A biodegradable biopolymer material consists of silk fibroin from domesticated silkworm; silk fibroin from wild silkworm; a composite material comprising silk fibroin from domesticated silkworm and silk fibroin from wild silkworm; or a composite material comprising either silk fibroin from domesticated silkworm or silk fibroin from wild silkworm and at least one secondary substance selected from the group consisting of cellulose, chitin, chitosan, chitosan derivatives, keratin from wool and polyvinyl alcohol. The material may be prepared by, for instance, casting an aqueous solution of domesticated silkworm silk fibroin on the surface of a substrate and then cast drying the applied solution. The biodegradable biopolymer material is effectively used as, for instance, a metal ion-adsorbing material, a sustained release substrate for a useful substance such as a medicine, a biological cell-growth substrate and a biodegradable water-absorbing material.

Description of the Invention

SUMMARY OF THE INVENTION

Accordingly, it is generally an object of the present invention to solve the problems associated with the foregoing conventional techniques and more specifically to provide a biodegradable biopolymer material consisting of a silk protein excellent as a polymeric substrate; a hybridized biodegradable biopolymer material comprising the silk protein and a specific secondary substance hybridized together and having unique characteristic properties, which are not observed for the silk protein alone; a method for the preparation of the same; and functional materials consisting of the foregoing biodegradable biopolymer materials, such as a metal ion-adsorbing material, a sustained release carrier for a useful substance, a biological cell-growth substrate and a biodegradable and water absorbable material.

The silk fibers from domesticated silkworm and those from wild silkworm are fibrous materials produced and spun by silkworm and they have strong resistance to chemicals even to the action of, for instance, chemical agents and enzymes since they have fibrous structures as determined by the X-ray diffraction analysis. This is the reason why the silk fiber from domesticated silkworm is classified as the biologically non-absorbent material. Thus, the inventors of this invention have conducted various studies to provide a material comprising such a silk protein having good biodegradability while making the most use of the excellent biochemical properties of the silk protein and to develop a technique for preparing a novel material whose biodegradability can be controlled by using silk fibroin from domesticated silkworm as a starting material and combining the starting material with a specific secondary substance. The inventors have further inspected for the degradation behavior observed for a novel composite material obtained during the process for the development when acting an enzyme on the composite material, have found that a biopolymer material possessing biodegradability can be provided and have thus completed the present invention.

The biodegradable biopolymer material of the present invention is characterized in that it consists of silk fibroin from domesticated silkworm; silk fibroin from wild silkworm; a composite material comprising silk fibroin from domesticated silkworm and silk fibroin from wild silkworm; or a composite material comprising either silk fibroin from domesticated silkworm or silk fibroin from wild silkworm and at least one secondary substance selected from the group consisting of cellulose, chitin, chitosan, chitosan derivatives, keratin from wool and polyvinyl alcohol.

In this respect, the biodegradable biopolymer material may be one capable of being biologically degraded by the action of at least one enzyme selected from the group consisting of proteases, collagenases and chymotrypsin.

The shape of the biodegradable biopolymer material may be any one such as a fibrous, membrane-like, powdery, gel-like or porous shape.

The method for the preparation of a biodegradable biopolymer material according to the present invention comprises the steps of applying, onto the surface of a substrate, an aqueous solution of silk fibroin from domesticated silkworm, an aqueous solution of silk fibroin from wild silkworm, an aqueous mixed solution containing an aqueous solution of silk fibroin from domesticated silkworm and an aqueous solution of silk fibroin from wild silkworm or an aqueous mixed solution comprising either an aqueous solution of silk fibroin from domesticated silkworm or an aqueous solution of silk fibroin from wild silkworm and an aqueous solution of at least one secondary substance selected from the group consisting of cellulose, chitin, chitosan, chitosan derivatives, keratin from wool and polyvinyl alcohol; and then drying the applied solution to dryness to form a film-like biodegradable biopolymer material, wherein if using the aqueous mixed solution, the aqueous solutions as the constituents of the aqueous mixed solution are uniformly admixed together by stirring them such that they do not undergo any gelation, precipitation and/or coagulation reaction to thus prepare the aqueous mixed solution.

Moreover, a powdery biodegradable biopolymer material of the present invention can be prepared by freezing the foregoing aqueous solution of silk fibroin from domesticated silkworm, the foregoing aqueous solution of silk fibroin from wild silkworm or the foregoing aqueous mixed solution and then drying the frozen aqueous solution under a reduced pressure. In this connection, the mixed aqueous solution is prepared by the same mixing method used above. Further, a gel-like biodegradable biopolymer material of the present invention can be prepared by adjusting the pH value of the foregoing aqueous solution of silk fibroin from domesticated silkworm, the foregoing aqueous solution of silk fibroin from wild silkworm or the foregoing aqueous mixed solution to a level falling within the acidic region and then coagulating the entire aqueous solution to thus give a gel-like biodegradable biopolymer material. Incidentally, a porous substance can be prepared by subjecting the gel-like product of the biodegradable biopolymer material thus prepared to lyophilization.

In the preparation of the foregoing aqueous mixed solution, the concentrations of the aqueous solution of silk fibroin from domesticated silkworm, the aqueous solution of silk fibroin from wild silkworm and the aqueous solution of the secondary substance preferably range from 0.1 to 5% w/v, respectively. This is because if the concentration is less than 0.1% w/v, the amount of the aqueous solutions required for the preparation of the composite material increases and this is not preferred from the viewpoint of the operation efficiency, while if it exceeds 5% w/v, it is difficult to uniformly admix two solutions and as a result, it is likewise impossible to prepare any composite material having uniform quality.

