JPH01118530A - Molded item of fibroin and its preparation - Google Patents

Abstract

PURPOSE:To obtain a molded item of fibroin having excellent flexibility, stretchability, bio-compatibility etc. and being suitable for sewing fibers, artificial blood vessels, sensor membranes etc., by adding a crosslinking agent into a fibroin soln. and solidifying and molding the fibroin. CONSTITUTION:An aq. fibroin soln. is prepd. by refining and removing sericin from a raw material such as cocoons, raw silk etc., dissolving it into an aq. calcium chloride soln. and desalting by means of dialysis. Then, a proper amt. of a crosslinking agent (e.g., water-soluble carbodiimide and glutar aldehyde) is added to this fibroin soln. to crosslink fibroin and to insolubilize it in water. Then, a molded item of fibroin is obtd. by molding into a required shape such as film, fiber etc. and drying it. Moreover, a method wherein, after the fibroin soln. is solidified and molded, the molded item is reacted with a crosslinking agent (e.g., diisocyanate) is also used.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a fibroin molded product that has excellent stretchability, flexibility, and biocompatibility, and a method for producing the same.

<Conventional technology> Silk thread has been used since ancient times for clothing as a natural fiber with good texture, texture, and luster, but in recent years its main component, fibroin, has been used in cosmetics as a moldable natural protein. It has come to be used as a material for industrial and medical purposes. For example, an enzyme-containing membrane (Japanese Patent Application Laid-Open No. 55-8
1589), immobilized enzyme carrier (JP-A-56-1568)
7), Porous membrane (JP-A-56-40156), solid fragrance (JP-A-56-163658), hydrophilic porous body (JP-A-56-166235), pack agent (JP-A-57-77812) ), lotion (JP-A-57-175
11!1), water-absorbing agents (Japanese Patent Publication No. 58-244), poultices (Japanese Patent Publication No. 59-27821), foams (Japanese Patent Publication No. 58-27821),
9-21339), immobilized antibody carrier (Japanese Patent Application Laid-Open No. 80-1989)
42259), immobilized antigen/antibody carrier (Japanese Patent Application Laid-open No. 1983
-155129) Drug carrier (JP-A-6O-15512
5), Make-up Cosmetics I (JP-A-62-415), etc. Fibroin is used alone or in combination with other compounds.

The fibroin used for these purposes is obtained by removing sericin from cocoon, raw silk, cocoon waste, raw silk waste, etc., dissolving it in an aqueous solution containing alkali metals and alkaline earth metals, and then desalting it by a method such as dialysis. The raw material is an aqueous solution.

This aqueous fibroin alone or together with other compounds is molded into the desired form and properties to produce the target substance, but in any case, water-soluble fibroin must be transferred to water-insoluble fibroin.

As one method for this purpose, treatment to increase the degree of crystallinity has been carried out. In other words, the molecular forms of fibroin are random coil, 5ilk I
Type crystals (α type structure) and 5ilkll type crystals (β type structure) are known (0, Kratky).

et, al, Nature, 165.475 (195
0), 5ilk Since all crystals other than type II crystals are water-soluble,
The crystallization fraction of the 5ilkII type crystal must be increased by some means to make it insoluble in water.

Examples of such methods include adding diols, polyhydric alcohols, etc. (Japanese Unexamined Patent Publication No. 55-81589), freeze-drying at low temperature (Japanese Unexamined Patent Publication No. 56-40156), and adjusting the pH to 3.0 or more.
5.0, add lower alcohol, stir at high shear deformation speed, and perform ultrasonic treatment.
-163658, etc.).

In addition, instead of increasing the degree of crystallinity, depending on the application, synthetic polymers may be grafted (e.g., JP-A-57-77612), combined with synthetic polymers (e.g., JP-A-58-244), or the main component, rubber. Methods such as binding fibroin (Japanese Patent Publication No. 59-21339) may also be considered.

However, these techniques had the following drawbacks.

That is, fibroin with increased crystallinity has high strength but low elongation and flexibility. Furthermore, when fibroin is combined with a synthetic polymer, the biocompatibility of fibroin is reduced by half due to the effect of the synthetic polymer.

