U.S. patent application number 13/122837 was filed with the patent office on 2011-09-15 for modified silk films containing glycerol.
This patent application is currently assigned to TRUSTEES OF TUFTS COLLEGE. Invention is credited to David Kaplan, Shenzhou Lu, Fiorenzo Omenetto, Xiaoqin Wang.
Application Number | 20110223153 13/122837 |
Document ID | / |
Family ID | 42101226 |
Filed Date | 2011-09-15 |
United States Patent
Application |
20110223153 |
Kind Code |
A1 |
Lu; Shenzhou ; et
al. |
September 15, 2011 |
MODIFIED SILK FILMS CONTAINING GLYCEROL
Abstract
The present invention provides for compositions and methods for
preparing aqueous insoluble, ductile, flexible silk fibroin films.
The silk films comprise silk fibroin and about 10% to about 50%
(w/w) glycerol, and are prepared by entirely aqueous processes. The
ductile silk film may be further treated by extracting the glycerol
from and re-drying the silk film. Active agents may be embedded in
or deposited on the glycerol modified silk film for a variety of
medical applications. The films may be into 3-dimentional
structures, or placed on support surfaces as labels or coatings.
The glycerol modified silk films of the present invention are
useful in variety of applications such as tissue engineering,
medical devices or implants, drug delivery, and edible
pharmaceutical or food labels.
Inventors: |
Lu; Shenzhou; (Suzhou,
CN) ; Wang; Xiaoqin; (Winchester, MA) ;
Omenetto; Fiorenzo; (Wakefield, MA) ; Kaplan;
David; (Concord, MA) |
Assignee: |
TRUSTEES OF TUFTS COLLEGE
Medford
MA
|
Family ID: |
42101226 |
Appl. No.: |
13/122837 |
Filed: |
October 9, 2009 |
PCT Filed: |
October 9, 2009 |
PCT NO: |
PCT/US09/60135 |
371 Date: |
May 31, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61104135 |
Oct 9, 2008 |
|
|
|
Current U.S.
Class: |
424/130.1 ;
264/2.6; 424/93.6; 424/93.7; 424/94.1; 435/395; 514/1.1; 514/44R;
514/773 |
Current CPC
Class: |
C08J 2389/00 20130101;
A61L 27/54 20130101; B05D 3/0254 20130101; B05D 1/30 20130101; A61L
2420/06 20130101; C08K 5/053 20130101; A61L 27/3804 20130101; A61P
31/00 20180101; A61L 27/3604 20130101; B05D 3/107 20130101; C08K
5/053 20130101; A61L 27/50 20130101; C08J 5/18 20130101; C08J 3/18
20130101; C12N 11/02 20130101; A61P 31/04 20180101; C08L 89/00
20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/130.1 ;
435/395; 514/773; 424/93.7; 514/1.1; 514/44.R; 424/94.1; 424/93.6;
264/2.6 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 5/071 20100101 C12N005/071; A61K 47/42 20060101
A61K047/42; A61K 35/12 20060101 A61K035/12; A61K 38/02 20060101
A61K038/02; A61K 31/7088 20060101 A61K031/7088; A61K 38/43 20060101
A61K038/43; A61K 35/76 20060101 A61K035/76; A61P 31/00 20060101
A61P031/00; A61P 31/04 20060101 A61P031/04; A61P 35/00 20060101
A61P035/00; B29D 11/00 20060101 B29D011/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
no. EB002520 awarded by the National Institutes of Health and No.
FA9550-07-1-0079 awarded by the Air Force Office of Scientific
Research. The U.S. federal government has certain rights in the
invention.
Claims
1. A silk film comprising silk fibroin and about 10% (w/w) to about
50% (w/w) glycerol.
2. The silk film of claim 1, wherein the glycerol content of the
silk film is about 20% (w/w) to about 40% (w/w)
3. The silk film of claim 1, wherein the glycerol content of the
silk film is about 30% (w/w).
4. The silk film of claim 1, further comprising at least one active
agent.
5. The silk film of claim 1, further comprising silk microspheres
or silk nanospheres embedded in the silk film.
6. The silk film of claim 1, wherein said film is layered or folded
into a sponge or a block.
7. The silk film of claim 4, wherein the at least one active agent
is selected from the group consisting of cells, proteins, peptides,
nucleic acid analogues, nucleotides or oligonucleotides, peptide
nucleic acids, aptamers, antibodies or fragments or portions
thereof, hormones, hormone antagonists, growth factors or
recombinant growth factors and fragments and variants thereof,
cytokines, enzymes, antibiotics or antimicrobial compounds,
viruses, antivirals, toxins, prodrugs, chemotherapeutic agents,
small molecules, drugs, and combinations thereof.
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. The silk film of claim 1, further comprising an optical pattern
on the silk film.
13. The silk film of claim 12, wherein the optical pattern is a
holographic image.
14. A method for preparing a silk film, comprising: blending a silk
fibroin solution with glycerol, wherein the concentration of
glycerol in the silk fibroin/glycerol blend solution is about 10%
to about 50% (w/w); casting the silk fibroin/glycerol blend
solution onto a film-supporting surface; and drying the silk
film.
15. The method of claim 14, further comprising the steps of
immersing the silk film in a liquid in which glycerol dissolves for
a period of time to deplete glycerol from the silk film; and drying
the glycerol-depleted film.
16. The method of claim 14, further comprising annealing said
film.
17. A method for covering a surface of a substrate with a silk
composition comprising: providing a film-support substrate; and
covering the film-support substrate with a silk fibroin/glycerol
blend film comprising about 10% to 50% glycerol (w/w).
18. The method of claim 17, wherein the silk fibroin/glycerol blend
film further comprises at least one biopolymer.
19. The method of claim 18, wherein the biopolymer is PVA or
PEO.
20. The method of claim 19, wherein the silk fibroin/glycerol blend
film further comprises at least one active agent.
21. (canceled)
22. (canceled)
23. (canceled)
24. A method of embedding at least one active agent in a silk film,
comprising: blending a silk fibroin solution with at least one
active agent and glycerol, wherein the concentration of glycerol in
the silk blend solution is about 10% to 50% (w/w); casting the silk
blend solution onto a film-supporting surface; and drying the
film.
25. The method of claim 24, wherein the at least one active agent
is selected from the group consisting of cells, proteins, peptides,
nucleic acid analogues, nucleotides or oligonucleotides, peptide
nucleic acids, aptamers, antibodies or fragments or portions
thereof, hormones, hormone antagonists, growth factors or
recombinant growth factors and fragments and variants thereof,
cytokines, enzymes, antibiotics or antimicrobial compounds,
viruses, antivirals, toxins, prodrugs, chemotherapeutic agents,
small molecules, drugs, and combinations thereof.
26. The method of claim 24, further comprising the steps of
immersing the silk film in a liquid in which glycerol dissolves for
a period of time to deplete glycerol from the silk film; and drying
the glycerol-depleted film.
27. The method of claim 24, further comprising annealing said film.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. .sctn.119(e)
of U.S. Provisional Application No. 61/104,135 filed Oct. 9, 2008,
the contents of which are incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention provides for compositions and methods
for preparing silk fibroin films containing glycerol and having
improved mechanical properties.
BACKGROUND
[0004] Silk fibroin has excellent film-forming capabilities and is
also compatible for use in the human body. Silk fibroin films,
without further manipulation or treatment, are soluble in water
because of dominating random coil protein structures. The
structural features of the protein can be transformed from random
coil to .beta.-sheet structure by several treatments, including
mechanical stretching, immersion in polar organic solvents, or
curing in water vapor. This structural transition results in
aqueous insolubility, thus providing options for the use of the
material in a range of biomedical and other applications. Some pure
silk fibroin films tend, over time, to become stiff and brittle in
the dry state, however, exhibiting impressive tensile strength but
low ductility. There remains a need to modify the physical and
mechanical properties of silk fibroin films to improve mechanical
properties and provide for more flexible silk fibroin-based systems
for biomedical and other applications.
