U.S. patent application number 13/320036 was filed with the patent office on 2012-03-22 for vortex-induced silk fibroin gelation for encapsulation and delivery.
This patent application is currently assigned to TRUSTEES OF TUFTS COLLEGE. Invention is credited to David L. Kaplan, Tuna Yucel.
Application Number | 20120070427 13/320036 |
Document ID | / |
Family ID | 43429736 |
Filed Date | 2012-03-22 |
United States Patent
Application |
20120070427 |
Kind Code |
A1 |
Kaplan; David L. ; et
al. |
March 22, 2012 |
VORTEX-INDUCED SILK FIBROIN GELATION FOR ENCAPSULATION AND
DELIVERY
Abstract
The present invention provided for a novel process of forming
silk fibroin gels, and controlling the rate of .beta.-sheet
formation and resulting hydrogelation kinetics, by vortex treatment
of silk fibroin solution. In addition, the vortex treatment of the
present invention provides a silk fibroin gel that may be
reversibly shear-thinned, enabling the use of these approach for
precise control of silk self-assembly, both spatially and
temporally. Active agents, including biological materials, viable
cells or therapeutic agents, can be encapsulated in the hydrogels
formed from the processes, and be used as delivery vehicles. Hence,
the present invention provide for methods for silk fibroin gelation
that are useful for biotechnological applications such as
encapsulation and delivery of active agents, cells, and bioactive
molecules.
Inventors: |
Kaplan; David L.; (Concord,
MA) ; Yucel; Tuna; (Medford, MA) |
Assignee: |
TRUSTEES OF TUFTS COLLEGE
Medford
MA
|
Family ID: |
43429736 |
Appl. No.: |
13/320036 |
Filed: |
June 1, 2010 |
PCT Filed: |
June 1, 2010 |
PCT NO: |
PCT/US2010/036841 |
371 Date: |
December 8, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61182794 |
Jun 1, 2009 |
|
|
|
61219952 |
Jun 24, 2009 |
|
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Current U.S.
Class: |
424/130.1 ;
424/184.1; 424/93.1; 424/93.6; 424/93.7; 424/94.1; 514/20.9;
514/21.2; 514/7.6 |
Current CPC
Class: |
A61K 9/06 20130101; A61K
9/5089 20130101; A61K 9/5052 20130101; A61K 9/0024 20130101; A61K
47/42 20130101; A61P 31/00 20180101; A61K 38/00 20130101 |
Class at
Publication: |
424/130.1 ;
514/21.2; 424/93.1; 514/7.6; 514/20.9; 424/184.1; 424/94.1;
424/93.6; 424/93.7 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 39/395 20060101 A61K039/395; A61K 38/18 20060101
A61K038/18; A61K 39/00 20060101 A61K039/00; A61K 38/43 20060101
A61K038/43; A61K 35/76 20060101 A61K035/76; A61K 35/12 20060101
A61K035/12; A61K 35/34 20060101 A61K035/34; A61K 35/23 20060101
A61K035/23; A61K 35/407 20060101 A61K035/407; A61K 35/28 20060101
A61K035/28; A61K 35/54 20060101 A61K035/54; A61K 35/48 20060101
A61K035/48; A61P 31/00 20060101 A61P031/00; A61K 35/00 20060101
A61K035/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under
contract No. P41 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. government has certain rights in the
invention.
Claims
1. A method of forming silk fibroin gel, comprising vortexing a
silk fibroin solution for a sufficient period of time to initiate
intermolecular self-assembly of silk fibroin .beta.-sheet
structure, wherein substantial silk fibroin gelation occurs in less
than about 16 hours after the vortexing.
2. The method of claim 1, wherein said vortexing yields a solid
phase and an aqueous phase, and wherein the method further
comprises removing said solid phase and allowing gelation of the
aqueous phase.
3. The method of claim 1 or 2, wherein the silk fibroin in the
solution has a concentration about 6 wt % or lower.
4. The method of claim 3, wherein the silk fibroin in the solution
has a concentration ranging from about 1.0 wt % to about 5.2 wt
%.
5. The method of claim 1, wherein the gelation time is controlled
by adjusting one or more of (a) the time period of the vortex
treatment; (b) the concentration of the silk fibroin in solution;
or (c) the temperature of the silk fibroin solution after the
vortex treatment.
6. (canceled)
7. The method of claim 1, further comprising introducing at least
one active agent to the silk fibroin solution before substantial
gelation occurs in the silk fibroin solution; allowing the
silk-fibroin to gel, forming a silk fibroin gel-encapsulated active
agent.
8. The method of claim 7, wherein the active agent is a therapeutic
agent or a biological material, selected from the group consisting
of cells, proteins, peptides, nucleic acids, nucleic acid analogs,
nucleotides or oligonucleotides, peptide nucleic acids, aptamers,
antibodies or fragments or portions thereof, antigens or epitopes,
hormones, hormone antagonists, growth factors or recombinant growth
factors and fragments and variants thereof, cell attachment
mediators, cytokines, enzymes, antibiotics or antimicrobial
compounds, viruses, toxins, prodrugs, chemotherapeutic agents,
small molecules, drugs, and combinations thereof.
9. The method of claim 7, wherein the active agent is a cell
selected from the group consisting of progenitor cells or stem
cells, smooth muscle cells, skeletal muscle cells, cardiac muscle
cells, epithelial cells, endothelial cells, urothelial cells,
fibroblasts, myoblasts, oscular cells, chondrocytes, chondroblasts,
osteoblasts, osteoclasts, keratinocytes, kidney tubular cells,
kidney basement membrane cells, integumentary cells, bone marrow
cells, hepatocytes, bile duct cells, pancreatic islet cells,
thyroid, parathyroid, adrenal, hypothalamic, pituitary, ovarian,
testicular, salivary gland cells, adipocytes, precursor cells, and
combinations thereof.
10. The method of claim 9, the active agent further comprises a
cell growth media.
11. The method of claim 7, wherein the silk fibroin
gel-encapsulated active agent is suitable for a biodelivery
device.
12. The method of claim 7, wherein the silk fibroin
gel-encapsulated active agent is suitable for a medical implant or
a tissue repair material.
13. A method of delivering a reversibly shear-thinned silk fibroin
gel to a target site, comprising: vortexing a silk fibroin solution
for a sufficient period of time to initiate gelation, wherein the
silk fibroin undergoes substantial gelation after the vortex
treatment to form a silk fibroin gel; introducing the silk fibroin
gel through a shear-inducing delivery device to the target site
while applying a shear force to shear-thin the silk fibroin gel;
and removing the shear force, whereupon the shear-thinned silk
fibroin gel recovers from shear-thinning and re-gels.
14. The method of claim 13, further comprising adding an active
agent to said silk fibroin solution.
15. The method of claim 13 wherein said reversible shear-thinning
silk fibroin gel is delivered locally to said target site with high
spatial precision.
16. The method of claim 15, wherein said reversible shear-thinning
silk fibroin gel is delivered locally to said target site by
injection through a needle.
17. The method of claim 15, wherein the method is suitable for
implanting a medical implant or a tissue repair material.
18. A method for homogeneous delivery of at least one active agent
to a target site, comprising: vortexing a silk fibroin solution for
a sufficient period of time to initiate gelation; introducing at
least one active agent to the silk fibroin solution either before
vortexing or before substantial gelation occurs in the silk fibroin
solution, thereby forming a silk fibroin gel-encapsulated active
agent that may be shear-thinned reversibly; introducing to the
target site the active agent-encapsulated silk fibroin gel through
a shear-inducing delivery device to the target site while applying
a shear force to shear-thin the agent-encapsulated silk fibroin
gel; and removing the shear force, whereupon the shear-thinned silk
fibroin gel-encapsulated agent recovers gel form, thereby
distributing the active agent in the gel form at the target site
homogeneously.
19. The method of claim 18, wherein said reversible shear-thinning
silk fibroin gel is delivered locally to said target site with high
spatial precision.
20. The method of claim 19, wherein said reversible shear-thinning
silk fibroin gel is delivered locally to said target site by
injection.
21. The method of claim 18, wherein the at least one silk fibroin
gel-encapsulated active agent is delivered to the target site in
vivo.
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn.119(e) of U.S. Provisional Application No. 61/182,794
filed Jun. 1, 2009, and U.S. Provisional Application No. 61/219,952
filed Jun. 24, 2009, the contents of which are incorporated herein
by reference in their entirety.
FIELD OF THE INVENTION
[0003] This invention provides for methods of forming silk fibroin
gels comprising vortexing, and methods of delivering active agents
encapsulated in vortex-induced silk hydrogels.
BACKGROUND OF THE INVENTION
[0004] Biocompatible and biodegradable polymer hydrogels are useful
carriers for delivering active agents and cells for biomedical
applications, such as in tissue engineering and controlled drug
release. Purified native silk fibroin protein forms
.beta.-sheet-rich crosslinked hydrogel structures from aqueous
solution, with the mechanics of the process and gel properties
influenced by environmental parameters. Traditional gelation
methods using aqueous native silk protein solutions, under
physiologically relevant conditions, often range from days to weeks
for gelation: too slow for the incorporation of cells and labile
active agents. High temperature, low pH, or high ionic strength may
reduce the gelation time to a few hours, but these conditions may
potentially alter cell or bioactive molecule function and limit
cell viability. Moreover, biological and some physical properties
of silk hydrogel scaffolds are crucial for cell
encapsulation/delivery applications. For example, hydrogelation
kinetics should be controlled to enable homogeneous 3-dimensional
(3-D) encapsulation of cells/active agents and prevention of
cell/active agent sedimentation. The ease of application of
hydrogel/active molecules scaffolds into the target cite with high
spatial precision is also of practical consideration for
encapsulation/delivery applications. Thus, there remains need in
the art for a process of initiating silk fibroin gelation at mild
physiological conditions, with controllable kinetics and properties
of the silk hydrogel, as well as the delivery of the gels with
spatial precision.
SUMMARY OF THE INVENTION
[0005] The present invention provides for methods of inducing silk
fibroin gelation and forming silk fibroin gels. The method
comprises vortexing a silk fibroin solution for a sufficient period
of time to initiate intermolecular self-assembly of silk fibroin
.beta.-sheet structure. For example, under particular conditions,
substantial silk fibroin gelation occurs within 16 hours of the
vortex treatment.
