JPH0694518B2 - Method for producing silk fibroin porous body - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a porous body made of silk fibroin.

[Prior Art] Polymer porous membranes are used in the electronics industry, food industry,
It is used in a wide range of industrial fields such as the chemical industry, pharmaceutical industry, medical field, and fermentation industry. For example, a cellulosic porous body is used as a support for oxygen immobilization, and in this case, the concentration of the stock solution for forming the porous body, the composition of the coagulation bath, the temperature of the pores in the porous body can be adjusted by adjusting the temperature. The shape and size must be controlled, and it cannot be said that the method is easy. Other materials for the synthetic polymer porous material include polyurethane, polyacrylonitrile, polypropylene, and ethylene-vinyl alcohol copolymer, but these materials are difficult to prepare porous materials having various pore sizes. Therefore, the development of new materials is desired.

According to JP 56-166235 A, raw silk is used as a raw material, which is scoured and dissolved in an aqueous solution of calcium salt, ethanol and water, and the resulting solution is dialyzed to obtain an aqueous silk fibroin solution. After making a starch dispersion into this aqueous solution and gelling it, the gelled product is heated under saturated steam to gelatinize the starch contained in the gelled product, and then the gelatinized starch is decomposed with amylase. Then, a method for producing a silk fibroin porous body has been proposed.

However, this method has complicated steps and is still unsatisfactory as an industrial method.

〔Purpose〕

An object of the present invention is to provide an industrially advantageous method for producing a silk fibroin porous body which does not have the above problems.

〔Constitution〕

According to the present invention, an aqueous solution of unmodified silk fibroin obtained from the silkworm body is allowed to gel under the condition of pH 6 or less to cause silk fibroin to gel, or the aqueous solution acts as a poor solvent for silk fibroin. There is provided a method for producing a porous silk fibroin body, which comprises adding a water-soluble organic solvent to gel silk fibroin, and lyophilizing the resulting hydrous silk fibroin gel under vacuum conditions.

The silk fibroin aqueous solution used in the present invention is a diluted aqueous solution obtained by dissolving an undenatured silk fibroin solution obtained from the body of domestic silkworms or wild silkworms in water (the silk fibroin concentration is usually 10% by weight or less). In the invention, the silk fibroin contained in this aqueous solution is gelled. The gelation of silk fibroin is performed by adjusting the pH of the silk fibroin aqueous solution to 6.0.
Hereinafter, it is preferably adjusted to 2 to 4. By this gelation treatment, water-insoluble hydrous silk fibroin gel is precipitated in the aqueous solution, and the precipitate is separated and recovered and used as a molding material to obtain a porous body. As the pH adjuster, organic and inorganic acid aqueous solutions such as acetic acid and hydrochloric acid are used. The hydrated silk fibroin gel is converted into a silk fibroin porous body by freeze-drying it under vacuum. The porous body thus obtained has inside thereof fine pores continuous with the surface formed by evaporation and permeation of liquid water contained in the hydrous silk fibroin gel.

In addition, gelation of silk fibroin can be performed by adjusting the pH of the aqueous solution and by adding a water-soluble organic solvent such as alcohol, which acts as a poor solvent for fibroin, to the aqueous solution.

In the porous body of the present invention, the size of the pores (pore diameter) is usually 0.1 to 500 μm, preferably 1 to 100 μm. Further, the porous body may have any shape such as a block shape in addition to a film shape or a tubular film body. In the present invention, the size of the pores can be adjusted by the concentration of the silk fibroin solution, the pH condition for gelation, the type and concentration of the poor solvent used for gelation, the freezing temperature, and the like.

In the present invention, such a porous body may contain various enzymes and pharmacologically active substances such as pharmaceuticals. Porous materials containing such enzymes and pharmacologically active substances are
These enzymes or pharmacologically active substances may be dissolved or dispersed in the silk fibroin aqueous solution used for the production.

〔effect〕

The silk fibroin porous material of the present invention can be used in various fields like conventional polymer membranes, for example, a blood purification system, a solute separation membrane, a filter, a cell growth carrier, a drug, and a sustained release of agricultural chemicals. Carrier, enzyme-immobilized support,
It can be used as a fragrance holder, a medical prosthesis, an artificial skin, a synthetic leather, a soil modifier, etc.

〔Example〕

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

Example 1 A posterior silk gland was taken out from a heat silkworm body of a mulberry reared domestic silkworm, liquid silk fibroin in the silk gland was dispersed in distilled water, and this was used as a raw material. A 0.8% aqueous solution of liquid silk fibroin was prepared by increasing the sample concentration by blast drying.
This solution was put into a dialysis membrane device made of cellulose and dialyzed with an acetic acid aqueous solution at 5 ° C adjusted to pH 2.65 for 15 hours. In the cellulose dialysis device, the gelled precipitate part and the supernatant part were separated. Happened. The supernatant portion was removed by decantation, and the precipitated portion of the gel-state precipitate was placed on a polyethylene film and frozen at -80 ° C. When this was vacuum dried in a frozen state, a silk fibroin porous material was obtained.

