JPH01119261A - Heparin fixing carrier and its manufacture - Google Patents

Abstract

PURPOSE:To lengthen the time of sustaining anti-thrombic ability as well as to improve the bio-compatibility, by fixing heparin to fibroin with a coupling agent. CONSTITUTION:A fibroin solution of a desired concentration is prepared. Both adequate amounts of an coupling agent and heparin are added to the fibroin solution prepared to make heparin fixed to fibroin via the coupling agent. The reaction is preferably held at a room temperature to prevent denaturation. The coupling agent applied to the fibroin solution may be 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (water-soluble carbodiimide: EDC), glutaraldehyde (GA), an epoxy compound or an isoxazolium salt.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a heparin-immobilized carrier that has excellent antithrombotic duration and excellent biocompatibility, and a method for producing the same.

<Conventional technology> Silk thread has been used for clothing since ancient times as a natural fiber with good texture, warmth, and luster, but in recent years its main component, fibroin, has been developed as a moldable natural protein. It came to be used as a material for cosmetics, medical purposes, etc. For example, an enzyme-containing membrane (Japanese Patent Application Laid-Open No. 55-8
1589), immobilized enzyme carrier (JP-A-56-1588)
7), Porous membrane (JP-A-56-40158), Solid fragrance agent (JP-A-56-163658), Hydrophilic porous body (JP-A-56-166235), Packing agent (JP-A-57-77612) ), lotion (JP-A-57-175
110'J, Water-absorbing agent (Japanese Patent Publication No. 58-244), Poultice (Japanese Patent Application Publication No. 59-27821), Foam (Japanese Patent Publication No. 58-27821)
9-21339), immobilized antibody carrier (Japanese Patent Application Laid-Open No. 1986-1
42259), immobilized antigen/antibody carrier (Japanese Patent Application Laid-open No. 1983
-155129) Drug carrier (JP-A-6O-15512
5) Fibroin is used alone or in combination with other compounds in makeup cosmetics (Japanese Patent Application Laid-open No. 82-415).

However, since fibroin is a bio-derived material, these techniques are used with the expectation that it will have biocompatibility.
Alternatively, it was simply a matter of mixing raw material fibroin with enzymes or drugs to express its functions.

In other words, it is difficult to say that biocompatibility and functionality were actively added.

Furthermore, many materials in which heparin is immobilized have been considered as antithrombotic materials. For example, tridodecyl
A method of heparinizing a polymer surface by ionic bonding using a surfactant such as methyl ammonium chloride ("Heparin", Kodansha Scientific, P, P, 219-223), or a method of fixing it to a polymer surface by a covalent bond. It was also considered that it could become

However, with ionic bonding, heparin dissolves into the blood relatively quickly and is finally gone in a short time.Also, with both covalent bonding and ionic bonding, there are limitations on the carrier polymer from the viewpoint of biocompatibility. It becomes a drawback.

<Object of the Invention> Therefore, the object of the present invention is to provide a heparin-immobilized carrier that eliminates the drawbacks of the above-mentioned prior art, has excellent antithrombotic duration, and also has excellent biocompatibility, and a method for producing the same. I'm trying.

(Structure of the Invention) According to the first aspect of the present invention, there is provided a heparin-immobilized carrier characterized in that heparin is immobilized on fibroin with a coupling agent.

According to a second aspect of the present invention, heparin immobilization is characterized in that after insolubilizing fibroin from fibroin m t&, treatment is performed with a coupling agent and heparin to immobilize heparin to fibroin via the coupling agent. A method of manufacturing a carrier is provided.

According to a third aspect of the present invention, the heparin-immobilized carrier is characterized in that a coupling agent and heparin are added to a fibroin solution to immobilize heparin to the fibroin via the coupling agent, and then the fibroin is insolubilized. A manufacturing method is provided.

In the above invention, the coupling agent is preferably carbodiimide or glutaraldehyde.

<Specific Structure of the Invention> The heparin-immobilized carrier of the present invention and the method for producing the same will be explained in more detail below.

First, the method for producing the heparin-immobilized carrier of the present invention will be described. In the present invention, first, an aqueous solution of fibroin is obtained. Raw materials for the fibroin aqueous solution include cocoon,
Fibroin sources such as raw silk, cocoon waste, raw silk waste, silk spinning, and secondary silk thread are used. After removing sericin by scouring these raw materials according to a conventional method, for example, a copper-ammonia aqueous solution, a copper hydroxide-ethylenediamine aqueous solution, a rhodanate aqueous solution,
Fibroin is dissolved in an aqueous lithium bromide solution, a calcium chloride solution, a calcium nitrate solution, or a magnesium nitrate aqueous solution, and then dialyzed against water or the like and desalted to obtain an aqueous fibroin solution. This aqueous fibroin solution can be adjusted to a desired concentration.