SUMMARY OF THE INVENTION
[0005] The present invention provides for films comprising silk
fibroin and glycerol, which have distinct properties compared with
silk fibroin films lacking glycerol. More specifically, the aqueous
solubility and biocompatibility are enhanced with the use or
inclusion and use of glycerol as a plasticizer. Processing silk
fibroin in water also enhances both biocompatibility and the
potential to load bioactive compounds without loss of function, and
adds "green chemistry" value to these biomaterials. For example,
blends of silk fibroin and glycerol with glycerol concentrations
above 30% (w/w) cast into films resulted in the conversion of silk
secondary structure from random coil to a-helix, prevented silk
from dissolution upon hydration, provided distinct film
nanostructure morphology, improved film flexibility in either dry
(as-cast film) or wet (after leaching out the glycerol)
environments, and preserved cell biocompatibility. Mechanistically,
glycerol may replace water in silk fibroin chain hydration,
resulting in initial stabilization of helical structures as opposed
to random coil or .beta.-sheet structures. The impact of glycerol
on stabilizing film structure, aqueous insolubility and function
apparently occurs above a glycerol concentration of about 20 wt %
glycerol. The use of glycerol in combination with silk fibroin in
materials processing expands the functional features attainable
with this fibrous protein, and the formation of more flexible films
with potential utility in biomaterial and device applications.
[0006] The present invention provides for a silk film comprising
silk fibroin and from about 10% (w/w) to about 50% (w/w) glycerol,
in which the film is prepared by entirely aqueous processes, and
the silk film is ductile and substantially aqueous-insoluble. Many
embodiments of the silk/glycerol blend films of the present
invention exhibit higher ductility than silk films lacking
glycerol, optionally following methanol treatment or
water-annealing. The glycerol in the silk fibroin film, without
being bound by theory, appears to stabilize the .alpha.-helical
structure of the silk fibroin. Thus, in one embodiment, the ductile
silk fibroin film may be converted from .alpha.-helical structure
to .beta.-sheet structure by extracting glycerol from the silk film
and re-drying the film.
[0007] In one embodiment, a composition comprising glycerol
modified silk film may be used as a 2-dimensional or 3-dimensional
construct for tissue engineering, and may further comprise at least
one active agent. Such tissue engineered construct may be used for
organ repair, organ replacement, or other regenerated tissue
materials such as cardiac muscle or cornea tissues. A 3-dimensional
tissue engineering embodiment may be made by wrapping or shaping a
ductile silk/glycerol film around a device or implant, such as a
dental implant, and allowing the film to dry. Silk/glycerol blends
may be formed, or the films folded or shaped, into sponges or
blocks or other 3-dimensional structures. Optionally, the glycerol
may then be leached out from the silk. Thus, the silk film may also
be used as coatings on biomedical materials such as medical
devices, tissue-engineered materials or implants, by coating the
surfaces of such structures with a silk/glycerol ductile film.
Coating from such modified silk film provides for improved
compatibility and conforms well to the contours of the
substrate.
[0008] In another embodiment, the glycerol-containing silk fibroin
film is a composite material comprising a silk-based structure,
such as silk fibroin nanospheres or microspheres, optionally
containing active agents. Additionally, the silk composite material
may include a silk-based composite support surface, such as a
3-dimensional structure of a medical implant or device, on which
the ductile glycerol/silk film is shaped.
[0009] The embodiments of the prevent invention also provide for
methods of preparing a silk film which is substantially
aqueous-insoluble, by blending a silk fibroin solution with
glycerol, wherein the concentration of glycerol in the silk
fibroin/glycerol blend solution ranges from about 10% to 50% (w/w);
casting the silk fibroin/glycerol blend solution onto a
film-supporting surface; and drying the film. Silk films prepared
by this process exhibit increased ductility compared with silk
films lacking glycerol.
[0010] At least one active agent may be embedded in the ductile
silk film by blending a silk fibroin solution with at least one
active agent and glycerol before casting and drying the film.
Similarly, cells or tissues may be embedded in the silk/glycerol
blend films.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows data on the dissolution of silk and glycerol
from blend films. *Significant differences between groups
(P<0.01). Data represent the ave.+-.SD (n=4).
[0012] FIGS. 2A-2C show FTIR determination of silk secondary
structures in blend films with different glycerol content. FIG. 2A:
blend films directly after film casting. FIG. 2B: blend films after
90% (v/v) methanol treatment for 1 hour. FIG. 2C, 20% (w/w)
glycerol film with and without water treatment for 1 hour.
*significant differences between groups (P<0.01). Data represent
the ave.+-.SD (n=4).
[0013] FIGS. 3A-3D present mechanical properties of blend films
with different glycerol content. FIG. 3A, tensile strength. FIG.
3B, elongation at break. FIG. 3C, tensile modulus of dry blend
films. FIG. 3D, tensile modulus of wet blend films after water
treatment for 1 hour. *significant differences between groups
(P<0.01). Data represent the average.+-.SD (n=5).
[0014] FIGS. 4A-4D show SEM images of blend films. FIG. 4A,
glycerol content 10% (w/w). FIG. 4B, glycerol content 20% (w/w).
FIG. 4C, glycerol content 30% (w/w), water treated for 1 hour. FIG.
4D, glycerol content 0%, methanol treated for 1 hour. Scale bar=200
nm.
[0015] FIGS. 5A-5D are micrographs showing nano-filament structures
in water-treated silk films containing 30% (w/w) glycerol. FIGS. 5A
and 5D show different regions in the film. FIG. 5B, high
magnification of 5A. FIG. 5C, side view of 5A. FIG. 5E, high
magnification of 5D. Scale bar=200 nm in 5A, 5C, 5D; 100 nm in 5B,
5E.
[0016] FIGS. 6A and 6B demonstrate attachment and proliferation of
fibroblasts on different surfaces. FIG. 6A shows microscopic images
of cultured fibroblasts on 30% (w/w) glycerol/silk film, pure silk
film, and tissue culture plastic (TCP). FIG. 6B depicts attachment
of fibroblasts on different films. FIG. 6C shows proliferation of
fibroblasts on different films. Data represent the average.+-.SD
(n=6). Bar=50 .mu.m.
[0017] FIG. 7 is a schematic illustration of silk structural
transitions in glycerol-blended silk films.
DETAILED DESCRIPTION
[0018] It should be understood that this invention is not limited
to the particular methodology, protocols, and reagents, etc.,
described herein and as such may vary. The terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention, which
is defined solely by the claims.
[0019] As used herein and in the claims, the singular forms include
the plural reference and vice versa unless the context clearly
indicates otherwise. Other than in the operating examples, or where
otherwise indicated, all numbers expressing quantities of
ingredients or reaction conditions used herein should be understood
as modified in all instances by the term "about."
[0020] All patents and other publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that might be used in connection with the present
invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0021] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as those commonly understood to
one of ordinary skill in the art to which this invention pertains.
Although any known methods, devices, and materials may be used in
the practice or testing of the invention, the methods, devices, and
materials in this regard are described herein.
[0022] Silk fibroin has excellent film-forming capabilities and is
also compatible for use in the human body. Altman et al., 24
Biomats. 401-16 (2003); Vepari & Kaplan, 32 Prog. Polym. Sci.
991-1007 (2007). Silk fibroin films have good dissolved oxygen
permeability in the wet state, similar to that of human skin, which
suggests potential applications for these films in wound dressing
and artificial skin systems. Minoura et al., 11 Biomats., 430-34
(1990); Minoura et al., 31 Polymer, 265-69 (1990a). Films formed
from silk fibroin, without further manipulation, are soluble in
water, however, because of dominating random coil protein
structures. The structural features of the protein can be
transformed from random coil to .beta.-sheet form by treatment with
heating (Hu et al., 41 Macromolecules 3939-48 (2008)), mechanical
stretching (Jin & Kaplan, 424 Nature 1057-61 (2003)), immersion
in polar organic solvents (Canetti et al., 28 Biopolymers--Peptide
Sci. .sctn.1613-24 (1989)), and curing in water vapor (Jin et al.,
15 Adv. Funct. Mat. 1241-47 (2005)). This structural transition
results in aqueous insolubility, thus providing options for the use
of the material in a range of biomedical and other applications
such as sensor platforms. Zhang, 16 Biotechnol. Adv. 961-71 (1998).
Some pure silk fibroin films tend, over time, to become stiff and
brittle in the dry state, however, exhibiting impressive tensile
strength but low elongation. Jin et al., 2005. Therefore, there
remains a need to modify the physical and mechanical properties of
silk films to control properties, mainly towards more flexible
systems.
[0023] Blending polymers with plasticizers is a traditional
approach to address ductility and tensile strength as outlined
above. For example, some studies have suggested that silk film
properties can be modified by blending silk with other synthetic or
natural polymers, such as alginate, polyallylamine, chitosan,
cellulose, poly(caprolactone-co-D,L-lactide), S-carboxymethyl
keratin, poly(vinyl alcohol) (PVA), poly(ethylene glycol), and
poly(ethylene oxide). See Liang & Hirabayashi, 45 J. Appl.