The metal ion-adsorbing material according to the present invention consists of a biodegradable biopolymer material, which is silk fibroin from domesticated silkworm; silk fibroin from wild silkworm; a composite material comprising silk fibroin from domesticated silkworm and silk fibroin from wild silkworm; or a composite material comprising either silk fibroin from domesticated silkworm or silk fibroin from wild silkworm and at least one secondary substance selected from the group consisting of cellulose, chitin, chitosan, chitosan derivatives, keratin from wool and polyvinyl alcohol. In this connection, the metal ions may be anti-bacterial metal ions such as silver, copper and cobalt ions or metal ions present in waste water.

The sustained release carrier for a useful substance according to the present invention is characterized in that it consists of a biodegradable biopolymer material, which is silk fibroin from domesticated silkworm; silk fibroin from wild silkworm; a composite material comprising silk fibroin from domesticated silkworm and silk fibroin from wild silkworm; or a composite material comprising either silk fibroin from domesticated silkworm or silk fibroin from wild silkworm and at least one secondary substance selected from the group consisting of cellulose, chitin, chitosan, chitosan derivatives, keratin from wool and polyvinyl alcohol and that it can gradually release the useful substance supported on the biodegradable biopolymer material while being biodegraded by the action of a protease, chymotrypsin or a collagenase. The biodegradable biopolymer material is preferably a porous substance.

The living cell-growth substrate according to the present invention consists of a biodegradable biopolymer material, which is silk fibroin from domesticated silkworm; silk fibroin from wild silkworm; a composite material comprising silk fibroin from domesticated silkworm and silk fibroin from wild silkworm; or a composite material comprising either silk fibroin from domesticated silkworm or silk fibroin from wild silkworm and at least one secondary substance selected from the group consisting of cellulose, chitin, chitosan, chitosan derivatives, keratin from wool and polyvinyl alcohol. The substrate is used for effectively and economically growing living cells.

The biodegradable water absorbable material according to the present invention consists of a biodegradable biopolymer material, which is silk fibroin from domesticated silkworm; silk fibroin from wild silkworm; a composite material comprising silk fibroin from domesticated silkworm and silk fibroin from wild silkworm; or a composite material comprising either silk fibroin from domesticated silkworm or silk fibroin from wild silkworm and at least one secondary substance selected from the group consisting of cellulose, chitin, chitosan, chitosan derivatives, keratin from wool and polyvinyl alcohol.

The term "biodegradation" herein used means any reaction including, for instance, a digestion or hydrolysis reaction of silk fibroin and/or the secondary substance into small molecules through the action of an enzyme and a digestion reaction thereof into amino acids. Accordingly, an enzyme may degrade the substrate into small molecules through reactions other than digestion in the present invention, but the enzyme may likewise conveniently be referred to as a protease (proteolytic enzyme).

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, raw materials for use in the preparation of an aqueous solution containing silk fibroin from silk protein fibers may, for instance, be silk fibers from domesticated or wild silkworms. The silk fibroin of the silk fiber per se obtained from domesticated silkworm usable herein may be, for instance, silk fibroin as a silk protein from, for instance, larvae of domesticated silkworm (Bombyx mori) reared in farmhouses and larvae of KUWAGO (Bombyx mandarina or mulberry wild silkworm) as a relative species of the domesticated silkworm. Examples of silk fibroin from wild silkworm usable herein are silk fibroin obtained from larvae of Antheraea pernyi, Antheraea yamamai, Antheraea militta, Antheraea assama, Philosamia cynthia ricini and Philosamia cynthia pryeri. Alternatively, raw materials for preparing the silk fibroin aqueous solution may likewise be, for instance, by-products from domesticated and wild silkworms, silk fibers, silk fiber products and aggregates of silk fibers, in addition to the foregoing silk fibers.

As has been described above, the secondary substance to be hybridized with the silk fibroin from domesticated or wild silkworm is at least one member selected from the group consisting of cellulose, chitin, chitosan, chitosan derivatives, keratin from wool and polyvinyl alcohol.

An aqueous solution of silk fibroin from domesticated silkworm is admixed with an aqueous solution of silk fibroin from wild silkworm or either an aqueous solution of silk fibroin from domesticated silkworm or an aqueous solution of silk fibroin from wild silkworm is admixed with an aqueous solution of such a secondary substance, followed by extending the resulting mixed aqueous solution over the surface of a substrate of, for instance, polyethylene and then solidifying the extended solution through drying to thus produce a biodegradable biopolymer material, which is a composite material (or a hybrid material) of the silk fibroin from domesticated silkworm and the silk fibroin from wild silkworm or a composite material of the silk fibroin from domesticated silkworm or the silk fibroin from wild silkworm with the secondary substance. Biodegradable biopolymer materials may likewise be prepared from an aqueous solution containing silk fibroin from domesticated silkworm alone and an aqueous solution containing silk fibroin from wild silkworm alone by repeating the same procedures used above.

The preparation of an aqueous solution of silk fibroin from domesticated silkworm and an aqueous solution of silk fibroin from wild silkworm as well as the preparation of a membrane of silk fibroin from domesticated silkworm and a membrane of silk fibroin from wild silkworm will hereunder be described in detail and a method for the preparation of a hybrid (composite material) using an aqueous mixed solution comprising an aqueous solution of silk fibroin from domesticated silkworm or an aqueous solution of silk fibroin from wild silkworm and an aqueous solution of each secondary substance or cellulose, chitin, chitosan, chitosan derivatives, keratin from wool or polyvinyl alcohol will be detailed below.

