U.S. patent application number 10/458277 was filed with the patent office on 2004-01-08 for biodegradable biopolymers, method for their preparation and functional materials constituted by these biopolymers.
This patent application is currently assigned to NATIONAL INSTITUTE OF AGROBIOLOGICAL SCIENCES. Invention is credited to Arai, Takayuki, Tsukada, Masuhiro.
Application Number | 20040005363 10/458277 |
Document ID | / |
Family ID | 29996509 |
Filed Date | 2004-01-08 |
United States Patent
Application |
20040005363 |
Kind Code |
A1 |
Tsukada, Masuhiro ; et
al. |
January 8, 2004 |
Biodegradable biopolymers, method for their preparation and
functional materials constituted by these biopolymers
Abstract
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.
Inventors: |
Tsukada, Masuhiro;
(Ibaraki-ken, JP) ; Arai, Takayuki; (Ibaraki-ken,
JP) |
Correspondence
Address: |
ARENT FOX KINTNER PLOTKIN & KAHN
1050 CONNECTICUT AVENUE, N.W.
SUITE 400
WASHINGTON
DC
20036
US
|
Assignee: |
NATIONAL INSTITUTE OF
AGROBIOLOGICAL SCIENCES
|
Family ID: |
29996509 |
Appl. No.: |
10/458277 |
Filed: |
June 11, 2003 |
Current U.S.
Class: |
424/537 ;
106/124.4; 106/136.1; 106/156.5; 435/408 |
Current CPC
Class: |
C12N 2533/50 20130101;
C12N 5/0068 20130101; A61L 27/3604 20130101; A61K 38/1767 20130101;
A61L 27/48 20130101; A61L 27/227 20130101 |
Class at
Publication: |
424/537 ;
435/408; 106/136.1; 106/124.4; 106/156.5 |
International
Class: |
A61K 035/24; A61K
035/37; C12N 005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 19, 2002 |
JP |
178126/2002 |
Claims
What is claimed is:
1. A biodegradable biopolymer material characterized in that the
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.
2. The biodegradable biopolymer material as set forth in claim 1,
wherein the biodegradable biopolymer material is one capable of
being digestively degraded by the action of an enzyme selected from
the group consisting of proteases, collagenases and
chymotrypsin.
3. The biodegradable biopolymer material as set forth in claim 1,
wherein the biodegradable biopolymer material possesses a fibrous,
membrane-like, powdery, gel-like or porous shape.
4. The biodegradable biopolymer material as set forth in claim 2,
wherein the biodegradable biopolymer material possesses a fibrous,
membrane-like, powdery, gel-like or porous shape.
5. A method for the preparation of a biodegradable biopolymer
material comprising 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 containing
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 cast drying the applied solution to form a membrane-like
biodegradable biopolymer material, wherein if using the mixed
aqueous solution, the aqueous solutions as the constituents of the
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.
6. A method for the preparation of a biodegradable biopolymer
material comprising the steps of freezing 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 containing 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 frozen aqueous
solution under a reduced pressure to thus form a powdery
biodegradable biopolymer material, wherein if using the mixed
aqueous solution, the aqueous solutions as the constituents of the
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.
7. A method for the preparation of a biodegradable biopolymer
material comprising the steps of adjusting, to a level falling
within the acidic range, the pH value of 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 containing 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 entirely coagulating the
aqueous solution to give a gel-like biodegradable biopolymer
material.
8. A method for the preparation of a biodegradable biopolymer
material comprising the step of subjecting, to lyophilization, the
gel-like biodegradable biopolymer material prepared according to
the method as set forth in claim 6 to thus obtain a porous
body.
9. The method as set forth in claim 5, wherein the concentrations
of the aqueous solution of the silk fibroin from domesticated
silkworm, the aqueous solution of the silk fibroin from wild
silkworm and the aqueous solution of the secondary substance used
in the preparation of the mixed aqueous solution range from 0.1 to
5% w/v, respectively.
10. The method as set forth in claim 6, wherein the concentrations
of the aqueous solution of the silk fibroin from domesticated
silkworm, the aqueous solution of the silk fibroin from wild
silkworm and the aqueous solution of the secondary substance used
in the preparation of the mixed aqueous solution range from 0.1 to
5% w/v, respectively.
11. The method as set forth in claim 7, wherein the concentrations
of the aqueous solution of the silk fibroin from domesticated
silkworm, the aqueous solution of the silk fibroin from wild
silkworm and the aqueous solution of the secondary substance used
in the preparation of the mixed aqueous solution range from 0.1 to
5% w/v, respectively.
12. The method as set forth in claim 8, wherein the concentrations
of the aqueous solution of the silk fibroin from domesticated
silkworm, the aqueous solution of the silk fibroin from wild
silkworm and the aqueous solution of the secondary substance used
in the preparation of the mixed aqueous solution range from 0.1 to
5% w/v, respectively.
13. A metal ion-adsorbing material comprising a biodegradable
biopolymer material, which comprises a biodegradable biopolymer
material 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.
14. The metal ion-adsorbing material as set forth in claim 13,
wherein the metal ions are antibacterial metal ions or metal ions
present in waste water.
15. A sustained release carrier for a useful substance comprising a
biodegradable biopolymer material, which comprises a biodegradable
biopolymer material 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.
16. The sustained release carrier for a useful substance as set
forth in claim 15, wherein the biodegradable biopolymer material is
a porous substance.
17. A substrate for growth of biological cells comprising a
composite material containing 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, wherein the substrate is used for the growth of biological
cells.
18. A biodegradable water-absorbing material consisting of a
biodegradable biopolymer material, which comprises a biodegradable
biopolymer material 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.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a biodegradable biopolymer
material, which is degraded while being decomposed by the action of
an enzyme and which is thus converted into small molecules and a
method for the preparation of the biodegradable biopolymer material
as well as a functional material containing the material such as a
metal ion-adsorbing material, a sustained release carrier for a
useful substance, a biological cell-growth substrate and a
biodegradable water-absorbing material.
[0002] It is quite a long time since materials consisting of
organic polymers and possessing biodegradability came on to the
market. In the medical field, there have frequently been used
materials, which are biologically decomposed and degraded through
the action of an enzyme to thus form small molecules. Materials
recently widely put into practical use include, as typical
examples, poly(oxy-acids) such as poly(lactic acid) and
poly(glycolic acid) among the biodegradable organic polymers and
these materials have widely been used as implanting materials to be
embedded in the living bodies, materials for the in vivo delivery
or carriers for sustained release of medicines.
[0003] These poly(lactic acid) and poly(glycolic acid) show
excellent resistance to chemicals. Poly(lactic acid) and
poly(glycolic acid) are non-toxic and quite susceptible to
hydrolysis and accordingly, they have widely been used as materials
capable of being decomposed and absorbed in vivo. Moreover,
poly(glycolic acid) can be prepared as a very high molecular weight
polymer and therefore, it is useful as a material, which should
have excellent mechanical or dynamic characteristic properties such
as high tensile strength. Specifically, poly(lactic acid) and
poly(glycolic acid) have been used as, for instance, biodegradable
and bioabsorbable suture.
[0004] Moreover, in the medical field, silk sutures from
domesticated silkworm have long been used as sutures for surgical
operations. The use of the silk fiber from domesticated silkworm as
sutures for surgical operations, for the first time, dates away
back to the beginning of the eleventh century. The total volume of
the sutures traded in this country is equivalent to about six
billion yens a year (in 1985), 46% of which corresponds to the
volume of the silk sutures. The silk fiber is excellent in, for
instance, tensile strength and knot strength and can easily be
sterilized. For this reason, the silk fiber has favorably been used
as sutures. Therefore, even when judging from the actual conditions
of the use of the conventional silk sutures, the silk fiber can
quite easily be sterilized, it is never biologically decomposed
within a short period of time when embedded in the living body and
when it is implanted in the living body, it only insignificantly
causes an antigen-antibody reaction with the biological
tissues.
[0005] The cocoon fiber (the silk fiber) is a protein fiber
produced and spun by matured larvae of silkworm. The silkworms are
divided into two groups or domesticated silkworms reared in
farmhouses and wild type ones. Silk fibroin fibers are those
obtained by removing sericin as an adhesive substance, which covers
the surface of the cocoon fiber, by treating the cocoon fiber with,
for instance, an alkali.
[0006] The silk fibers from wild silkworm in general mean those
produced and spun by, for instance, Antheraea pernyi, Antheraea
yamamai, Antheraea militta, Antheraea assama, Philosamia cynthia
ricini and Philosamia cynthia pryeri.
[0007] The foregoing silk suture is a non-absorbent material, it is
never decomposed within a short period of time and accordingly, it
would remain in the living body even after the suture. For this
reason, it has been used for the purposes different from those of
the threads for suture made from poly(oxy-acids) such as
poly(lactic acid) and poly(glycolic acid), which are absorbed in
the body and decomposed into water and carbon dioxide within
several weeks after the suture.
[0008] With respect to the foregoing metal ion-adsorbing material
and sustained release carrier for useful substances consisting of
the aforementioned biodegradable biopolymer, there has not yet been
proposed any product having satisfactory characteristic
properties.
[0009] As has been discussed above in detail, poly(lactic acid) has
widely been used as a biodegradable and bioabsorbable material, but
it suffers from a problem in that the production cost thereof is
too high. Moreover, poly(glycolic acid) has been used as a
biodegradable and bioabsorbable material because of the advantages
described above. On the other hand, it is too expensive, has high
crystallizability, is too hard and is inferior in the compatibility
with soft tissues. Moreover, it also suffers from problems such
that the rate of decomposition thereof cannot easily be controlled
and that the control of the biodegradability thereof is likewise
difficult even if this material is chemically modified.
[0010] Further, fibrous poly(lactic acid) has a glass transition
temperature similar to that of, for instance, polyethylene
terephthalate fiber and accordingly, the poly(lactic acid) fibers
possess mechanical properties quite resemble to those observed for
the polyethylene terephthalate fibers. In this respect, however,
poly(lactic acid) or the like has a crystallization velocity slower
than that observed for polyethylene terephthalate and fibers of,
for instance, poly(lactic acid) are not sufficiently oriented and
are not satisfactorily crystallized even when they are passed
through the usual spinning and/or orientation steps. For this
reason, additional problems arise when putting them into practical
use, for instance, the tensile strength and dimensional stability
of poly(lactic acid) are insufficient.
[0011] In addition, the higher the molecular weight of the
foregoing poly(oxy-acids), the slower the rate of the decomposition
thereof. In this connection, it is necessary to produce poly(lactic
acid) and poly(glycolic acid) whose molecular weight is controlled
for the control of the decomposition speed of these polymers, but
the production of such polymers requires much labor and the use of
highly advanced techniques requiring a great deal of skill. For
this reason, the use of poly(oxy-acids) has presently been limited
to medical applications such as absorbent sutures and cosmetic
applications and accordingly, there has strongly been desired for
the establishment of a production process, which is not expensive
or is economical and does not require any skilled technique.
[0012] As has been discussed above, the suture of silk differs from
sutures of poly(oxy-acids) such as poly(lactic acid) and
poly(glycolic acid), which are finally decomposed into water and
carbon dioxide in the living body. Accordingly, there has strongly
been desired for the development of a biodegradable material whose
biodegradability in vivo can be controlled, which does not suffer
from any problem concerning the biological safety and whose
production cost is very low and which can biologically be
decomposed without producing any cytotoxic products, does not form
any harmful substance such as formaldehyde as a by-product and
which is thus safe to the biological tissues.