Polymer Sci. 1937-43 (1992); Arai et al., 84 J. Appl. Polymer Sci.
1963-70 (2002); Kitagawa & Yabuki, 80 J. Appl. Polymer Sci.
928-34 (2001); Noishiki et al., 86 J. Appl. Polymer Sci. 3425-29
(2002); Kesenci et al., 12 J. Biomats. Sci. Polymer Ed. 337-51
(2001); Lee et al., 9 J. Biomats. Sci. Polymer Ed. 905-14 (1998);
Tsukada et al., 32 J. Polymer Sci. B, 243-48 (1994); Gotoh et al.,
38 Polymer 487-90 (1997); Jin et al., 5 Biomacromols. 711-17
(2004). For example, blends of silk fibroin and PEO show materials
stabilization (Jin et al., 2004; Jin et al., 3 Biomacromol. 1233-39
(2002)), and the use of water as a plasticizer may improve film
properties (Jin et al., 2005).
[0024] In many cases, however, improving blends to effect
mechanical properties remains a challenge. In particular, avoiding
additions of other polymers while generating systems that maintain
stability for extended time frames remains a goal. Thus, the
present invention provides for alternative plasticizer options: in
particular glycerol. Previously, silk fibroin films were immersed
in 10% glycerin (10 minutes at 95.degree. C.), and conditioned in a
humidity rich drier to effect crystal transformation from of silk
Ito II. Kawahara et al., 291 Macromol. Mater. Eng. 458-62 (2006).
Also, the addition of 3%-8% glycerin reduced phase separation of
silk fibroin/PVA blends. Dai et al., 86 J. Appl. Polymer Sci.
2342-47 (2002). In both of these approaches, silk fibroin solution
was generated by dissolving degummed silk in the ternary solvent
system of CaCl.sub.2/CH.sub.3CH.sub.2OH/H.sub.2O.
[0025] In the methods of the present invention, glycerol was
blended with an aqueous-dissolved silk fibroin solution and then
cast into films. These films were assessed for mechanical
properties and structural features to better understand the
interactions between the silk fibroin and glycerol. Specific
interactions between silk fibroin and glycerol provide benefits to
the film properties, perhaps enacted by affecting silk fibroin
crystallization behavior in the formation of the .beta.-sheets as
the stabilizing physical cross-links in the films, without the
necessary addition of other polymers.
[0026] The present invention also provides for silk films with
distinct aqueous dissolution properties, and methods for adjusting
the dissolution properties of silk films by blending silk fibroin
solution with the suitable amount of glycerol. In particular, the
dissolution in water of silk fibroin from silk/glycerol blend films
was measured by UV absorbance, because silk fibroin has significant
tyrosine content (>5 mole %) that, unlike glycerol, absorbs at
280 nm wavelength. After a rapid initial weight loss in the first
hour, no further significant difference was found for the residual
mass and dissolved silk content over time (FIG. 1). When the
glycerol content in the silk/glycerol blend films was 2% and 5%
(w/w), the films completely dissolved in water, similar to the
control silk films that contained no glycerol (FIG. 1). Therefore,
glycerol at concentrations lower than about 5% (w/w) did not appear
to have significantly changed silk film properties.
[0027] When the glycerol content in the films was increased from
about 10% to 20% (w/w), the residual mass of the films that
remained insoluble increased from about 10% to about 75%,
respectively (p<0.01, FIG. 1). Further increases in glycerol to
about 30% (w/w) reduced solubility further, although the results
were not statistically significant when compared to the 20%
glycerol data. These results indicated that 20% (w/w) glycerol is a
concentration that induces significant changes in silk film
properties, resulting in substantial insolubility of the material
in water (i.e., about 75% residual mass retained after soaking in
aqueous solution). When the glycerol content was significantly
below 20% (w/w), the amount of silk that dissolved in water
decreased as the glycerol content increased. At 20% (w/w) glycerol,
less than 5% of the total silk mass was soluble in water, much
lower than that from 10% (w/w) glycerol films (p<0.01, FIG. 1).
From the residual mass determinations, the 20% (w/w) glycerol film
lost approximately 25% of the total mass in water. Thus, in
comparing masses, initial glycerol contents and UV absorbance of
the solubilized material, blend films containing more than about
30% (w/w) glycerol lost almost all the glycerol in water while the
silk fibroin protein remained stable in the films, likely due to
glycerol-induced change in silk structure. This result was not
observed at the lower glycerol contents.
[0028] Changes in film solubility due to glycerol content indicate
that glycerol induces structural changes in the silk fibroin. The
self-assembly process of silk fibroin protein into water-insoluble
fibers is accompanied by increased .beta.-sheet structure content,
or silk II, or crystalline structures. Kaplan et al., in PROTEIN
BASED MATS., 103-31 (McGrath, ed., Birkhauser, Boston, Mass.,
1998); Motta et al., 203 Macromol. Chem. Phys. 1658-65 (2002); Chen
et al., 89 Biophys. Chem. 25-34 (2001). In vitro, the silk II
structure can be obtained by solvent treatment, such as with
methanol and ethanol. Cast silk films after water annealing
(exposure of cast films in water vapor for 24 hours), exhibit
stable silk I structure with increased type II .beta.-turns. Jin et
al., 2005. Once formed, the silk I structure in water-annealed
films does not transition to the silk II structure, even with
methanol treatment. In the silk fibroin/glycerol films of the
present invention, the .alpha.-helical structure content is
apparently increased up to approximately 50%, while the 13-turn
content decreased in the blend films having a glycerol content
higher than 10% (w/w) (p<0.01, FIG. 2A). These structural
changes were distinguished from the changes observed in
methanol-treated and water-annealed silk films prepared in the
absence of glycerol.
[0029] The secondary structure content remained relatively
unchanged when the glycerol content in the films was increased from
about 10% to 20% (w/w). Thus, stable .alpha.-helical structures
apparently dominate the glycerol blended material. A three-fold
helical crystal structure (silk III) has been reported previously
for silk at air-water interfaces using the Langmuir-Blodgett
technique, reflecting the amphilicity features of silk (Valluzzi et
al., 24 Int'l J. Biol. Macromol. 237-42 (1999)), but not in
glycerol modified silk materials. The silk III structure can be
transformed into the more stable silk II if the compression force
was more than 35 mNm.sup.-1. The amino acid side chain
distributions along the helix and the orientation of the chain axis
have been well-characterized in these studies. Valluzzi et al.,
1999. For the glycerol/fibroin blend silk films, after methanol
treatment, .beta.-sheet structure content increased to about
50%-60%, while .alpha.-helical structure content decreased to about
20%, regardless of the glycerol content in the films (FIG. 2B).
This response, in terms of structural transitions induced by
methanol, is different from that observed with the water-annealed
silk films where no conformational transition from .alpha.-helical
to .beta.-sheet occurs upon methanol treatment. Jin et al., 2005.
Furthermore, after the 20% (w/w) glycerol blended silk films were
rinsed with water and re-dried in the air, .alpha.-helical
structure content decreased while .beta.-sheet and .beta.-turn
structure content increased to approximately 45% and 20%,
respectively (p<0.01, FIG. 2C). Therefore, for glycerol blend
silk films, stable silk II structures (crystalline, .beta.-sheets)
can be obtained by leaching out the glycerol and re-drying the
film.
[0030] Mechanistically, glycerol appears to alter the silk fibroin
intramolecular and intermolecular interactions and result in a
conformational transition from random coil to .alpha.-helices,
typically regarded as an unstable intermediate state toward stable
.beta.-sheet structure formation. The presence of glycerol appears
to stabilize the .alpha.-helical structure, however, preventing
further transition toward .beta.-sheet structures. It appears that
the concentration of glycerol may reach a critical level to achieve
this extent of structural control. For 20% and 50% (w/w)
glycerol/fibroin blend films, the molar ratios between glycerol and
silk fibroin are approximately 1000:1 and 4000:1, respectively.
After immersion in an aqueous solution where the glycerol leaches
out, the blend film may still contain some .alpha.-helical
structure, most likely due to the stabilizing effect of residual
bound glycerol molecules. This could be the reason that the wet
films (immersed in water) remained flexible when compared to
non-glycerol containing films after methanol treatment. The silk
structural transition from .alpha.-helix to .beta.-sheet may occur
during the film re-drying process, due to increased silk
concentration and intermolecular interactions between silk fibroin
molecules. As a result, the re-dried films become somewhat brittle,
similar to methanol-treated silk fibroin films.