At the time of dissolution, alcohols may be added to increase solubility, and the aqueous fibroin solution may be gelled.

In the present invention, an appropriate amount of a coupling agent and heparin are added to the aqueous fibroin solution prepared in this way, so that heparin is immobilized on the fibroin via the coupling agent. The reaction temperature at this time is preferably room temperature to prevent denaturation. As a coupling agent to be applied to an aqueous fibroin solution, 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide hydrochloride (water soluble carbodiimide: EDC),
Glutaraldehyde (GA), epoxy compounds, isoxazolium salts, etc. can be used. In addition,
EDC is commercially available in the form of hydrochloride, but it is thought to dissociate in water and react with fibroin in the form of carpodiimide.

After heparin is bonded to fibroin via a coupling agent in this manner, it is molded into a desired shape.

In another method of the present invention, water-soluble fibroin is rendered water-insoluble without adding a coupling agent or heparin to the fibroin aqueous solution prepared as described above. Thereafter, a coupling agent and heparin are added to the water-insolubilized fibroin and reacted to bond heparin to fibroin via the coupling agent. Methods for this water insolubilization include methods such as addition of lower alcohols such as ethanol and methanol aqueous solution, stretching, and heat treatment.

When a coupling agent and heparin are added to an aqueous fibroin solution to bind heparin to fibroin, a portion of the heparin will be incorporated into the fibroin crystals.

However, if a coupling agent and heparin are added after making fibroin water-insoluble, heparin will be bound to the surface of fibroin, and heparin can be effectively utilized.

Therefore, it is preferable to once make the fibroin insoluble in water and then apply the cabling agent and heparin to the fibroin.

The heparin-immobilized carrier obtained as described above may be subjected to lower alcohol treatment, heat treatment, etc. Depending on the purpose, fibroin may be chemically modified, mixed with additives, etc. in advance. However, the molded product may be subjected to chemical modification, additive extraction, etc.

The heparin-immobilized carrier formed from the aqueous fibroin solution is dried by heat treatment, air drying, or the like.

Fibroin with heparin fixed thereon can be molded by the above-mentioned method or various other methods.
It can be molded into various shapes depending on the purpose.

The crosslinked fibroin molded product of the present invention has excellent antithrombotic activity for a long time, as shown in the Examples below. In addition, silk thread has excellent biocompatibility since it is also used as a suture thread, and the fibroin molded article on which heparin is fixed according to the present invention also has excellent biocompatibility.

The heparin-fixed fibroin molded product can be processed into various forms depending on its use.

For example, the molded product can be made into a film, and casting is suitable for forming the film from the above-mentioned aqueous fibroin solution, but other methods such as extrusion molding can also be used.

The film-like fibroin molded product can be used for blood containers and the like.

Further, the molded product can be made into a fibrous form, and forming a fibroin form from an aqueous fibroin solution can be carried out in a conventional manner. This is used in filters, sutures, etc.

Moreover, the molded product can be made into a film. The membrane is for osmosis;
It is applied to films on other substrates, and the application is blood IA.
There are physical membranes, etc. As a method of forming a film on other substrates,
Examples include coating and drying.

Moreover, the molded product can also be processed into a block shape such as a porous body, microbeads, or pellets.

The formation of porosity can be achieved by adding a foaming agent, freeze drying, etc., and the formation of microbeads can be performed by a solvent suspension method. When these drugs are loaded with drugs, sustained release properties are improved.

Other uses include adsorbent carriers.

Although the heparin-fixed fibroin molded product of the present invention is water-insolubilized, it is thought that the β-type structure is small. Random coil and α-type structures melt even when molded because they are water-soluble, but the crosslinked fibroin molded product of the present invention swells but does not melt when immersed in water. Since heparin fixed in fibroin is gradually released (dissociated),
The function (antithrombotic) of heparin can be maintained over a long period of time.

(Examples) Next, the present invention will be specifically explained by giving examples and comparative examples.