[0013] The silk protein as a biopolymer from an insect, which can
be used as a raw material for the foregoing silk suture is a
naturally occurring polymer material produced through the
biosynthesis of silkworms, excellent in the biological
compatibility with the biological tissues and has good molding
properties. Therefore, if by-products of silk obtained in the
process for preparing raw silk and silk products are used as
starting material for the sutures, one can save the cost of raw
materials. Moreover, silk proteins include a large number of active
sites rich in chemical reactivity and therefore, the fields of
applications thereof (such as the use as medical materials) can
considerably and widely be extended if a technique, which permits
the control of the biodegradability or biochemical properties of
silk fibroin through, for instance, hybrid processings and/or
chemical modification treatments, can be developed. For this
reason, there has strongly been desired for the development of a
novel biodegradable material, which can effectively be used in the
medical field, using such biopolymers from insects as starting
materials and secondary substances capable of being combined
(hereunder also referred to as ybrid or hybridized with the former
(composite (materials)).
SUMMARY OF THE INVENTION
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] The shape of the biodegradable biopolymer material may be
any one such as a fibrous, membrane-like, powdery, gel-like or
porous shape.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] (A) Preparation of Aqueous Solution of Silk Fibroin from
Domesticated Silkworm and Membrane of Silk Fibroin from
Domesticated Silkworm
[0032] An aqueous solution of pure silk fibroin from domesticated
silkworm may be prepared by the following method:
[0033] 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 fibroid aqueous solution with
water.
[0034] 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.
[0035] 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.
[0036] (B) Preparation of Aqueous Solution of Silk Fibroin from
Wild Silkworm and Membrane of Silk Fibroin from Wild Silkworm
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] (C) Hybrid Membrane of Domesticated Silkworm Silk Fibroin
and Wild Silkworm Silk Fibroin
[0042] 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.
[0043] 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.
[0044] 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.
[0045] (D) Hybrid Membrane of Domesticated or Wild Silkworm Silk
Fibroin and Cellulose
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] (E) Hybrid Membranes of Domesticated or Wild Silkworm Silk
Fibroin and Chitin, Chitosan and Chitosan Derivatives
[0051] 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.
[0052] 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.).
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] (F) Hybrid Membrane of Domesticated or Wild Silkworm Silk
Fibroin and Wool Keratin
[0058] 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.
[0059] 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%.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] (G) Hybrid Membrane of Domesticated Silkworm Silk Fibroin or
Wild Silkworm Silk Fibroin and Polyvinyl Alcohol
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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, (August) :
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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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).
[0090] The present invention will hereunder be described in more
detail with reference to the following Examples and Comparative
Examples, but the present invention is not limited to these
specific Examples at all. In the following descriptions, the term
"%" means w/v unless otherwise specified.
[0091] First of all, various test methods used in the following
description will be described in detail.
[0092] (1) Evaluation of Mechanical Properties
[0093] The strength and elongation at break of each silk fiber upon
the breakage thereof were determined using INSTRON (Autograph
AGS-5D available from Shimadzu Corporation) under the following
measurement conditions: the length of a sample to be tested of 50
mm, the rate of extension of 10 mm/min and the chart full scale of
250 g. In this connection, each measured value means the average of
20 measurements repeatedly carried out.
[0094] (2) Methods for the Adsorption of Metal Ions on
Biodegradable Biopolymer Material and for Quantitative
Determination Thereof
[0095] Each sample to be examined was immersed in a 0.5 mM aqueous
metal salt solution containing potassium nitrate (the pH value
thereof was adjusted to 11.4 by the addition of aqueous ammonia) at
room temperature for 30 hours to thus adsorb metal ions on the
sample. In this respect, metal ions were adsorbed on the sample by
immersing the latter in an aqueous metal salt solution (the pH
value thereof was controlled to 8.5).
[0096] The metal ions adsorbed on each test sample were analyzed
using a plasma atomic absorption spectrometer (ICP-AES) available
from Perkin-Elmer Company. More specifically, each test sample (5
to 10 mg) was completely hydrolyzed with 2 mL of a 65% aqueous
nitric acid solution in a microwave hydrolysis furnace
(MDS-81DCCEM), 10 mL of water was additionally added to the
hydrolyzed sample prior to the analysis and then the resulting
mixture was subjected to the analysis in the ICP-AES. The amount of
metal ions adsorbed on each sample is expressed in terms of the
amount of metal ions (in mM unit) per unit mass of the sample.
[0097] (3) Decomposition Treatment with Enzyme
[0098] An enzyme used in the biodegradation experiment was
dissolved in a buffering solution optimum for the digestion. This
solution was charged into a 100 mL volume sterilized glass beaker,
followed by the addition of each test sample and decomposition
thereof with the enzyme at 37.degree. C. for a predetermined time.
The degree of biodegradation of each test sample observed after the
treatment over a predetermined time is expressed in terms of the
rate of the residual sample (by weight) (hereunder referred to as
"rate of remaining weight") irrespective of the presence of the
enzyme. More specifically, the rate of remaining mass is given by
the following equation: [(Wi-We)/Wi].times.100 (%) wherein Wi and
We represent the masses of each sample determined before and after
the biodegradation test, respectively.
[0099] Thus, the term "rate of remaining mass" herein used means
the rate (%) of the residual sample (by weight) even after the
digestion to the sample weight prior to the biodegradation. In this
respect, the smaller the value, the greater the amount of the
sample hydrolyzed or the higher the biodegradability of the
sample.
[0100] (4) Biodegradation Rate
[0101] The digestion rate observed when hydrolyzing each test
sample with an enzyme was evaluated by the following method. The
digestion rate is herein defined to be the amount (%) of the sample
biodegraded during the biodegradation procedure carried out over 50
hours relative to the initial mass of each test sample or the mass
of the test sample at the initiation of the biodegradation test,
which is defined to be 100. Therefore, the higher the digestion
rate observed for a specific sample, the higher the
biodegradability of the sample.
[0102] (5) Fourier Transform Infrared Absorption Spectra
[0103] The absorption spectra of each test sample concerning the
molecular shape thereof were analyzed using an FT-IR (Fourier
Transform Infrared Absorption Spectra) measuring device available
from Perkin-Elmer Company. This analysis was carried out over a
wave number range of from 2000 to 400 cm.sup.-1 and the number of
scanning was 20.
[0104] (6) Test for Wear Resistance
[0105] A GAKUSHIN Type color fastness to rubbing tester Model II
was used as a rub tester, there was fitted to this rub tester a
polyethylene terephthalate (PET) substrate coated with a thin
membrane of each sample selected from a variety of silk proteins
and the wear resistance test was carried out by reciprocating a
friction element having an applied load of 500 g over 10 times in
such a manner that the sample fitted to the friction element is
lightly rubbed with that fixed to a test table under the action of
a predetermined load. The sample was subjected to the FT-IR
spectroscopic measurement prior to and after the wear resistance
test to thus determine the wave numbers of absorption peaks. A
sample thin membrane on the PET substrate whose FT-IR absorption
peak shows reduction of its intensity after the friction operation
is judged to be one easily peelable.
[0106] (7) Crystallinity Index of Domesticated Silkworm Silk
Fibroin Membrane
[0107] The crystallinity index of a domesticated silkworm silk
fibroin membrane hydrolyzed with a variety of enzymes was evaluated
according to the following method: In this method, the Fourier
transform infrared absorption spectra (FT-IR) measuring device used
was a Nicolet-150P measuring device available from Nicolet
Instruments, Madison, Wis. equipped with an ATR diamond cell
(SPECAC). In the IR spectroscopy, the peak strengths of amide band
III at wave numbers of 1230 cm.sup.-1 and 1260 cm.sup.-1 were
determined and the crystallinity index (CI) was determined
according to the following formula. In this respect, the value of
CI is a numerical value corresponding to the ratio of the peak
strengths and does not have any unit.
CI=I [1230 cm.sup.-1]/I [1260 cm.sup.-1]
[0108] (8) Amino Acid Analysis
[0109] Various protein materials corresponding to various
biodegradation times were subjected to the following amino acid
analysis. Each test sample used herein was prepared by hydrolyzing
each protein material with a 6N hydrochloric acid solution at
105.degree. C. for 24 hours. The amino acid analysis was carried
out using RP-HPLC.
[0110] (9) Test for Antibacterial Activity Against Vegetative
Pathogenic Bacteria:
[0111] As vegetative pathogenic bacteria, there was selected the
bacterial canker of tomato (scientific name: Corynebacterium
michiganense pv. michiganense), which is a typical of the universal
vegetative pathogenic bacteria, whose resistant bacteria may easily
induced, which may attack various kinds of plants or which is a
polyxeny putrefactive bacterium and which is one of gram positive
bacteria quite rarer in the vegetative pathogenic bacteria, and the
antibacterial activity of each test sample (composite membrane) was
evaluated on the basis of the growth-inhibitory effect thereof on
the vegetative pathogenic bacterium.
[0112] The evaluation of antibacterial effects in the following
Examples was carried out according to the following method.
[0113] Method for Examining Antibacterial Activity Against
Bacterium
[0114] There were admixed 25 mL of semi-synthesized Wakimoto Medium
or King Medium B, which had been dissolved with heating and then
maintained at 55.degree. C. and 2 mL of the bacterium to be assayed
(concentration: 109/mL) and then the mixture was poured into a
petri dish to thus solidify the same in a plate-like shape. A
sample membrane of about 1 cm square was placed on this plate-like
medium containing the bacterium and the whole sample was closely
adhered to the culture medium. The resulting assembly was
maintained at a temperature ranging from 20 to 25.degree. C. and
the size of the inhibitory circle appearing at the periphery of the
sample was practically determined in the unit of mm in
predetermined intervals to thus evaluate the bacterial
growth-inhibitory effect observed on the culture medium in the
proximity to the sample to be assayed and to thus confirm the
presence of any antibacterial activity or evaluate the relative
superiority on the basis of the change in the size of the
inhibitory circle observed.
[0115] (10) Test for Insect's Cell Growth
[0116] Using a culture medium comprising Grace Medium (G8142
available from Sigma Company) containing 5% powdered body fluid of
silkworm and 5% fetal calf serum (available from Gibco Company), to
which 1% penicillin-streptomycin mixed antibiotic had been
supplemented, Ae cells from Antheraea pernyi or Bm cells from
domesticated silkworm were cultivated. After 2 days, the number of
insect cells present in the cell culture medium was determined
using a hemocytometer to thus analyze the conditions of the Ae and
Bm cells proliferated on the surfaces of various kinds of composite
materials whose silk fibroin contents were different from one
another.
[0117] (11) Determination of Molecular Weight
[0118] Each fibrous or membrane-shaped sample used in the
biodegradation test was dissolved in a small amount of a 50% (w/v)
aqueous solution of lithium thiocyanate at 40.degree. C. over 30
minutes. Then the resulting solution was diluted with 50 mM sodium
phosphate buffering solution, a 0.15 M aqueous solution of
potassium chloride (pH 7.2) and further a 5M aqueous solution of
urea, the diluted solution was digestion into a dialysis membrane
of cellulose (Spectra/Por 6, MW 10=3.5 kDa, available from Spectrum
Company) and then the solution in the membrane was dialyzed against
the same buffering solution over 48 hours. After the dialysis, the
dialyzate was diluted with distilled water to a silk fibroin
concentration of 1 mg/mL, filtered through a 0.2 .mu.m porous
filter immediately thereafter and then analyzed by the size
exclusion chromatography technique. Waters chromatographic system
used herein is equipped with a pump (mod. 510) provided with a
temperature control device, an injector (mod. U6K) and a refractive
index detector (mod. 410). This system is provided with software
for chromatography (Maxima 820 (Waters)) and GPC Lanter software.