[0031] As defined herein, `dry blend films` refers to silk films
prepared by directly casting the silk fibroin/glycerol blend
solutions to form films and then drying the films overnight. `Wet
blend films` refers to the same cast and dried films that are
subsequently immersed and extracted in ultrapure water at
37.degree. C. for 1 hour, which dissolves out glycerol, and dried
again in the air. Accordingly, the dry environment refers to the
environment leading to the `as-cast` silk fibroin/glycerol blend
film, and the wet environment refers to the steps comprising a
further treatment of the `as-cast` silk fibroin/glycerol blend film
to withdraw glycerol from the film.
[0032] The mechanical properties of the silk fibroin/glycerin films
of the present invention were also examined. The tensile strength
of dry blend films changed with a change in glycerol content in the
films. When the glycerol content increased from 0% to about 20%
(w/w), the tensile strength significantly increased from about 8
MPa to 13 MPa (p<0.01, FIG. 3A). When the glycerol content was
increased above 20%, tensile strength significantly decreased. At
40% glycerol, the tensile strength was about 4 MPa, significantly
lower than that of the 0% and 20% (w/w) glycerol films (p<0.01,
FIG. 3A). The tensile strength of glycerol-depleted films (films
after glycerol has leached out) did not change significantly with
change in glycerol content, with less than 2 MPa determined for all
samples (p>0.05, FIG. 3A). For the dry blend films, elongation
at break remained low (below 3%) when the glycerol content was
below 20% (w/w). These values significantly increased to
approximately 150% when the glycerol content was increased to 30%
and 40% (w/w). At 50% (w/w) glycerol, the elongation at break
values decreased to less than 20%. The trend was similar for that
of tensile strength except that the highest elongation at break was
obtained at 30%-40% (w/w) glycerol rather than 20% (w/w) glycerol
with highest tensile strength. For the wet blend films, the
elongation at break of the 20% (w/w) glycerol films was about 27%,
significantly higher than that of the 0% and 40% glycerol films
(14% and 8%, respectively) (p<0.01, FIG. 3B). Therefore,
compared to methanol-treated silk films without glycerol, the
glycerol blend films had higher ductility in both the dry and wet
states, a useful property for many applications.
[0033] The ductility of the glycerol-silk films was also greater
than that of water-annealed silk films, as water-annealed films
exhibited elongation at break of about 6% (Jin et al., 2005), which
is 25-times lower than that of the 30% glycerol silk films
presented herein. Free-water content may also influence the
flexibility of silk films. Kawahara et al., 2006. Blends with
glycerol may preserve the free-water content in the silk films and,
therefore, improved film flexibility. The role of glycerol in
helical content of the silk fibroin may also play a role in the
mechanical behavior of the films. When the glycerol content was
increased from 0% to 40% (w/w), the tensile modulus decreased about
17-fold in the dry blend films, and about 2.5-fold for the wet
blend films (FIGS. 3C and 3D). Apparently, with more glycerol in
the blends the films became mechanically weaker, and this effect
was more pronounced for the dry blend films. The tensile modulus of
dry silk fibroin/glycerol blend films was more than 100-times
higher than the corresponding wet blend, glycerol-depleted films
from which glycerol had been leached-out.
[0034] The nano-structures of silk fibroin in the silk blend films
were analyzed by morphological characterization to further assess
the impact of glycerol on film properties. Silk films were
fractured in liquid nitrogen and the cross sections of the films
examined by SEM. Silk fibroin protein formed globular
nano-structures with diameters of 100 nm-200 nm when the glycerol
content was 10% (w/w) (FIG. 4A). The globules, however, were not
observed when 20% (w/w) glycerol was blended in the film: the blend
films had relatively smooth morphologies when viewed by SEM (FIG.
4B). These results indicate that a high content of glycerol
(>20% w/w) influences silk fibroin self-assembly and
nano-structure features. Interestingly, when the 20% (w/w) glycerol
silk films were treated with water to leach out the glycerol and
then re-dried in the air, the silk fibroin self-assembled into
nano-filaments, similar to those observed in methanol-treated pure
silk films (FIGS. 4C and 4D). This observation is consistent with
the secondary structure transitions with .beta.-sheet structure
formation in both water-treated and methanol-treated glycerol silk
films (FIGS. 2B and 2C). Therefore, the formation of
nano-structures in glycerol-blended films correlated with the
structural features in the films, and was likely influenced by silk
secondary structural changes.
[0035] The silk nano-filament structures that had formed in the 30%
(w/w) glycerol films after water treatment were further studied by
SEM (FIGS. 5A, 5D). The nano-filament structures were more clearly
visible at higher magnification (FIGS. 5B and 5E) and in side view
(FIG. 5C). In different regions of the film, distinguished
morphologies and organization of nano-filaments was observed
(compare FIGS. 5A, 5B and 5D, 5E), probably due to inhomogeneous
drying rates during silk film casting. The size of the
nano-filaments, however, was consistently about 10 nm-20 nm
throughout the film.
[0036] The glycerol content in silk films may be important for
controlling silk secondary structural transitions and influencing
the mechanical properties of the films. Glycerol molecules may
interact with silk fibroin chains via intermolecular forces, most
likely hydrogen bonds between hydroxyl groups of glycerol and amide
groups of silk. Dai et al., 86 J. Appl. Polymer Sci. 2342-47
(2002). This interaction may alter the hydrophobic hydration state
of protein chains, as these are hydrophobic proteins due to the
high content of glycine-alanine repeats (Bini et al., 335 J. Mol.
Biol. 27-40 (2004)), and therefore induce silk secondary structural
change from predominant random coils (silk solution state or as
cast film) to .alpha.-helices (FIG. 7). This interaction may
stabilize the helical stage of silk unless the film has been
treated by solvents, such as water and methanol. Upon solvent
treatment, some glycerol molecules solubilize from the films and
diffuse into the surrounding medium, although tightly bound
glycerol molecules likely stay associated with the silk fibroin
chains, stabilizing silk .alpha.-helical structures and preserving
film flexibility. Water molecules that replace leached-out glycerol
and form weaker hydrogen bonds with fibroin molecules might also
contribute to maintaining silk structure and mechanical properties.
When these glycerol-depleted films are re-dried, the strong
intermolecular interactions between silk molecules may dominate,
promoting a structural transition from .alpha.-helices to the more
thermodynamically stable .beta.-sheets (FIG. 7). Such process is
similar to the previously reported mechanism of silk structural
transitions based on the change in hydrophobic hydration state of
the protein chains. Matsumoto et al., 110 J. Phys. Chem. B 21630-38
(2006).
[0037] Although some of the interactions of glycerol in silk film
mechanics have been explored (Kawahara et al., 291 Macromol. Mater.
Eng. 458-62 (2006); Dai et al., 86 J. Appl. Polymer Sci. 2342-47
(2002)), the particular formulation and, importantly, the function
of the glycerol in the present invention is distinct from those
reported previously. For instance, the tensile properties of
silk/PVA blend films were modified by inclusion of up to 8%
glycerol in the silk/PVA blend. The tensile strength and elongation
at break for the silk/PVA films were about 350 kg/cm.sup.3 and 10%,
respectively. When 5% glycerol was blended with PVA/silk film to
reduce phase separation, the resulting film tensile strength and
elongation at break were 426 kg/cm.sup.2 and 53%, respectively.
Increasing the concentration of glycerol to >5%, however,
significantly reduced the tensile strength of silk/PVA blend films.
Dai et al., 2002. By contrast, in one embodiment of the present
invention, incorporating 30% glycerol in the fibroin silk film
significantly improved both the tensile strength (to about 12 MPa)
and elongation at break (150%), without the incorporation of
PVA.
[0038] In another study, glycerol solution was used as a
post-treatment of pure silk film to convert the silk structure from
silk Ito silk II (.beta.-sheet structure). More specifically, silk
film was immersed in 10% glycerol solution, heated at 95.degree.
C., and dried at 50% relative humidity. Although the
glycerol-soaked film underwent self-expansion after the soaking
treatment, its ductility was not assessed. Kawahara et al., 2006.
In contrast, in some embodiments of the present invention, silk
fibroin solution is blended with glycerol and cast into highly
ductile films, as demonstrated by the improved tensile strength and
elongation at break of the silk films containing about 10% to 50%
glycerol.