(A) Preparation of Aqueous Solution of Silk Fibroin from Domesticated Silkworm and Membrane of Silk Fibroin from Domesticated Silkworm

An aqueous solution of pure silk fibroin from domesticated silkworm may be prepared by the following method:

First, cocoon fibers produced and spun by domesticated silkworm are boiled in an alkaline aqueous solution of a neutral salt such as sodium carbonate to remove sericin and to thus prepare silk fibroin fibers as the entity of the domesticated silkworm silk fibers. Then the resulting silk fibroin fiber is dissolved in a concentrated aqueous solution of a neutral salt and heated to form a silk fibroin aqueous solution. This silk fibroin aqueous solution contains the silk fibroin and a large quantity of ions originated from the neutral salt used above. Thus, the aqueous solution is poured into a cellulose membrane for dialysis, the both ends of the membrane are tied up with sawing threads and dialyzed against tap water or pure water for a desired period of time ranging from 2 to 5 days to thus give an aqueous solution of pure domesticated silkworm silk fibroin. Aqueous solutions of silk fibroin having a variety of concentrations can be prepared by partially evaporating the water of the resulting silk fibroin aqueous solution or diluting the resulting silk fibroin aqueous solution with water.

The aqueous solution of domesticated silkworm silk fibroin thus prepared can be extended over a substrate such as a polyethylene membrane, followed by solidification of the extended layer of the silk fibroin solution through evaporation to dryness at room temperature to thus give a domesticated silkworm silk fibroin membrane.

In addition, the domesticated silkworm silk fibroin aqueous solution can be prepared by adding domesticated silkworm silk protein fibers (silk fibers) to a concentrated aqueous solution of a neutral salt such as calcium chloride, calcium nitrate, lithium bromide or lithium thiocyanate and then heating the mixture to thus dissolve the silk fibers in the aqueous solution. The concentration of the neutral salt in the aqueous solution ranges from about 5 to 9M and the it is sufficient to heat the mixture to a temperature ranging from about 25 to 70.degree. C. and preferably 25 to 60.degree. C. for the dissolution of the silk fibers. In this respect, if the dissolution temperature exceeds 70.degree. C., the molecular weight of the silk protein is reduced, the resulting material loses its polymeric characteristics and as a result, the molding properties of the material may considerably impaired. The dissolution time is preferably set at a level on the order of 1 to 20 minutes. Among the foregoing neutral salts, those satisfactorily dissolving the domesticated silkworm silk protein fibers are lithium salts excellent in the ability of solubilizing the domesticated silkworm silk fibroin fibers, with lithium bromide being in general preferred. For instance, an aqueous lithium bromide solution having a concentration of not less than 8M and preferably not less than 8.5M would permit dissolution of domesticated silkworm silk protein fibers by the treatment at a temperature of not less than 55.degree. C. for a time of not less than 15 minutes.

(B) Preparation of Aqueous Solution of Silk Fibroin from Wild Silkworm and Membrane of Silk Fibroin from Wild Silkworm

To prepare an aqueous solution of wild silkworm silk fibroin from silk fibers from wild silkworms such as those from Antheraea pernyi and Antheraea yamamai, the wild silkworm cocoon fibers is first immersed in an aqueous solution of sodium peroxide in a predetermined amount with respect to the mass thereof, boiled for a desired time period therein to thus form wild silkworm silk fibroin fibers and the resulting silk fibroin fibers are dissolved in an aqueous solution of a neutral salt having a high solubilization ability. Then the resulting aqueous solution is dialyzed in the same manner used above in connection with the domesticated silkworm silk fiber to thus give an aqueous solution of pure wild silkworm silk fibroin. This preparation method will hereunder be described in more detail.

In the preparation of an aqueous solution of wild silkworm silk fibroin by dissolving wild silkworm silk fibers, the silk sericin covering the surface of the wild silkworm cocoon fibers should be removed by a method different from that for refining the domesticated silkworm silk sericin. This is because tannin is also adhered to the surface of the wild silkworm silk fibers other than sericin and the sericin is insolibilized due to the cross-linking action of the tannin. It is, for instance, necessary to immerse the wild silkworm cocoon fibers in about 50 volumes of a 0.1% sodium peroxide aqueous solution on the basis of the mass of the cocoon fibers and to then subject the cocoon fibers to a boiling treatment therein, for instance, at 98.degree. C. for one hour in order to remove these sericin and tannin. The wild silkworm silk fibroin fibers from which the sericin and tannin have been removed in advance are then dissolved in an aqueous solution of a neutral salt having a high solubilization ability such as lithium thiocyanate. The solution of the wild silkworm silk fibroin fibers in the aqueous neutral salt solution is poured into a cellulose membrane for dialysis, the both ends of the membrane are tied up with sawing threads and dialyzed against tap water or pure water maintained at room temperature for a desired period of time ranging from 2 to 5 days to completely remove the lithium ions present therein and to thus give an aqueous solution of pure wild silkworm silk fibroin.

The aqueous solution of wild silkworm silk fibroin thus prepared can be extended over a substrate such as a polyethylene membrane, followed by solidification of the extended layer of the silk fibroin solution through evaporation to dryness at room temperature to thus give a wild silkworm silk fibroin membrane.

If the silk proteins from domesticated silkworm and those from wild silkworm can sufficiently be admixed together in the form of aqueous solutions, a composite material of these silk proteins may be prepared by solidifying an aqueous mixed solution of these components through evaporation to dryness and the resulting hybrid membrane may possess characteristic properties such as biodegradability, transparency, adhesion stability and other biochemical properties different from those observed for the materials from domesticated silkworm silk fibroin alone and wild silkworm silk fibroin alone. Thus, we will hereunder explain the method for preparing a hybrid membrane of the domesticated silkworm silk fibroin and the wild silkworm silk fibroin starting from the aqueous solutions of domesticated silkworm silk fibroin and wild silkworm silk fibroin.

(C) Hybrid Membrane of Domesticated Silkworm Silk Fibroin and Wild Silkworm Silk Fibroin

Desired amounts of the aqueous solution of domesticated silkworm silk fibroin and the aqueous solution of wild silkworm silk fibroin prepared above are digestion into a beaker, followed by extremely carefully and gently mixing them with stirring using a glass rod in such a manner that the aqueous solution never undergoes gelation. The mixed aqueous solution thus prepared can be extended over a substrate such as a polyethylene membrane, followed by solidification of the extended layer of the silk fibroin solution through evaporation to dryness at room temperature to thus give a transparent hybrid membrane. In this respect, the concentrations of the aqueous solution of domesticated silkworm silk fibroin and the aqueous solution of wild silkworm silk fibroin are preferably on the order of 0.1 to 3% w/v and particularly preferably 0.4 to 2% w/v, respectively.