[0006] Some embodiments of the invention provide for methods of
controlling the gelation time of silk fibroin initiated by
vortexing a silk fibroin solution for a sufficient period of time
to initiate gelation. For example, the gelation time may be
controlled by adjusting the time period of the vortex treatment,
the concentration of the silk fibroin in solution, or the
temperature of the silk fibroin solution after the vortex
treatment.
[0007] Another embodiment provides for methods of embedding or
encapsulating at least one active agent in a silk fibroin hydrogel.
The method comprises vortexing a silk fibroin solution for a
sufficient period of time to initiate gelation, introducing the
agent(s) to the silk fibroin solution before substantial gelation
occurs in the silk fibroin solution, and allowing the silk-fibroin
to complete gelation to form a silk fibroin gel-embedded active
agent. The active agent may be a therapeutic agent, such as a small
molecule or drug, or biological materials such as cells.
[0008] The present invention also provides for methods of preparing
reversible shear-thinning silk fibroin gels. For example, after
vortexing a silk fibroin solution for a sufficient period to
initiate gelation of the silk fibroin and allowing substantial
gelation, the silk gel may be subjected to shear force (e.g.,
forced through a needle) to induce reversible thinning. Thus, the
present invention also provides for a silk gel that may be
reversibly shear-thinned.
[0009] Some embodiments of the invention relate to methods of
delivering a reversibly shear-thinned silk fibroin gel to a target
site. One method comprises vortexing a silk fibroin solution for a
sufficient period of time to initiate gelation, allowing
substantial gelation to occur after the vortex treatment to form a
silk fibroin gel, introducing the silk fibroin gel through a
shear-inducing delivery device to the target site while applying a
shear force to shear-thin the silk fibroin gel, and removing the
shear force, whereupon the shear-thinned silk fibroin gel recovers
from shear-thinning and re-gels.
[0010] Another embodiment of the invention also relates to a method
of embedding active agents in a reversible shear-thinned silk
fibroin gel, and delivering the reversibly shear-thinned silk
fibroin gel-encapsulated active agent to a target site. For
example, one method comprises vortexing a silk fibroin solution for
a sufficient period of time to initiate gelation, introducing at
least one active agent to the silk fibroin solution before
substantial gelation occurs in the silk fibroin solution, thereby
forming a silk fibroin gel-embedded active agent that may be
shear-thinned reversibly, introducing to the target site the active
agent-encapsulated silk fibroin gel through a shear-inducing
delivery device to the target site while applying a shear force to
shear-thin the agent-embedded silk fibroin gel, and removing the
shear force, whereupon the shear-thinned silk fibroin gel-embedded
active agent recovers gel form.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 depicts the dynamic silk fibroin .beta.-sheet
structure formation during the gelation process. FIG. 1a shows
far-UV Circular Dichroism (CD) spectra collected immediately after
vortexing silk solutions for different durations, t.sub.V. FIG. 1b
demonstrates correlation between the time evolution of the increase
in [.theta.].sub.216 measured by CD and the shear storage modulus,
G' measured by dynamic oscillatory rheology during post-vortex
incubation. FIGS. 1C and 1D depict rheology frequency sweeps of
storage (FIG. 1C) and loss (FIG. 1D) modulus as a function of
post-vortex assembly time, with t.sub.a under the same assembly
conditions as in (FIG. 1D). The frequency sweep data collected from
non-vortexed silk solution was also given in (FIG. 1C) and (FIG.
1D) for comparison (+).
[0012] FIG. 2 depicts the kinetics of silk fibroin hydrogelation
under various conditions. The kinetics of silk fibroin
hydrogelation are examined by varying vortex time (FIGS. 2A and
2B), assembly temperature (FIGS. 2C and 2D) and protein
concentration (FIGS. 2E and 2F). Power law exponents in FIG. 2A
were obtained from fits to the blue-colored G' data. t.sub.V=7 min
for (FIGS. 2C-2F).
[0013] FIGS. 3a and 3b depict strain sweeps collected from
vortex-induced hydrogels with different silk concentrations (arrows
show apparent yielding). FIGS. 3c and 3d show frequency sweeps
collected from the hydrogels before (open symbols) and immediately
after shear-thinning by injection through a 21 gauge needle (closed
symbols).
[0014] FIG. 4 is a scheme depicting the possible mechanism of silk
fibroin hydrogelation and the resulting injectable silk
hydrogels.
DETAILED DESCRIPTION
[0015] 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.
[0016] 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."
[0017] 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.
[0018] 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.
[0019] In the present invention, a vortexing technique that causes
shear gradient is used to induce changes in silk fibroin structure
and solution viscoelastic properties to control the post-vortex
self-assembly and hydrogelation kinetics of silk fibroin. The
process exposes silk fibroin solution to a treatment comprising
vortexing for a sufficient period of time to initiate gelation.
Gelation time can be controlled within physiological relevant
conditions, ranging from minutes to hours, based on the process
parameters used; such as the duration time of vortexing, silk
fibroin concentration, and post-vortex assembly temperature of silk
fibroin. After vortexing, the silk fibroin undergoes a rapid
structural change from random coil to .beta.-sheet, corresponding
to gelation. The vortexing technique of the present invention is
also used to prepare silk fibroin gels that may be shear-thinned.
After the applied shear force is removed, the shear-thinned silk
fibroin recovers from shear-thinning and reforms a hydrogel. A
active agent, such as for example a therapeutic agent, a biological
agent, cells, or the combination of these, can be added to the
silk. The present invention thus provides methods for various
biomedical applications, such as encapsulation/delivery of cells
and bioactive molecules. For example, a shear-thinning hydrogel
material could be implanted by minimal invasion to the delivery
site, such as by injection through a needle, and such material
would recover immediately to a stiff network after removal of
applied shear, facilitating localization of a uniform density of
cells/bioactive molecules at the delivery site.
[0020] Hydrogel materials, both synthetic and natural, are
considered useful scaffolds for encapsulation and delivery of cells
and active agents, such as for tissue engineering (Lee et al., 101
J. Chemical Reviews 1869-79. (2001)), drug/growth factor release
(Langer, 33 Acc. Chem. Res. 94-101 (2000)), and cell therapeutic
applications. Hydrogels used in these types of applications have
mechanical and structural properties similar to some tissues and
extracellular matrices (ECM); therefore, they can be implanted for
tissue restoration or local release of therapeutic factors. To
encapsulate and deliver cells, hydrogels should, preferably, be
formed without damaging cells, be nontoxic to the cells and the
surrounding tissue, be biocompatible, have suitable mass transport
capability to allow diffusion of nutrients and metabolites, have
sufficient mechanical integrity and strength to withstand
manipulations associated with implantation, have controllable
lifetimes, and should maintain gel volume after implantation for a
reasonable period of time depending on the application (Drury &
Mooney, 24 Biomaterials 4337-51 (2003)).
[0021] A variety of synthetic materials, such as poly(ethylene
oxide) (PEO), poly(vinyl alcohol) (PVA), poly(acrylic acid) (PAA),
poly(propylene furmarate-co-ethylene glycol) (P(PF-co-EG)), and
naturally derived materials, such as agarose, alginate, chitosan,
collagen, fibrin, gelatin, and hyaluronic acid (HA) have been used
to form hydrogels. Gelation occurs when the polymer chains
crosslink either chemically or physically into networks, triggered
by chemical reagents (e.g., cross-linkers) or physical stimulants
(e.g., pH and/or temperature). Hydrogels formed from synthetic
polymers offer the benefit of gelation and gel properties that are
controllable and reproducible, through the use of specific
molecular weights, block structures, and crosslinking modes.
[0022] Synthetic hydrogelating systems can be classified into
polymer-based hydrogels (Anseth et al., 10TH INTL. SYMP. RECENT
ADV. DRUG DELIV. SYS. 199-209 (Elsevier Sci. By, Salt Lake City,
Utah, 2001)), polymer-peptide hybrid hydrogels (Lutolf et al., 23
Natl. Biotechnol. 47-55 (2005)), and peptidic self-assembling
hydrogels (Pochan et al., 125 J. Am. Chem. Soc. 11802-03 (2003);
Schneider et al., 124 J. Am. Chem. Soc. 15030-37 (2002)). Peptidic
hydrogel systems are promising synthetic materials because they
generally show low immunogenicity and controllable assembly
kinetics, nanostructure formation and hydrogel mechanical
properties (Haines-Butterick et al., 104 P.N.A.S. 7791-96 (2007);
Yucel et al., 41 Macromolecules 5763-72 (2008)).
[0023] Additionally, natural biopolymeric systems generally show
better compatibility for hosting cells and bioactive molecules
(Lutolf et al., 23 Natl. Biotechnol. 47-55 (2005)). Among naturally
derived biomaterials, silk fibroin protein, the self-assembling
structural protein in natural silkworm fibers, has been studied
because of its excellent mechanical properties, biocompatibility,
controllable degradation rates, and self assembly into .beta.-sheet
rich networks (Altman et al., 24 Biomats. 401-16 (2003); Horan et
al., 26 Biomats. 3385-93 (2005); Ishida et al., 23 Macromolecules
88-94 (1990); Jin et al., 424 Nature, 1057-61 (2003); Kim et al. 26
Biomats. 2775-85 (2005)). Silk fibroin hydrogels are of interest
for many biomedical applications, such as bone-filling materials
(Fini et al., 26 Biomats. 3527-36 (2005)), and cell encapsulation
for 3-D cell culture (Wang et al., 29 Biomats. 1054-64 (2008)).
[0024] Silk fibroin is a high-molecular weight block copolymer
consisting of a heavy (.about.370 kDa) and a light chain (.about.26
kDa) with varying amphiphilicity linked by a single disulfide bond
(Inoue et al., 275 J. Biol. Chem. 40517-18 (2000)). The heavy chain
contains hydrophobic, repetitive oligo-peptides rich in alanine and
glycine amino acids interspersed with small, more hydrophilic,
charged and amorphous regions that give the chain a polyelectrolyte
nature. The sequence of the light chain is less repetitive and has
a high content of glutamic and aspartic acid residues. Silk fibroin
has been processed into a variety of material formats, such as
films, electrospun fibers, 3-D porous scaffolds, microspheres and
hydrogels, mainly for tissue engineering and cell/drug delivery
applications (Kim et al., 5 Biomacromol. 786-92 (2004); Matsumoto
et al., 110 J. Phys. Chem. B 21630-38 (2006); Wang et al., 36 Intl.