[0039] The glycerol blended silk films presented herein demonstrate
unique features of diverse and controllable silk structure
transitions, desired mechanical properties, and ease of fabrication
(one-step film casting without further treatments). These features
suggest that these films have utility in biomedical
applications.
[0040] The present invention thus provides for methods of preparing
silk films with increased tensile strength and ductility. The
methods comprise blending a silk fibroin solution with glycerol,
where the concentration of glycerol in the silk fibroin/glycerol
blend solution is about 10% to 50% (w/w); casting the silk
fibroin/glycerol blend solution onto a film-supporting surface; and
drying the film. This simple process confers the silk films of
present invention with designable tensile strength and ductility,
depending on the concentration of glycerol, offering an alternative
to silk films prepared silk fibroin solution in absence of
glycerol. In addition, silk blend films comprising other
biopolymers, such as PVA and PEO, may also be modified by glycerol
to enhance the flexibility or ductility of the silk/biopolymer
blend film, employing the same process as described above.
[0041] Additionally, the glycerol silk blends of the present
invention may be combined with other silk-based structures to form
3-dimensional silk scaffolds, silk sponges, or other silk composite
structures having 3-dimensional structures, for applications such
as drug delivery systems, tissue engineered materials or other
biomedical devices. For example, the ductile silk film of the
present invention may be combined with silk fibroin nanospheres or
microspheres carrying an active agent to provide sustained release
of the active agent. As another example, silk fiber-based composite
comprising silk fibers optionally coated with silk fibroin solution
or silk gel may be combined with the ductile silk film of the
present invention to provide flexible fibrous materials for use as
optical fiber or muscle fibers. Glycerol can be easily blended with
any silk composite to alter the mechanical properties of the
silk-based structure. Alternatively, silk-based composite may be
wrapped or shaped with a ductile silk/glycerol film around the
contour of the silk-based structure. All of the silk composites
described herein can be easily functionalized with drugs,
antibiotics, cell responses molecules, dyes, enzymes and other
small and large molecules, with retention of function.
[0042] With improved flexibility of silk film or silk blend film by
glycerol modification, the processes of the present invention may
be used to modify a variety of silk blend films or coatings in a
variety of medical applications such as wound closure systems,
including vascular wound repair devices, hemostatic dressings,
sponges, patches and glues, sutures, drug delivery (WO
2005/123114), biopolymer sensor (WO 2008/127402), and in tissue
engineering applications, such as, for example, tissue-engineered
organs or other biodegradable implantation into the human body
(WO2004/0000915; WO2008/106485). The improved flexibility of silk
film is advantageous as it may provide flexible expandability or
contractibility to the biomedical material as required by some
applications such as functional dressing materials or tissue
materials such as muscle tissue. For example, a ductile silk film
of the present invention may be shaped around a structure (such as
an implant). The silk film may comprise additional active agents
selected to further the purpose of the device, such as tissue or
bone promoting agents in a dental device. Additionally, once the
ductile film has been shaped to the structure, glycerol may be
removed by leaching as described herein.
[0043] The silk fibroin/glycerol blend films of the present
invention also provide a suitable platform for the attachment and
proliferation of fibroblasts. Because of the modified and
potentially useful mechanical properties for these silk blend
films, the potential utility of such biomaterials in cell and
tissue culture is important to assess. Thus, in preliminary
studies, the attachment and proliferation of fibroblast cells on
30% (w/w) glycerol-silk films was compared with methanol-treated
pure silk films and tissue culture plastic (TCP) as controls.
Initial cell attachment (3 hours) on all three surfaces was similar
(first row in FIG. 6A) and as quantified by Alamar Blue staining
(FIG. 6B). Cell proliferation in fourteen days of culture, however,
was different on the different surfaces. After four days culture,
fibroblasts on TCP grew faster than those on pure silk films and
blend silk films, an observation consistent with prior studies on
pure silk films. (Sofia et al., 54 J. Biomed. Mater. Res. 139-48
(2001); Wang et al., 29 Biomats. 894-903 (2008). After fourteen
days culture, the number of cells on TCP was about 1.8-times more
than that on the silk films, and there was no significant
difference between the pure silk films and blend silk films, as
determined by Alamar Blue staining (FIG. 6C). The 30% (w/w)
glycerol silk film only differed from the methanol-treated silk
film for fibroblasts proliferation in the time period from six days
to eleven days, in which cells grew faster on the methanol-treated
film than on the glycerol film (p<0.01, FIG. 6C). RGD-modified
silk films exhibit excellent surface properties to promote rapid
attachment and proliferation of fibroblasts, osteoblast-like cells,
and human bone marrow-derived mesenchymal stem cells. Chen et al.,
67 J. Biomed. Mater. Res. A, 559-70 (2003). Thus, similar
strategies could be employed with the silk-glycerol blend
films.
[0044] The embodiments of the present invention thus provides for
silk/glycerol film that may be suitable for a tissue engineered
constructs that can be used for organ repair, organ replacement or
regeneration strategies that may benefit from these modified silk
materials. A tissue engineered construct comprising silk
fibroin/glycerol blending material and optionally at least one
bioactive agent such as a cell, may be used for organ repair, organ
replacement or regeneration strategies including, but not limited
to, spinal disc, cranial tissue, dura, nerve tissue, liver,
pancreas, kidney, bladder, spleen, cardiac muscle, skeletal muscle,
tendons, ligaments, cornea tissues, and breast tissues. Any type of
cell can be added to the tissue-engineered construct for culturing
and possible implantation, including cells of the muscular and
skeletal systems, such as chondrocytes, fibroblasts, muscle cells
and osteocytes, parenchymal cells such as hepatocytes, pancreatic
cells (including Islet cells), cells of intestinal origin, and
other cells such as nerve cells, bone marrow cells, skin cells,
pluripotent cells and stem cells (including, e.g., embryonic stems,
adult stem cells, and induced pluripotent stem cells), and
combination thereof, either as obtained from donors, from
established cell culture lines, or even before or after molecular
genetic engineering. Pieces of tissue can also be used, which may
provide a number of different cell types in a single structure.
[0045] Alternatively, the flexible silk/glycerol film may also be
used as coatings on biomedical materials such as medical device,
tissue-engineered materials or implants. As discussed above, the
improved flexibility of the glycerol modified silk film may provide
flexible expandability or contractibility to match the contractible
properties of the biomedical material as required by some
applications such as functional dressing materials or tissues such
as muscle tissue. Because the modified silk film is less prone to
break in elongation, contraction, stretch or deformation, coating
from such film will provide for improved compatibility and will
conform well to the contours of the substrate. The substrates or
articles for coating of the modified silk film may include any
number of tissues, regenerated tissue, medical device, medical
implant, veterinary device, or veterinary implant. For example, a
ductile silk/glycerol film may be wrapping around a device or
implant, such as spine cages, coronary stents, dental implants or
hip and knee prostheses.
[0046] As noted, silk/glycerol blend film may be modified to
contain at least one active agent. The agent may be mixed with a
silk fibroin solution prior to forming the silk blend film, or
loaded into the silk blend film after it is formed. The variety of
active agents that can be used in conjunction with the silk blend
film of the present invention is vast. For example, the active
agent may be a therapeutic agent or biological material, such as
cells (including stem cells), proteins, peptides, nucleic acids
(DNA, RNA, siRNA), nucleic acid analogues, nucleotides,
oligonucleotides or sequences, peptide nucleic acids, aptamers,
antibodies, hormones, hormone antagonists, growth factors or
recombinant growth factors and fragments and variants thereof,
cytokines, or enzymes, antibiotics, viruses, antivirals, toxins,
prodrugs, chemotherapeutic agents, small molecules, drugs and
combinations thereof. Exemplary active agent suitable for modifying
the silk blend film of the present invention includes cells
(including stem cells), erythropoietin (EPO), YIGSR peptides,
glycosaminoglycans (GAGs), hyaluronic acid (HA), integrins,
selectins and cadherins; analgesics and analgesic combinations;
steroids; antibiotics; insulin; interferons a and y; interleukins;
adenosine; chemotherapeutic agents (e.g., anticancer agents); tumor
necrosis factors .alpha. and .beta.; antibodies; cell attachment
mediators, such as RGD or integrins, or other naturally derived or
genetically engineered proteins, polysaccharides, glycoproteins,
cytotoxins, prodrugs, immunogens, or lipoproteins.