The blending of either an aqueous solution of domesticated silkworm silk fibroin or an aqueous solution of wild silkworm silk fibroin with an aqueous solution of a secondary substance as will be detailed below may likewise be carried out in the same manner used above.

In this respect, a method for the preparation of a hybrid of domesticated silkworm silk fibroin and silk fibroin from Antheraea pernyi has already been reported by the inventors of this invention (M. Tsukada et al., Journal of Applied Polymer Science, 1994, 32: 1175-1181). However, this article never includes any description, which teaches and/or suggests the biodegradability of the hybrid.

(D) Hybrid Membrane of Domesticated or Wild Silkworm Silk Fibroin and Cellulose

A hybrid membrane consisting of domesticated silkworm silk fibroin and cellulose can be prepared by admixing the foregoing aqueous solution of the domesticated silkworm silk fibroin and an aqueous solution of cellulose according to the following method.

First, domesticated silkworm silk fibroin fibers and commercially available powdery cellulose (available from Fluka Company) free of any particular purification treatment are separately dissolved in cuprammonium ([Cu(NH.sub.3).sub.4](OH).sub.2) aqueous solution to thus form respective aqueous solutions. Then these two kinds of aqueous solutions are admixed in a desired mixing ratio (domesticated silkworm silk fibroin fibers/cellulose) with extremely carefully and gently stirring in such a manner that the mixture never undergoes any gelation, precipitation and/or solidification. The mixed aqueous solution thus prepared is gently extended over a substrate such as a glass plate placed on a horizontal plane and a mixed solution containing acetone and acetic acid is carefully added to the surface of the extended mixed aqueous solution to thus remove the metal complex present in the mixed aqueous solution while solidifying the domesticated silkworm silk fibroin and cellulose. Thereafter, the solidified mixture is washed with a mixed solution of glycerin and water and then with water, followed by drying the mixture at room temperature to thus give a hybrid membrane containing domesticated silkworm silk fibroin and cellulose.

A hybrid membrane containing wild silkworm silk fibroin can likewise be prepared by the same procedures used above in connection with the preparation of the hybrid membrane containing the domesticated silkworm silk fibroin.

In this respect, the inventors of this invention and the collabs have already reported a method for the preparation of a hybrid of domesticated silkworm silk fibroin and cellulose. (see G. Freddi et al., Journal of Applied Polymer Science, 1995, 56: 1537-1545). However, this article never includes any description, which teaches and/or suggests the biodegradability of the hybrid.

(E) Hybrid Membranes of Domesticated or Wild Silkworm Silk Fibroin and Chitin, Chitosan and Chitosan Derivatives

A hybrid membrane comprising domesticated silkworm silk fibroin and chitin, chitosan or a chitosan derivative can be prepared by admixing an aqueous domesticated silkworm silk fibroin solution and an aqueous solution of chitin, chitosan or a chitosan derivative according to the following method. The chitosan derivative usable in the present invention is not restricted to any specific one and may be, for instance, chitin, carboxylated carboxy methyl chitin (hereunder also referred to as "CMK") from chitosan, Na salt of carboxy methyl chitin and glycol chitosan.

The chitin used in the present invention may be, for instance, one from a marine crustacean such as a prawn or a black tiger or chitin covering the crust of an insect. The crust of a crustacean or an insect comprises inorganic substances such as calcium carbonate and proteins and therefore, chitin may be isolated by the removal of contaminants other than chitin according to any currently known method. Moreover, it is also convenient to use a commercially available powdery product of chitin (Wako Pure Chemical Industries, Ltd.).

Chitin may be converted into water-soluble one according to the following method. First, powdery chitin is suspended in a concentrated aqueous caustic alkali (such as sodium hydroxide) solution and stirred over a desired period of time under reduced pressure. Then the resulting powdery chitin is charged into a concentrated aqueous caustic alkali solution containing a surfactant such as sodium dodecyl sulfate, stirred and allowed to stand overnight at a low temperature (for instance, -20.degree. C.); or the resulting powdery chitin is suspended in liquid ammonia (-33.degree. C.) and then metal potassium is added to the resulting suspension to thus prepare alkali chitin in which the hydrogen atoms on the C6 and C3 hydroxyl groups of chitin are substituted with sodium or potassium. The alkali chitin thus prepared is compressed and dispersed in ice crushed into fine pieces, followed by the sulfidation of the chitin through the addition of carbon disulfide to thus obtain a chitin sulfide. An aqueous solution can be prepared using this sulfide.

Moreover, the alkali chitin may be reacted with an epoxy compound, an allyl or an alkali halide to thus give an o-allyl derivative or an o-alkyl derivative. Further, the alkali chitin may be reacted with ethylene chlorohydrin (2-chloroethanol) to give ethylene glycol chitin and it may be reacted with chloroacetic acid to give o-(carboxymethyl) chitin. If ethylene glycol chitin is reacted with a concentrated caustic alkali aqueous solution (for instance, a 40% sodium hydroxide aqueous solution) under desired reaction conditions (for instance, 100.degree. C. for 5 hours) with stirring, the acetamide groups present on the chitin molecules are hydrolyzed into free amino groups to thus give water-soluble glycol chitosan. Chitosan derivatives including the glycol chitosan can easily be dissolved in an aqueous acid solution having a wide concentration range, such as an aqueous acetic acid solution.