The column temperature was set at 30.degree. C. The column used
herein was Shodex Protein KW-804 (Waters, 8.times.300 mm) packed
with porous silica gel coated with hydrophilic OH groups
(pre-column, Shodex Protein KW-G, 6.times.50 mm). The amount of the
sample loaded on the column ranged from 50 to 100 .mu.l and the
exit velocity was set at 0.5 mK/min. The analysis was carried out
using distilled water as the moving phase using or without using 50
mM sodium phosphate buffering solution, a 0.15 M aqueous solution
of potassium chloride (pH 7.2) and a 5M solution of urea.
[0119] The reference markers for molecular weight used herein were
kits for HMW and LMW gel filtration correction (available from
Pharmacia Biotech.). In this connection, the detection was carried
out at 254 nm using a UV detector.
[0120] Technical terms concerning the molecular weight
determination will hereunder be described:
[0121] Molecular Weight: This is a weight average molecular weight
or a value (molecular weight) determined by integrating the area
surrounded by the elution curve and this is dependent on the whole
peptides present in the sample and having a variety of molecular
weights.
[0122] Peak Molecular Weight: This is the molecular weight
corresponding to the peak of the elution curve and corresponds to
the molecular weight of peptides present in the sample and having
the maximum population. In this case, the molecular weight
distribution is not taken into consideration.
EXAMPLE 1
[0123] Preparation of Aqueous Solution of Bombyx mori Silk Fibroin
and Bombyx mori Silk Fibroin Membrane
[0124] First, cocoon fibers from Bombyx mori silkworm were immersed
into a mixed aqueous solution containing 0.2% Marcel Soap and 0.05%
sodium carbonate, followed by boiling the mixture at 98.degree. C.
for 30 minutes to thus remove the sericin adhering the outer layer
of the cocoon fibers and to thus prepare silk fibroin fibers. The
resulting silk fibroin fibers (10 g) were immersed in an 8.5M
lithium bromide aqueous solution at a temperature of not less than
55.degree. C. for 15 minutes to thus dissolve the silk fibroin
fibers. This aqueous neutral salt solution was poured in a dialysis
membrane of cellulose, the both ends of the membrane were tied up
with sawing threads and dialyzed against tap water maintained at
5.degree. C. for 2 days to completely remove the lithium ions and
bromide ions present therein and to thus give an aqueous solution
of pure Bombyx mori silk fibroin. Aqueous solutions of silk fibroin
having a variety of concentrations were prepared by partially
evaporating the water of the resulting silk fibroin aqueous
solution or diluting it with water and these aqueous solutions were
used in the following Examples.
[0125] The silk fibroin aqueous solution thus prepared was cast
dried on a polyethylene substrate at room temperature to thus form
a Bombyx mori silk fibroin membrane.
EXAMPLE 2
[0126] Preparation of Aqueous Solution of Silk Fibroin from
Antheraea pernyi and Membrane of Antheraea pernyi Silk Fibroin
[0127] First, cocoon fibers from Antheraea pernyi were immersed in
a 0.1% aqueous solution of sodium peroxide at 98.degree. C. for one
hour to thus remove the silk sericin and tannin covering the
surface of the cocoon fibers from Antheraea pernyi and to thus
prepare Antheraea pernyi silk fibroin fibers (material-to-liquor
ratio 1:50). The Antheraea pernyi silk fibroin fibers whose sericin
and tannin had been removed in advance were dissolved in an aqueous
lithium thiocyanate solution, the resulting aqueous solution was
poured into a dialysis membrane of cellulose, the both ends of the
membrane were tied up with sawing threads and dialyzed against tap
water maintained at room temperature for 2 days to completely
remove the lithium ions and thiocyanate ions present therein and to
thus give an aqueous solution of pure Antheraea pernyi silk
fibroin.
[0128] The Antheraea pernyi silk fibroin aqueous solution thus
prepared was cast dried on a polyethylene substrate at room
temperature to thus form an Antheraea pernyi silk fibroin
membrane.
EXAMPLE 3
[0129] Hybrid Membrane of Bombyx mori Silk Fibroin and Antheraea
pernyi Silk Fibroin
[0130] To a beaker, there were added predetermined amounts of the
aqueous solutions of Bombyx mori silk fibroin and Antheraea pernyi
silk fibroin prepared in Examples 1 and 2 respectively and these
solutions were carefully admixed together through gentle stirring
with a glass rod in such a manner that the aqueous solutions never
underwent any gelation (or precipitation). The mixed aqueous
solution thus prepared was cast dried on a polyethylene substrate
at room temperature to thus form a transparent hybrid membrane.
[0131] The concentrations of the both aqueous solutions of Bombyx
mori silk fibroin and Antheraea pernyi silk fibroin were set at a
level of 0.1 to 3 wt %. The use of aqueous solutions having a
concentration falling within the range would permit the appropriate
control of the amounts of these aqueous solution required for the
preparation of such a hybrid membrane and the efficient operations.
In addition, the use thereof permitted the uniform mixing of these
two liquids and as a result, a hybrid membrane having uniform
quality could be obtained.
EXAMPLE 4
[0132] Hybrid Membrane of Bombyx mori Silk Fibroin and
Cellulose
[0133] First, Bombyx mori silk fibroin fibers and commercially
available powdery cellulose free of any particular purification
(available from Fluka Company) were separately dissolved in a
cuprammonium solution ([Cu(NH.sub.3).sub.4](OH).sub.2) to prepare
corresponding aqueous solutions. Then these two kinds of aqueous
solutions were admixed together with gentle stirring such that the
Bombyx mori silk fibroin fibers/cellulose composition was equal to
80/20, 60/40, 40/60 or 20/80. The mixed aqueous solution thus
prepared was gently cast dried on a glass plate arranged on a
horizontal plane and a mixed solution containing acetone and acetic
acid (4:1 (v/v)) was carefully added to the surface of the mixed
aqueous solution to thus remove the metal complex present in the
mixed aqueous solution while solidifying or coagulating the Bombyx
mori silk fibroin and the cellulose. Thereafter, the resulting
membrane was washed with a mixed solution containing glycerin and
water (7:13 (v/v)) and then with water and then dried at room
temperature to give a hybrid membrane of Bombyx mori silk fibroin
and cellulose. The resulting membrane had a thickness ranging from
about 10 to 30 .mu.m.
EXAMPLE 5
[0134] Hybrid Membrane of Bombyx mori Silk Fibroin and Chitin,
Chitosan Derivatives
[0135] In this Example, the aqueous solution of Bombyx mori silk
fibroin prepared in Example 1 was admixed with an aqueous solution
of chitin or a chitosan derivative as a secondary substance to
prepare a hybrid membrane of Bombyx mori silk fibroin and chitin or
a chitosan derivative as a secondary substance.
[0136] First, powdery chitin was suspended in a 42% aqueous
solution of sodium hydroxide and stirred for 4 hours under reduced
pressure. Then the resulting powdery chitin was charged into a 60%
aqueous sodium hydroxide solution containing sodium
dodecyl-sulfate, stirred and then allowed to stand at -20.degree.
C. overnight, or the resulting powdery chitin was suspended in
liquid ammonia (-33.degree. C.) and then metal potassium was added
to the suspension to thus give alkali chitin. The alkali chitin
thus prepared was compressed, dispersed in finely divided ice and
then sulfurized by the addition of carbon disulfide to thus give
chitin sulfide. The aqueous solution of this sulfide was used as an
aqueous solution of a secondary substance.
[0137] Separately, the alkali chitin and ethylene chlorohydrin
(2-chloroethanol) were reacted under the known reaction conditions
to give ethylene glycol chitin, this chitin was treated in a 40%
aqueous sodium hydroxide solution at 100.degree. C. for 5 hours
with stirring to thus form water-soluble glycol chitosan. The
glycol chitosan was dissolved in an aqueous acetic acid solution
and the resulting solution was used as an aqueous solution of a
secondary substance.
[0138] Then, to each of the aqueous solution of sulfide and the
aqueous solution of glycol chitosan prepared according to the
foregoing methods, there was added an aqueous solution of Bombyx
mori silk fibroin prepared according to the method used in Example
1 to thus give each corresponding aqueous mixed solution. This
aqueous mixed solution was cast dried on a polyethylene substrate
to thus form a transparent, soft membrane-like composite material
comprising chitin sulfide or glycol chitosan and Bombyx mori silk
fibroin.
EXAMPLE 6
[0139] Hybrid Membrane of Bombyx mori Silk Fibroin and Wool
Keratin
[0140] To dissolve wool yarns, Cystine cross linkings thereof were
cleaved using mercapto-ethanol or thioglycollic acid in a nitrogen
gas atmosphere to thus solubilize the keratin molecules through
reduction. When using mercapto-ethanol, the reduction was carried
out in a urea solution having a concentration of 8M, while when
using thioglycollic acid, the reduction was carried out by the
addition of 4% NaCl.
[0141] More specifically, degreased wool yarns were immersed in a
urea solution having a concentration of 8M, followed by degassing,
addition of mercapto-ethanol in an amount of 5 mL per 10 g of wool
yarns at a temperature of 25.degree. C. in a nitrogen gas
atmosphere and stirring over 3 hours to reduce the wool keratin
molecules and to thus give wool keratin carrying SH groups. Then
the resulting wool keratin was dialyzed against pure water to
remove the urea and the excess mercapto-ethanol and to thus give an
aqueous solution of wool keratin. This aqueous wool keratin
solution was used as an aqueous solution of a secondary
substance.
[0142] Moreover, to 10 g of the reduced wool keratin obtained
according to the foregoing method, there was added 15 g of
iodoacetic acid at 25.degree. C. with stirring in a nitrogen gas
atmosphere to thus react them. After 2 hours, the pH value of the
reaction system was adjusted to about 8.5, the resulting reaction
solution was poured into a dialysis membrane of cellulose, the both
ends of the membrane were tied up with sawing threads and dialyzed
against pure water to remove the excess iodoacetic acid present
therein and to thus give an aqueous solution of S-carboxymethyl
keratin. This aqueous solution was used as an aqueous solution of a
secondary substance.
[0143] To each of the aqueous reduced wool keratin solution and the
aqueous solution of S-carboxymethyl keratin prepared according to
the foregoing procedures, there was added an aqueous solution of
Bombyx mori silk fibroin prepared according to the procedures used
in Example 1 to thus give each corresponding aqueous mixed
solution. This aqueous mixed solution was cast dried on a
polyethylene substrate to form a hybrid membrane of reduced keratin
or S-carboxymethyl keratin and Bombyx mori silk fibroin.
EXAMPLE 7
[0144] Hybrid Membrane of Bombyx mori Silk Fibroin and Polyvinyl
Alcohol
[0145] Polyvinyl alcohol (PVA) was added to hot water of 85.degree.