[0047] One or more active agents may be used to modify the
silk/glycerol blend film. For instance, when using silk blend film
of the present invention as a platform to support biological
material such as cells, it may be desirable to add other materials
to promote the growth of the agent, promote the functionality of
the agent after it is released from the silk blend film, or
increase the agent's ability to survive or retain its efficacy
during the processing period. Exemplary materials known to promote
cell growth include, but not limited to, cell growth media, such as
Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS),
non-essential amino acids and antibiotics, and growth and
morphogenic factors such as fibroblast growth factor (e.g., FGF
1-9), transforming growth factors (TGFs), vascular endothelial
growth factor (VEGF), epidermal growth factor (EGF), platelet
derived growth factor (PDGF), insulin-like growth factor (IGF-I and
IGF-II), bone morphogenetic growth factors (e.g., BMPs 1-7), bone
morphogenetic-like proteins (e.g., GFD-5, GFD-7, and GFD-8),
transforming growth factors (e.g., TGF-.alpha., TGF-.beta. I-III),
nerve growth factors, and related proteins. Growth factors are
known in the art, see, e.g., Rosen & Thies, CELLULAR & MOL.
BASIS BONE FORMATION & REPAIR(R.G. Landes Co.). Additional
material to be embedded in silk/glycerol film may include DNA,
siRNA, antisense, plasmids, liposomes and related systems for
delivery of genetic materials; peptides and proteins to active
cellular signaling cascades; peptides and proteins to promote
mineralization or related events from cells; adhesion peptides and
proteins to improve film-tissue interfaces; antimicrobial peptides;
and proteins and related compounds.
[0048] Embedding a bioactive agent in the silk/glycerol
blend-produced film enables the delivery of active agents in a
controlled released manner. Maintaining the bioactive agent in an
active form throughout the process of embedding the agent in the
silk enables it to be active upon release from the silk film.
Controlled release of the active agent permits active agent to be
released sustainably over time, with controlled release kinetics.
In some instances, the bioactive agent is delivered continuously to
the site where treatment is needed, for example, over several
weeks. Controlled release over time, for example, over several days
or weeks, or longer, permits continuous delivery of the bioactive
agent to obtain preferred treatments. The controlled delivery
vehicle is advantageous because it protects the bioactive agent
from degradation in vivo in body fluids and tissue, for example, by
proteases.
[0049] Controlled release of the active agent from the silk film
may be designed to occur over time, for example, over 12 hours or
24 hours. The time of release may be selected, for example, to
occur over a time period of about 12 hours to 24 hours; about 12
hours to 42 hours; or, e.g., about 12 to 72 hours. In another
embodiment, release may occur for example on the order of about 1
day to 15 days. The controlled release time may be selected based
on the condition treated. For example, longer times may be more
effective for wound healing, whereas shorter delivery times may be
more useful for some cardiovascular applications.
[0050] Controlled release of the active agent from the silk film in
vivo may occur, for example, in the amount of about 1 ng to 1
mg/day. In other embodiments, the controlled release may occur in
the amount of about 50 ng to 500 ng/day, or, in another embodiment,
in the amount of about 100 ng/day. Delivery systems comprising
therapeutic agent and a carrier may be formulated that include, for
example, 10 ng to 1 mg therapeutic agent, or about 1 .mu.g to 500
.mu.g, or, for example, about 10 .mu.g to 100 .mu.g, depending on
the therapeutic application.
[0051] The silk/glyerol blend-produced film of the present
invention may also be surface patterned for bio-optical device
application. The surface patterning technique are known in the art,
for example, ink jet printing of patterns, dip pen nanolithography
patterns, microcontact printing or soft lithographic techniques.
See Wilran et al., 98 P.N.A.S. 13660-64 (2001); Bettinger et al, 19
Adv. Mat. 2847-50 (2007). Also see PCT/US/07/83620;
PCT/US2008/082487. Topographic patterning on the surface of silk
film combined with silk film's optical transparent clarity may
provide high resolution surface features that are not only suitable
for bio-optical device such as an optical grating, a lens, a
microlen array (WO 08/127,404), but also suitable for tissue
engineered construct due to their ability to direct cellular
function and matrix deposition such as tissue alignment and
proliferation (WO 08/106,485).
[0052] Hence, particular embodiments described herein provide for
glycerol modified silk films that are useful for ocular biomedical
devices and ocular tissue engineering. For example, in the
application in corneal tissue engineering, the surface of silk film
supports the corneal fibroblast attachment and proliferation. The
optional surface patterning of the modified silk films provides
further guidance to cell alignment. The glycerol modified silk film
may be used for in vivo cornea tissue repair or in vitro cornea
tissue regeneration for subsequent implantation. Because of its
soft and flexible nature, the silk film modified by glycerol using
the method of the present invention provides for improved comfort
and compatibility to patient in need of such tissue implantation.
Additional exemplary applications of modified silk film in ocular
biomedical devices include, but not limited to, fabrication of soft
contact lenses, intraocular lenses, glaucoma filtration implants,
keratoprostheses, scleral buckles, and viscoelastic replacement
agents.
[0053] Another application of the glycerol modified silk film in
the present invention is to fabricate flexible optical device. As
noted, silk film surface may be further patterned with high
resolution features. Using the glycerol modified silk films of the
present invention, a flexible, expandable holographic label may be
provided that is easily elongated, stretched or deformed to match
the surface contour of the product in need of, for example, a
label. For example, silk film may be nanopatterned with high
resolution diffraction microrelief to confer a holographic image,
thus providing an edible holographic product identification label
that easily conforms to a capsule, tablet, or food product. See
PCT/US09/47751.
[0054] As noted herein, glycerol modified silk films are edible.
Coloring agents, releasing agents, coating agents, sweetening,
flavoring and perfuming agents, preservatives and antioxidants can
also be present in the silk film or formulation comprising silk
film. For example, a flavored silk film formulation or flavored
silk film coated formulation of vitamins, nutraceuticals, or other
pharmaceuticals may be produced for pediatric use.
[0055] In summary, silk films blended with glycerol (>10% w/w)
are apparently enriched in .alpha.-helical structure, which further
transitions to crystalline .beta.-sheet structures upon removal of
glycerol by methanol or water treatments and re-drying the film.
Silk/glycerol blend films rich in .beta.-sheet structure were
composed of characteristic nano-filaments, while those rich in
.alpha.-helical structure did not exhibit these morphologies. The
blend films, in either the as-cast or glycerol-depleted states,
were more ductile than both methanol-treated and water-annealed
pure silk fibroin films, even though they were less resistant to
stretch deformation. Both glycerol-blended (30% w/w) and
methanol-treated silk films supported fibroblast attachment and
growth. Mechanistically, the role of glycerol appears to mimic that
of water in controlling the structural transitions of the silk
fibroin chains, providing a new and useful control point in
regulating the structure and thus material properties of silk-based
biomaterials.
[0056] Thus, the embodiments of the present invention provide for a
silk film comprising silk fibroin and about 10% (w/w) to about 50%
(w/w) glycerol. This silk film may comprise about 20% (w/w) to
about 40% (w/w), or about 30% (w/w).
[0057] Additionally, the silk film may include at least one active
agent. The active agent may be cells, proteins, peptides, nucleic
acid analogues, nucleotides or oligonucleotides, peptide nucleic
acids, aptamers, antibodies or fragments or portions thereof,
hormones, hormone antagonists, growth factors or recombinant growth
factors and fragments and variants thereof, cytokines, enzymes,
antibiotics or antimicrobial compounds, viruses, antivirals,
toxins, prodrugs, chemotherapeutic agents, small molecules or
drugs, or combinations thereof. In a particular embodiment, the
active agent is a cell. The cell may be selected from hepatocytes,
pancreatic Islet cells, fibroblasts, chondrocytes, osteoblasts,
exocrine cells, cells of intestinal origin, bile duct cells,
parathyroid cells, thyroid cells, cells of the
adrenal-hypothalamic-pituitary axis, heart muscle cells, kidney
epithelial cells, kidney tubular cells, kidney basement membrane
cells, nerve cells, blood vessel cells, cells forming bone and
cartilage, smooth muscle cells, skeletal muscle cells, oscular
cells, integumentary cells, bone marrow cells, keratinocytes,
pluripotent cells, induced pluripotent stem cells, adult stem cells
or embryonic stem cells, or combinations thereof.