A mixed aqueous solution obtained by the addition of a domesticated silkworm silk fibroin aqueous solution to the foregoing aqueous glycol chitosan solution may be extended over, for instance, a polyethylene substrate and then solidified through evaporation to dryness to thus prepare a transparent and soft composite material (a hybrid membrane) comprising domesticated silkworm silk fibroin and glycol chitosan. Hybrid membranes may likewise be prepared by the use of aqueous solutions of other chitosan derivatives or an aqueous solution of water-solubilized chitin instead of the foregoing glycol chitosan aqueous solution according to the same procedures used above.

In the case of wild silkworm silk fibroin, hybrid membranes may be prepared by repeating the same procedures used above in connection with the domesticated silkworm silk fibroin.

(F) Hybrid Membrane of Domesticated or Wild Silkworm Silk Fibroin and Wool Keratin

Usable in the present invention may be, for instance, wool keratin fibers as well as aqueous keratin solutions and aqueous S-carboxy methyl keratin (CMK) solutions, which can be prepared as follows. These aqueous solutions may be prepared according to the conventionally known methods.

First of all, to solubilize wool yarns, the Cystine cross linkings are cleaved using a reducing agent (such as mercapto-ethanol or thioglycollic acid) in a nitrogen gas atmosphere or keratin molecules are reduced and solubilized. If mercapto-ethanol is used, it is preferred to carry out the reduction in a urea solution. In this case, the concentration of urea in general ranges from 7.5 to 8.8 M and preferably 7.8 to 8 M. Moreover, if thioglycollic acid is used, it is desirable to add NaCl to the reaction system in an amount of 1 to 4%.

For instance, when using mercapto-ethanol, which may act as a reducing agent, wool yarns are immersed in a urea solution having a concentration specified above, followed by degassing, adding mercapto-ethanol to the mixture in an amount of 3 to 5 mL per 10 g of wool yarns at a temperature of not more than 45.degree. C. and desirably 20 to 25.degree. C. in a nitrogen gas atmosphere and stirring the resulting mixture over a predetermined period of time (for instance, about 3 hours). Thus keratin molecules in wool yarns are reduced and keratin molecules having SH groups are correspondingly prepared. Then the reaction system containing the keratin molecules having SH groups is digestion into a cellulose membrane for dialysis, the both ends of the cellulose membrane are tied up with sawing threads and sufficiently dialyzed against pure water to remove the urea and the excess mercapto-ethanol present therein and to thus give an aqueous solution of the wool keratin. This aqueous wool keratin solution may be used as an aqueous solution of a secondary substance used in the present invention according to the same procedures used above.

Moreover, if the wool keratin carrying --SH groups obtained above is further reacted with an alkylation agent, for instance, any known alkylation agent such as an (substituted) alkyl halide to form an S-(substituted) alkyl keratin, the aqueous solution thereof may likewise be used in the present invention. This alkylation may be carried out according to any known method. The alkylation will hereunder be described using iodoacetic acid as an alkylation agent by way of example. To the foregoing reduced keratin, there is added iodoacetic acid (molecular weight: 185.95) in an amount ranging from 10 to 17 g per 10 g of the wool yarns in order to react them at a temperature ranging from 20 to 25.degree. C. in a nitrogen gas atmosphere with stirring. After 1 to 2 hours, the pH value of the reaction system is adjusted to about 8.5, followed by dialysis against pure water to remove the excess iodoacetic acid and to thus give an aqueous solution of S-carboxymethyl keratin.

To the aqueous solution of the reduced keratin or the aqueous solution of the S-carboxymethyl keratin, there can be added an aqueous solution of domesticated silkworm silk fibroin to give a mixed aqueous solution, followed by extending the mixed aqueous solution over the surface of a substrate such as a polyethylene substrate and then drying the extended aqueous layer to thus give a hybrid membrane of the reduced keratin or the S-carboxymethyl keratin and the domesticated silkworm silk fibroin.

In the case of the wild silkworm silk fibroin, a hybrid membrane can be prepared according to the same procedures used for preparing the hybrid membrane of the domesticated silkworm silk fibroin.

(G) Hybrid Membrane of Domesticated Silkworm Silk Fibroin or Wild Silkworm Silk Fibroin and Polyvinyl Alcohol

Polyvinyl alcohol (PVA having an average degree of polymerization of about 2000 available from Wako Pure Chemical Co., Ltd.) is charged into hot water, followed by careful dissolution using a stirring machine to thus form an aqueous PVA solution having a desired concentration (for instance, a 0.5% w/v PVA aqueous solution). An appropriate amount of an aqueous solution of domesticated silkworm silk fibroin is added to this PVA aqueous solution, followed by allowing the resulting mixture to stand at room temperature for not less than 30 minutes to form a complex aqueous solution of domesticated silkworm silk fibroin and PVA. The complex aqueous solution can be extended over the surface of a substrate such as a polyethylene substrate and the moisture of the extended aqueous layer is evaporated over a whole day and night to thus give a transparent hybrid membrane of PVA and the domesticated silkworm silk fibroin.

In the case of the wild silkworm silk fibroin, a hybrid membrane can be prepared according to the same procedures used for preparing the hybrid membrane of the domesticated silkworm silk fibroin.

In this connection, the inventor of this invention and the collaborators have already reported a method for the preparation of a hybrid membrane of PVA and domesticated silkworm silk fibroin (see, M. Tsukada et al., Journal of Applied Polymer Science, 1994, 32: 243-248). However, This article never includes any disclosure, which refers to or suggests the biodegradability of the hybrid membrane at all.