(Example 1) Raw silk was placed in 50 times the amount of 0.5% sodium carbonate aqueous solution, slowly stirred at 85 to 90°C for 20 minutes, and then
The sericin was removed by washing twice with the same amount of water and then with running water.

This raw silk was further subjected to Soxhlet extraction using a benzene/methanol ratio of 2/1 to remove wax, pigments, and the like.

3 g of the thus refined raw silk was immersed in 8 MLIBr aqueous solution 100° C. and allowed to stand at 37° C. for 48 hours to dissolve. Place this solution in a cellulose dialysis tube,
Dialysis was performed against water. The dialysate was changed three times a day.
Silver nitrate was dissolved in the dialysate until it no longer became cloudy.

The entire dialysis tube was taken out and air-dried with air to concentrate the fibroin aqueous solution. Furthermore, about 900
The solids were removed by centrifugation at 0 G for 10 minutes.

This fibroin solution was cast into a polystyrene Petri dish and dried at room temperature to obtain a fibroin film. Since this film was water-soluble as it was, it was treated with ethanol to make it insoluble and used for experiments.

In Example 1 of the present invention, a fibroin film was prepared using heparin and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter abbreviated as EDC).
Each sample was immersed in a solution in which 00 mg of heparin was dissolved in 50 mj2 of citric acid-phosphate buffer of pH 3.2, and in a solution in which 500 mg of heparin was similarly dissolved as Comparative Example 1.
Incubate for 5 hours at 25°C. Thereafter, the film was taken out and immersed in 250 mg of distilled water, and the change over time in the amount of heparin eluted was measured.

(Example 2) A fibroin film was prepared in the same manner as in Example 1, immersed in a pH 3,2 citric acid-phosphate buffer in which 200 tng of heparin was dissolved, and incubated at 37°C for 24 hours. Of these, half were taken out as Comparative Example 2, and heparin was eluted in a pH 7.4 citric acid-phosphate buffer. Glutaraldehyde (hereinafter referred to as GA) was added to the remaining half of the film as Example 2 of the present invention to a concentration of 0.025M, and further incubated for 24 hours. In addition, GA concentration 0.025M
The film was transferred to a pH 7.4 citrate-phosphate buffer, where it was again kept for 24 hours. After washing with water, transfer to 0.2% by weight dimedone solution and remove excess unreacted GA.
(24 hours). After washing with water, heparin was eluted in a citric acid-phosphate buffer at pH 7.4.
Changes in heparin elution amount over time were quantified.

Results〉 Heparin elution amount [IU] Original non-occurrence 1
13.00 Comparative Example 1 2200 Inventive Example 23 Comparative Example 2 300 In both Inventive Examples 1 and 2, the elution amount of heparin was controlled compared to Comparative Example 1.2, and it was immobilized on fibroin.

(Example 3) Changes over time in the amount of heparin eluted into water from the fibroin films obtained in Inventive Example 1 and Comparative Example 1 were measured. However, since the experiment was conducted on a smaller scale than in Example 1, the absolute amount of eluted heparin was small. The results are shown in the table below.

Heparin elution amount [10] > Elution time Present invention example 1 Comparative example 1 [Hr] 24 67
In 8B Comparative Example 1, the elution amount was saturated after 12 hours, but in Inventive Example 1, it was saturated after 72 hours, and the heparin elution duration was extended.

<Effects of the Invention> Since the heparin-immobilized carrier of the present invention immobilizes heparin on fibroin using carbodiimide or glutaraldehyde, heparin is easily eluted when the car contains heparin in fibroin. However, since the immobilized carrier of the present invention not only contains heparin but also binds it, its effect persists and is useful as an antithrombotic material. It is something.

Such a heparin-fixed carrier can be easily obtained by the production method of the present invention.

Claims (5)

[Claims] (1) A heparin-immobilized carrier characterized in that heparin is immobilized on fibroin using a coupling agent. (2) The heparin-immobilized carrier according to claim 1, wherein the coupling agent is a carbodiimide. (3) The heparin-immobilized carrier according to claim 1, wherein the coupling agent is glutaraldehyde. (4) A method for producing a heparin-immobilized carrier, which comprises insolubilizing fibroin from a fibroin solution and then treating it with a coupling agent and heparin to immobilize heparin to fibroin via the coupling agent. (5) A method for producing a heparin-immobilized carrier, which comprises adding a coupling agent and heparin to a fibroin solution to immobilize heparin to fibroin via the coupling agent, and then insolubilizing the fibroin.