C., carefully dissolved therein using a stirring machine and the
resulting solution was allowed to stand at room temperature for 30
minutes to thus give a 0.5% aqueous solution of PVA. To this PVA
aqueous solution, there was added a 0.3% w/v aqueous solution of
Bombyx mori silk fibroin prepared according to the procedures used
in Example 1, the resulting aqueous mixed solution was cast dried
on a polyethylene substrate over a whole day and night to form a
transparent hybrid membrane of PVA and Bombyx mori silk
fibroin.
EXAMPLE 8
[0146] Peel Resistance of Silk Fibroin Membrane with Respect to PET
Substrate
[0147] A silk fibroin membrane was adhered to the surface of a
polyethylene terephthalate (PET; trade name: Tetoron) membrane
according to the following method. The foregoing Tetoron membrane
was immersed in a 5% aqueous Bombyx mori silk fibroin (BF) solution
prepared according to the procedures used in Example 1, a 3%
aqueous Antheraea pernyi silk fibroin (TF) solution prepared
according to the procedures used in Example 2 or an equivalent
mixed aqueous solution of the 5% aqueous Bombyx mori silk fibroin
(BF) solution and the 3% aqueous Antheraea pernyi silk fibroin (TF)
solution, withdrawn therefrom and then dried at room temperature.
Each of these membranes (hereunder abbreviated as "PET/BF",
"PET/TF", "PET/(BF+TF)" respectively) was subjected to the
determination of ATR (Attenuated Total Reflection) spectra. This
ATR spectroscopic analysis was carried out using an ATR infrared
spectrophotometer (FT-IR5300 available from Nippon Bunko K. K.)
(Resolution 2; Number of Scanning: 32; gain: 100; using Apdization
CS). In addition, a friction element was reciprocated 10 times on a
membrane sample using an abrasion machine and then the membrane
sample was again subjected to the ATR spectroscopic analysis. The
results observed for the sample prior to the friction test are
listed in the following Table 1.
1TABLE 1 Sample Wave Number (cm.sup.-1) and Absorption Intensity
PET 1711 (vs), 1408 (s), 1338 (s) PET/BF 1687 (s), 1657 (vs), 1554
(s), 1408 (s), 1338 (s), 650 (s) PET/TF 1688 (s), 1655 (vs), 1408
(s), 1338 (s), 620 (s) PET/(BF + TF) 1789 (s), 1657 (vs), 1655
(vs), 1408 (s), 1338 (s), 650 (w), 620 (s)
[0148] In Table 1, symbols "vs", "s" and "w" indicate that the
spectral intensities are very strong, strong and weak,
respectively.
[0149] As will be clear from the data listed in Table 1, the ATR
spectra observed for PET/BF include absorption peaks ascribable to
PET (1408 and 1338 cm.sup.-1) and absorption peaks ascribable to
Bombyx mori silk fibroin (1657 and 650 cm.sup.-1), which are
superimposed to one another. Moreover, the ATR spectra observed for
PET/TF include absorption peaks ascribable to Antheraea pernyi silk
fibroin (1655 and 620 cm.sup.-1) in addition to those ascribable to
PET, which are overlapped to one another. The ATR spectra observed
for PET/BF and PET/TF obtained after the friction element was
reciprocated 10 times thereon were identical to those observed for
PET per se prior to the adhesion of these silk fibroin
membranes.
[0150] The foregoing results clearly indicate that when the PET/BF
and PET/TF laminate surface are treated in an abrasion machine, the
BF or TF membrane on the surface of PET substrate is scraped off
during the reciprocating motions of the friction element. In the
case of the PET/(BF+TF) laminate, however, the absorption peaks
ascribable to BF and the absorption peaks ascribable to TF still
remain in the ATR spectra observed for the PET/(BF+TF) laminate
even after the laminate surface is treated in an abrasion machine.
This clearly indicates that BF+TF is certainly adhered to the
surface of the PET substrate and scarcely peeled off therefrom even
when mechanically rubbed.
[0151] In addition, a PET substrate having a size of 2 cm.times.3
cm was immersed in an aqueous solution containing simply Bombyx
mori or Antheraea pernyi silk fibroin and a mixed aqueous solution
prepared by admixing an aqueous solution of Bombyx mori silk
fibroin and an aqueous solution of Antheraea pernyi silk fibroin by
carefully and gently stirring with a glass rod at room temperature
in a desired mixing ratio (at 25.degree. C. for 10 minutes). Then
the PET covered with each solution was withdrawn from each aqueous
solution and cast dried at room temperature. To make the silk
protein membrane on the PET substrate insoluble, the coated PET
substrate with silk fibroin was immersed in a 50% aqueous solution
of methanol for 5 minutes, withdrawn therefrom and then dried at
room temperature. The processed membrane thus prepared was
subjected to the same ATR spectrometric analysis described above in
connection with the foregoing membrane free of any insolubilization
treatment.
[0152] As a result, there were observed absorption peaks ascribable
to Bombyx mori silk fibroin membrane in addition to those
ascribable to the PET substrate in the case of the membrane simply
consisting of Bombyx mori silk fibroin; absorption peaks ascribable
to Antheraea pernyi silk fibroin membrane in addition to those
ascribable to the PET substrate in the case of the membrane simply
consisting of Antheraea pernyi silk fibroin; and absorption peaks
ascribable to Bombyx mori and Antheraea pernyi silk fibroin
membranes in addition to those ascribable to the PET substrate in
the case of the hybrid membrane. This fact clearly indicates that
the surface of the PET substrate is covered with a membrane
consisting of Bombyx mori silk fibroin, Antheraea pernyi silk
fibroin or a hybrid thereof.
[0153] Then the peel resistance of various coated membrane with
respect to a PET substrate were evaluated according to the
foregoing method using PET substrates whose surfaces were covered
with a membrane of Bombyx mori silk fibroin, a membrane of
Antheraea pernyi silk fibroin and a hybrid membrane of Bombyx mori
and Antheraea pernyi silk fibroins, respectively.
[0154] As a result, it was found that the membrane simply
consisting of Bombyx mori or Antheraea pernyi silk fibroin had such
a tendency that it was slightly easily peeled off from the PET
substrate, while the hybrid membrane had a tendency that it was
hardly peeled off therefrom and excellent in the adhesion
stability. In other words, it was found that the interaction
between the surface of the PET substrate and the hybrid membrane
(BF+TF membrane) is higher than those observed between the surface
of the PET substrate and the BF membrane and between the surface of
the PET substrate and the TF membrane. More specifically, a BF or
TF membrane adhered to a PET substrate is easily peeled off from
the PET substrate when any mechanical frictional force is applied
to each laminate, but a hybrid membrane is strongly interacted with
a PET substrate and therefore, the former is hardly peeled off from
the latter.
EXAMPLE 9
[0155] Cell-Growth Ability on Hybrid Membrane Surface
[0156] The cell-growth behaviors of Bombyx mori cells (Bm cells)
and Antheraea pernyi cells (Ae cells) on the membrane surface were
examined according to the method described above using a Bombyx
mori silk fibroin membrane (BF membrane), an Antheraea pernyi silk
fibroin membrane (TF membrane) and a hybrid membrane (BF+TF
membrane) prepared according to the procedures used in Examples 1,
2 and 3, respectively. The results obtained indicate that both of
these cells show almost the same tendency in the cell-growth
behavior and therefore, only the results observed for the Bm cells
are summarized in the following Table 2.
2 TABLE 2 Component of Membrane Degree of Cell-Growth BF .+-. TF
.+-. BF + TF +
[0157] In Table 2, ".+-." means that the degree of cell-growth is
almost identical to or slightly superior to that observed for the
polystyrene surface as a control substrate and "+" means that the
degree of cell-growth visually judged is superior to that observed
for the polystyrene surface.
[0158] As will be seen from the results listed in Table 2, the
degree of insect cell-growth observed on the surface of a culture
medium covered with the hybrid membrane of BF and TF is higher than
that observed on the surface of a culture medium covered with the
Bombyx mori silk fibroin membrane (BF membrane) or the wild
silkworm silk fibroin membrane (TF membrane).
EXAMPLE 10
[0159] Optimum Biodegradation Conditions
[0160] In this Example, it is intended to determine the optimum
conditions for the biodegradation test carried out in the following
Examples. This is because various conditions should be adjusted
such that the enzyme used can maintain its maximum activity even
when changing the kind and concentration of the enzyme, for
instance, the kind of the buffering solution and the pH value
should appropriately be selected.
[0161] The optimum conditions for evaluating the decomposition
behavior of biodegradable biopolymer material with enzymes are as
follows. The Optimum conditions such as the kinds of enzymes,
enzyme activities and optimum pH values of buffering solutions are
summarized in the following Table 3. The biodegradation temperature
was set at 37.degree. C. As enzymes, there were used three kinds of
enzymes or chymotrypsin, collagenase and protease. The
material-to-buffering solution ratio 1:250 was maintained and the
biodegradation behavior was monitored or examined over 570 hours.
The enzyme concentration was expressed in terms of the mass (mg) of
each enzyme per 1 mL of the culture medium. The chymotrypsin, the
collagenase (Type F) and the protease herein used were all
available from Sigma Aldrich Japan Co., Ltd.
[0162] The buffering solutions used in this Example were TES
(N-tris (hydroxymethyl)-methyl-2-aminoethane sulfonic acid
available from Wako Pure Chemical Industries, Inc.) for the
biodegradation tests using collagenase; tris
(hydroxymethyl)-amino-methane (2-amino-2-hydroxymethyl--
1,3-propanediol available from Wako Pure Chemical Industries, Inc.)
for the biodegradation tests using chymotrypsin; and potassium
phosphate buffering solution for the biodegradation tests using
protease.
3 TABLE 3 Enzyme Concn. Activity (units/ Enzyme (mg/mL) Buffering
Solution mg solid) pH Collagenase 0.2, 0.5 50 mL TES, 50 mM
CaCl.sub.2 1.8-2.2 7.4 Chymotrypsin 0.2, 0.5 50 mM Tris, 5 mM
CaCl.sub.2 40-60 7.8 Protease 0.2, 0.5 40 mM Potassium phosphate
(pH 7.5) 5.7 7.5
EXAMPLE 11
[0163] In this Example, the biodegradation behaviors of Bombyx mori
silk fibroin membrane or Antheraea pernyi silk fibroin membrane
were investigated.
[0164] The cocoon layer of Bombyx mori was cut into pieces having a
size of 1/4 time the original one and the waxes and dyestuffs
included in the sample were removed in a Soxhlet extractor using an
ethanol/benzene mixed solution (1:2 v/v). Then the cocoon fiber
sample was charged into a mixed solution containing 0.2% Marcel
Soap and 0.05% sodium carbonate, the mixture was then boiled at
98.degree. C. for 30 minutes to thus remove sericin as an adhesive
substance present in the outer layer of the cocoon fiber. At this
stage, the material-to-liquor ratio was set at 1:100. Ten grams of
Bombyx mori silk fibroin fibers thus prepared were immersed in a
8.5 M aqueous solution of lithium bromide at a temperature of not
less than 55.degree. C. for 15 minutes to solubilize the silk
fibers. This aqueous neutral salt solution was poured into a
dialysis membrane of cellulose, the both ends of the membrane were
tied up with sawing threads and dialyzed against pure water
maintained at room temperature for 4 days to completely remove the
lithium and bromide ions present therein and to thus give an
aqueous solution of Bombyx mori silk fibroin having a concentration
of 0.2%.
