U.S. patent application number 12/883139 was filed with the patent office on 2011-08-04 for dermal fillers comprising silk fibroin hydrogels and uses thereof.
This patent application is currently assigned to ALLERGAN, INC.. Invention is credited to Gregory H. Altman, Jingsong Chen, Adam L. Collette, Rebecca L. Horan, Pierre F. Lebreton, Sebastien Pierre.
Application Number | 20110189292 12/883139 |
Document ID | / |
Family ID | 44341900 |
Filed Date | 2011-08-04 |
United States Patent
Application |
20110189292 |
Kind Code |
A1 |
Lebreton; Pierre F. ; et
al. |
August 4, 2011 |
DERMAL FILLERS COMPRISING SILK FIBROIN HYDROGELS AND USES
THEREOF
Abstract
The present specification provides compositions useful as dermal
fillers and methods using such compositions to treat a condition of
skin.
Inventors: |
Lebreton; Pierre F.;
(Annecy, FR) ; Pierre; Sebastien; (Annecy, FR)
; Collette; Adam L.; (Westminister, MA) ; Horan;
Rebecca L.; (Arlington, MA) ; Chen; Jingsong;
(Virginia Beach, VA) ; Altman; Gregory H.;
(Arlington, MA) |
Assignee: |
ALLERGAN, INC.
Irvine
CA
|
Family ID: |
44341900 |
Appl. No.: |
12/883139 |
Filed: |
September 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12764039 |
Apr 20, 2010 |
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12883139 |
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61170895 |
Apr 20, 2009 |
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Current U.S.
Class: |
424/488 ;
514/626 |
Current CPC
Class: |
A61K 9/0019 20130101;
A61L 27/3604 20130101; A61P 17/02 20180101; A61P 17/00 20180101;
A61K 31/167 20130101; A61K 47/42 20130101; A61K 8/64 20130101; A61L
27/227 20130101; A61K 9/06 20130101; A61K 31/16 20130101; A61K
38/17 20130101; A61Q 19/08 20130101; A61L 27/3641 20130101; A61K
31/167 20130101; A61K 2300/00 20130101; A61K 38/17 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/488 ;
514/626 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 31/16 20060101 A61K031/16; A61P 17/00 20060101
A61P017/00 |
Claims
1. A composition comprising a gel phase, the gel phase including a)
hydrogel particles comprising a substantially sericin-depleted silk
fibroin and an amphiphilic peptide; and b) hydrogel particles
comprising a matrix polymerand,
2. The composition of claim 1, wherein the silk fibroin hydrogel
particles comprises about 1% (w/v) to about 10% (w/v) of silk
fibroin.
3. The composition of claim 1, wherein the final concentration of
the silk fibroin is from about 3 mg/g to about 30 mg/g.
4. The composition of claim 1, wherein the amphiphilic peptide
comprising a RGD motif or a non-RDG integrin.
5. The composition of claim 3, wherein the amphiphilic peptide is
23 RGD.
6. The composition of claim 1, wherein the amphiphilic peptide
comprises of a tail region, followed by a spacer region and finally
a RGD motif.
7. The composition of claim 1, wherein the silk fibroin hydrogel
particles comprises a molar ratio of 1:10 to 10:1 moles of the
amphiphilic peptide per mole of the silk fibroin.
8. The composition of claim 6, wherein the silk fibroin hydrogel
particles comprises a molar ratio of 3:1 moles of the amphiphilic
peptide per mole of the silk fibroin.
9. The composition of claim 1, wherein the silk fibroin hydrogel
particles comprises a protein structure having a .beta.-sheet
conformation of at least 80%.
10. The composition of claim 1, wherein the silk fibroin hydrogel
particles comprises a protein structure having a .beta.-sheet
conformation of at least 50%.
11. The composition of claim 1, wherein the silk fibroin hydrogel
particles comprises a protein structure having a .beta.-sheet
conformation of at least 20%.
12. The composition of claim 1, wherein the silk fibroin hydrogel
particles comprises a protein structure having an .alpha.-helical
and random coil conformation of at most 20%.
13. The composition of claim 1, wherein the silk fibroin hydrogel
particles further comprise a synthetic molecule having the formula:
(molecule X).sub.n--(spacer peptide).sub.0-300-(five-amino-acid
peptide tail) is conjugated to the silk fibroin.
14. The composition of claim 1, wherein the matrix polymer is a
glycosaminoglycan, a lubricin, a polysaccharide, or any combination
thereof.
15. The composition of claim 14, wherein the glycosaminoglycan is a
chondroitin sulfate, a dermatan sulfate, a keratan sulfate, a
heperan sulfate, a hyaluronan, or any combination thereof.