J. Biol. Macromol. 66-70 (2005); Wang et al., 29 Biomats. 1054-64
(2008); Hino et al., 266 J. Colloid & Interf. Sci. 68-73
(2003); Jin et al., 3 Biomacromol. 1233-39 (2002); Jin et al., 5
Biomacromol. 711-17 (2004); Nazarov et al., 5 Biomacromol. 718-26
(2004); Wang et al., 117 J. Control. Release 360-70 (2007)). See
also U.S. patent applications Ser. No. 11/020,650; No. 10/541,182;
No. 11/407,373; and No. 11/664,234; WO/2008/118133;
WO/2008/106485.
[0025] In nature, silk fibroin aqueous solution is produced in the
posterior section of silkworm gland and then stored in the middle
section at a high solution concentration and contains a high
content of random coil or .alpha.-helical structure. The silk
fibroin protein goes through a structural transition into
.beta.-sheet-containing fibers during fiber spinning into air, due
to elongational and shear forces and the concomitant changes in the
ionic concentration and pH (Chen et al., 3 Biomacromol. 644-48
(2002); Dicko et al., 5 Biomacromol. 704-10 (2004); Terry et al., 5
Biomacromol. 768-72 (2004); Vollrath et al., 410 Nature 541-48
(2001); Zhou et al., 109 J. Phys. Chem. B 16937-45 (2005)), leading
to the formation of solid fibers (Vollrath & Knight, 410
Nature, 541-48 (2001)). In vitro, purified silk fibroin aqueous
solutions undergo self-assembly into .beta.-sheet structures and
form hydrogels. This sol-gel transition may be influenced by
temperature, pH, and ionic strength (Wang et al., 2005; Kim et al.,
2004; Matsumoto et al., 2006). For example, the compressive
strength and modulus of silk hydrogels increases with an increase
in silk fibroin concentration and temperature (Kim et al.,
2004).
[0026] Both shear and elongation forces have been reported to
induce structural transition of silk fibroin into .beta.-sheet
containing fibers. In addition, shear forces have also been
reported to affect the rheological properties of aqueous silk
fibroin solutions. For example, an anomalous shear thickening
followed by shear thinning behavior was reported at or above 4.2 wt
% silk fibroin concentrations (Ochi et al., 3 Biomacromol. 1187-96
(2002)), similar to that observed for associating polymers (Cadix
et al., 38 Macromol. 527-36 (2005); Witten et al., 18 Macromol.
1915-18 (1985)). This behavior was related to the alignment and
stretching of polymer chains induced by the shear rate gradient and
alternating rupture and recovery of the crosslinks. At higher silk
concentrations of about 25 wt %, a phase separation between a
white, tough material and the surrounding clear liquid was reported
at steady shear rates above 2 s.sup.-1 (Terry et al., 5
Biomacromol. 768-72 (2004)). This apparent phase separation was
attributed to shear-induced crystallization into a .beta.-sheet
structure by stretching of fibroin molecules due to the applied
flow field and repulsion of bound water at relatively low shear
rates as compared to those within the silkworm duct. At higher silk
concentrations of about 25 wt %, a phase separation between a
white, tough material and the surrounding clear liquid was reported
at steady shear rates above 2 s.sup.-1 (Terry et al., 2004). This
apparent phase separation was attributed to shear-induced
crystallization into a .beta.-sheet structure by stretching of
fibroin molecules due to the applied flow field and repulsion of
bound water at relatively low shear rates as compared to those
within the silkworm duct.
[0027] Silk fibroin hydrogels are of interest for many biomedical
applications, such as bone-filling materials (Fini et al., 26
Biomats. 3527-36 (2005)), implantable medical devices, bioactive
molecules encapsulation, and cell encapsulation for 3-D cell
culture (Wang et al., 2008). See also WO 2008/150861;
PCT/US2009/058534.
[0028] For many cell-based applications, self-assembly into
.beta.-sheet structure and concomitant hydrogelation of silk
fibroin must be induced under mild conditions in a relatively short
period of time (within hours). Silk gelation is typically too slow
under physiologically relevant solution conditions, in the absence
of chemical modifications to the native silk fibroin protein, for
the realization of cell encapsulation applications. For silk
fibroin concentrations from 0.6% to 15% (w/v), days to weeks were
required for the sol-gel transition at room temperature or
37.degree. C. (Kim et al., 2004; Matsumoto et al., 2006; Fini et
al., 2005). Nonphysiological treatments, such as lowering pH,
increasing temperature or increasing high ionic strength may reduce
the self-assembly and hydrogelation time of silk fibroin to a few
hours (Kim et al., 2004); Matsumoto et al., 2006; Wang et al.,
2005), but these conditions both potentially alter cell function
and affect cell viability. One way to induce rapid and controlled
hydrogelation of the silk fibroin, in the absence of harsh solution
conditions, is by ultrasonication treatment (Wang et al. 29
Biomats. 1054-64 (2008)).
[0029] Additionally, for cell encapsulation/delivery applications,
several biological (e.g., cytocompatibility, cell adhesion and
subsequent cell morphological changes, cell proliferation, cell
phenotype maintenance, and, in some cases, cell proliferation, and
post-injection biodegradability of hydrogel matrix), and some
physical (bulk mechanical properties as determined by the local and
global structure) properties of hydrogel scaffolds are considered
crucial. Other materials criteria should also be met before
widespread use of these materials for cell delivery applications
(Haines-Butterick et al., 104 P.N.A.S. 7791-96 (2007); Yucel,
Early-Time, .beta.-Hairpin Self-Assembly & Hydrogelation:
Structure, Kinetics & Shear-Recovery (Ph.D. Dissertation, Univ.
Delaware, Newark, 2008). For example, hydrogelation kinetics should
be controlled precisely to enable homogeneous 3-D encapsulation of
cells and prevention of cell sedimentation.
[0030] Another practical consideration for injectable hydrogel/cell
scaffolds is the ease of application into the body with high
spatial precision (Haines-Butterick et al., 2007). For example, a
shear-thinning hydrogel material can be implanted by minimal
invasion to the delivery site, such as by injection through a
needle. Thus, a hydrogel that shear-thins into a sol-state during
injection enables a homogeneous delivery of cells to a wound site,
as compared with cell delivery in solution. In addition, it may be
important for the shear-thinned hydrogel material to recover
quickly or immediately into a stiff network after removal of
applied shear force, facilitating localization of a uniform density
of cells at the delivery site.
[0031] In the present invention, novel methods to induce and
control the formation of silk fibroin .beta.-sheet structure and
the concomitant silk hydrogelation are accomplished through vortex
treatment. More specifically, a new vortexing-based method is
presented that accelerates the sol-gel transition in a temporally
controllable manner. The method comprises vortexing a silk fibroin
solution for a sufficient period of time to initiate intermolecular
self-assembly of silk fibroin .beta.-sheet structure. For example,
under particular conditions, substantial silk fibroin gelation
occurs within 16 hours of the vortex treatment. After vortexing,
the silk fibroin undergoes a rapid structural change from random
coil to .beta.-sheet, corresponding to gelation. Gelation-time can
be controlled, ranging from minutes to hours, based on the process
parameters used. The methods further provide for manipulation of
the duration time of vortexing, silk fibroin concentration, and
post-vortex assembly temperature of silk fibroin to affect the
formation of silk hydrogelation, the dynamics structural changes
after gelation, and physical properties of silk gel.
[0032] A broad range of silk fibroin concentrations, in the aqueous
solution, are suitable for the vortex treatment in the present
invention. For example, the concentration of silk fibroin in
solution may be less than about 30 wt %. Typically, lowering the
initial silk protein concentration reduces the final hydrogel
stiffness and increases the silk gelation time. The vortex
treatment of the present invention allows induction of silk fibroin
gelation at a lower protein concentration than those studied
previously. For example, an aqueous solution having a concentration
about 6 wt % fibroin or lower may be used. A particular embodiment
is directed towards the use of an aqueous solution having a
concentration ranging from about 1 wt % to about 5.2 wt % fibroin.
Another embodiment is directed towards the use of an aqueous
solution having a concentration of about 1.3 wt % fibroin or lower,
for instance, from about 0.3 wt % fibroin to about 1.3 wt %
fibroin.
[0033] As used herein, the term "fibroin" includes silkworm fibroin
and insect or spider silk protein. See e.g., Lucas et al., 13 Adv.
Protein Chem. 107-242 (1958). Any type of silk fibroin may be used
according to the present invention. Silk fibroin produced by
silkworms, such as Bombyx mori, is the most common and represents
an earth-friendly, renewable resource. For instance, silk fibroin
used in a silk gel may be attained by extracting sericin from the
cocoons of B. mori. Organic silkworm cocoons are also commercially
available. There are many different silks, however, including
spider silk (e.g., obtained from Nephila clavipes), transgenic
silks, genetically engineered silks, such as silks from bacteria,
yeast, mammalian cells, transgenic animals, or transgenic plants
(see, e.g., WO 97/08315; U.S. Pat. No. 5,245,012), and variants
thereof, that may be used. An aqueous silk fibroin solution may be
prepared from silkworm cocoons using techniques known in the art.
Suitable processes for preparing silk fibroin solution are
disclosed, for example, in U.S. patent application Ser. No.
11/247,358; WO/2005/012606; and WO/2008/127401.
[0034] Vortex treatment is used in the present invention to apply
shear-gradient to silk fibroin solution to induce sol-gel
transition of silk fibroin and control the post-vortex
self-assembly and hydrogelation kinetics. Shear-induced gelation
has been reported for polymers in poor solvents (Onuki, 9 J.
Phys.-Condens. Matter 6119-57 (1997)), and amphiphilic associating
polymers (Cadix et al., 38 Macromol. 527-36 (2005); Witten et al.,
18 Macromol. 1915-18 (1985)). For polymers in poor solvents, shear
flow is believed to increase the concentration fluctuations, which
may lead to the assembly of macromolecules in the absence of
excluded volume in a poor solvent (Onuki, 1997). For associating
polymers, increased intermolecular interactions between
self-associating chains that undergo non-Gaussian stretching due to
flow were argued to lead to shear-induced gelation (Witten et al.,
18 Macromol. 1915-18 (1985)). The present invention provides for a
novel technique for causing shear gradient to induce a sol-gel
transition of silk fibroin and control the post-vortex
self-assembly and hydrogelation kinetics.