[0165] Alternatively, cocoon fibers from Antheraea pernyi were
degummed in a 0.1% aqueous sodium peroxide solution in an amount of
50 times the mass of the cocoon fibers at a temperature of
98.degree. C. for one hour to remove sericin and tannin. The
Antheraea pernyi silk fibroin fibers from which sericin and tannin
had been removed were dissolved in an aqueous solution of lithium
thiocyanate maintained at 55.degree. C., the resulting aqueous
solution was poured into a dialysis membrane of cellulose, the both
ends of the membrane were tied up with sawing threads and dialyzed
against pure water to thus give an aqueous solution of Antheraea
pernyi silk fibroin having a concentration of 0.3%.
[0166] The 0.2% aqueous solution of Bombyx mori silk fibroin and
the 0.3% aqueous solution of Antheraea pernyi silk fibroin prepared
according to the procedures used above were separately cast dried
on a polyethylene substrate at room temperature to thus form a
Bombyx mori silk fibroin membrane (BF membrane) and an Antheraea
pernyi silk fibroin membrane (TF membrane), respectively.
[0167] The Bombyx mori silk fibroin membrane and the Antheraea
pernyi silk fibroin membrane thus prepared were proteolyticaly
digested for the relation between the biodegradation behaviors by
the action of a variety of enzymes and the biodegradation time (0,
24, 72, 240, 576 hours). The rates (%) of the residual sample
weight as a function of the biodegradation time are summarized in
the following Table 4. The enzyme concentration was set at 0.2 and
0.5 mg/mL.
4 TABLE 4 Biodegradation Time (hr.) Sample Enzyme (Concn.) 0 24 72
240 576 Bombyx mori Silk Control 100 98 -- -- -- Fibroin Membrane
Collagenase (0.2 mg/mL) 100 98 95.5 93.5 90 Collagenase (0.5 mg/mL)
100 97.8 96.5 91.0 87.5 Chymotrypsin (0.5 mg/mL) 100 90.9 90.5 89.7
90.6 Protease (0.2 mg/mL) 100 81.8 75.6 71.6 54.5 Protease (0.5
mg/mL) 100 72.7 43.5 44.4 27.9 Antheraea pernyi Silk Protease (0.2
mg/mL) 100 96.9 88.6 88 86.2 Fibroin Membrane Protease (0.5 mg/mL)
100 88.8 89.8 87.2 76.1
[0168] The enzyme concentrations listed in Table 4 are expressed in
terms of the amount (mg) of each enzyme added to the biodegradation
medium per 1 mL of the latter.
[0169] The data listed in Table 4 clearly indicate that the Bombyx
mori silk fibroin membrane is quite susceptible to the digestion
with protease and that in the biodegradation experiment at a
protease concentration of 0.2 mg/mL, the rate of the residual
sample weight is found to be 54.5% after the biodegradation time of
576 hours. On the other hand, when acting the same concentration of
protease on the Antheraea pernyi silk fibroin membrane, the rate of
the residual sample weight is found to be 86.2% after the
biodegradation time of 576 hours. This indicates that the Bombyx
mori silk fibroin membrane is more susceptible to the digestion
with protease as compared with the Antheraea pernyi silk fibroin
membrane.
EXAMPLE 12
[0170] Crystallinity Index
[0171] When the Bombyx mori silk fibroin membrane is biodegraded,
the mass of the membrane undergoes changes and the digestion
reaction thereof is advanced with the elapse of the biodegradation
time. In this Example, the crystallinity index of each membrane was
investigated in order to make clear any change in the fine
structure of the Bombyx mori silk fibroin membrane per se during
the biodegradation process.
[0172] The Bombyx mori silk fibroin membrane, which had been
biodegraded with a variety of enzymes for a predetermined period of
time, was evaluated for the crystallinity index (CI) according to
the foregoing method. In this connection, CI has no dimension. The
results thus obtained are listed in the following Table 5.
5 TABLE 5 Biodegradation Time (hr.) Enzyme (Concn.) 0 72 240 408
Collagenase (0.2 mg/mL) 0.547 0.55 0.56 0.568 Chymotrypsin (0.2
mg/mL) 0.55 0.557 0.568 0.569 Protease (0.2 mg/mL) 0.547 0.585
0.594 0.601 Protease (0.5 mg/mL) 0.547 0.607 0.617 0.615
[0173] As will be clear from the data listed in Table 5, the
amorphous region of the Bombyx mori silk fibroin membrane is
digested through the digestion reaction with the enzyme and as a
result, the crystalline region thereof increases.
EXAMPLE 13
[0174] Biodegradation Behavior of Silk Fibers
[0175] The biodegradation behaviors of Bombyx mori silk fibers and
Antheraea pernyi silk fibers were investigated on the basis of the
rate of the residual sample weight according to the same procedures
used in Example 11. The results obtained are summarized in the
following Table 6.
6 TABLE 6 Biodegradation Time (hr.) Sample Enzyme (Concn.) 0 24 72
240 576 Bombyx mori Silk Control 100 100 -- -- -- Fiber Collagenase
(0.2 mg/mL) 100 100 100 100 99 Collagenase (0.5 mg/mL) 100 99.5 99
99 98 Bombyx mori Silk Control 100 99 -- -- -- Fiber Chymotrypsin
(0.2 mg/mL) 100 99.5 99.5 99.3 99 Chymotrypsin (0.5 mg/mL) 100 99
99 99 99 Bombyx mori Silk Control 100 100 -- -- -- Fiber Protease
(0.2 mg/mL) 100 98.6 99.3 99.3 99.3 Protease (0.5 mg/mL) 100 100
100 99.8 99.3 Antheraea pernyi Control 100 100 -- -- -- Silk Fiber
Collagenase (0.2 mg/mL) 100 100 100 98 97 Collagenase (0.5 mg/mL)
100 100 100 100 99 Antheraea pernyi Control 100 100 -- -- -- Silk
Fiber Chymotrypsin (0.2 mg/mL) 100 100 99 99 98 Chymotrypsin (0.5
mg/mL) 100 100 100 100 100 Antheraea pernyi Control 100 100 -- --
-- Silk Fiber Protease (0.2 mg/mL) 100 100 100 99.9 99.5 Protease
(0.5 mg/mL) 100 99.2 99.0 98.4 99.2
[0176] The results listed in Table 6 indicate that the Bombyx mori
silk fiber and the Antheraea pernyi silk fiber are scarcely
biodegraded on the basis of the decreasing rate of the residual
sample weight. However, the silk fiber may undergo changes in fine
structures and may suffer deterioration in addition to weight
changes during the biodegradation process. For this reason, the
Bombyx mori silk fiber was evaluated for the deterioration during
the biodegradation process on the basis of the strength and
elongation according to the following method.
[0177] Bombyx mori silk fibers were added to a culture medium
containing protease, collagenase or chymotrypsin, they were thus
biodegraded over a predetermined time period and then changes, with
time, of the strength and elongation at break of the silk fibers
after the biodegradation were determined. The results thus obtained
are summarized in the following Table 7.
7 TABLE 7 Biodegradation Time (hr.) Strength (N) 0 24 72 240 408
Collagenase (0.2 mg/mL) 4.68 3.60 4.12 3.86 3.64 Chymotrypsin (0.2
mg/mL) 4.68 3.83 3.72 3.97 3.78 Protease (0.2 mg/mL) 4.68 3.74 3.52
-- 3.14 Protease (0.5 mg/mL) 4.68 3.83 3.77 -- 3.23 Biodegradation
Time (hr.) Elongation (%) 0 24 72 240 408 Collagenase (0.2 mg/mL)
32.7 26.3 25.1 25.7 23.5 Chymotrypsin (0.2 mg/mL) 32.7 28.1 27.8
26.4 25.8 Protease (0.2 mg/mL) 32.7 21.6 20.5 -- 18.1 Protease (0.5
mg/mL) 32.7 24.1 23.2 -- 19.0
[0178] In Table 7, the enzyme concentration is given in parentheses
behind each corresponding enzyme name and it is expressed in terms
of the amount of the enzyme (mg) per 1 mL of the culture medium.
Moreover, the strength of the silk fiber expressed in N can be
converted into that in kg on the basis of the equation: (numerical
value of each strength)/9.81. In this Table, for instance, 4.68 N
corresponds to 477.1 g.
[0179] The data listed in Table 7 clearly indicate that both of the
strength and elongation of the silk fiber decrease with increasing
of biodegradation time. The data listed in Table 6 indicate that
the silk fiber is not biodegraded at first view, but it is clear
that the deterioration of the silk fiber is in fact advanced due to
the biodegradation.
EXAMPLE 14
[0180] Biodegradability of Hybrid Membrane
[0181] A hybrid membrane sample prepared from Bombyx mori silk
fibroin (BF) and Antheraea pernyi silk fibroin (TF) according to
the procedures used in Example 3 and a hybrid membrane prepared
from Bombyx mori silk fibroin (BF) and cellulose (Cell) according
to the procedures used in Example 4 were examined for the relation
between the biodegradation time and the rate (%) of the residual
sample weight observed when they are hydrolyzed with protease. The
results thus obtained are listed in the following Table 8.
8 TABLE 8 Biodegradation Time (hr.) Sample Presence of Enzyme 24 72
240 408 572 BF:TF (8:2) present 85.4 81.8 77.3 75.0 75.0 BF:TF
(8:2) absent 100 100 100 100 100 BF:TF (6:4) present 85 84.2 84.2
84.2 78.9 BF:TF (6:4) absent 95.4 95.4 94.4 100 100 BF:TF (4:6)
present 100 91.3 86.9 86.4 86.4 BF:TF (4:6) absent 100 100 100 100
95.5 BF:TF (2:8) present 100 100 96.9 97.1 96.9 BF:TF (2:8) absent
100 100 100 100 95.5 BF:Cell (8:2) present 86.4 87.9 51.7 50 52.1
BF:Cell (8:2) absent 88.8 89.5 89.5 90 90 BF:Cell (6:4) present
77.3 72 65.4 60.8 61.5 BF:Cell (6:4) absent 94.1 96.7 97.1 100 96.9
BF:Cell (4:6) present 78.6 75 73.0 73.3 73.3 BF:Cell (4:6) absent
100 100 100 100 100 BF:Cell (2:8) present 94.1 94.1 94.1 87.5 82.4
BF:Cell (2:8) absent 94.4 100 100 100 100 BF:Cell (0:10) present
97.5 94.1 100 97.1 97.7 BF:Cell (0:10) absent 93.7 95.7 96.7 97.7
96.3
[0182] In the column entitled "Presence of Enzyme" in Table 8, each
section specified by "absent" means the biodegradation experiment,
which is conducted in an aqueous solution containing only a
buffering solution and free of any proteolytic enzyme, while each
section specified by "present" means the biodegradation experiment,
which is carried out in an enzyme-containing decomposition solution
containing both protease and a buffering solution. In addition,
"BF:TF (4:6)" means a hybrid membrane prepared by admixing an
aqueous solution of Bombyx mori silk fibroin and an aqueous
solution of Antheraea pernyi silk fibroin such that the resulting
mixed solution contains 40% Bombyx mori silk fibroin and 60%
Antheraea pernyi silk fibroin and then cast drying. "BF:Cell (8:2)"
means a hybrid membrane prepared in such a manner that the
resulting hybrid membrane comprises 80% Bombyx mori silk fibroin
and 20% cellulose.