16. The composition of claim 14, wherein the polysaccharide is a
dextran, a dextrin, a starch, a hetastarch, a glycogen, a polyvinyl
acetate, a polyvinyl pyrrolidone, a polyethylene glycol, a
polyethylene imine, a cellulose, a methyl cellulose, a
carboxymethyl cellulose, a hydroxyethyl cellulose, a hydroxypropyl
cellulose, a hydroxyethyl methyl cellulose, a hydroxypropyl methyl
cellulose, or any combination thereof.
17. The composition of claim 1, wherein the matrix polymer is
crosslinked.
18. The composition of claim 17, wherein the crosslinked matrix
polymer has a degree of crosslinking of at least 1%.
19. The composition of claim 17, wherein the crosslinked matrix
polymer has a degree of crosslinking of at most 17%.
20. The composition of claim 17, wherein the crosslinked matrix
polymer has a degree of crosslinking of about 1% to about 17%.
21. The composition of claim 1, wherein the uncrosslinked matrix
polymer represents about 90% or more by weight of the total matrix
polymer present in the composition.
22. The composition of claim 1, wherein the final concentration of
the matrix polymer is from about 12 mg/g to about 22.8 mg/g.
23. The composition of claim 1, wherein the silk fibroin hydrogel
particles and matrix polymer hydrogel particles have a
cross-sectional area from about 0.1 .mu.m.sup.2 to about 1000
.mu.m.sup.2.
24. The composition of claim 1, wherein the silk fibroin hydrogel
particles and matrix polymer hydrogel particles have a
cross-sectional area from about 0.1 .mu.m.sup.2 to about 10
.mu.m.sup.2.
25. The composition of claim 1, wherein the silk fibroin hydrogel
particles and matrix polymer hydrogel particles have a
cross-sectional area from about 20 .mu.m.sup.2 to about 50
.mu.m.sup.2.
26. The composition of claim 1, wherein the composition further
comprises a carrier phase.
27. The composition of claim 26, wherein the carrier phase
comprises saline.
28. The composition of claim 26, wherein the carrier phase
comprises a surfactant solution.
29. The composition of claim 26, wherein the gel phase is 50% to
99% of the total formulation volume, the remainder being a carrier
solution.
30. The composition of claim 29, wherein the gel phase is 75% of
the total formulation volume, the remainder being a carrier
solution.
31. The composition of claim 1, wherein the composition further
comprising lidocaine.
32. The composition of claim 1, wherein, upon injection, the
hydrogel particles remains substantially at the injection site for
one month to eighteen months.
33. A composition comprising a gel phase, the gel phase including
hydrogel particles comprising a substantially sericin-depleted silk
fibroin and a matrix polymer.
34. The composition of claim 33, wherein the matrix polymer is a
glycosaminoglycan, a lubricin, a polysaccharide, or any combination
thereof.
35. The composition of claim 34, wherein the glycosaminoglycan is a
chondroitin sulfate, a dermatan sulfate, a keratan sulfate, a
heperan sulfate, a hyaluronan, or any combination thereof.
36. The composition of claim 34, wherein the polysaccharide is a
dextran, a dextrin, a starch, a hetastarch, a glycogen, a polyvinyl
acetate, a polyvinyl pyrrolidone, a polyethylene glycol, a
polyethylene imine, a cellulose, a methyl cellulose, a
carboxymethyl cellulose, a hydroxyethyl cellulose, a hydroxypropyl
cellulose, a hydroxyethyl methyl cellulose, a hydroxypropyl methyl
cellulose, or any combination thereof.
37. The composition of claim 33, wherein the matrix polymer is
crosslinked.
38. The composition of claim 33, wherein the crosslinked matrix
polymer has a degree of crosslinking of at least 1%.
39. The composition of claim 33, wherein the crosslinked matrix
polymer has a degree of crosslinking of at most 17%.
40. The composition of claim 33, wherein the crosslinked matrix
polymer has a degree of crosslinking of about 1% to about 17%.
41. The composition of claim 33, wherein the uncrosslinked matrix
polymer represents about 90% or more by weight of the total matrix
polymer present in the composition.
42. The composition of claim 33, wherein the final concentration of
the matrix polymer is from about 12 mg/g to about 22.8 mg/g.
43. The composition of claim 33, wherein the hydrogel particles
comprises about 1% (w/v) to about 10% (w/v) of silk fibroin.
44. The composition of claim 33, wherein the final concentration of
the silk fibroin is from about 3 mg/g to about 30 mg/g.
45. The composition of claim 33, wherein the hydrogel particles
further comprise an amphiphilic peptide.
46. The composition of claim 33, wherein the silk fibroin hydrogel
particles and matrix polymer hydrogel particles have a
cross-sectional area from about 0.1 .mu.m.sup.2 to about 1000
.mu.m.sup.2.
47. The composition of claim 33, wherein the silk fibroin hydrogel
particles and matrix polymer hydrogel particles have a
cross-sectional area from about 0.1 .mu.m.sup.2 to about 10
.mu.m.sup.2.