[0035] Generally speaking, vortex treatments may be performed in
any manner known in the art. A commercially available vortexer may
be used for the vortex treatment. The vortex treatment may involve
exposing the silk fibroin to vortexing one time, or may involve
multiple separate exposures. The vortex treatment should last for a
period of time sufficient to initiate the gelation process, but not
so long as to compromise the mechanical properties of the hydrogel.
Typically, vortex treatments may last from 2 minutes to 15 minutes
depending on the rotational speed of the vortexer, the amount of
silk fibroin used, the concentration of the solution, and other
factors appreciated by those of ordinary skill in the art. For
example, the vortex treatments last from about 2 minutes to about
11 minutes.
[0036] Gelation typically begins at the onset of the vortex
treatment and continues after the treatment ends. The vortex
treatment initiate structural transition of silk fibroin from
random coil and .alpha.-helical rich structures into .beta.-sheet
structures and form hydrogels. This process is sol-gel transition.
The dynamics of gelation of silk fibroin solution may be
characterized by measuring the .beta.-sheet content through
Circular Dichroism (CD) spectroscopy and dynamic oscillatory
rheology under the same assembly conditions at the same time points
after triggering hydrogelation by vortexing. This process could
capture the isotemporal evolution of the overall protein structure,
correlated to concomitant changes in viscoelastic properties, due
to vortex-induced hydrogelation. For example, FIG. 1A shows far-UV
CD spectra collected from aqueous native silk solutions with a
protein concentration .phi.=1.3 wt % at 25.degree. C. immediately
after vortex treatment. Non-vortexed silk solution did not show any
local minima attributable to .alpha.-helical or .beta.-sheet
conformations within the observed wavelength range (210
nm<.lamda.<260 nm), indicating that the molecular
conformation is predominantly random-coil in solution. Vortexing
the silk solution for 2 minutes (t.sub.V=2 min) lead to a
detectable increase in the apparent .beta.-sheet content, as
observed by the formation of a local minimum at .lamda.=216 nm in
the CD signal ([.theta.].sub.216). FIG. 1B shows the dependence of
.beta.-sheet content detected by CD spectroscopy and the shear
storage modulus, G' measured by dynamic oscillatory rheology on
post-vortex assembly time, t.sub.a (25.degree. C., .phi.=2.6 wt %,
and t.sub.V=7 min). Here, [.theta.].sub.216* was calculated by
subtracting the [.theta.].sub.216 value obtained from non-vortexed
silk solution rich in random coil content from the
[.theta.].sub.216 value measured from the sample to observe the
evolution of .beta.-sheet content due to silk self-assembly.
[0037] The time progress of [.theta.].sub.216* and G' were very
similar, showing a gradual increase with increasing assembly time.
In a double logarithmic scale, G' initially increased gradually,
followed by a rapid increase in G' after t.sub.a.about.100 minutes.
This orders of magnitude increase in G' may indicate a
percolation-like transition due to increasing connectivity of
.beta.-sheet rich macromolecule clusters to form a hydrogel
network. Overall, a strong correlation was apparent between the
increasing .beta.-sheet content due to changes in molecular
conformation and intermolecular self-assembly possibly leading to
macromolecular cluster formation and the subsequent increase in the
elastic-like behavior, presumably due to increasing intercluster
interactions. CD spectroscopy indicates that vortexing aqueous
solutions of silkworm silk lead to a transition from an overall
protein structure that is initially rich in random coil to that
rich in .beta.-sheet content. Dynamic oscillatory rheology
experiments collected under the same assembly conditions as the CD
experiments indicates that the increase in .beta.-sheet content due
to intramolecular conformational changes and intermolecular
self-assembly of the silk fibroin was directly correlated with the
subsequent changes in viscoelastic properties due to hydrogelation.
With increasing vortex time (t.sub.V=5 min), the absolute value of
[.theta.].sub.216 increased further, suggesting an increase in the
overall .beta.-sheet content.
[0038] Formation of a hydrogel with substantial stiffness
corresponds to the formation of substantial .beta.-sheet content
and the substantial gelation of silk fibroin. For example, samples
with an initial protein concentration .gtoreq.1.3 wt % (G' ca. 100
Pa) formed self-supporting gels while lower concentration samples
apparently flowed after inversion of sample vials. Moreover, the
solid-like, opaque phase that formed during vortexing precipitated
out during subsequent incubation for protein concentrations lower
than 1.3 wt %. Therefore, G' values .gtoreq.100 Pa could be
considered to represent "substantial hydrogel stiffness" for
practical purposes. Substantial gelation usually occurs within 16
hours after the vortex treatment. For example, the silk fibroin gel
forms in less than 2 hours after the vortex treatment. In a
particular embodiment, the silk fibroin undergoes gelation at a
time period ranging from about 5 minutes to about 2 hours after the
vortex treatment. Thus, depending on requirements, gelation time
can occur from minutes to hours, based on the vortex treatment of
the silk fibroin solution. A strictly defined lower threshold for a
vortex time may not be obtainable due to the complication of
measurements. When the gelation kinetics of silk fibroin solution
is characterized by CD spectroscopy, even 1 minute of vortex
treatment of 1 mL silk solutions at a vortex speed of 3,200 rpm
could result in an apparent increase in the overall .beta.-sheet
content. This apparent .beta.-sheet content increase could
presumably speed the gelation kinetics compared to non-vortexed
solutions. Note that in measuring the stiffness of gelation for
practical "substantial hydrogel stiffness," characterized by
dynamic oscillatory rheology (e.g., G' of about 100 Pa for 1.3 wt %
hydrogels), vortex times less than 5 minutes at 25.degree. C. may
not induce gelation fast enough for rheological measurements.
[0039] FIGS. 1C and 1D show the time evolution of the dynamic
frequency sweeps of the shear storage (G') and loss modulus (G'')
during post-vortex self-assembly collected under the same assembly
conditions as in FIG. 1B. Non-vortexed silk fibroin solution
essentially behaved as a low viscosity, Newtonian fluid within the
measured frequencies (dynamic complex viscosity, .eta.*.about.3
mPas). There was a significant increase in the elastic-like
behavior immediately after vortexing: Both G' and G'' increased by
orders of magnitude, with G'>G'' at all measured frequencies. G'
showed a weaker frequency dependence than G''
(G'.about..omega..sup.0.16 and G''.about..omega..sup.0.22),
suggesting that the macromolecule clusters could essentially behave
as viscoelastic fluids displaying gel-like behavior within the
observed frequency range. The apparent gel-like behavior may be
attributed to the relatively large size of the micron-scale
macromolecule clusters. With increasing assembly time after
vortexing, there was a gradual increase in both G' and G'' which
showed progressively weaker frequency dependence, especially after
the apparent percolation transition of the clusters at t.about.100
minutes. G' and G'' became essentially frequency independent within
the measured frequency range after t.sub.a*.about.1000 min of
assembly (G'.about..omega..sup.0.02 and
G''.about..omega..sup.0.08), suggesting the formation of a hydrogel
network consisting of permanent intercluster physical
crosslinks.
[0040] Without being bound by theory, the mechanism of
vortex-induced hydrogelation of the silk fibroin may be analogous
to shear-gradient induced hydrogelation. Silk fibroin protein is an
amphiphilic, block copolymer that consists of segments with
predominantly hydrophobic domains that are phase-separated in the
nanometer scale to enable solubilization in water (Bini et al., 335
J. Mol. Biol. 27-40 (2004)). Based on overall hydropathy of silk
fibroin (in the absence of the nanophase-separated, folded
molecular arrangement), water can be considered as a poor solvent
for the high molecular weight silk molecules. For example,
viscoelastic characterization of silk fibroin solutions in
LiBr.H.sub.2O/H.sub.2O/C.sub.2H.sub.5OH mixed solvents showed that
the solution dynamic viscosity and flow activation energy decrease
with increasing water content, while the dissolution time and the
concentration of LiBr necessary to dissolve silk fibroin increased
with increasing water content, suggesting that water acts as a poor
solvent in this solvent system (Matsumoto & Uejima, 35 J.
Polym. Sci. Pol. Chem. 1949-54 (1997); Matsumoto et al., 35 J.
Polym. Sci. Pol. Chem. 1955-59 (1997)). The shear-gradient caused
by vortex treatment increases the concentration fluctuations in the
aqueous silk solution, which could lead to self-assembly of silk
fibroin into .beta.-sheet rich silk macromolecule clusters and
increased intercluster interactions in the absence of excluded
volume. Perhaps the spatial heterogeneity of concentration
fluctuations or a shear gradient may be responsible for controlling
the kinetics of native silk hydrogelation: The shear-gradient may
cause non-Gaussian stretching (unfolding) of silk fibroin molecular
domains and formation of macromolecule clusters rich in
.beta.-sheet content due to increased exposure of hydrophobic
domains to water. Increasing size and concentration of .beta.-sheet
macromolecule clusters, and subsequent increase in the
concentration and overall lifetime of intercluster crosslinks and
the physical entanglements between dangling fibroin chains, could
eventually lead a percolation-like transition into a permanent,
physical hydrogel network.
[0041] The vortex treatment described herein may include other
treatment(s) to assist in the gelation process. In one embodiment,
increasing the vortex time increases the silk fibroin solution
turbidity and may yield a bulk phase separation of a white,
solid-like material from the aqueous phase, especially at lower
silk fibroin concentrations. The treatment therefore may further
comprise removing the solid phase and allow gelation of the
remaining aqueous phase. This visible, solid-like, sticky phase may
form for all vortex times (t.sub.V.gtoreq.5 min) and silk protein
concentrations that provide suitable gelation kinetics. Such solid
phase could potentially be useful, for example, for muco-adhesive
applications.
[0042] As mentioned, the aqueous silk fibroin solution under vortex
treatment may have a broad range of concentration. The
concentration may be less than about 30 wt %. Typically, lowering
initial protein concentration reduces the final hydrogel stiffness
and increases the time of silk gelation. The vortex treatment of
the present invention allows initiation of silk fibroin gelation at
a lower protein concentration than those previously studied. For
example, an aqueous solution having a silk fibroin concentration of
about 6 wt % or less may be used. A particular aspect is directed
towards the use of an aqueous solution having a silk fibroin
concentration about 1 wt % or higher.