[0183] As will be seen from the data listed in Table 8, in the case
of the hybrid membrane of Bombyx mori silk fibroin and Antheraea
pernyi silk fibroin (BF:TF membrane) the hybrid membrane is hardly
biodegradable as a whole, as the content of the Antheraea pernyi
silk fibroin increases. Moreover, in the case of the hybrid
membrane of Bombyx mori silk fibroin and cellulose (BF:Cell
membrane), the hybrid membrane is likewise hardly
biodegradable.
EXAMPLE 15
[0184] Hybrid Membrane of Bombyx mori Silk Fibroin and Wool
Keratin
[0185] First of all, an aqueous solution of wool keratin was
prepared as follows.
[0186] Pigments and greases contained in wool (64' S) from sheep
belonging to the Merino species were removed by treating the same
with a benzene/ethanol (50/50 (% by volume)) mixed solvent for 2.5
hours using a Soxhlet extractor.
[0187] A three-necked flask was herein used. The first neck thereof
was connected to one end of a rubber tube, the other end of which
was connected to a nitrogen gas cylinder for drying through a
three-way cock, the second neck thereof was always occupied by a pH
electrode assembly for the control of the pH value of the reaction
system and the third or remaining neck or port was used for the
introduction of any necessary reagent into the system. Wool yarns
(8.18 g) from sheep of Merino species, which had been cut into
short yarns having a yarn length of about 1 cm, were charged into
the three-necked flask and then 450 mL of an 8M aqueous urea
solution was added to the flask. The flask was purged with nitrogen
gas, the pressure in the flask was reduced to about 45 mmHg for 15
minutes using an aspirator and then the pressure in the flask was
abruptly returned to the atmospheric pressure, these operations
being repeated three to four times. Thus, the air contained in the
wool yarns present in the three-necked flask was completely removed
so that the reaction of the aqueous urea solution with keratin
molecules would efficiently be advanced. After the completion of
the displacement with nitrogen gas, 4.8 mL of mercapto-ethanol as a
reducing agent was added to the three-necked flask and the wool
yarns were allowed to stand in the 8M aqueous urea solution over 2
to 3 hours. Then about 100 mL of a 5N-KOH aqueous solution was
added to the flask in small portions to thus adjust the pH value of
the mixed aqueous solution in the flask to 10.5. The content of the
flask was allowed to stand at room temperature for 3 hours till the
wool yarns were completely dissolved in the aqueous solution to
thus give an aqueous keratin solution. The resulting aqueous
keratin solution was poured into a dialysis membrane of cellulose,
the both ends of the membrane were tied up with sawing threads and
dialyzed against pure water for 2 days. The resulting aqueous
keratin solution was subjected to drying through ventilation or it
was if desired diluted with pure water to thus give an aqueous
keratin solution having a desired keratin concentration.
[0188] The keratin in the 0.01% aqueous keratin solution thus
prepared was subjected to an S-carboxy-methylation reaction at room
temperature for one hour by the addition of 9.5 g of iodoacetic
acid to 450 mL of the aqueous keratin solution. The pH value of the
aqueous keratin solution was adjusted to 8.5 by the addition of
5N-KOH aqueous solution to thus give an aqueous solution of
S-carboxymethyl keratin solution. This aqueous solution was poured
into a dialysis membrane of cellulose, the both ends of the
membrane were tied up with sewing yarns and dialyzed against pure
water for 2 days.
EXAMPLE 16
[0189] Weight-Average Molecular Weight of Bombyx mori Silk Fibroin
Membrane and Bombyx mori Silk Fiber
[0190] A Bombyx mori silk fibroin membrane and Bombyx mori silk
fibers were enzymatically decomposed, sufficiently washed with
water and then dried. The resulting sample was subjected to HPLC
measurements to determine the weight-average molecular weight
thereof. The results thus obtained are listed in the following
Table 9.
9 TABLE 9 Weight-Average Molecular Weight (kD) Enzyme Processing
Time (hr.) Sample (Concn: mg/mL) 0 72 240 408 576 Bombyx mori
Collagenase (0.2) 119.8 98.5 96.8 94.3 -- Silk Fibroin Membrane
Chymotrypsin (0.2) 119.8 77.4 65.7 53.7 -- Protease (0.2) 119.8
109.6 105.6 102.4 -- Bombyx mori Collagenase (0.2) 233.1 184.3 --
-- 204.4 Silk Fiber Protease (0.2) 233.1 256.5 -- -- 247.4 Protease
(0.5) 233.1 261.7 -- -- 241.7
[0191] In Table 9, each numerical value given in parentheses
appearing in the column entitled "Enzyme" means the concentration
of an enzyme used and the numerical value "0.2" means that the
culture medium contains 0.2 mg of the enzyme per 1 mL of the
medium.
[0192] As will be clear from the data listed in Table 9, the
molecular weight of the Bombyx mori silk fibroin membrane was found
to be gradually reduced from its initial value of 120,000 D after
the proteolytic reaction with the enzyme. More specifically, it was
confirmed that the weight-average molecular weight of the membrane
was reduced to about 94,000 D and 100,000 D after 408 hours from
the initiation of the biodegradation with collagenase and protease,
respectively and that the weight average molecular weight of the
membrane was rapidly reduced from its initial value of 120,000 D to
about 54,000 D at the same biodegradation time. The molecular
weight of the untreated Bombyx mori silk fiber is about 230,000 D.
In this respect, it was found that the weight-average molecular
weight thereof was only slightly reduced even after the enzymatic
digestion.
Example 17
[0193] Changes in Weight-Average Molecular Weight Associated with
Biodegradation Treatment
[0194] After hydrolyzing the Bombyx mori silk fibroin membrane
prepared according to the same procedures used in Example 1 with
three kinds of enzymes or collagenase, chymotrypsin and protease
for a predetermined period of time (72, 240 or 408 hours), the
membrane was sufficiently washed with water to give a sample. Each
sample obtained after the biodegradation over a predetermined time
was evaluated for the weight-average molecular weight and peak
molecular weight. In this respect, the weight-average molecular
weight thereof was determined by the high performance liquid
chromatography (HPLC) technique. The results thus obtained are
summarized in the following Table 10.
10 TABLE 10 Weight-Average Peak Molecular Enzyme Molecular Weight
(kD) Weight (kD) Control 119.8 79.8 Collagenase: 0.2 mg/mL (72
hrs.) 98.5 53.9 (240 hrs.) 96.8 52.9 (408 hrs.) 94.3 45.8
Chymotrypsin: 0.2 mg/mL (72 hrs.) 77.4 41.1 (240 hrs.) 65.7 37.6
(408 hrs.) 53.7 34.8 Protease: 0.2 mg/mL (72 hrs.) 109.6 36.4 (240
hrs.) 105.6 33.4 (408 hrs.) 102.4 31.4
[0195] In Table 10, the time given in parentheses appearing in the
column entitled "Enzyme" means the elapsed biodegradation time.
[0196] The data listed in Table 10 clearly indicate that when an
enzyme digests on the Bombyx mori silk fibroin membrane, the
weight-average molecular weight of the membrane is reduced from the
initial level of 120,000 D with the progress of the biodegradation
time, but after 72 hours from the initiation of the biodegradation,
the rate of the molecular weight change of the membrane was
reduced. In particular, in the case of collagenase and
chymotrypsin, the weight-average molecular weight and the peak
molecular weight were reduced with the progress of the
biodegradation time. On the other hand, in the case of protease,
the weight-average molecular weight was reduced with the progress
of the biodegradation time, but the extent of the molecular weight
reduction was insignificant and the peak molecular weight was
reduced to an extent almost identical to that observed for the
chymotrypsin.
COMPARATIVE EXAMPLE 1
[0197] Molecular Weight Change of Bombyx mori Silk Fiber Associated
with Biodegradation Treatment
[0198] The changes in the molecular weight of a Bombyx mori silk
fiber sample with the elapsed biodegradation time when digesting
the silk fiber with collagenase, chymotrypsin and protease were
monitored and evaluated by the high performance liquid
chromatography technique. The results thus obtained are listed in
the following Table 11.
11 TABLE 11 Weight-Average Peak Molecular Enzyme Molecular Weight
(kD) Weight (kD) Control 233.1 179.3 Collagenase: 0.2 mg/mL (72
hrs.) 184.3 93.7 (576 hrs.) 204.4 109.7 Chymotrypsin: 0.2 mg/mL (72
hrs.) 129.4 66.0 (576 hrs.) 174.4 92.8 Protease: 0.2 mg/mL (72
hrs.) 256.5 176.9 (576 hrs.) 247.4 155.2 Protease: 0.5 mg/mL (72
hrs.) 261.7 173.5 (576 hrs.) 241.7 148.7
[0199] In Table 11, the time given in parentheses appearing in the
column entitled "Enzyme" means the elapsed biodegradation time.
[0200] The data listed in Table 11 clearly indicate that the Bombyx
mori silk fiber hardly causes any reduction of the weight-average
molecular weight and the peak molecular weight even when an enzyme
acts on the thread, as compared with the Bombyx mori silk fibroin
membrane (Example 17). For this reason, the silk fiber is not
useful in the fields wherein biodegradability is required, while
materials excellent in biodegradability are, for instance, Bombyx
mori silk fibroin membranes and Antheraea pernyi silk fibroin
membranes.
EXAMPLE 18
[0201] Biodegradation Rate
[0202] The following samples were digested for the biodegradation
rate observed when these samples were hydrolyzed with protease,
collagenase or chymotrypsin: the Bombyx mori silk fiber and the
Antheraea pernyi silk fiber; the Bombyx mori silk fibroin membrane
(BF membrane) prepared according to the procedures used in Example
1; the Antheraea pernyi silk fibroin membrane (TF membrane)
prepared according to the procedures used in Example 2; the hybrid
membrane (BF+TF membrane) consisting of Bombyx mori silk
fibroin/Antheraea pernyi silk fibroin prepared according to the
procedures used in Example 3; the hybrid membrane (BF+Cell
membrane) consisting of Bombyx mori silk fibroin/cellulose prepared
according to the procedures used in Example 4; and the hybrid
membrane (BF+CMK membrane) consisting of Bombyx mori silk
fibroin/carboxymethyl chitin prepared according to the method
described above. The results thus obtained are summarized in the
following Table 12. In this connection, the term "biodegradation
rate" is herein defined to be a value obtained by dividing the
sample weight observed after 50 hours from the initiation of the
biodegradation by the original sample weight (the sample weight
prior to the biodegradation) and expressed in terms of "%".
Accordingly, when a sample is not biodegraded at all, the
biodegradation rate of the sample corresponds to 0%/50 hours.