48. The composition of claim 33, wherein the silk fibroin hydrogel
particles and matrix polymer hydrogel particles have a
cross-sectional area from about 20 .mu.m.sup.2 to about 50
.mu.m.sup.2.
49. The composition of claim 33, wherein the composition further
comprises a carrier phase.
50. The composition of claim 49, wherein the carrier phase
comprises saline.
51. The composition of claim 49, wherein the carrier phase
comprises a surfactant solution.
52. The composition of claim 49, wherein the gel phase is 50% to
99% of the total formulation volume, the remainder being a carrier
solution.
53. The composition of claim 52, wherein the gel phase is 75% of
the total formulation volume, the remainder being a carrier
solution.
54. The composition of claim 33, wherein the composition further
comprising lidocaine.
55. The composition of claim 33, wherein, upon injection, the
hydrogel particles remains substantially at the injection site for
one month to eighteen months.
56. A method of treating a soft tissue condition in an individual
in need thereof, the method comprising the step of administering a
composition of claim 1 into a skin region of the individual,
wherein the administration improves the condition.
57. The method of claim 56, wherein the soft tissue condition is a
breast tissue condition, a facial tissue condition, a neck
condition, a skin condition, an upper arm condition, a lower arm
condition, a hand condition, a shoulder condition, a back
condition, a torso including abdominal condition, a buttock
condition, an upper leg condition, a lower leg condition including
calf condition, a foot condition including plantar fat pad
condition, an eye condition, a genital condition, or a condition
effecting another body part, region or area.
58. The method of claim 57, wherein the breast tissue condition is
a breast imperfection, a breast defect, a breast augmentation, or a
breast reconstruction.
59. The method of claim 57, wherein the facial tissue condition is
a facial imperfection, a facial defect, a facial augmentation, or a
facial reconstruction.
60. The method of claim 57, wherein the facial tissue condition is
a dermal divot, a sunken check, a thin lip, a nasal imperfection or
defect, a retro-orbital imperfection or defect, a facial fold, a
facial line, a facial wrinkle, or other size, shape or contour
imperfection or defect of the face.
61. The method of claim 60, wherein the wrinkle is a glabellar
line, a nasolabial line, a perioral line, or a marionette line.
62. The method of claim 57, wherein the facial tissue condition is
skin dehydration, a lack of skin elasticity, skin roughness, a lack
of skin tautness, a skin stretch line or mark, or skin
paleness.
63. The method of claim 56, wherein the soft tissue condition is
Parry-Romberg syndrome or lupus erythematosus profundus.
64. The method of claim 56, wherein the soft tissue condition is
urinary incontinence, fecal incontinence, or gastroesophageal
reflux disease (GERD).
65. A method of treating a soft tissue condition in an individual
in need thereof, the method comprising the step of administering a
composition of claim 33 into a skin region of the individual,
wherein the administration improves the condition.
66. The method of claim 65, wherein the soft tissue condition is a
breast tissue condition, a facial tissue condition, a neck
condition, a skin condition, an upper arm condition, a lower arm
condition, a hand condition, a shoulder condition, a back
condition, a torso including abdominal condition, a buttock
condition, an upper leg condition, a lower leg condition including
calf condition, a foot condition including plantar fat pad
condition, an eye condition, a genital condition, or a condition
effecting another body part, region or area.
67. The method of claim 66, wherein the breast tissue condition is
a breast imperfection, a breast defect, a breast augmentation, or a
breast reconstruction.
68. The method of claim 66, wherein the facial tissue condition is
a facial imperfection, a facial defect, a facial augmentation, or a
facial reconstruction.
69. The method of claim 66, wherein the facial tissue condition is
a dermal divot, a sunken check, a thin lip, a nasal imperfection or
defect, a retro-orbital imperfection or defect, a facial fold, a
facial line, a facial wrinkle, or other size, shape or contour
imperfection or defect of the face.
70. The method of claim 69, wherein the wrinkle is a glabellar
line, a nasolabial line, a perioral line, or a marionette line.
71. The method of claim 66, wherein the facial tissue condition is
skin dehydration, a lack of skin elasticity, skin roughness, a lack
of skin tautness, a skin stretch line or mark, or skin
paleness.
72. The method of claim 65, wherein the soft tissue condition is
Parry-Romberg syndrome or lupus erythematosus profundus.
73. The method of claim 65, wherein the soft tissue condition is
urinary incontinence, fecal incontinence, or gastroesophageal
reflux disease (GERD).
Description
CROSS REFERENCE
[0001] This patent application is a continuation-in-part that
claims priority under 35 U.S.C. .sctn.120 to U.S. Non-Provisional
patent application Ser. No. 12/764,039, filed Apr. 20, 2010, a
patent application that claims priority pursuant to 35 U.S.C.