[0043] The present invention also provides for methods of
controlling gelation time of silk fibroin initiated by vortexing a
silk fibroin solution for a sufficient period of time to initiate
gelation under conditions that gelation occurs within minutes to
hours. Hydrogelation kinetics can be controlled easily by changing
the processing parameters (FIG. 2), such as vortex time (FIGS. 2A
and 2B), post-vortex assembly temperature (FIGS. 2C and 2D), and
silk concentration (FIGS. 2E and 2F). By controlling these
parameters, self-assembly and hydrogelation kinetics of silk
fibroin may be adjusted from minutes to hours. This may bring many
advantages to using silk fibroin hydrogels in biomedical
applications, such as homogeneous encapsulation of cells in 3-D,
namely, cells can be introduced immediately before the rapid
gelation due to the apparent percolation transition to prevent cell
sedimentation.
[0044] Additionally, understanding the hydrogelation kinetics of
silk fibroin and controlling the parameters accordingly to control
the gelation time may also help the homogeneous encapsulation of
cells/active agents. For example, the shear storage modulus (G')
values at the apparent percolation may be in a rather narrow range,
for example, between 30 Pa and 100 Pa (at 10 rad/s) for many
self-assembly conditions. Therefore, the solution viscoelastic
properties of silk fibroin can be estimated accordingly and
decisions can be made in terms of when the cells/active agents can
be introduced to silk fibroin for homogeneous encapsulation.
[0045] As noted, the gelation time may be controlled by adjusting
the time period of the vortex treatment. Adjusting the time period
of the vortex treatment can effectively change the formation time
of the silk gelation without significantly altering the final
mechanical properties of the self-assembled silk hydrogels. For
example, FIGS. 2A and 2B show the time evolution of the shear
storage (G') and loss modulus (G''), respectively after vortexing
the silk fibroin solutions for different times, t.sub.V (25.degree.
C., .phi.=5.2 wt %). There was a noted increase in the initial G'
values immediately after vortexing with increasing vortex time,
presumably due to increasing concentration of macromolecule
clusters. Moreover, the apparent jump in G' attributed to
increasing cluster connectivity shifted to shorter assembly times,
i.e., t.sub.a* decreases with increasing vortex time
(t.sub.a*.about.35 min, 50 min, 100 min and >1000 min for
t.sub.V=11 min, 9 min, 7 min and 0 min, respectively). The increase
in stiffness after t.sub.a* was slower with increasing vortex time
and the apparent equilibrium stiffness of the final hydrogel was
essentially independent of the vortex time.
[0046] The gelation time may be controlled by the temperature of
the silk fibroin solution after the vortex treatment. Within
certain range of post-vortex temperature, hydrogelation kinetics of
vortexed silk solutions increases by increasing the post-vortex
assembly temperature. For example, FIGS. 2C and 2D show the time
evolution of rheological properties at different post-vortex
self-assembly temperatures of silk fibroin (.phi.=5.2 wt %,
t.sub.V=7 min). A master curve of the G' data shown in FIG. 2C
could be constructed by normalizing the assembly time by a time
shift factor, t.sub.0, indicating that the self-assembly mechanism
at different temperatures may be similar within the studied
temperature range. As a further note, an Arrhenius type plot shows
a linear dependence of the time shift factor as a function of
reciprocal assembly temperature (ln(t.sub.0)=4801/T-11.2, shown as
in the inset of FIG. 2D). A detailed analysis of the temperature
dependence of the gelation time demonstrates that the logarithm of
"substantial gelation time, t.sub.a* (for G' ca. 100 Pa)" shows
approximately linear dependence on reciprocal T, with
t.sub.a*(min)=exp(5403/T(K)-13.4) (for t.sub.V=7 min and .phi.=5.2
wt %). For comparison, the time evolution of G' and G'' for the
non-vortexed solution at 50.degree. C. were also given in FIG. 2A,
which shows no detectable change in the viscoelastic behavior after
1,000 min, indicating the significant effect of vortexing on
hydrogelation kinetics.
[0047] The gelation time may also be controlled by the
concentration of the silk fibroin solution. The final hydrogel
stiffness may be dependent on the initial protein concentration.
For example, FIGS. 2E and 2F show the time evolution of the
viscoelastic properties for different silk concentrations
(25.degree. C. for t.sub.V=7 min). The initial G' values after
vortexing apparently increased with decreasing silk concentration.
This observation could be attributed to the increasing effect of
vortex-induced concentration fluctuations and formation of a higher
concentration of macromolecule clusters with decreasing protein
concentration. At low protein concentrations
(0.32<<.phi.<1.3 wt %) G'.about..phi..sup.1.5, while at
higher protein concentrations (1<.phi.<5.2 wt %)
G'.about..phi..sup.4. The concentration dependence of network
stiffness of vortexed silk hydrogels in the high concentration
regime resembles that in shear-induced gelation of amphiphilic
polymers in the entangled regime (G'.about..phi..sup.3.7) (Cadix et
al., 38 Macromol. 527-36 (2005)). A much weaker theoretical
concentration dependence (G'.about..phi..sup.2.5) was observed for
highly crosslinked, semiflexible biopolymer chain networks
(Mackintosh et al., 75 Phys. Rev. Lett. 4425-28 (1995)).
[0048] In addition, various other factors may also affect the
gelation time. For example, the rotational speed of vortexer. Those
of ordinary skill in the art, in light of the present application,
are able to alter the rotational speed of vortex treatment, to
produce the desired level of gelation and the desired time frame in
which gelation occurs. Further, vortex speed and sample volume may
also affect the vortex time (t.sub.v) dependence and silk
concentration dependence of the gelation time.
[0049] The invention also relates to a method of embedding at least
one active agent in silk fibroin gel. The method comprises, for
example, vortexing a silk fibroin solution for a sufficient period
of time to initiate gelation, introducing the agent(s) to the silk
fibroin solution before substantial gelation occurs in the silk
fibroin solution, and allowing the silk-fibroin gel to complete
gelation to form a silk fibroin gel-embedded active agent.
[0050] The active agent can represent any material capable of being
embedded in the silk fibroin gel. For example, the agent may be a
therapeutic agent, or a biological material, such as cells
(including stem cells), proteins, peptides, nucleic acids (e.g.,
DNA, RNA, siRNA), nucleic acid analogs, nucleotides,
oligonucleotides, peptide nucleic acids (PNA), aptamers, antibodies
or fragments or portions thereof (e.g., paratopes or
complementarity-determining regions), antigens or epitopes,
hormones, hormone antagonists, growth factors or recombinant growth
factors and fragments and variants thereof, cell attachment
mediators (such as RGD), cytokines, enzymes, small molecules,
drugs, dyes, amino acids, vitamins, antioxidants, antibiotics or
antimicrobial compounds, anti-inflammation agents, antifungals,
viruses, antivirals, toxins, prodrugs, chemotherapeutic agents, or
combinations thereof. See, e.g., PCT/US09/44117; U.S. Patent
Application Ser. No. 61/224,618). The agent may also be a
combination of any of the above-mentioned agents. Encapsulating
either a therapeutic agent or biological material, or the
combination of them, is desirous because the encapsulated product
can be used for numerous biomedical purposes.
[0051] In some embodiments, the active agent may also be an
organism such as a fungus, plant, animal,bacterium, or a virus
(including bacteriophage). Moreover, the active agent may include
neurotransmitters, hormones, intracellular signal transduction
agents, pharmaceutically active agents, toxic agents, agricultural
chemicals, chemical toxins, biological toxins, microbes, and animal
cells such as neurons, liver cells, and immune system cells. The
active agents may also include therapeutic compounds, such as
pharmacological materials, vitamins, sedatives, hypnotics,
prostaglandins and radiopharmaceuticals.
[0052] Exemplary cells suitable for use herein may include, but are
not limited to, progenitor cells or stem cells, smooth muscle
cells, skeletal muscle cells, cardiac muscle cells, epithelial
cells, endothelial cells, urothelial cells, fibroblasts, myoblasts,
oscular cells, chondrocytes, chondroblasts, osteoblasts,
osteoclasts, keratinocytes, kidney tubular cells, kidney basement
membrane cells, integumentary cells, bone marrow cells,
hepatocytes, bile duct cells, pancreatic islet cells, thyroid,
parathyroid, adrenal, hypothalamic, pituitary, ovarian, testicular,
salivary gland cells, adipocytes, and precursor cells. The active
agents can also be the combinations of any of the cells listed
above. See also WO 2008/106485; PCT/US2009/059547; WO
2007/103442.
[0053] Exemplary antibodies that may be incorporated in silk
fibroin include, but are not limited to, abciximab, adalimumab,
alemtuzumab, basiliximab, bevacizumab, cetuximab, certolizumab
pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, ibritumomab
tiuxetan, infliximab, muromonab-CD3, natalizumab, ofatumumab
omalizumab, palivizumab, panitumumab, ranibizumab, rituximab,
tositumomab, trastuzumab, altumomab pentetate, arcitumomab,
atlizumab, bectumomab, belimumab, besilesomab, biciromab,
canakinumab, capromab pendetide, catumaxomab, denosumab,
edrecolomab, efungumab, ertumaxomab, etaracizumab, fanolesomab,
fontolizumab, gemtuzumab ozogamicin, golimumab, igovomab,
imciromab, labetuzumab, mepolizumab, motavizumab, nimotuzumab,
nofetumomab merpentan, oregovomab, pemtumomab, pertuzumab,
rovelizumab, ruplizumab, sulesomab, tacatuzumab tetraxetan,
tefibazumab, tocilizumab, ustekinumab, visilizumab, votumumab,
zalutumumab, and zanolimumab. The active agents can also be the
combinations of any of the antibodies listed above.