If it is crystallized by stirring, most of it will precipitate, resulting in poor yield. Fibroin is denatured when the pl is set to 3 to 5 or when subjected to ultrasonic treatment.

In addition, the fibroin molded product obtained in this way is
Since the β-type crystallization fraction is high, the molecular backing is denser than that of the one with a low β-type crystallization fraction. Therefore, properties such as water absorption and substance permeability are also not good.

<Object of the Invention> Therefore, the object of the present invention is to eliminate the drawbacks of the above-mentioned conventional techniques and provide a fibroin molded product with excellent flexibility, stretchability, and biocompatibility, and a method for producing the same. There is something to do.

<Structure of the invention> Conventionally, the β-type crystal fraction was increased to make it water insoluble.
β-type crystals have a dense molecular backing, so when used as a membrane, they have low permeability to water and solutes. Furthermore, when used as a drug carrier, there was less space for the drug to enter.

On the other hand, the present inventors have discovered that even a random coil state or an α-type crystal state can be rendered water insoluble by crosslinking, leading to the present invention.

According to a first aspect of the present invention, there is provided a fibroin molded article consisting essentially of fibroin, the fibroin being treated with a crosslinking agent.

According to a second aspect of the present invention, there is provided a method for producing a fibroin molded article, which comprises adding a crosslinking agent to a fibroin solution and then solidifying the fibroin.

According to a third aspect of the present invention, there is provided a method for producing a fibroin molded article, which comprises solidifying and molding fibroin from a fibroin solution and then treating the product with a crosslinking agent.

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

Moreover, various forms of the molded product can be applied, such as cast film, recycled fibers, permeable membranes, coated films, porous bodies, and microbeads.

<Specific Structure of the Invention> The fibroin molded product of the present invention and the method for producing the same will be explained in more detail below.

First, the method for producing a fibroin molded article 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.

During dissolution, alcohols may be added to increase solubility. Further, the fibroin aqueous solution may be a gelled one.

In the present invention, an appropriate amount of crosslinking agent is added to the aqueous fibroin solution prepared in this way to crosslink the fibroin. The reaction temperature at this time is preferably room temperature to prevent denaturation. Examples of crosslinking agents to be applied to the fibroin aqueous solution that make water-soluble fibroin water-insoluble include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (water-soluble carbodiimide: EDC), glutaraldehyde (GA) , epoxy compounds, bisimidates, diazides, dinitrenes, cybarides, dialdehydes, simalimides, diisocyanates, jepoxies, disulfonyl chlorides, carbodiimides, isoxazolium salts, di(1゜10-phenanthroline) ) Copper, dithiobis(succinimidylpropionic acid)
, Woodward reagent, etc. can be used. Na
EDC is commercially available in the form of hydrochloric acid, but it is thought to dissociate in water and react with fibroin in the form of carbodiimide.

After crosslinking the fibroin in this way, it is molded into a desired shape.

In another method of the present invention, the aqueous fibroin solution prepared as described above is molded into a desired shape without adding a crosslinking agent, and then crosslinked to form a molded body containing water-soluble fibroin with a low degree of crystallinity. agent may be reacted. This makes the water-soluble fibroin water-insoluble.

The crosslinking agents used at this time include diisocyanate,
Jepoxy compounds and the like can be used.

The crosslinked molded product obtained as described above may be subjected to lower alcohol treatment, heat treatment, etc.

Depending on the purpose, the fibroin may be subjected to chemical modification, additive mixing, etc., or the molded product may be subjected to chemical modification, additive extraction, etc.

A molded article formed from an aqueous fibroin solution is dried using a dehydrating agent such as alcohol, heat treatment, or the like.

Fibroin that has been crosslinked by the above-mentioned method or various other methods is molded, and it can be molded into various shapes depending on its use, purpose, etc.

The crosslinked fibroin molded product of the present invention has excellent flexibility and elasticity, as shown in the examples below. Also,
Silk thread originally has excellent biocompatibility as it is also used for sutures, and the fibroin molded product of the present invention also has excellent biocompatibility.

The 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 preferred for forming the film from the aqueous fibroin solution, but other methods such as regeneration coagulation can also be used.

The film-like fibroin molded product can be used for filters, sensor membranes, and the like.