Observation of the scanning electron micrograph of the sample surface and cross section of the silk porous film revealed that the sample surface had a relatively smooth and dense structure, with a layered structure of approximately 100 μm in the longitudinal section, and between the layered structures. , The porosity having a diameter of 30 to 80 μm was confirmed. The prepared silk fibroin porous material was placed at 25 ° C.
It was dipped in distilled water for 12 hours to allow it to hydrate, lightly remove the water droplets on the surface, and measure the water content. As a result, about 8 times as much water as the dry weight of the sample was contained.

Example 2 The same 0.8% sample aqueous solution as in Example 1 was placed in a dialysis membrane apparatus made of cellulose, and this was dialyzed with a phthalate solution of pH 4.01 at 5 ° C. for 15 hours. The obtained gel-like sample and the supernatant were separated by decantation, the gel-like sample was frozen at −80 ° C., and then freeze-dried for 12 hours under a reduced pressure of 10 mmHg or less to produce a porous body. The surface of the porous body was harder than that of the porous body obtained in Example 1. Although the layered structure was slightly collapsed, a three-dimensional layered structure was observed and the pore size of the porous body was 30 μm.

Comparative Example 1 In the same manner as in Examples 1 and 2, distilled water having a pH of 7.76 at 5 ° C was used.
It was dialyzed for 15 hours. In this case, a gel-like sample was not obtained, and it remained in an aqueous solution state. This sample was frozen at −80 ° C. and then freeze-dried for 12 hours under a reduced pressure of 10 ⁻³ mmHg or less to prepare a non-woven fabric-like soft porous body. Even when the solution was adjusted to pH 9.52 with an aqueous sodium carbonate solution, a gel-like sample could not be obtained, and a soft porous body was obtained after freeze-drying.

Example 3 Without adjusting the pH of the 0.7% silk fibroin aqueous solution derived from silkworm silkworm glands, 2.3 ml of methanol was added to 50 ml of the sample solution, and the mixture was left at 5 ° C. for 10 hours to precipitate a gel-like substance. Further, the supernatant was removed by decantation, and the gel-like material was once freeze-solidified at −30 ° C. and freeze-dried under a reduced pressure of 10 ⁻³ mmHg to obtain a porous body. The pore diameter of the obtained porous body was about 30 to 100 μm.

Example 4 A 0.6% aqueous silk fibroin solution was placed in a dialysis membrane apparatus made of cellulose, and the solution was added to a solution obtained by adding 8 parts of water to 2 parts of methanol.
The gel was precipitated by dialysis at 10 ° C for 10 hours, and the gel was separated from the supernatant by centrifugation at 3500 rpm. The gel was freeze-solidified at −45 ° C. and dried under reduced pressure in the same manner as in Example 3 to obtain a porous body having a diameter of 2 to 10 μm.

Example 5 A 0.6% aqueous silk fibroin solution was placed in a dialysis membrane apparatus made of cellulose, and the solution was added to a solution prepared by adding 6 parts of water to 4 parts of methanol.
The gel was precipitated by dialysis at 10 ° C for 10 hours, and the gel was separated from the supernatant by centrifugation at 3500 rpm. By drying under reduced pressure in the same manner as in Example 3 once freeze-solidified at −45 ° C.
A porous body having a diameter of 50 to 100 μm was obtained.

Example 6 A 0.6% aqueous silk fibroin solution was placed in a dialysis membrane apparatus made of cellulose, and the solution was added with 5 parts of a solution prepared by adding 4 parts of water to 6 parts of methanol.
The gel was precipitated by dialysis at 10 ° C for 10 hours, and the gel was separated from the supernatant by centrifugation at 3500 rpm. Once frozen and solidified at −45 ° C. and dried under reduced pressure as in Example 3, 5
A porous body with a diameter of -30 μm was obtained.

Example 7 A 0.6% aqueous silk fibroin solution was placed in a dialysis membrane apparatus made of cellulose, and 5 parts were added in a solution prepared by adding 1 part of water to 9 parts of methanol.
It was dialyzed at 10 ° C. for 10 hours to precipitate a gel. Without separating the gel-like substance from the supernatant by centrifugation, it was freeze-solidified once at −80 ° C. and dried under reduced pressure as in Example 3.
A porous body with a diameter of -30 μm was obtained.

Example 8 0.5 ml of acetic acid solution was added to 50 cc of 0.7% silk fibroin aqueous solution, and the mixture was allowed to stand at 5 ° C. for 8 hours to precipitate a gel-like substance,
The gel was separated from the supernatant by centrifugation at 3500 rpm. By freeze-solidifying at −30 ° C. and drying under reduced pressure in the same manner as in Example 3, a porous body having a diameter of 50 to 100 μm was obtained.