[0054] Exemplary antibiotic agents include, but are not limited to,
actinomycin; aminoglycosides (e.g., neomycin, gentamicin,
tobramycin); .beta.-lactamase inhibitors (e.g., clavulanic acid,
sulbactam); glycopeptides (e.g., vancomycin, teicoplanin,
polymixin); ansamycins; bacitracin; carbacephem; carbapenems;
cephalosporins (e.g., cefazolin, cefaclor, cefditoren,
ceftobiprole, cefuroxime, cefotaxime, cefipeme, cefadroxil,
cefoxitin, cefprozil, cefdinir); gramicidin; isoniazid; linezolid;
macrolides (e.g., erythromycin, clarithromycin, azithromycin);
mupirocin; penicillins (e.g., amoxicillin, ampicillin, cloxacillin,
dicloxacillin, flucloxacillin, oxacillin, piperacillin); oxolinic
acid; polypeptides (e.g., bacitracin, polymyxin B); quinolones
(e.g., ciprofloxacin, nalidixic acid, enoxacin, gatifloxacin,
levaquin, ofloxacin, etc.); sulfonamides (e.g., sulfasalazine,
trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole),
sulfadiazine); tetracyclines (e.g., doxycyline, minocycline,
tetracycline, etc.); monobactams such as aztreonam;
chloramphenicol; lincomycin; clindamycin; ethambutol; mupirocin;
metronidazole; pefloxacin; pyrazinamide; thiamphenicol; rifampicin;
thiamphenicl; dapsone; clofazimine; quinupristin; metronidazole;
linezolid; isoniazid; piracil; novobiocin; trimethoprim;
fosfomycin; fusidic acid; or other topical antibiotics. Optionally,
the antibiotic agents may also be antimicrobial peptides such as
defensins, magainin and nisin; or lytic bacteriophage. The
antibiotic agents can also be the combinations of any of the agents
listed above. See also PCT/US2010/026190.
[0055] Exemplary enzymes suitable for use herein include, but are
not limited to, peroxidase, lipase, amylose, organophosphate
dehydrogenase, ligases, restriction endonucleases, ribonucleases,
DNA polymerases, glucose oxidase, laccase, and the like.
Interactions between components may also be used to functionalize
silk fibroin through, for example, specific interaction between
avidin and biotin. The active agents can also be the combinations
of any of the enzymes listed above. See U.S. Patent Application
Ser. No. 61/226,801.
[0056] When an agent is introduced into the silk fibroin solution
after the vortex treatment, the conditions of the vortex treatment
may be adjusted so that gelation occurs some period of time after
the vortex treatment. If gelation occurs during the vortex
treatment or immediately thereafter, an insufficient amount of time
may exist to introduce the agent into the silk fibroin solution.
For example, when the agent is introduced after the vortex
treatment, the silk fibroin undergoes gelation at a time period
ranging from about five minutes to about two hours after the vortex
treatment.
[0057] When introducing therapeutic agents or biological material
into the silk fibroin, other materials known in the art may also be
added with the agent. For instance, it may be desirable to add
materials to promote the growth of the agent (for biological
materials), promote the functionality of the agent after it is
released from the silk gel, or increase the agent's ability to
survive or retain its efficacy during the period it is embedded in
the silk. Materials known to promote cell growth include 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 (FGF), transforming growth factors (TGFs), vascular
endothelial growth factor (VEGF), epidermal growth factor (EGF),
insulin-like growth factor (IGF-I), bone morphogenetic growth
factors (BMPs), nerve growth factors, and related proteins may be
used. Growth factors are known in the art, see, e.g., Rosen &
Thies, CELLULAR & MOLECULAR BASIS BONE FORMATION & REPAIR
(R.G. Landes Co., Austin, Tex., 1995). Additional options for
delivery via the gels include DNA, siRNA, antisense, plasmids,
liposomes and related systems for delivery of genetic materials;
peptides and proteins to activate cellular signaling cascades;
peptides and proteins to promote mineralization or related events
from cells; adhesion peptides and proteins to improve gel-tissue
interfaces; antimicrobial peptides; and proteins and related
compounds.
[0058] Additional biocompatible material may also be blended into
the silk fibroin hydrogels, such as polyethylene oxide (see, e.g.,
U.S. Patent Application Ser. No. 61/225,335), polyethylene glycol
(see PCT/US09/64673), collagen, fibronectin, keratin, polyaspartic
acid, polylysine, alginate, chitosan, chitin, hyaluronic acid,
pectin, polycaprolactone, polylactic acid, polyglycolic acid,
polyhydroxyalkanoates, dextrans, polyanhydrides, glycerol (see
PCT/US2009/060135), and other biocompatible polymers, see WO
2004/0000915. Alternatively, the silk may be mixed with
hydroxyapatite particles, see PCT/US08/82487. As noted herein, the
silk fibroin may be of recombinant origin, which provides for
further modification of the silk such as the inclusion of a fusion
polypeptide comprising a fibrous protein domain and a
mineralization domain, which are used to form an organic-inorganic
composite. These organic-inorganic composites can be constructed
from the nano- to the macro-scale depending on the size of the
fibrous protein fusion domain used, see WO 2006/076711. See also
U.S. patent application Ser. No. 12/192,588.
[0059] The silk-fibroin embedded active agents or biological
materials may be suitable for long term storage and stabilization
of the cells and/or active agents. Cells and/or active agents, when
incorporated in the silk hydrogel of the present invention, can be
stable (i.e., maintaining at least 50% of residual activity) for at
least 30 days at room temperature (i.e., 22.degree. C. to
25.degree. C.) and body temperature (37.degree. C.). Hence,
temperature-sensitive active agents, such as some antibiotics, can
be stored in silk fibroin hydrogels without refrigeration.
Importantly, temperature-sensitive bioactive agents can be
delivered (e.g., through injection) into the body in silk hydrogels
and maintain activity for a longer period of time than previously
imagined. See, e.g., PCT/US2010/026190.
[0060] The silk-fibroin embedded active agents (e.g., therapeutic
agents) or biological materials are suitable for a biodelivery
device. Techniques for using silk fibroin as a biodelivery device
may be found, for example, in U.S. patent applications Ser. No.
10/541,182; No. 11/628,930; No. 11/664,234; No. 11/407,373;
PCT/US07/020789; PCT/US08/55072; PCT/US09/44117. Some embodiments
of the present invention relate to the utility of silk-fibroin
embedded therapeutic agents or biological materials as drug
delivery systems for potential utility in medical implants, tissue
repairs and for medical device coatings.
[0061] The silk fibroin hydrogel structure enables the biodelivery
vehicle to have a controlled release. Controlled release permits
dosages to be administered over time, with controlled release
kinetics. In some instances, delivery of the therapeutic agent or
biological material is continuous 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 therapeutic agent or biological material
to obtain preferred treatments. The controlled delivery vehicle is
advantageous because it protects the therapeutic agent or
biological material from degradation in vivo in body fluids and
tissue, for example, by proteases. See, e.g., PCT/US09/44117.
[0062] Controlled release of the bioactive agent from the silk
hydrogel may be designed to occur over time, for example, for
greater than about 12 hours or 24 hours, inclusive; greater than 1
month or 2 months or 5 months, inclusive. The time of release may
be selected, for example, to occur over a time period of about 12
hours to 24 hours, or about 12 hours to 1 week. In another
embodiment, release may occur for example on the order of about 1
month to 2 months, inclusive. The controlled release time may be
selected based on the condition treated. For example, a particular
release profile may be more effective where consistent release and
high local dosage are desired.
[0063] A pharmaceutical formulation may be prepared that contains
the silk fibroin hydrogel having encapsulated bioactive agents. The
formulation can be administered to a patient in need of the
particular active agent that has been encapsulated in the silk
fibroin. The pharmaceutical formulation may be administered by a
variety of routes known in the art including topical, oral, ocular,
nasal, transdermal or parenteral (including intravenous,
intraperitoneal, intramuscular and subcutaneous injection as well
as intranasal or inhalation administration), and implantation. The
delivery may be systemic, regional, or local. Additionally, the
delivery may be intrathecal, e.g., for delivery to the central
nervous system.
[0064] When desired, the active agent-containing silk hydrogel may
include a targeting ligand or precursor targeting ligand. Targeting
ligand refers to any material or substance which may promote
targeting of the pharmaceutical formulation to tissues and/or
receptors in vivo and/or in vitro with the formulations of the
present invention. The targeting ligand may be synthetic,
semi-synthetic, or naturally-occurring. Materials or substances
which may serve as targeting ligands include, for example,
proteins, including antibodies, antibody fragments, hormones,
hormone analogues, glycoproteins and lectins, peptides,
polypeptides, amino acids, sugars, saccharides, including
monosaccharides and polysaccharides, carbohydrates, vitamins,
steroids, steroid analogs, hormones, cofactors, and genetic
material, including nucleosides, nucleotides, nucleotide acid
constructs, peptide nucleic acids (PNA), aptamers, and
polynucleotides. Other targeting ligands in the present invention
include cell adhesion molecules (CAM), among which are, for
example, cytokines, integrins, cadherins, immunoglobulins and
selectin. A precursor to a targeting ligand refers to any material
or substance which may be converted to a targeting ligand. Such
conversion may involve, for example, anchoring a precursor to a
targeting ligand. Exemplary targeting precursor moieties include
maleimide groups, disulfide groups, such as ortho-pyridyl
disulfide, vinylsulfone groups, azide groups, and iodo acetyl
groups.
[0065] In preparation for in vivo application, the silk fibroin of
the present invention may be formulated to include excipients.
Exemplary excipients include diluents, solvents, buffers, or other
liquid vehicle, solubilizers, dispersing or suspending agents,
isotonic agents, viscosity controlling agents, binders, lubricants,
surfactants, preservatives, stabilizers and the like, as suited to
particular dosage form desired. The formulations may also include
bulking agents, chelating agents, and antioxidants. Where
parenteral formulations are used, the formulation may additionally
or alternately include sugars, amino acids, or electrolytes.
[0066] More specifically, examples of materials which can serve as
pharmaceutically acceptable carriers include, but are not limited
to, sugars such as lactose, glucose and sucrose; starches such as
corn starch and potato starch; cellulose and its derivatives such
as sodium carboxymethyl cellulose, ethyl cellulose and cellulose
acetate; powdered tragacanth; malt; gelatine; talc; oils such as
peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil;
corn oil and soybean oil; esters such as ethyl oleate and ethyl
laurate; agar; non-toxic compatible lubricants such as sodium
lauryl sulfate and magnesium stearate; polyols, for example, of a
molecular weight less than about 70,000 kD, such as trehalose,
mannitol, and polyethylene glycol. See, e.g., U.S. Pat. No.
5,589,167. Exemplary surfactants include nonionic surfactants, such
as Tween surfactants, polysorbates, such as polysorbate 20 or 80,
etc., and the poloxamers, such as poloxamer 184 or 188, pluronic
polyols, and other ethylene/polypropylene block polymers, etc.