[0058] The silk film may also include silk microspheres or silk
nanospheres embedded in the silk film. The silk film may be film is
a layered or folded into a sponge or block. The silk films also
provide for constructs for tissue engineering. In particular, the
tissue engineered construct may be a corneal tissue construct in
which the cell is a corneal fibroblast. In some embodiments, the
silk film may further comprise a cell growth medium.
[0059] The silk films of the present invention may also include a
pattern on the silk film, such as an optical pattern, in particular
a holographic image.
[0060] The embodiments of the present invention also provide for a
method for preparing a silk film, comprising blending a silk
fibroin solution with glycerol, wherein the concentration of
glycerol in the silk fibroin/glycerol blend solution is about 10%
to about 50% (w/w), casting the silk fibroin/glycerol blend
solution onto a film-supporting surface, and drying the silk film.
The method may also include additional steps of immersing the silk
film in a liquid in which glycerol dissolves for a period of time
to deplete glycerol from the silk film; and drying the
glycerol-depleted film. The method may also further comprise
annealing the film, for example treating the film with methanol or
water vapor.
[0061] The present embodiments also provide for a method of
covering a surface of a substrate with a silk composition by
providing a film-support substrate; and covering the film-support
substrate with a silk fibroin/glycerol blend film comprising about
10% to 50% glycerol (w/w). The silk fibroin/glycerol blend film may
further comprise at least one biopolymer, such as PVA or PEO. the
silk fibroin/glycerol blend film may further comprise at least one
active agent.
[0062] Another embodiment of the invention is a silk film-covered
substrate prepared according to the method of covering a surface of
a substrate with a silk composition by providing a film-support
substrate; and covering the film-support substrate with a silk
fibroin/glycerol blend film comprising about 10% to 50% glycerol
(w/w). The substrate may be a tissue, regenerated tissue, medical
device, medical implant, veterinary device, or veterinary implant,
such as a dental implant. The substrate may also be a silk-based
composite.
[0063] Another embodiment of the present invention is a method of
embedding at least one active agent in a silk film, comprising
blending a silk fibroin solution with at least one active agent and
glycerol, wherein the concentration of glycerol in the silk blend
solution is about 10% to 50% (w/w); casting the silk blend solution
onto a film-supporting surface; and drying the film. In this
method, the active agent may be cells, proteins, peptides, nucleic
acid analogues, nucleotides or oligonucleotides, peptide nucleic
acids, aptamers, antibodies or fragments or portions thereof,
hormones, hormone antagonists, growth factors or recombinant growth
factors and fragments and variants thereof, cytokines, enzymes,
antibiotics or antimicrobial compounds, viruses, antivirals,
toxins, prodrugs, chemotherapeutic agents, small molecules, drugs,
or combinations thereof. This method may also further include the
steps of immersing the silk film in a liquid in which glycerol
dissolves for a period of time to deplete glycerol from the silk
film; and drying the glycerol-depleted film. The method may also
include the further step of annealing the film.
[0064] In some embodiments of the present invention may be defined
in any of the following numbered paragraphs:
1. A silk film comprising silk fibroin and about 10% (w/w) to about
50% (w/w) glycerol. 2. The silk film of paragraph 1, wherein the
glycerol content of the silk film is about 20% (w/w) to about 40%
(w/w) 3. The silk film of any of paragraphs 1 to 2, wherein the
glycerol content of the silk film is about 30% (w/w). 4. The silk
film of any of paragraphs 1 to 3, further comprising at least one
active agent. 5. The silk film of any of paragraphs 1 to 4, further
comprising silk microspheres or silk nanospheres embedded in the
silk film. 6. The silk film of any of paragraphs 1 to 5, wherein
said film is a layered or folded into a sponge or block. 7. The
silk film of any of paragraphs 1 to 6, wherein the at least one
active agent is selected from the group consisting of cells,
proteins, peptides, nucleic acid analogues, nucleotides or
oligonucleotides, peptide nucleic acids, aptamers, antibodies or
fragments or portions thereof, hormones, hormone antagonists,
growth factors or recombinant growth factors and fragments and
variants thereof, cytokines, enzymes, antibiotics or antimicrobial
compounds, viruses, antivirals, toxins, prodrugs, chemotherapeutic
agents, small molecules, drugs, and combinations thereof. 8. A
construct for tissue engineering comprising the silk film of any of
paragraphs 1 to 7, wherein at least one active agent is a cell. 9.
The construct for tissue engineering of paragraph 8, wherein the
cell is selected from the group consisting of hepatocytes,
pancreatic Islet cells, fibroblasts, chondrocytes, osteoblasts,
exocrine cells, cells of intestinal origin, bile duct cells,
parathyroid cells, thyroid cells, cells of the
adrenal-hypothalamic-pituitary axis, heart muscle cells, kidney
epithelial cells, kidney tubular cells, kidney basement membrane
cells, nerve cells, blood vessel cells, cells forming bone and
cartilage, smooth muscle cells, skeletal muscle cells, oscular
cells, integumentary cells, bone marrow cells, keratinocytes,
pluripotent stem cells, induced pluripotent stem cells, adult stem
cells and embryonic stem cells, and combinations thereof. 10. The
construct for tissue engineering of paragraph 9, wherein the tissue
engineered construct is a cornea tissue construct and the cell is
corneal fibroblast. 11. The construct for tissue engineering of any
of paragraphs 8 to 10, further comprising a cell growth medium. 12.
The silk film of any of paragraphs 1 to 7, further comprising an
optical pattern on the silk film. 13. The silk film of paragraph
12, wherein the optical pattern is a holographic image. 14. A
method for preparing a silk film, comprising: blending a silk
fibroin solution with glycerol, wherein the concentration of
glycerol in the silk fibroin/glycerol blend solution is about 10%
to about 50% (w/w); casting the silk fibroin/glycerol blend
solution onto a film-supporting surface; and drying the silk film.
15. The method of paragraph 14, further comprising the steps of
immersing the silk film in a liquid in which glycerol dissolves for
a period of time to deplete glycerol from the silk film; and drying
the glycerol-depleted film. 16. The method of paragraphs 14 or 15,
further comprising annealing said film. 17. A method for covering a
surface of a substrate with a silk composition comprising:
providing a film-support substrate; and covering the film-support
substrate with a silk fibroin/glycerol blend film comprising about
10% to 50% glycerol (w/w). 18. The method of paragraph 17, wherein
the silk fibroin/glycerol blend film further comprises at least one
biopolymer. 19. The method of paragraph 18, wherein the biopolymer
is PVA or PEO. 20. The method of paragraph 19, wherein the silk
fibroin/glycerol blend film further comprises at least one active
agent. 21. A silk film-covered substrate prepared according to the
method of paragraphs 17-20. 22. The silk film-covered substrate of
paragraph 21, wherein the substrate is a tissue, regenerated
tissue, medical device, medical implant, veterinary device, or
veterinary implant. 23. The silk film-covered substrate of
paragraphs 20 or 22, wherein the substrate is a silk-based
composite. 24. A method of embedding at least one active agent in a
silk film, comprising: blending a silk fibroin solution with at
least one active agent and glycerol, wherein the concentration of
glycerol in the silk blend solution is about 10% to 50% (w/w);
casting the silk blend solution onto a film-supporting surface; and
drying the film. 25. The method of paragraph 24, wherein the at
least one active agent is selected from the group consisting of
cells, proteins, peptides, nucleic acid analogues, nucleotides or
oligonucleotides, peptide nucleic acids, aptamers, antibodies or
fragments or portions thereof, hormones, hormone antagonists,
growth factors or recombinant growth factors and fragments and
variants thereof, cytokines, enzymes, antibiotics or antimicrobial
compounds, viruses, antivirals, toxins, prodrugs, chemotherapeutic
agents, small molecules, drugs, and combinations thereof. 26. The
method of paragraph 24 or 25, further comprising the steps of
immersing the silk film in a liquid in which glycerol dissolves for
a period of time to deplete glycerol from the silk film; and drying
the glycerol-depleted film. 27. The method of any of paragraphs 24
to 26, further comprising annealing said film.
EXAMPLES
Example 1
Silk Fibroin Purification
[0065] Silk fibroin aqueous stock solutions were prepared as
previously described. Sofia et al., 54 J. Biomed. Mater. Res.