As has been discussed above, the silk proteins from domesticated silkworm, those from wild silkworm and secondary substances may be well admixed together in their aqueous solution states and hybrid membranes can be prepared from the resulting aqueous mixed solutions. The resulting hybrid membranes may show biochemical characteristic properties such as biodegradability, transparency (light transmission properties) and a cell-growth ability, which are different from those observed for a material simply comprising domesticated silkworm silk fibroin or wild silkworm silk fibroin. In addition, the hybrid membrane also possesses, for instance, excellent metal ion-adsorbing properties and resistance to peeling. To obtain a hybrid membrane from an aqueous solution of domesticated silkworm silk fibroin, an aqueous solution of wild silkworm silk fibroin and aqueous solutions of secondary substances in this case, it is sufficient that the concentration of each aqueous solution falls within the range of from 0.1 to 5% w/v, as has been specified above, and preferably 0.4 to 3% w/v and thus hybrid membranes having uniform quality can be obtained. In this connection, the aqueous solution of domesticated silkworm silk fibroin and the aqueous solution of wild silkworm silk fibroin; and the aqueous solution of domesticated silkworm silk fibroin or the aqueous solution of wild silkworm silk fibroin and the aqueous solution of secondary substances may be admixed together in any rate and therefore, the mixing ratio of these components in the resulting composite may, if desired, be set at an arbitrarily level.

To admix domesticated silkworm silk fibroin and wild silkworm silk fibroin, or an aqueous solution of domesticated silkworm silk fibroin or an aqueous solution of wild silkworm silk fibroin with an aqueous solution of a secondary substance, it is sufficient to gently admix these aqueous solutions with stirring using a glass rod. This is because if these solutions are rapidly admixed together or they are admixed vigorously or violently, a shear stress is applied to the silk fibroin molecules, the aqueous solutions undergo coagulation and it is sometimes observed that these solutions are not uniformly admixed.

The biodegradable biopolymer material of the present invention may have any shape such as a sheet-like, membrane-like, powdery, bead-like, gel-like, fibrous, tubular or hollow thread-like one.

In the present invention, the biodegradability of a biodegradable biopolymer material can be evaluated by treating it with a buffering solution containing a peptidase in a predetermined concentration for a predetermined period of time. More specifically, the biodegradable biopolymer material is digested (or hydrolyzed) through the treatment thereof with an enzyme-containing aqueous dissociation solution prepared by dissolving an enzyme having a desired activity in a desired buffering solution at 37.degree. C. for a predetermined period of time. The degree of biodegradation is evaluated by calculating the extent of the biodegradable biopolymer material digested by the enzyme on the basis of the weight change of the sample.

The degree of digestion is greatly influenced by the kinds of enzymes used, the concentrations of the enzyme, the time required for the enzyme-decomposition and/or the kinds of materials to be treated. Moreover, the degree of digestion also greatly varies depending on whether the material is silk protein fibers or silk protein membranes. The silk protein fiber produced by silkworm has a fibrous structure peculiar thereto and a large density of hydrogen bonds formed between fibrous molecules and therefore, it is hardly hydrolyzed even when introducing it into an aqueous solution of a peptidase. For this reason, the silk protein fiber can be used as a sample for a biodegradation test without any pre-treatment. Contrary to this, a silk fibroin membrane or the like as a silk protein membrane prepared after once dissolving the silk protein fibers in a neutral salt solution gets swollen through the absorption of moisture and is ultimately dissolved therein. In the biodegradation test, the dissociation behavior of the material in a buffering solution containing an enzyme is examined and therefore, the silk fibroin membrane per se thus prepared cannot directly be subjected to such a biodegradation test. It is thus necessary to subject the membrane to an insolubilization treatment in order to use the same as a test sample. The material or membrane may be insolibilized by, for instance, immersion thereof in an aqueous solution of an alcohol such as methanol or ethanol; or by the use of a conventionally known epoxy compound or an aldehyde such as formalin. For instance, the membrane may be insolibilized by immersing it in a 20 to 80% methanol aqueous solution for a time usually ranging from 5 to 10 minutes and preferably by immersing it in a 40 to 60% methanol aqueous solution for 5 to 10 minutes. More specifically, it is sufficient to lightly immerse the membrane in a 50% (v/v) methanol aqueous solution at room temperature for not less than one minute and then dry it in air at room temperature.

Moreover, almost all of the composite materials other than the foregoing silk fibroin membrane, immediately after the preparation thereof by the process for evaporation to dryness are insoluble in water. Usually, these materials are desirably insoluble in water in many applications and it is sufficient, in such cases, to make them insoluble in water by the treatment with methanol. The composite material of domesticated silkworm silk fibroin and cellulose or that of domesticated silkworm silk fibroin and polyvinyl alcohol is water-soluble immediately after the preparation thereof. If the composite material is treated with methanol, the silk fibroin thus becomes insoluble in water, but the cellulose and polyvinyl alcohol components are never converted into water-insoluble ones through such a methanol treatment. Accordingly, it is preferred for such composite materials to subject them to a cross-linking reaction with a reagent having a strong cross-linking ability such as formalin.

The peptidase (digestive enzyme) usable in the present invention may be any one. The peptidase may likewise be one, which cleaves a distinct site of a substrate or one whose cleaving site on a substrate cannot be specified. The biodegradable biopolymer material of the present invention may be biodegraded by the action of an enzyme such as proteases, collagenases, and chymotrypsin. As has been described above, it is desirable for the evaluation of the biodegradability using these enzymes to use a buffering solution having a desired pH value capable of maintaining the maximum enzyme activity. The combination of an enzyme and a buffering solution used in the enzymatic decomposition is not restricted to any specific one. Examples of preferred combinations of enzymes and buffering solutions are a collagenase and 50 mM TES (buffering solution) or 50 mM CaCl.sub.2 (pH 7.4); chymotrypsin and 50 mM Tris (buffering solution) or 5 mM CaCl.sub.2 (pH 7.8); and a protease and 40 mM potassium phosphate (buffering solution) (pH 7.5). A borate buffering solution having a low ionic strength is preferably used as such a buffering solution and the pH thereof roughly ranges from 7 to 8.