Suitable buffers include Tris, citrate, succinate, acetate, or
histidine buffers. Suitable preservatives include phenol, benzyl
alcohol, metacresol, methyl paraben, propyl paraben, benzalconium
chloride, and benzethonium chloride. Other additives include
carboxymethylcellulose, dextran, and gelatin. Suitable stabilizing
agents include heparin, pentosan polysulfate and other heparinoids,
and divalent cations such as magnesium and zinc. Coloring agents,
releasing agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives and antioxidants can also be
present in the composition, according to the judgment of the
formulator or ordinary skill
[0067] The present invention also provides for a method of
preparing reversible shear-thinning silk fibroin gels. The method
comprises vortexing a silk fibroin solution for a sufficient period
of time to initiate gelation of the silk fibroin. The silk fibroin
then undergoes substantial gelation after the vortex treatment
thereby forming a reversible shear-thinning silk fibroin gel. An
important practical consideration for encapsulation/delivery
application of hydrogel/bioactive molecules scaffolds is the ease
of application into the target cite not only with high temporal
precision by also with high spatial precision. For example, a
shear-thinning hydrogel material can be implanted with minimal
invasion to the delivery site, such as by injection through a
needle. A hydrogel that shear-thins into a sol during injection may
enable more homogeneous delivery of active agent and/or cells
(e.g., to the wound site) as compared with cell delivery in
solution. In addition, it is advantageous for the shear-thinned
hydrogel material to recover quickly, even immediately, to form a
stiff network after removal of applied shear force, facilitating
localization of a uniform density of cells and/or active agent at
the delivery site.
[0068] Thus, the silk fibroin gels prepared by the vortex treatment
of the present invention may be shear-thinned. In one embodiment,
the shear-thinning behavior of vortex-induced hydrogels formed on
the rheometer plate has been examined. FIGS. 3a and 3b show the
dependence of G' and G'' on the amplitude of the applied shear
force as a function of protein concentration. Hydrogels display a
linear viscoelastic regime and apparently yield above the shear
amplitude, .gamma..sub.B (.gamma..sub.B.about.2%, .about.40$ and
.about.10% for 1.3 wt %, 2.6 wt % and 5.2 wt % hydrogels,
respectively) followed by apparent shear thinning. Shear thinning
could be attributed to the rupture of dangling chain entanglement
crosslinks and the breaking apart of clusters from the hydrogel
network at high shear amplitude. From a practical viewpoint, shear
thinning of the hydrogel (e.g., during injection to an in vivo
site) could enable a more homogeneous delivery of cells and/or
active agents in the shear-thinned sol to the wound site as
compared with cells/active molecules delivery in solution. The
apparent decrease in .gamma..sub.B from 2.6 wt % to 5.2 wt % may be
due to formation of a slip layer at the hydrogel/rheometer plate
interface at high shear for very stiff hydrogels leading to an
underestimation of .gamma..sub.B.
[0069] The stiffness of vortex-induced silk fibroin gels of the
invention may recover from shear-thinning after removal of applied
shear. For example, the recovery of crosslinks in vortexed
hydrogels after shear-thinning may be examined by comparing
frequency sweeps collected before and immediately after directly
injecting the gels through a 21 gauge needle onto the rheometer
plate (FIGS. 3c and 3d). Injection through a needle was chosen to
demonstrate the possibility of injection of the hydrogels in a
minimally invasive manner to a delivery site. For all hydrogels,
there was significant recovery of stiffness immediately after
injection (for .phi.=1.3 wt %, G' recovered almost to the
pre-injection value, while for higher protein concentrations
G'.about.1/3 of the pre-injection value). All hydrogels essentially
displayed frequency independent shear modulus immediately after
cessation of applied shear due to injection. It is unlikely that
the shear-recovery of vortexed hydrogels after shear thinning is
due to the disruption and reformation of permanent, intermolecular
.beta.-sheet crosslinks. Therefore, the presence of another
crosslinking mechanism in addition to the intermolecular
.beta.-sheet crosslinks could be hypothesized similar to that
proposed for .beta.-hairpin peptide hydrogels (Haines-Butterick et
al., 2007; Yucel et al., 2008; Yucel, Ph.D. Dissertation, 2008).
For example, large, .beta.-sheet rich clusters could slide past
each other during shear thinning by the temporary release of
intercluster, dangling chain entanglements at high shear
amplitudes, which may reform permanently after removal of shear,
with no significant change in the overall .beta.-sheet content.
Overall, considering the high stiffness values and the frequency
independence of viscoelastic properties immediately after
injection, these materials may be used to facilitate localization
of cells at the injection site with high spatial precision.
[0070] The present invention also provides methods of delivering a
reversibly shear-thinned silk fibroin gel to a target site. The
method comprises vortexing a silk fibroin solution for a sufficient
period of time to initiate gelation, allowing substantial gelation
to occur after the vortex treatment to form a silk fibroin gel,
introducing the silk fibroin gel through a shear-inducing delivery
device to the target site while applying a shear force to
shear-thin the silk fibroin gel, and removing the shear force,
whereupon the shear-thinned silk fibroin gel recovers from
shear-thinning and re-gels. In one embodiment, such reversibly
shear-thinned silk fibroin gel is delivered locally to the target
site with high spatial precision. The delivery devices may be any
delivery devices known in the art that can produce shear force to
the hydrogels. In another embodiment, the reversibly shear-thinned
silk fibroin gel is delivered locally to the target site by
injection through a needle.
[0071] Another embodiment of the invention relates to a method of
encapsulating or embedding bioactive agents to a reversible
shear-thinned silk fibroin gel, and delivering the reversibly
shear-thinned silk fibroin gel-encapsulated/embedded active agent
to a target site. Such method may be used for homogeneous delivery
of one or more active agents to a target site. The method comprises
vortexing a silk fibroin solution for a sufficient period of time
to initiate gelation, introducing at least one bioactive agent to
the silk fibroin solution before substantial gelation occurs in the
silk fibroin solution, thereby forming a silk fibroin gel-embedded
active agent that may be shear-thinned reversibly, introducing to
the target site the active agent-encapsulated silk fibroin gel
through a shear-inducing delivery device to the target site while
applying a shear force to shear-thin the silk gel-embedded
bioagent, and removing the shear force, whereupon the shear-thinned
silk fibroin gel-embedded bioagent recovers gel form. The method
hence may distribute the active agent contained in the silk gel at
the target site homogeneously.
[0072] The novel techniques based on vortexing to induce silk
fibroin gelation, described herein, form silk fibroin gels, and
also control the rate of .beta.-sheet formation and the concomitant
hydrogelation kinetics of aqueous silk fibroin solutions. Shear
gradient-induced changes in silk fibroin structure and solution
viscoelastic properties post-vortex treatment are presented herein.
The novel technique controls the post-shear self-assembly and
hydrogelation kinetics of silk fibroin for cell encapsulation and
delivery applications. The silk fibroin gels may be characterized
by various techniques. CD spectroscopy indicates that vortexing
aqueous solutions of silk leads to a transition from an overall
protein structure that is initially rich in random coil to protein
structure that is rich in .beta.-sheet content. Dynamic oscillatory
rheology experiments collected under the same assembly conditions
as the CD experiments indicate that the increase in .beta.-sheet
content due to intramolecular conformational changes and
intermolecular self-assembly of the silk fibroin is directly
correlated with the subsequent changes in viscoelastic properties
due to hydrogelation. Vortexing low-viscosity silk solutions leads
to orders of increase in the complex shear modulus, G* and
formation of rigid hydrogels (G* .apprxeq.70 kPa for 5.2 wt %
protein concentration). The vortex-induced, .beta.-sheet-rich silk
hydrogels consist of permanent, physical, intermolecular
crosslinks, although the sol-gel may be reversibly manipulated by
shear stress. The novel vortexing technique is simple, yet
versatile. The hydrogelation kinetics can be controlled easily
(from minutes to hours) by changing the vortex time, assembly
temperature and/or protein concentration, providing a useful
timeframe for cell encapsulation. The shear thinning and recovery
behavior of vortex-induced silk hydrogels are studied using
rheology to highlight the suitability of these materials as
versatile cell delivery scaffolds. Vortex-induced hydrogels may be
shear-thinned by injection through needles, and the hydrogel
stiffness recovered immediately after removal of applied shear.
Physicochemical characterization can be further correlated with
cell behavior to study the applicability of these materials for
homogeneous 3-D cell encapsulation, homogeneous delivery in vivo,
and localization of hydrogel/cell scaffolds at the injection
site.
[0073] The invention will be further characterized by the following
examples which are intended to be exemplary of the embodiments.
[0074] The present invention can be defined in any of the following
numbered paragraphs:
[0075] A method of forming silk fibroin gel, comprising vortexing a
silk fibroin solution for a sufficient period of time to initiate
intermolecular self-assembly of silk fibroin .beta.-sheet
structure, wherein substantial silk fibroin gelation occurs in less
than about 16 hours after the vortexing.
[0076] The method of paragraph 75, wherein said vortexing yields a
solid phase and an aqueous phase, and wherein the method further
comprises removing said solid phase and allowing gelation of the
aqueous phase.
[0077] The method of paragraph 75 or 76, wherein the silk fibroin
in the solution has a concentration about 6 wt % or lower.
[0078] The method of paragraph 77, wherein the silk fibroin in the
solution has a concentration ranging from about 1.0 wt % to about
5.2 wt %.
[0079] A method of controlling gelation time of silk fibroin
initiated by vortexing a silk fibroin solution for a sufficient
period of time to initiate gelation, wherein the gelation time is
controlled by adjusting one or more of (a) the time period of the
vortex treatment; (b) the concentration of the silk fibroin in
solution; or (c) the temperature of the silk fibroin solution after
the vortex treatment.
[0080] A method of preparing a reversible shear-thinning silk
fibroin gel, comprising vortexing a silk fibroin solution for a
sufficient period of time to initiate gelation, wherein the silk
fibroin undergoes substantial gelation after the vortex treatment
thereby forming a reversibly shear-thinning silk fibroin gel.
[0081] A method of preparing a silk fibroin gel-encapsulated active
agent, comprising:
[0082] vortexing a silk fibroin solution for a sufficient period of
time to initiate gelation; introducing at least one active agent to
the silk fibroin solution before substantial
[0083] gelation occurs in the silk fibroin solution; allowing the
silk-fibroin to gel, forming a silk fibroin gel-encapsulated active
agent.