12 TABLE 12 Enzyme Biodegradation Concn. Rate Sample Enzyme (mg/mL)
(%/50 hrs.) Bombyx mori Silk fiber Protease 0.2 1.4 " " 0.4 0
Bombyx mori Silk fiber Collagenase 0.2 0 " " 0.5 0.5 Bombyx mori
Silk fiber Chymotrypsin 0.2 0.5 " " 0.5 1.0 Antheraea pernyi Silk
fiber Protease 0.2 0 " " 0.5 0 Antheraea pernyi Silk fiber
Collagenase 0.2 0 " " 0.5 0 Antheraea pernyi Silk fiber
Chymotrypsin 0.2 0 " " 0.5 0 BF Membrane Protease 0.2 18.2 " " 0.5
27.3 BF Membrane Collagenase 0.2 2.0 " " 0.5 2.2 BF Membrane
Chymotrypsin 0.5 9.1 TF Membrane Protease 0.2 3.1 " " 0.5 11.2 TF
Membrane Chymotrypsin 0.2 14.5 BF:TF (10:0) Protease 0.2 18.2 BF:TF
(8:2) " 0.2 15 BF:TF (6:4) " 0.2 15 BF:TF(4:6) " 0.2 0 BF:TF (2:8)
" 0.2 0 BF:TF (0:10) " 0.2 3.1 BF:Cell (10:0) Protease 0.2 18.2
BF:Cell (8:2) " 02 13.6 BF:Cell (6:4) " 0.2 22.7 BF:Cell (4:6) "
0.2 21.4 BF:Cell (2:8) " 0.2 5.9 BF:Cell (0:10) " 0.2 2.5 BF:CMK
(6:4) Protease 0.2 12.1 BF:CMK (2:8) " 0.2 8.5
[0203] In Table 12, "BF:TF" and "BF:Cell" represent the hybrid
membrane of Bombyx mori silk fibroin (BF) and Antheraea pernyi silk
fibroin (TF) and the hybrid membrane of Bombyx mori silk fibroin
(BF) and cellulose (Cell), respectively and the corresponding
numerical values given in parentheses mean that the mixing ratios
of BF to TF and those of BF to Cell are 100/0, 80/20, 60/40, 40/60,
20/80 and 0/100.
[0204] The data listed in Table 12 also clearly indicate that when
digesting a hybrid membrane of Bombyx mori silk fibroin and
Antheraea pernyi silk fibroin with protease, the hybrid membrane
containingAntheraea pernyi silk fibroin in an amount ranging from
60 to 80% does not substantially undergo any biodegradation from
the viewpoint of the biodegradation rate and this is substantially
consistent with the conclusion deduced from the data concerning the
rate of residual sample weight discussed in Example 13. This is a
property, which is never observed for the membrane simply
consisting of Bombyx mori silk fibroin or Antheraea pernyi silk
fibroin.
EXAMPLE 19
[0205] Composite Materials Having Different Shapes
[0206] A mixture of an aqueous solution of Bombyx mori silk fibroin
and an aqueous solution of Antheraea pernyi silk fibroin was poured
into a beaker, a dilute aqueous acetic acid solution was gradually
added to the mixed aqueous solution in small portions to control
the pH value of the whole aqueous solution to 2.5 and to thus form
a gel-like product of Bombyx mori silk fibroin and Antheraea pernyi
silk fibroin. Moreover, the gel-like product thus obtained was
directly subjected to lyophilization without removing any moisture
from the product according to a known method to prepare a porous
product of Bombyx mori silk fibroin and Antheraea pernyi silk
fibroin.
[0207] Alternatively, a mixture of an aqueous solution of Bombyx
mori silk fibroin and an aqueous solution of Antheraea pernyi silk
fibroin was cast dried on a polyethylene substrate to thus give a
transparent composite membrane.
[0208] Moreover, acetic acid was added to a mixture of an aqueous
solution of Bombyx mori silk fibroin and an aqueous solution of
Antheraea pernyi silk fibroin, followed by the control of the pH
value thereof to 3.0 and the lyophilization of the aqueous mixture
by a known method to thus form a powdery hybrid consisting of
Bombyx mori silk fibroin and Antheraea pernyi silk fibroin.
EXAMPLE 20
[0209] Amino Acid Analysis
[0210] A Bombyx mori silk fibroin membrane was digested with
protease and then the biodegraded sample was subjected to amino
acid analysis to thus examine the relationship between the results
of the amino acid analysis and the biodegradation time. The results
thus obtained are summarized in the following Table 13. In Table
13, the amount of each amino acid residue is expressed in terms of
"mole %".
13 TABLE 13 Biodegradation Time (day) Amino Acid 0 3 10 17 Cyst
0.03 0.04 0.02 0.00 Asp 1.28 0.58 0.83 0.59 Glu 1.09 0.54 0.77 0.51
Ser 10.89 9.96 10.47 10.68 Gly 45.00 46.49 45.95 46.73 His 0.15
0.10 0.10 0.10 Arg 0.48 0.32 0.36 0.29 Thr 0.78 0.50 0.59 0.54 Ala
29.43 31.52 30.36 31.71 Pro 0.35 0.27 0.28 0.26 Tyr 5.76 5.74 5.80
5.05 Val 2.31 2.17 2.24 1.85 Met 0.09 0.07 0.06 0.27 Cyst 0.02 0.00
0.00 0.00 Ile 0.66 0.46 0.59 0.41 Leu 0.51 0.31 0.53 0.28 Phe 0.81
0.71 0.77 0.56 Lys 0.36 0.22 0.28 0.17 Total 100.00 100.00 100.00
100.00 Gly 45.00 45.95 46.49 46.73 Ala 29.43 30.36 31.52 31.71 Ser
10.89 10.47 9.96 10.68 Total-1 85.32 86.78 87.97 89.12 Tyr 5.76
5.80 5.74 5.05 Acid 2.40 1.62 1.16 1.10 Basic 0.99 0.74 0.64 0.56
Others 5.53 5.06 4.49 4.18 Total-2 14.68 13.22 12.03 10.88
[0211] In Table 13, "Total" means the amount of the whole amino
acids residues obtained in the amino acid analysis expressed in the
unit of mole %, "Total-1" means the total amount of the amino acid
residues or Gly, Ala and Ser constituting the crystalline region,
while "Total-2" means the total amount of acidic amino acid
residues (acid) such as Glu and Asp, basic amino acid residues
(basic) such as Lys, Arg and His as well as other amino acids.
[0212] As will be seen from the results listed in Table 13, there
is observed a distinct tendency in the results obtained by the
amino acid analysis of the biodegraded Bombyx mori silk fibroin
membrane. More specifically, the total amount of Gly, Ala and Ser,
which are principal amino acids constituting the crystalline region
of the silk fibroin, gradually increases with the lapse of the
digestion time, while the total amount of bulky polar side
chain-containing amino acids such as Tyr, acidic amino acids and
basic amino acids is reduced. In other words, the amino acid
composition of the silk fibroin is shifted to that observed for the
crystalline region as the biodegradation proceeds as has been
described above. Therefore, it would be considered that when an
enzyme acts on silk fibroin, the enzyme first acts on the amorphous
region quite susceptible to the enzymatic attack to thus induce the
digestion of the fibroin. The crystalline region of the fibroin
hardly susceptible to the attack of the enzyme still remains even
after the biodegradation and accordingly, the principal chemical
structure of the fibroin is shifted to that mainly comprising
crystalline amino acids as the biodegradation proceeds.
EXAMPLE 21
[0213] Adsorption of Metal Ions
[0214] Experiments were conducted according to the method for
adsorbing metal ions, which had been described above. The metal
salt aqueous solutions used herein were a 0.5 mM aqueous solution
of silver nitrate (AgNO.sub.3) and a 0.5 mM aqueous solution of
copper nitrate (Cu(NO.sub.3).sub.2). There were immersed, in each
of these aqueous solutions, a Bombyx mori silk fibroin membrane (BF
membrane) prepared according to the same procedures used in Example
1, an Antheraea pernyi silk fibroin membrane (TF membrane) prepared
according to the same procedures used in Example 2 and composite
membranes (80:20 and 50:50 (weight ratio) BF+TF membranes)
consisting of Bombyx mori silk fibroin and Antheraea pernyi silk
fibroin prepared according to the same procedures used in Example 3
at a temperature of 25.degree. C. for 30 minutes to adsorb silver
ions or copper ions on each sample and to thus determine the
quantity of metal ions adsorbed on each sample.
[0215] Moreover, various kinds of biodegradable biopolymer
membranes on which silver ions had been adsorbed according to the
foregoing method were evaluated for the antibacterial activity
against tomato canker causal bacterium: Corynebacterium
michiganense pv. michiganense.
[0216] The amount of the metal ion adsorption and the antibacterial
activity thus obtained are listed in the following Table 14.
14 TABLE 14 Amt. of Ag.sup.+ Amt. of Cu.sup.2+ Antibacterial
Activity Sample (mmol/g) (mmol/g) (mm) BF 0.18 0.23 2 TF 0.24 0.30
2.5 BF + TF (80:20) 0.72 0.95 8 BF + TF (50:50) 0.93 1.25 8.7
[0217] The data listed in Table 14 clearly indicate that the
composite membrane obtained by combining Bombyx mori silk fibroin
and Antheraea pernyi silk fibroin may absorb metal ions in an
amount greater than that observed for the membrane simply
consisting of Bombyx mori silk fibroin or Antheraea pernyi silk
fibroin and as a result, the former displays antibacterial activity
toward plant pathogenic fungi or bacteria. Thus, the silk
fibroin-containing composite material can be used as a metal
adsorbent and an antibacterial material.
EXAMPLE 22
[0218] Rate of Light Transmission of Silk Protein Membrane
[0219] A Bombyx mori silk fibroin membrane (BF membrane), an
Antheraea pernyi silk fibroin membrane (TF membrane) and composite
membranes (80:20 and 50:50 (weight ratio) BF+TF membranes) obtained
by hybridizing Bombyx mori silk fibroin and Antheraea pernyi silk
fibroin were evaluated for the transmittance spectra using a
self-recording (or autographic) spectrophotometer (Model: W-2100S)
available from Shimadzu Corporation. In this connection, the
spectra thus determined were transmittance spectra including those
originated from surface reflection. The resulting rates of light
transmission are listed in the following Table 15.
15 TABLE 15 Sample Rate of Light Transmission (%) BF Membrane 87.3
TF Membrane 84.7 BF:TF (80:20) Membrane 93.4 BF:TF (50:50) Membrane
90.1
[0220] The data listed in Table 15 clearly indicate that the
composite membrane is highly permeable to light rays as compared
with the membrane simply consisting of Bombyx mori silk fibroin or
Antheraea pernyi silk fibroin or the former has a degree of
clearness higher than that observed for the latter.
EXAMPLE 23
[0221] Drug-Sustained Release Properties
[0222] To an equivalent mixture of a 2% aqueous solution of Bombyx
mori silk fibroin and a 2% aqueous solution of Antheraea pernyi
silk fibroin prepared according to the same procedures used in
Example 3, there was gently added an aqueous solution prepared by
dissolving 5 mg of acetyl salicylic acid in 50 mL of water. The
aqueous mixed solution allowed to stand at room temperature was
gradually converted into a gel. The resulting gel was once frozen
at a temperature of -10.degree. C. and then dried in vacuo to thus
form a porous composite material containing acetyl salicylic acid.
The porous composite material was digested in an enzyme solution
containing 2 mg/mL of protease over a predetermined period of time
(24 and 72 hours) and the amount of the agent gradually released
from the porous composite material during the digestion process was
evaluated on the basis of the UV absorbance at 206.9 nm, which was
determined using a UV absorbance meter available from Shimadzu
Corporation. The results thus obtained are listed in the following
Table 16. Each UV absorbance value listed in Table 16 is obtained
by subtracting the UV absorption observed for the initial enzyme
solution or observed at the biodegradation time of 0 from the UV
absorption observed for the enzyme solution containing the agent
gradually released from the porous composite material.