The concentration of the protein hydrolase (or peptidase) aqueous solution may vary depending on the kinds of proteins as substrates and in general ranges from 0.1 to 0.8 mg/mL and preferably 0.2 to 0.5 mg/mL. This is because if the enzyme concentration is less than 0.1 mg/mL, the efficiency of the digestion is insufficient, while if it exceeds 0.8 mg/mL, the biodegradation experiment becomes less advantageous from the economical standpoint.

One of the inventors of this invention has previously prepared domesticated silkworm silk fibroin membrane and domesticated silkworm silk fibers by dissolving domesticated silkworm silk fibroin fibers, followed by the biodegradation of them to make clear the biodegradation behavior thereof with time (see N. Minoura et al., Biomaterials, 1990, 11 (Aug.): 430-434). In this article, it is confirmed that this domesticated silkworm silk fibroin membrane is hydrolyzed to a significant extent in a protease solution, while the domesticated silkworm silk fibers are not hydrolyzed to any significant degree. However, a silk material from wild silkworm is one of silk proteins having a primary structure completely different from the chemical structure of these domesticated silkworm silk fibers and there have not yet been reported any information concerning the biodegradability of wild silkworm silk fibers and wild silkworm silk fibroin membrane.

According to the present invention, a powdery biodegradable biopolymer material can be prepared by lyophilizing an aqueous solution of domesticated silkworm silk fibroin, an aqueous solution of wild silkworm silk fibroin, an aqueous mixed solution containing an aqueous solution of domesticated silkworm silk fibroin and an aqueous solution of wild silkworm silk fibroin or an aqueous mixed solution comprising either an aqueous solution of domesticated silkworm silk fibroin or an aqueous solution of wild silkworm silk fibroin and an aqueous solution of a secondary substance such as cellulose according to any known method. More specifically, these aqueous solutions are frozen at a temperature of about -10.degree. C. and then frozen solutions are allowed to stand in an atmosphere maintained at a reduced pressure to remove the moisture present in the sample and to thus form a powdery material. In addition, a gel-like biodegradable biopolymer material may be obtained by adjusting the pH value of the aqueous solution of each sample so as to fall within the acidic region, for instance, not more than 4.4 to coagulate the entire aqueous solution and to thus convert it into a gel. A membrane-like biodegradable biopolymer material may be obtained by extending the aqueous solution of each sample over a substrate such as a polyethylene substrate or a glass plate, followed by evaporating the extended layer to dryness for a sufficient period of time.

All of the foregoing powdery, gel-like and membrane-like biodegradable biopolymer materials are soluble in water and therefore, they can, if desired, be insolibilized in water by immersing in an aqueous alcohol solution as has been discussed above.

The easiness of the biodegradability of the biodegradable biopolymer material of the invention through the action of a hydrolase is determined by the concentration of the enzyme, the buffering solution used, the digestion time, the degree of water-insolubilization and the content of the domesticated silkworm silk fibroin. For this reason, the easiness of the biodegradability of a material can be improved by reducing the water-insolubility or increasing the water-solubility and increasing the content of the domesticated silkworm silk fibroin in the material. A silk material free of any fibrous structure such as silk fibroin membrane is quite susceptible to digestion with an enzyme unlike the silk fibroin fibers. In particular, the easiness of the biodegradability of a composite material (hybrid) is determined by the degree of water insolubility of the domesticated and wild silkworm silk fibroins, the kind of the secondary substance selected, the mixing ratio of the domesticated or wild silkworm silk fibroin to the secondary substance, the kind of the enzyme selected, the enzyme concentration and the treating time and therefore, the conditions for preparing hybrids, the mixing ratios or the biodegradation conditions can appropriately be changed or selected depending on the desired purposes.

A biodegradable biopolymer material having good biocompatibility can be prepared by hybridizing or blending silk fibroin with an organic polymer (secondary substance), which is excellent in the affinity to biological tissues, but is hardly decomposed with a protein hydrolase.

The biodegradable biopolymer material of the present invention may be a hybrid of materials, both of which serve as substrates for enzymes such as proteases, collagenases and chymotrypsin; or a hybrid of a polymer material capable of serving as a substrate and a secondary substance, which cannot serve as a substrate. Examples of proteins capable of serving as substrates for these three kinds of enzymes are domesticated silkworm silk fibroin, wild silkworm silk fibroin and wool keratin. When hybridizing these materials capable of serving as substrates for the enzymes with naturally occurring polymers, which cannot serve as substrates of these enzymes, such as cellulose, chitin, chitosan, chitosan derivatives and polyvinyl alcohol, there is observed such a tendency that the amount of the hybrid biodegraded is gradually reduced as the content of the naturally occurring polymer in the hybrid increases.

For instance, in the case of a hybrid membrane consisting of domesticated silkworm silk fibroin and cellulose, the domesticated silkworm silk fibroin is easily decomposed by the action of a protease and therefore, the higher the content of the domesticated silkworm silk fibroin, the easier the control of the degree of biodegradation of the hybrid. However, the behavior of the domesticated silkworm silk fibroin for a cellulase is completely contrary to the behavior discussed above and accordingly, the higher the content of the cellulose, the smaller the amount of the hybrid biodegraded as a whole. Thus, a biodegradable biopolymer material having a desired rate of biodegradation can be prepared by variously changing the mixing ratio of the protein capable of serving as a substrate for an enzyme used to a secondary substance, which can never serve as a substrate for the enzyme.

The biopolymer usable herein is not restricted to any specific one and may be, for instance, silk proteins from domesticated and wild silkworms (such as silk fibroins and silk sericin) or keratins from animals (such as wool keratin); collagen; and gelatin. Usable herein include, for instance, silk proteins from domesticated and mulberry wild silkworms, or silk proteins from Antheraea yamamai, Antheraea pernyi, Philosamia cynthia ricini and Philosamia cynthia pryeri Silkworms as wild silkworms. Such biopolymers may likewise be silk fibers, silk fiber products from domesticated and wild silkworms or fibrous aggregates thereof, or keratin fibers from animals and keratin fiber products.