[0084] The method of paragraph 81, wherein the active agent is a
therapeutic agent or a biological material, selected from the group
consisting of cells, proteins, peptides, nucleic acids, nucleic
acid analogs, nucleotides or oligonucleotides, peptide nucleic
acids, aptamers, antibodies or fragments or portions thereof,
antigens or epitopes, hormones, hormone antagonists, growth factors
or recombinant growth factors and fragments and variants thereof,
cell attachment mediators, cytokines, enzymes, antibiotics or
antimicrobial compounds, viruses, toxins, prodrugs,
chemotherapeutic agents, small molecules, drugs, and combinations
thereof.
[0085] The method of paragraph 81, wherein the active agent is a
cell selected from the group consisting of progenitor cells or stem
cells, smooth muscle cells, skeletal muscle cells, cardiac muscle
cells, epithelial cells, endothelial cells, urothelial cells,
fibroblasts, myoblasts, oscular cells, chondrocytes, chondroblasts,
osteoblasts, osteoclasts, keratinocytes, kidney tubular cells,
kidney basement membrane cells, integumentary cells, bone marrow
cells, hepatocytes, bile duct cells, pancreatic islet cells,
thyroid, parathyroid, adrenal, hypothalamic, pituitary, ovarian,
testicular, salivary gland cells, adipocytes, precursor cells, and
combinations thereof.
[0086] The method of paragraph 83, the active agent further
comprises a cell growth media.
[0087] The method of paragraph 81, wherein the silk fibroin
gel-encapsulated active agent is suitable for a biodelivery
device.
[0088] The method of paragraph 81, wherein the silk fibroin
gel-encapsulated active agent is suitable for a medical implant or
a tissue repair material.
[0089] A method of delivering a reversibly shear-thinned silk
fibroin gel to a target site, comprising:
[0090] vortexing a silk fibroin solution for a sufficient period of
time to initiate gelation, wherein the silk fibroin undergoes
substantial gelation after the vortex treatment to form a silk
fibroin gel;
[0091] introducing the silk fibroin gel through a shear-inducing
delivery device to the target site while applying a shear force to
shear-thin the silk fibroin gel; and
[0092] removing the shear force, whereupon the shear-thinned silk
fibroin gel recovers from shear-thinning and re-gels.
[0093] The method of paragraph 87, further comprising adding an
active agent to said silk fibroin solution.
[0094] The method of paragraph 87 or paragraph 88 wherein said
reversible shear-thinning silk fibroin gel is delivered locally to
said target site with high spatial precision.
[0095] The method of paragraph 89, wherein said reversible
shear-thinning silk fibroin gel is delivered locally to said target
site by injection through a needle.
[0096] The method of paragraph 89, wherein the method is suitable
for implanting a medical implant or a tissue repair material.
[0097] A method for homogeneous delivery of at least one active
agent to a target site, comprising:
[0098] vortexing a silk fibroin solution for a sufficient period of
time to initiate gelation;
[0099] introducing at least one active agent to the silk fibroin
solution either before vortexing or before substantial gelation
occurs in the silk fibroin solution, thereby forming a silk fibroin
gel-encapsulated active agent that may be shear-thinned
reversibly;
[0100] introducing to the target site the active agent-encapsulated
silk fibroin gel through a shear-inducing delivery device to the
target site while applying a shear force to shear-thin the
agent-encapsulated silk fibroin gel; and
[0101] removing the shear force, whereupon the shear-thinned silk
fibroin gel-encapsulated agent recovers gel form, thereby
distributing the active agent in the gel form at the target site
homogeneously.
[0102] The method of paragraph 92, wherein said reversible
shear-thinning silk fibroin gel is delivered locally to said target
site with high spatial precision.
[0103] The method of paragraph 93, wherein said reversible
shear-thinning silk fibroin gel is delivered locally to said target
site by injection.
[0104] The method of paragraph 92, wherein the at least one silk
fibroin gel-encapsulated active agent is delivered to the target
site in vivo.
[0105] A method of preparing a silk fibroin gel-encapsulated active
agent, comprising:
[0106] introducing at least one active agent to a silk fibroin
solution; and
[0107] vortexing the silk fibroin solution for a sufficient period
of time to initiate gelation.
[0108] The method of paragraph 96, further comprising adding a cell
population to said vortexed solution before significant onset of
gelation.
[0109] The method of paragraph 96 or paragraph 97, further
comprising reversibly shear-thinning the gel by passing it through
a hypodermic needle.
[0110] A silk fibroin gel-encapsulated active agent prepared by the
method of any one of paragraph 96 to paragraph 98.
EXAMPLES
Example 1
Preparation of Aqueous Silk Fibroin Solutions
[0111] Silk fibroin aqueous solutions were prepared as previously
described in the literature. See Sofia et al., 54 J. Biomed. Mater.
Res. 139-48 (2001). Briefly, Bombyx mori cocoons were boiled for 40
min in an aqueous solution of 0.02 M NaCo.sub.3 and then rinsed
thoroughly with deionized water. After overnight drying, the
extracted silk fibroin was dissolved in an aqueous solution of 9.3
M LiBr at 60.degree. C. overnight. The resulting solution was
dialyzed against deionized water using Slide-A-Lyzer dialysis
cassettes (MWCO 3,500, Pierce, Thermo Scientific, Waltham, Mass.)
for two days to remove the residual salt. The final concentration
of the silk fibroin was approximately 5.3 wt %. Lower concentration
silk solutions were prepared by diluting the 5.3 wt % stock
solution with deionized water. Additionally, the silk fibroin
solution may be concentrated, for example, to about 30% (w/v).
Briefly, the silk fibroin solution with a lower concentration may
be dialyzed against a hygroscopic polymer, such as PEG, amylose or
sericin, for a time period sufficient to result in a desired
concentration. See, e.g., WO2005/012606.
[0112] The silk fibroin solution can be combined with one or more
biocompatible polymers such as polyethylene oxide, polyethylene
glycol, collagen, fibronectin, keratin, polyaspartic acid,
polylysin, alginate, chitosan, chitin, hyaluronic acid, and the
like; or one or more active agents, such as cells, enzymes,
proteins, nucleic acids, antibodies and the like, as described
herein. See, e.g., WO2004/062697 and WO2005/012606. Silk fibroin
can also be chemically modified with active agents in the solution,
for example through diazonium or carbodiimide coupling reactions,
avidin-biodin interaction, or gene modification and the like, to
alter the physical properties and functionalities of the silk
protein. See, e.g., PCT/US09/64673; U.S. Applications Ser. No.
61/227,254; Ser. No. 61/224,618; Ser. No. 12/192,588.
Example 2
Vortex-Induced Hydrogelation
[0113] A 1 ml aliquot of silk solution was equilibrated at
25.degree. C. in a vial kept in a water bath for 10 min and mixed
for predetermined times at 3,200 rpm using a vortexer (Fisher
Scientific, Pittsburg, Pa.) to induce silk self-assembly and
hydrogelation. Such prepared silk hydrogelation was characterized
by CD and rheology experiment. Increasing the vortex time increased
the solution turbidity and eventually bulk phase separation of a
white and solid-like material was observed, especially at lower
protein concentrations. Both CD and rheology data were collected
from turbid solutions after removal of the solid phase.
Example 3
Circular Dichroism Spectroscopy
[0114] Circular dichroism (CD) spectra were collected using an Aviv
Model 410 CD spectrometer (Aviv Biomedical, Inc., Lakewood, N.J.).
After vortexing, aqueous silk solutions were immediately loaded in
a 0.1 mm quartz cell within a temperature controlled cell holder.
CD wavelength scans between 210 nm and 260 nm or time sweeps at 216
nm were collected at 25.degree. C. Due to the high silk
concentrations, the high dynode voltages below 210 nm lead to
erroneous data. The CD spectrum of water collected immediately
before each measurement was used for background correction.
[0115] The mean residual ellipticity was calculated from
[ .theta. ] = .theta. M 10 c l n ( deg cm 2 dmol ) ##EQU00001##
where .theta. is the measured ellipticity (deg), M is the mean
molecular mass (g/mol), c is the protein concentration
(g/cm.sup.3), 1 is the path length (cm), and n is the number of
residues. M and n for B. mori heavy chain were taken as 391,563
g/mol and 5263, respectively (Zhou et al, 28 Nucleic Acids Res.
2413-19 (2000)). For protein concentration measurements, a 0.5 mL
aliquot of silk solution was dried at 60.degree. C. overnight and
the solution concentration was calculated from the weight of the
dried film.
Example 4
Dynamic Oscillatory Rheology
[0116] Dynamic oscillatory time, frequency and strain sweeps were
performed using an ARES strain-controlled rheometer (TA
Instruments, New Castle, Del.) with 25 mm or 50 mm diameter
stainless steel parallel plate geometries at 0.5 mm measuring gap
distance. In a typical experiment, the silk solution was applied
slowly via a syringe on the rheometer plate to prevent shearing of
the sample immediately after vortexing. The normal force applied on
the sample during lowering of the top plate was limited to 0.1 N. A
low viscosity mineral oil and the solvent trap supplied by TA
instruments were used to prevent sample evaporation from the sides
of the plate. Dynamic oscillatory time sweeps were collected at a
low strain amplitude (.gamma.=1%, .omega.=10 rad/s) to prevent
possible sample manipulation due to applied shear during
measurements. Frequency sweeps were collected over a wide frequency
range (.gamma.=1%, .omega.=0.1-100 rad/s) after each time sweep. To
observe the time evolution of the frequency dependence of
viscoelastic properties, frequency sweeps were collected
continuously over a narrower range (.gamma.=1%, .omega.=0.5-100
rad/s). At the applied shear amplitude, continuous collection of
frequency sweeps had no detectable effect on the measured
rheological properties. Strain sweep measurements were performed
from .gamma.=0.01-1000% (.omega.=10 rad/s) at the end of each
experiment to determine the linear viscoelastic regime of the final
hydrogel.
Example 5
Injection Studies
[0117] To study the injectability of vortexed hydrogels and the
shear-recovery behavior, vortexed silk solutions were either loaded
into 1 ml syringes immediately after vortexing and allowed to gel
overnight or loaded into syringes after overnight hydrogelation in
vials. A 21 gauge needle was connected to the syringe and the
hydrogel was injected through the needle directly onto the
rheometer plate. A frequency sweep was collected over a wide
frequency range (.gamma.=1%, .omega.=0.1-100 rad/s) within 10 min
of injection of the sample.
* * * * *