[0223] Moreover, as a control, a porous material simply consisting
of Bombyx mori silk fibroin (BF) or Antheraea pernyi silk fibroin
(TF) was likewise evaluated for the sustained release
characteristics according to the same procedures used above in
connection with the foregoing porous composite material. The
results are likewise summarized in the following Table 16.
16 TABLE 16 Biodegradation Time (hr.) Sample 0 24 72 BF Porous
Material 0.10 0.259 0.259 TF Porous Material 0.10 0.270 0.271
Porous Composite Material 0.10 0.265 0.293
[0224] The data listed in Table 16 clearly indicate that the porous
composite material containing acetyl salicylic acid gradually
releases the drug over a long period of time as compared with the
drug-release behavior of the porous material simply consisting of
Bombyx mori silk fibroin or Antheraea pernyi silk fibroin. This
clearly indicates that the composite membrane possesses
drug-sustained release characteristics.
EXAMPLE 24
[0225] FT-IR of Composite Membrane
[0226] The following membranes were subjected to the FT-IR
measurements: a Bombyx mori silk fibroin membrane (BF membrane)
prepared according to the procedures used in Example 1; an
Antheraea pernyi silk fibroin membrane (TF membrane) prepared
according to the procedures used in Example 2; a composite membrane
(BF+TF membrane) consisting of Bombyx mori silk fibroin and
Antheraea pernyi silk fibroin prepared according to the procedures
used in Example 3; a composite membrane (BF+CMK membrane)
consisting of Bombyx mori silk fibroin and carboxymethyl chitin
used in Example 18; a composite membrane (TF+CMK membrane)
consisting of Antheraea pernyi silk fibroin and carboxymethyl
chitin; a membrane consisting of CMK alone; and a composite
membrane (BF+PVA membrane) consisting of Bombyx mori silk fibroin
and polyvinyl alcohol, to thus determine wave numbers appearing
within the range of from 2000 to 500 cm.sup.-1. The results thus
obtained are summarized in the following Table 17.
17TABLE 17 Sample Wave Number (cm.sup.-1) and Absorption Intensity
BF Membrane 1654, 1539, 1455, 1414, 1383, 1334, 1240, 1170, 1071,
1061, 1016, 950, 670, 532 TF Membrane 1650, 1546, 1274, 615 BF + TF
Membrane 1654, 1650, 1274, 950, 670, 615, 533 BF + CMK 1654, 1542,
1528, 1451, 1412, 1381, 1333, 1242, 1162, 1109, 1070, 949, 666, 559
TF + CMK (2:8) Membrane 1657, 1548, 1451, 1378, 1316, 1156, 1113,
1069, 1037, 951, 900, 618, 526 TF + CMK (8:2) Membrane 1653, 1542,
1282, 1334, 1307, 659, 617, 525 CMK Membrane 1654, 1592, 1570,
1413, 1374, 1318, 1155, 1111, 1071, 1038, 946, 901, 685, 615, 571
BF + PVA (2:8) Membrane 1420-1440, 1326, 1232, 1093, 913, 849
[0227] In Table 17, the term "TF+CMK (2:8)" means a composite
membrane prepared by blending Antheraea pernyi silk fibroin and
carboxymethyl chitin in a weight ratio of 20:80. In addition, the
term "BF+PVA (2:8)" means a composite membrane prepared by blending
Bombyx mori silk fibroin and polyvinyl alcohol in a weight ratio of
20:80.
[0228] The data listed in Table 17 indicate that the IR spectra
observed for the composite materials consisting of Bombyx mori silk
fibroin and secondary substances and the composite materials
consisting of wild silkworm silk fibroin and secondary substances
include only spectra ascribable to two kinds of constituents, which
are superimposed to one another and are free of any spectrum other
than those ascribable to the two constituents. This clearly
indicates or suggests that any new linkage is not formed between
the Bombyx mori silk fibroin or the wild silkworm silk fibroin and
the secondary substance.
[0229] The foregoing indicates that in the composite materials
consisting of domesticated or wild silkworm silk fibroin and
secondary substances selected from the group consisting of
cellulose, chitin, chitosan, chitosan derivatives, wool keratin and
polyvinyl alcohol, there is not any chemical or covalent bond
between the domesticated or wild silkworm silk fibroin and the
secondary substance, but these two components are simply coagulated
through the action of hydrogen bonds formed therebetween. This is
because the composite material is simply prepared by casting a
mixture of aqueous solutions of respective constituents on the
surface of a substrate and then solidification through evaporation
to dryness, without using any particular agent for cross-linking
molecules. In this connection, the aqueous mixture is allowed to
stand and, if desired, gently stirred so as not to cause any
coagulation of these two kinds of molecules while taking care not
to cause solidification due to any abrupt mixing mechanical
operation prior to the casting on the substrate surface.
[0230] As has been discussed above in detail, the biodegradable
biopolymer material of the present invention comprises an insect's
biopolymer alone such as domesticated silkworm silk fibroin or wild
silkworm silk fibroin, or a composite material comprising
domesticated or wild silkworm silk fibroin and a secondary
substance or at least one compound selected from the group
consisting of cellulose, wool keratin, chitin, chitosan, chitosan
derivatives and polyvinyl alcohol and the biodegradation of these
materials may be controlled.
[0231] The biodegradable biopolymer material of the present
invention should be insolibilized in water prior to the
biodegradation experiments, but it is also possible to use any
conventionally known agent for cross-linking protein molecules such
as formaldehyde or epoxy compounds. In addition, the silk protein
membrane or the composite material may likewise be insolubilized in
water by simple treatments, for instance, by lightly immersing the
membrane in an aqueous alcohol solution such as an aqueous methanol
or ethanol solution and then drying at room temperature.
[0232] The susceptibility of a hybrid to the biodegradation may be
determined by the degree of insolubilization of a domesticated or
wild silkworm silk fibroin membrane, the choice of the secondary
substance, the mixing ratio of the domesticated or wild silkworm
silk fibroin to the secondary substance, the kind of enzyme
selected, the enzyme concentration used and the processing time and
therefore, a hybrid having a desired biodegradability can be
prepared by appropriately selecting the conditions for producing
the same, the mixing ratio of the constituents and/or the
conditions for biodegradation.
[0233] The composite material made of domesticated or wild silkworm
silk fibroin with secondary substances would permit the achievement
of such a significant effect that the surface of the resulting
blending shows excellent biochemical characteristics, which are
never observed for the surface of the membrane comprising
domesticated or wild silkworm silk fibroin alone, or the secondary
substance alone. For instance, the surface of the hybrid is
excellent in the rate of biological cell-growth as compared with
the surface of the membrane comprising the domesticated silkworm
silk fibroin alone, or the secondary substance alone. In addition,
the hybrid is also excellent in the ability of coating the surface
of general organic polymers such as PET and the use of a hybrid
material would permit the improvement of the resistance of a
membrane to mechanical friction.
[0234] If a useful substance such as a water-soluble medicine or a
pharmacological component is included in the biodegradable
biopolymer material of the present invention, the medicine or the
pharmacological component can gradually be released while
biodegrading the biodegradable biopolymer material in the living
body and therefore, the material can be used as a sustained release
material.
[0235] The biodegradability can be reduced by the use of the silk
fibroin fibers from domesticated or wild silkworms and if an easily
biodegradable material is desired, a membrane-like material may be
used, such a membrane being able to be prepared by dissolving
domesticated or wild silkworm silk fibers in a neutral salt
solution, desalting the resulting solution using a dialysis
membrane of cellulose and then solidifying the resulting aqueous
solution through drying. The domesticated silkworm silk fibroin
membrane can easily be biodegraded as compared with the wild
silkworm silk fibroin membrane and therefore, a hardly
biodegradable composite material comprising domesticated and wild
silkworm silk fibroins may be obtained by increasing the content of
the wild silkworm silk fibroin present in the composite
material.
[0236] When the biodegradable biopolymer material of the present
invention is used while it is embedded in the living body, the
material is ultimately decomposed into lower molecules such as
water and carbon dioxide by the action of enzymes present in the
body such as protease and then excreted outside the body. The
easily biodegradable domesticated silkworm silk fibroin membrane
may be biodegraded within a relatively short period of time even
when it is embedded in the body unlike the hardly biodegradable
domesticated silkworm silk fibroin fibers and therefore, the
membrane can be used for temporarily helping the repairable damaged
biological tissues in their healing or for the preparation of a
sustained release drugs as has been discussed above. The absorptive
material of the present invention 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.
[0237] The biodegradable biopolymer material of the present
invention is digested and deteriorated through digestion with a
protease and therefore, it may likewise be used as a sustained
release carrier for a useful substance such as a medicine or a
physiologically active substance. For instance, when embedding, in
the biodegradable biopolymer material, a product obtained by taking
the useful substance in the biopolymer material or by fixing the
useful substance to the biopolymer material, the useful substance
is gradually released within the living body, while the biopolymer
material is digested with enzymes present in the body.
[0238] Cellulose derivatives have effectively been utilized in
various fields such as food additives, cosmetics, additives for
medicines and medicines such as an antithrombotic agent and
therefore, the composite material consisting of domesticated
silkworm silk fibroin and cellulose may be used in fields identical
to those listed above in connection with cellulose. Moreover, the
hybridization of domesticated silkworm silk fibroin with cellulose
would permit the mechanical properties of the silk fibroin, in
particular, in its dried conditions. In addition, the hybridization
of domesticated or wild silkworm silk fibroin with a secondary
substance such as cellulose would permit the production of a
material having improved moldability and transparency as well as
cell adhesion properties, as compared with those observed for a
membrane simply consisting of domesticated or wild silkworm silk
fibroin.
[0239] The domesticated silkworm silk fibroin membrane may
relatively easily be biodegraded with protease. For this reason, if
hybridizing the domesticated silkworm silk fibroin with hardly
biodegradable wild silkworm silk fibroin, the resulting hybrid
membrane would have a controlled degree of biodegradation and may
likewise have an improved film-forming ability and enhanced
transparency.
[0240] The extent of biodegradation of the biodegradable biopolymer
membrane according to the present invention can be controlled by a
simple treatment. Moreover, in a hybrid material, the biodegradable
biopolymer moiety is firmly adhered to the surface of a secondary
substance, the hybrid material is likewise excellent in the wear
resistance and therefore, the substrate coated with a hybrid
material is improved in the biological cell-growth properties on
the surface thereof as compared with the substrate coated only with
a protein. Accordingly, the hybrid material is useful as a
cell-growth substrate capable of being used in the field of
biotechnology.
[0241] When immersing the biodegradable biopolymer material of the
present invention in an aqueous solution containing antibacterial
metal ions, a large amount of such metal ions are adsorbed on the
biopolymer material and therefore, the biopolymer material carrying
such metal ions adsorbed thereon is useful as an antibacterial
fiber material. Moreover, when immersing the biopolymer material in
waste water, it can adsorb metal ions present in the waste water
and accordingly, the biopolymer material is also effective as a
fibrous material for adsorbing metal ions in waste water.
[0242] 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 coagulating sludge), foods,
industries, and agriculture and horticulture (for instance, the use
as a soil conditioner and a water-retention agent).
* * * * *