Example 9 A 0.6% aqueous silk fibroin solution was put into a dialysis membrane made of cellulose and treated with methanol at 5 ° C. for 10 hours to precipitate a gel. Without separating the gel from the supernatant by centrifugation, it was freeze-solidified once at -45 ° C and dried under reduced pressure in the same manner as in Example 3 to obtain a porous body having a diameter of 20 to 30 µm.

Example 10 A 0.6% silk fibroin aqueous solution was placed in a dialysis membrane apparatus made of cellulose, 8 parts of water was added to 2 parts of methanol, and the mixture was added at 10 ° C. at 10 ° C.
It was treated for a time to precipitate a gel. Without separating the gel-like substance from the supernatant by centrifugation, it is freeze-solidified once at -45 ° C. and dried under reduced pressure in the same manner as in Example 3 to obtain 30 to 60 μm.
A porous material having a diameter of m was obtained.

Example 11 A posterior silk gland was taken out from the body of a silkworm one day before spitting, washed with water, and liquid silk fibroin from which silk gland cells had been removed was dispersed in distilled water to prepare a 1.5% silk fibroin aqueous solution by a blast drying method. . In 100 ml of distilled water, 9.2 mg of acetylsalicylic acid was dissolved in the same silk fibroin aqueous solution, injected into a cellulose dialysis membrane device, and dialyzed with an aqueous acetic acid solution adjusted to pH 4.0 for 12 hours. The gel and the supernatant thus obtained are centrifuged at 3000 rpm for 20 minutes at a temperature of 25 ° C., and the precipitate is once frozen at a temperature of −80 ° C., then 10
^The product was dried under reduced pressure of ^-3 mmHg to obtain a drug-containing silkworm white porous material. 36 mg of the porous body was placed in a glass cell for ultraviolet spectrophotometer, and 3 ml of distilled water was added to it to reduce the amount of acetylsalicylic acid released from the porous body into the distilled water by 20%.
It was examined as a change in UV absorbance at 6.9 nm, and the results are shown in Table 1.

Example 12 Phosphate solution 6 having a pH of 6.86 was added to 33 ml of an aqueous solution of silk fibroin (0.5%) derived from a posterior silk gland taken out from a matured silkworm body of a silkworm.
26 ml to a pH 6.86 phosphate solution (6 ml) with addition of 26 ml.
3 mg of enzyme glucose oxidase was dissolved and added to the above silk fibroin aqueous solution. The same silk fibroin / glucose oxidase mixed solution left standing at 25 ° C for 3 hours
10 ml was collected, 0.1 ml of acetic acid was added thereto, and the mixture was allowed to stand at 5 ° C. for 10 hours to promote precipitation of a gel-like substance. 3500rp
The supernatant was removed by centrifugation at m for 25 minutes to obtain a water-containing gel. After freezing it at -80 ℃,
A silk fibroin porous material containing glucose oxidase was prepared by freeze-drying for 12 hours under a reduced pressure of ^-3 mmHg or less. The prepared porous body was stored at -30 ° C.

Example 13 A silk fibroin / glucose oxidase mixed solution prepared in the same manner as in Example 12 was placed in a cellulose dialysis membrane device, and a phthalate solution having a pH of 4.01 was used as a dialysis solution at 10 ° C at 5 ° C.
It was dialyzed for a period of time to precipitate a gel. 3500 rpm, 25
The gel-like product obtained by removing the supernatant by centrifugation for
After frozen at 80 ° C., a silk fibroin porous material containing the enzyme glucose oxidase was prepared by freeze-drying for 12 hours under reduced pressure of 10 ⁻³ mmHg or less.

Example 14 The enzyme-containing silk fibroin porous material obtained in Example 12 or Example 13 was attached to the electrode part of a dissolved oxygen concentration meter, and a phosphate buffer solution having different glucose concentrations (pH 6.4, temperature 37
When the electrode was charged at (° C), a change in current corresponding to glucose concentration, that is, a decrease in oxygen concentration was observed. Example
For porous materials obtained in 12 and 13, 1 × 10 ^-6 M to 2 × 1
A linear relationship was obtained between glucose concentration and current change in the range of 0 ^-3 M.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Tsukada ▲ Masuda Yu 1-2 Owashi, Yatabe-cho, Tsukuba-gun, Ibaraki Prefectural Government, Ministry of Agriculture, Forestry and Fisheries, Silkworm Test Station (56) Reference JP-A-56-40156 (JP, A) JP-A-56-166235 (JP, A)

Claims (2)

[Claims] 1. An aqueous solution of unmodified silk fibroin obtained from a silkworm body is allowed to gel under the condition of pH 6 or less, or the aqueous solution acts as a poor solvent for silk fibroin. A method for producing a porous silk fibroin body, which comprises adding a volatile organic solvent to gel silk fibroin, and freeze-drying the obtained hydrous silk fibroin gel under vacuum conditions. 2. The method according to claim 1, wherein the silk fibroin aqueous solution contains an enzyme or a drug.