139-48 (2001). Briefly, cocoons of Bombyx mori were boiled for 20
min in an aqueous solution of 0.02 M sodium carbonate, and then
rinsed thoroughly with pure water. After drying, the extracted silk
fibroin was dissolved in 9.3 M LiBr solution at 60.degree. C. for 4
hr, yielding a 20% (w/v) solution. This solution was dialyzed
against distilled water using SLIDE-A-LYZER.RTM. Dialysis
Cassettes, 3,500 MWCO (Pierce, Rockford, Ill.) for 3 days to remove
the salt. The solution was optically clear after dialysis and was
centrifuged to remove the small amounts of silk aggregates that
formed during the process, usually from environment contaminants
that are present on the cocoons. The final concentration of silk
fibroin aqueous solution was approximately 6% (w/v). This
concentration was determined by weighing the residual solid of a
known volume of solution after drying.
[0066] The 6% silk fibroin solution was stored at 4.degree. C.
before use and may be diluted to a lower concentration with
ultrapure water. To obtain a silk fibroin solution with a higher
concentration, the 6% silk fibroin solution may be dialyzed against
a hygroscopic polymer, such as polyethylene glycol (PEG), amylase,
or sericin. For example, a 6% silk fibroin solution may be exposed
to a 25%-50% wt % PEG (MW 8,000 to 10,000) solution on the outside
of a SLIDE-A-LYZER.RTM. 3,500 MWCO Dialysis Cassettes for 2 to 12
hr by osmotic pressure, and the final concentration of aqueous silk
solution concentrated to between 8%-30% wt % or greater.
Example 2
Preparation of Silk/Glycerol Blend Films
[0067] The purified silk fibroin solution was mixed with glycerol
at weight ratios of 0%, 5%, 10%, 20%, 30%, 40%, 50% (w/w). The
mixed solutions were poured into Petri dishes and dried at room
temperature in a laminar flow hood overnight. Unless otherwise
stated, the `dry blend films` refers to the films prepared by this
direct casting and overnight drying, and the `wet blend films`
refers to the same cast and dried films from which the glycerol is
subsequently extracted in ultrapure water at 37.degree. C. for 1
hr, after which the films are dried again in the air. For
additional variables in the treatment groups, methanol treatments
were used, and in these cases the films (with and without glycerol)
were immersed in 90% (v/v) methanol for 1 hr and then
air-dried.
Example 3
Dissolution of silk/glycerol films
[0068] Blend films were cut into approximately 5 mm.times.5 mm
squares, and one square film was weighed and immersed in ultrapure
water in a 2 ml tube to a concentration of 1% (weight of
film/volume of water), and kept at 37.degree. C. for 1 hr or 1 day.
After the incubation, the silk films were removed from the
solution, air-dried overnight, weighed, and compared with the mass
of original film to obtain residual mass (%). The remaining
solution was subjected to UV absorbance measurement at 280 nm. The
absorbance values were converted to the amount of silk solubilized
in water using purified silk fibroin solution at various
concentrations as standards. The amount of dissolved silk was then
compared with the total silk mass in the film to obtain the
percentage of the film dissolved silk in water.
Example 4
Analysis of Silk/Glycerol Films by Fourier Transform Infrared
(FTIR) Spectroscopy
[0069] The secondary structures present in the films, including
random coil, alpha-helices, beta-pleated sheets and turns, were
evaluated using Fourier Self-Deconvolution (FSD) of the infrared
absorbance spectra. FTIR analysis of treated samples was performed
with a Bruker Equinox 55/S FTIR spectrometer (Bruker Optics Inc.,
Billerica, Mass.), equipped with a deuterated triglycine sulfate
detector and a multiple-reflection, horizontal MIRacle.RTM. ATR
attachment with a Germanium (Ge) crystal, from Pike Tech. (Madison,
Wis.). A 5 mm.times.5 mm square-shape silk film was placed in the
Ge crystal cell and examined with the FTIR microscope in the
reflection mode. Background measurements were taken with an empty
cell and subtracted from the sample reading. For each measurement,
sixty-four scans were recorded with a resolution of 4 cm.sup.-1,
and the wavenumber ranged from 400 m.sup.-1 to 4000 cm.sup.-1.
[0070] FSD of the infrared spectra covering the amide I region
(1595 cm.sup.-1-1705 cm.sup.-1) was performed by Opus 5.0 software
(Opus Software, Inc., San Francisco, Calif.) as previously
described. Hu et al., 39 Macromolecules, 6161-70 (2006). Absorption
bands in the frequency range 1616 cm.sup.-1-1637 cm.sup.-1 and 1695
cm.sup.-1-1705 cm.sup.-1 represented enriched O-sheet structure;
bands in the range 1638 cm.sup.-1-1655 cm.sup.-1 were ascribed to
random coil structure; bands in the range 1656 cm.sup.-1-1663
cm.sup.-1 ascribed to alpha-helices; and bands in the range 1663
cm.sup.-1-1695 cm.sup.-1 to turns. Id.
Example 5
Mechanical Properties of Silk/Glycerol Films
[0071] Tensile tests were performed on an Instron 3366 testing
frame equipped with a 10 N capacity load cell and BIOPULS.TM.
testing system (Instron.RTM., Norwood, Mass.), including
submersible pneumatic clamps and temperature-controlled liquid
bath. Film samples were cast into silicone molds based on ASTM
standard D638-02a, and scaled up 2.times., resulting in an overall
length of 80 mm to accommodate the large surfaces needed for
clamping and gauge length necessary for video extensometry (28 mm).
For a dry environment, the films were conditioned in an
environmental chamber at 25.degree. C. and 50% relative humidity
for two days. For a wet environment, the silk/glycerol film samples
were hydrated in 0.1 M phosphate buffered saline (PBS) for 1 hr,
and then submerged in a BIOPULS.TM. bath (37.+-.0.3.degree. C.)
filled with PBS for at least 5 min prior to testing. The pure silk
fibroin films (0% glycerol) were pre-treated with 90% v/v methanol
for 1 hr, and then treated in the same way as glycerol samples. All
films were tested at a strain control rate of 0.1% s.sup.-1, based
on the initial clamp-to-clamp length (nominal length .about.47 mm,
nominal elongation rate .about.2.82 mm/min). Load and video
extensometer strain data were captured at 20 Hz., the latter based
on two fiducial painted markers placed at a nominal distance of
.about.1 cm on the surface of the thinnest portion of each film.
Five replicates of each film were tested. The original cross
sectional area was determined by measuring the film thickness by
SEM and multiplying by the specimen width (10 mm). The nominal
stress and strain were graphed, and the initial "linear elastic
modulus", strain to failure, and ultimate tensile strength (UTS)
were determined. UTS was determined as the highest stress value
attained during the test. The initial "linear elastic modulus" was
calculated by using a least-squares' fitting between the point
corresponding to 0.1 N load and the point corresponding to 50% of
the UTS. This was deemed sufficient to objectively capture the
linear portion of the stress/strain curve for all samples tested.
The elongation to failure was determined as the last data point
before a >10% decrease in load.
Example 6
Scanning Electron Microscopy (SEM)
[0072] Silk films were fractured in liquid nitrogen and sputtered
with platinum. The cross-section and surface morphologies of the
different silk films were imaged using a Zeiss SUPRA.TM. 55 VP SEM
(Carl Zeiss, Inc., Jena, Germany).
Example 7
Fibroblast Culture and Adhesion on Silk Films
[0073] Fibroblast cells were expanded in a growth medium containing
90% DMEM, 10% fetal bovine serum (FBS), 100 U/ml penicillin, 1000
U/ml streptomycin. Cell cultures were maintained at 37.degree. C.
in an incubator with 95% air and 5% CO.sub.2. The cultures were
replenished with fresh medium at 37.degree. C. every two days. For
adhesion, cells were seeded on silk films that were pre-cast in
24-well plates with 50,000 cells per well in 1 ml of
serum-containing medium. Empty wells with tissue culture plastic
(TCP) and no silk served as controls. Cell attachment was evaluated
3 hr after cell seeding by adding 50 .mu.l of alamar blue to the
culture medium, culturing for another 6 hr, and determining the
medium fluorescence (Ex=560 nm, Em=590 nm). During the culture,
cell proliferation was determined using alamar blue staining and
cell morphology was monitored by phase contrast light microscopy
(Carl Zeiss, Inc., Jena, Germany).
[0074] All experiments were performed with a minimum of N=3 for
each data point. Statistical analysis was performed by one-way
analysis of variance (ANOVA) and Student-Newman-Keuls Multiple
Comparisons Test. Differences were considered significant when
p.ltoreq.0.05, and very significant when p.ltoreq.0.01.
* * * * *