The biodegradable biopolymer material of the present invention is useful as a metal ion-adsorbing material. In particular, when immersing a composite material (hybrid) as a biodegradable biopolymer material of the present invention in an aqueous solution containing antibacterial metal ions such as silver, copper and/or cobalt ions, the composite material adsorbs a large quantity of these metal ions and therefore, the composite material carrying metal ions adsorbed thereon can be useful as an antibacterial material. Alternatively, when immersing the biodegradable biopolymer material in waste water, the material adsorbs various kinds of metal ions present in the waste water (for instance, base metal ions such as Cu.sup.2+, Ni.sup.2+, Vo.sup.2+, Zn.sup.2+, Co.sup.2+ and Al.sup.3+, and ions of rare earth metals such as Yb, Nd, Pr and La) and accordingly, the material is also useful as a material for adsorbing metal ions present in waste water. The metal ions thus adsorbed on the material may be recovered or disposed, according to circumstances.

A useful substance such as a water-soluble medicine or a pharmaceutically active substance can be included in or immobilized on the biodegradable biopolymer material, in particular, the composite material of the present invention and the resulting product may be implanted or embedded in, for instance, a living body so that the product implanted may gradually release the medicine or pharmaceutical component, while the material is decomposed and/or deteriorated through digestion with, for instance, a protease present in the body fluid. Therefore, the material of the present invention can be used as a sustained release carrier for useful substances. In this connection, the silk fibroin fiber from domesticated or wild silkworm may be used for making the biodegradability thereof light, or a membrane-like sample obtained by dissolving domesticated or wild silkworm silk fibers using a neutral salt, desalting the resulting solution using a cellulose dialysis membrane and then evaporating the dialyzed solution to dryness in order to obtain an easily decomposable material. The membrane of domesticated silkworm silk fibroin is more easily biodegraded than the membrane of wild silkworm silk fibroin and therefore, it is sufficient to increase the content of the wild silkworm silk fibroin to form a hardly biodegradable composite material comprising domesticated and wild silkworm silk fibroins.

As has been described above, when using the biodegradable biopolymer material, in particular, the composite material of the present invention while embedding it in the living body, the material is ultimately decomposed into small molecules such as water and carbon dioxide by the action of enzymes present in the body such as proteases, chymotrypsin and collagenases and finally excreted outside the body. A hybrid membrane with easily biodegradable domesticated silkworm silk fibroin may be biodegraded within a relatively short period of time even when embedding the same in the living body unlike hardly biodegradable domesticated silkworm silk fibroin fibers and therefore, the hybrid may be used for the temporal assist of the healing of remediable damaged biological tissues or as a sustained release carrier for drugs as has been discussed above. Such in vivo degradable and absorbable material may be used in a variety of applications such as the suture of incised and/or wound portions, arrest of hemorrhage, bone fixation, a clue for tissue-regeneration and a means for preventing adhesion.

The hybridization of domesticated or wild silkworm silk fibroin with a secondary substance would provide such a conspicuous effect that the resulting hybrid shows, on it surface, excellent biochemical properties, which have never been observed for the surface of the domesticated or wild silkworm silk fibroin or the secondary substance. For instance, the rate of cell-growth on the surface of the hybrid is higher than that observed on the surface of a product simply consisting of domesticated or wild silkworm silk fibroin or a secondary substance. Moreover, the hybridization of domesticated silkworm silk fibroin with wild silkworm silk fibroin or the hybridization of a secondary substance such as cellulose with domesticated or wild silkworm silk fibroin would provide a hybrid or composite material having improved moldability and transparency as compared with those observed for a membrane simply consisting of domesticated or wild silkworm silk fibroin and possessing excellent cell adhesion properties. In addition, the composite material also has a high wear resistance and the rate of cell-growth on the composite surface is improved as compared with that observed on the surface of a membrane consisting of a single protein. Accordingly, such a composite material may likewise be used as cell-growth materials in the field of biochemistry.

Moreover, cellulose derivatives may be used in food additives, cosmetics, additives for drugs and pharmaceutical preparations such as anti-thrombotic agents and therefore, the composite materials consisting of domesticated silkworm silk fibroin and cellulose may be used in applications similar to those for the cellulose.

The biodegradable biopolymer material of the present invention possesses water-absorbing properties, which make the material applicable as a water-absorbable resin used in, for instance, disposable hygienic goods and household goods, water cut-off agents, soil conditioners, dewing inhibitors, water-retention agent for agriculture and horticulture and the present invention would permit the supply of a water-absorbing material having such biodegradability in a low price without requiring any complicated steps. For this reason, the material of the present invention can be applied to any fields of applications identical to those for the conventionally known water-absorbing resins. For instance, the material of the present invention can be used in a wide variety of fields such as hygiene (typically the use as a diaper and a sanitary good), medical service (for instance, the use in cataplasms), civil engineering and architecture (for instance, the use as an agent for gelling sludge), foods, industries, and agriculture and horticulture (for instance, the use as a soil conditioner and a water-retention agent).
 

Claim 1 of 1 Claim

1. A method for the preparation of a biodegradable biopolymer material comprising the steps of: (A) applying onto the surface of a substrate, an aqueous mixed solution containing: (i) a 0.1 to 5% w/v aqueous solution of silk fibroin from a wild silkworm; and (ii) an aqueous solution of reduced wool keratin or an aqueous solution of S-carboxymethyl keratin, wherein the aqueous mixed solution of (A) is prepared by uniformly admixing the aqueous solutions by stirring such that they do not undergo any gelation, precipitation and/or coagulation reaction; (B) cast drying the applied solution to form a membrane-like biodegradable biopolymer material; and (C) subjecting the membrane-like biodegradable biopolymer material to a water-insolubilization treatment by immersion in a 20 to 80% aqueous solution of an alcohol.

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