Further, the molded product can be made into a fibrous form, and forming a fibrous form from an aqueous solution of buproin can be carried out in a conventional manner. This is used for sutures, artificial blood vessels, non-woven fabrics, etc.

Moreover, the molded product can be made into a film. The membrane is for osmosis;
It is applied to coatings on other substrates, and its uses include dialysis membranes,
This includes adding ecological compatibility to the substrate. Other methods for forming a film on a substrate include coating and drying.

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

Making it porous can be done by adding a foaming agent, freeze drying, etc., and making it into microbeads can be done by drying and pulverizing. When these drugs are loaded with drugs, sustained release properties are improved.

Other uses include adsorbent base materials.

It is considered that the fibroin molded product of the present invention is treated with a crosslinking agent to be insolubilized and does not have a β-type structure. ra
Although a ndom coil, α-type structure melts even if it is made into a molded product because it is water-soluble, the crosslinked fibroin molded product of the present invention is a random coil, α-type structure.
Since the mold structure is simply cross-linked and does not have a β-type structure, it will swell but will not dissolve when immersed in water.

<Examples> Next, the present invention will be specifically described with reference to 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 100 g of an 8 M LiBr aqueous solution 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
Centrifuged in 'OG for 10 minutes to remove solids. The concentration of this fibroin aqueous solution was 1.5 wt%.

Next, the liquid was divided into two parts, and one part of the peptide synthesis reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter referred to as EDC) as a crosslinking agent was added to one part along with fibroin and dissolved. The sample to which EDC was added is referred to as Invention Example 1, and the sample to which EDC was not added is referred to as Comparative Example 1.

Both were cast into a polystyrene Petri dish and dried at room temperature for 3 days. A fibroin film was formed on the Petri dish, and both could be easily peeled off from the Petri dish. When a portion of both was cut out and placed in water, the one to which EDC was added swelled but did not dissolve. The one without EDC was easily dissolved in water.

Next, both were soaked in ethanol overnight and dried.

When I cut out parts of both again and put them into water, this time neither of them dissolved in water.

Next, a tensile test was conducted on both. sample length 80
It was cut into pieces 10 mm wide and 0.04 mm thick, and heated at 23°C using Strograph T manufactured by Toyo Seiki.
The breaking strength and breaking elongation were measured at an initial length of 50 mm and a tensile speed of 50 mm/min.

The results are shown below.

Invention example 1 4.3X10' 60 Comparative example 1
11.6X10' 2 In this way, although the strength of Inventive Example 1 is 37% lower than that of Comparative Example 1, the elongation is 30 times that of Comparative Example 1, whereas Comparative Example 1 is rigid and brittle. It can be seen that the material of Invention Example 1 is flexible and has good elongation.

<Effects of the Invention> The fibroin molded product of the present invention has 30 times more elongation than conventional fibroin molded products that lacked elongation and flexibility. Fibroin molded products can be easily obtained.

Claims (11)

[Claims] (1) A fibroin molded article consisting essentially of fibroin, the fibroin being treated with a crosslinking agent. (2) The fibroin molded article according to claim 1, wherein the crosslinking agent is carbodiimide. (3) The fibroin molded article according to claim 1, wherein the crosslinking agent is glutaraldehyde. (4) The fibroin molded product according to any one of claims 1 to 3, wherein the fibroin molded product is a cast film. (5) The fibroin molded product according to any one of claims 1 to 3, wherein the fibroin molded product is a recycled fiber. (6) The fibroin molded product according to any one of claims 1 to 3, wherein the fibroin molded product is a permeable membrane. (7) The fibroin molded product according to any one of claims 1 to 3, wherein the fibroin molded product is a coat film. (8) The fibroin molded product according to any one of claims 1 to 3, wherein the fibroin molded product is a porous body. (9) The fibroin molded product according to any one of claims 1 to 3, wherein the fibroin molded product is a microbead. (10) A method for producing a fibroin molded article, which comprises adding a crosslinking agent to a fibroin solution and then solidifying the fibroin. (11) A method for producing a fibroin molded product, which comprises solidifying and molding fibroin from a fibroin solution and then treating it with a crosslinking agent.