.2 x 10-5 (gr
/crtf) The heparin binding amount was determined by measuring the sulfur content by hydrochloric acid hydrolysis of the heparinized product and then measuring the turbidity of the precipitate using BaCl2 (500 mm).

Example 2 By the same method as Example 1, various EvAs shown in Table 1 below were obtained.
created a film.

Furthermore, the solvents shown in Table 1 were used depending on the content of vinyl acetate.

Each film was saponified under the conditions shown in Table 1 while being placed on a glass plate, and it was confirmed by infrared spectrum measurement that each film had a degree of saponification of 90 factors or higher.

Using the above saponified film, GA or GMA and various polymerizable vinyl monomers were graft-polymerized using the same method as in Example 1 using a 4-tube flask with an internal volume of 500 m1, and further washed to heparinize. The reaction was carried out in the same manner.

The weight and sulfur content of this film were measured in the same manner as described in Example 1, and the Lindholm test was also carried out using the same amount of blood.

These results are shown in Table 2.

However, Ce(NH4)2(NO3)62H2 is used as a polymerization initiator.
When 0 was used, nitric acid and cerium salt were added in an equimolar amount.

The H2O3, //Fe5O- initiator was added in exactly the same way as the cerium salt.

From the results in Table 2, it can be seen that the method of the present invention is effective in improving anticoagulability.

Example 3 Soarex DH■ was made into a 10% (W/V) solution of ethyl acetate, and after filling a soft vinyl chloride tube (for dialysis, inner diameter 45 mm, outer diameter 7 mm, length 1 m), both ends were left open. It was hung in a fume hood to allow the internal liquid to fall naturally, and then left overnight to dry.

The same operation was repeated once again, but with the hanging direction reversed, to completely dry the inside.

Next, 1N-NaO containing 60 parts of ethanol inside this
H solution was added, both ends were closed, and the mixture was left at 30°C for 3 hours.

Thereafter, both ends were opened to let out the NaOH solution inside, and the solution was washed with running water all day and night, and then thoroughly replaced with nitrogen.

A graft polymerization solution having the following formulation was introduced into the above-mentioned internal saponification tube, which had been previously flushed with nitrogen, and left at 20° C. for 30 hours.

<Blended graft polymerization solution> Distilled water 10100 mlG 0.1 gr V-pd (see Table 2) 2.0 grCe (NH
4) 2(NO3)6・2H200,06grHNO3(
1/1 ON) 1.5 ml After that, the internal graft polymerization solution was taken out, distilled water was flowed for 10 minutes, and then the tube was filled with 20 parts (W/V) heparin soda solution, both ends were closed, and the tube was heated to 60 m Leave it at ℃ for 24 hours.

After that, remove the sodium heparate solution from the tube and add 1
After washing with running water in the evening, make 25% saline solution to 500ml, connect it to a pump and circulate it through the tube at a rate of 100 ml/min for 10 hours to extract free heparin soda, and then rinse thoroughly to remove physiological saline. The inside was filled with water and stored at 4°C.

This tube was cut into a length of 30 cm and filled with blood that had just been collected from the jugular vein of a beagle dog using the double syringe method.

Clamp both ends with forceps and place in a constant temperature bath at 37°C, and after 60 minutes, at predetermined intervals, clamp the ends with forceps at a new location approximately 2 cm from the end that was clamped with forceps, until the length is approximately 2 cm. The blood contained in the tube was taken out by cutting the tube, dropped into physiological saline, and the presence or absence of clots was visually determined.

Initially, blood sampling intervals should be preliminarily tested every 1 to 2 hours to find out the approximate time of clot formation and the next day, using blood from the same dog, the clot time should be within ±10 minutes. It was precisely determined that

The above samples, those coated with EVA, those with saponification progressed, and soft PVC tubes are shown below.

The data are average values measured using blood from three dogs.

(Clot generation time) Heparinized tube: 6 hrs or more EVA-coated tube: 135 minutes EVA, :]-Ti'g/1□.

Saponified tube Soft PVC tube 52 minutes ・The graft chain weight and heparin binding amount of this heparinized tube were measured using the same method as in Example 1, and the graft chain weight was 1.5×10-4 (gr 7 cm2). , heparin binding amount was 0.73×10 −5 (gr/cm 2 ).

From the above results, it can be seen that the method of the present invention is significantly effective in improving the anticoagulability of blood conduits.

Example 4 Soarex DH■ was dissolved in ethyl acetate at 10%
(2) The solution was cast onto a glass plate to obtain a 25 μm thick film by solvent evaporation.

This was immersed in a 60% (v7v) ethanol aqueous solution of 1N-NaOH at 30°C for 2 hours to obtain a saponification rate of 98% or more (infrared method).

This membrane was washed with water and subjected to kraft polymerization at 20° C. for 2 hours in the same manner as in Example 1 using the following formulation.

<Formulation> Distilled water 300ml GMA 0.1 gr AAm (acrylamide) 1.0 grCe
(NH4)2 (NO3)a 2H200,1grHN
Os (so) 3 ml! Saponified EVA film 250d (4CTLφ10 sheets) Next, 60 d in 15% (W/V) heparin soda aqueous solution.
℃ for 20 hours, washed with a large amount of water, and further Example 1
Free heparin was extracted with saline, washed, and stored in the same manner as described above.

The above film was subjected to a Lindholm blood coagulation test, a measurement of the amount of graft polymerization, and a measurement of the amount of heparin binding in the same manner as in Example 1.

The amount of graft polymerization is 1.5X10-4 (gr/cm2),
Heparin binding amount is 1.7X10-5 (gr/cm2)
Met.

Furthermore, in order to check the performance as a membrane, a pressure difference of 30 mmH was applied to the membrane.
Measurement of water permeation rate by loading g, and urea, vitamin B
Mass transfer coefficients were measured using 100 mg/dl and 10 mg/d7 solutions of I2, respectively.

The results are divided into unreacted saponified EVA film, unsaponified EVA film, and unsaponified EVA film.
In comparison with the VA film, a heparin fixation experiment with geltal aldehyde was conducted using the saponified film No. 8 (Soarex DH ■) of Comparative Example 2 shown in Table 3.

The conditions were to add 5g of heparin soda and 2g of Kurtal aldehyde to 100 volumes of water, add the above film Igr, immediately heat it to 50° for 10 minutes, then take it out, wash it with a large amount of water, and then add 500ml of 25% salt water. 10
The soaking process was repeated twice with the saline solution replaced, and after rinsing with a large amount of water, the sample was stored in physiological saline.

The heparin-fixed film using gel tar aldehyde described above and the heparinized film of NT18 of Example 2 were compared in a Lindholm test, and the results were as follows.

(Clotting time) Geltar aldehyde 45 minutes Example 2 %8 120 minutes control film (-Not converted to valine) 24 minutes

Claims (1)

[Scope of Claims] 1. An ethylene-vinyl acetate copolymer is saponified with an alkali, and the saponified product is prepared in the presence of a polymerization initiator to produce one or more vinyl components in which glycidyl acrylate or glycidyl methacrylate is necessarily one component. 1. A method for producing an anti-blood coagulant polymer material, which comprises graft polymerizing monomers and binding heparin to the graft polymer. 2. The manufacturing method according to claim 1, wherein the ethylene-vinyl acetate copolymer or a saponified product thereof is a molded article. 3 Claim 1 in which the polymerization initiator is hydrogen peroxide-ferrous sulfate type or ceric ammonium nitrate
Manufacturing method described in section. 4 As vinyl monomers, methyl acrylate, methyl methacrylate, N-bidulpyrrolidone, acrylamide, acrylic acid or its salt, 2-hydroxyethyl mequaacrylate, acrylonitrile, p-styrenesulfonic acid sorta, sodium allylsulfonate, The manufacturing method according to claim 1, which contains at least one component of vinyl acetate. 5. The manufacturing method according to claim 2, wherein the molded article is in the form of a film. 6. The manufacturing method according to claim 2, wherein the molded product is tubular.