.sctn.119(e) to U. S. Provisional Patent Application Ser. No.
61/170,895 filed Apr. 20, 2009, each of which is hereby
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present specification discloses purified silk fibroin
and method for purifying silk fibroins, hydrogels comprising silk
fibroin with or without an amphiphilic peptide and methods for
making hydrogels comprising silk fibroin and the use of silk
fibroin hydrogels in a variety of medical uses, including, without
limitation fillers for tissue space, templates for tissue
reconstruction or regeneration, scaffolds for cells in tissue
engineering applications and for disease models, a surface coating
to improve medical device function, or as a platform for drug
delivery.
BACKGROUND
[0003] Silk refers to a filamentous product secreted by an organism
such as a spider or silkworm. Fibroin is the primary structural
component of silk. It is composed of monomeric units comprising an
about 350 kDa heavy chain and an about 25 kDa light chain, and
interspersed within the fibroin monomers is another about 25 kDa
protein derived from the P25 gene. The ratio of heavy chain:light
chain:P25 protein is about 6:6:1. Fibroin is secreted by the silk
glands of the organism as a pair of complementary fibrils called
"brins". As fibroin brins leave the glands, they are coated with
sericin, a glue-like substance which binds the brins together.
Sericin is often antigenic and may be associated with an adverse
tissue reaction when sericin-containing silk is implanted in
vivo.
[0004] Silkworm silk fibers traditionally available in the
commercial market are often termed "degummed", which refers to the
loosening and removal of a portion of the sericin coat surrounding
the two fibroin brins through washing or extraction in hot soapy
water. This degummed silk often contains or is recoated with
sericin and other impurities in order to bind the plied
multifilament together into a single fiber. Therefore, degummed
silk, unless explicitly stated to the contrary, typically contains
twenty percent to twenty-eight percent (by weight) sericin and can
not be assumed to be sericin-free.
[0005] Silk fibers have historically been valued in surgery for
their mechanical properties, particularly in the form of braided
filaments used as a suture material. Residual sericin that may be
contained in these materials stands as a potential obstacle to its
use as a biomaterial as it does present the possibility for a
heightened immune response. This sericin contamination may be
substantially removed though, resulting in a virtually sericin-free
fibroin which may be used either as fibers or dissolved and
reconstituted in a number of forms. For example, natural silk from
the silkworm Bombyx mori may be subjected to sericin extraction,
spun into yarns then used to create a matrix with high tensile
strength suitable for applications such as bioengineered ligaments
and tendons. Use of regenerated silk materials has also been
proposed for a number of medical purposes including wound
protection, cell culture substrate, enzyme immobilization, soft
contact lenses, and drug-release agents.
[0006] Silk fibroin devices whether native, dissolved, or
reconstituted, do not typically contain cell-binding domains such
as those found in collagen, fibronectin, and many other
extracellular matrix (ECM) molecules. Fibroin is also strongly
hydrophobic due to the .beta.-sheet-rich crystalline network of the
core fibroin protein. These two factors couple to severely limit
the capacity of native host cells to bind to and interact with
implanted silk devices, as neither inflammatory cells like
macrophages or reparative cells like fibroblasts are able to attach
strongly, infiltrate and bioresorb the silk fibroin devices. In the
case of virgin silk and black braided (wax or silicone coated) silk
sutures, this is typically manifested in a harsh foreign-body
response featuring peripheral encapsulation. Substantially
sericin-free silk experiences a similar, though substantially fess
vigorous response when implanted. In essence, the host cells
identify silk as a foreign body and opt to wall it off rather than
interact with it. This severely limits the subsequent long-term
potential of the device particularly relating to tissue in-growth
and remodeling and potentially, the overall utility of the device.
If it is possible to provide a more effective biomaterial
formulation for mediating host-device interactions whereby cells
are provided with a recognizable, acceptable and hence
biocompatible surface, the biological, medicinal and surgical
utility of silk is dramatically improved.
[0007] One possible means of introducing this improved
cell-material interaction is to alter the silk fibroin material
format into a more biocompatible matrix. Manipulating the silk
fibroin to make it into a silk hydrogel formulation is one
particularly intriguing option because it consists of a silk
protein network which is fully saturated with water, coupling the
molecular resiliency of silk with the biocompatibility of a "wet"
material. Generation of a silk hydrogel may be accomplished in
short by breaking apart native silk fibroin polymers into its
individual monomeric components using a solvent species, replacing
the solvent with water, then inducing a combination of inter- and
intra-molecular aggregation. It has been shown that the sol-gel
transition can be selectively initiated by changing the
concentration of the protein, temperature, pH and additive (e.g.,
ions and hygroscopic polymers such as poly(ethylene oxide) (PEO),
poloxamer, and glycerol). Increasing the silk concentration and
temperature may alter the time taken for silk gelation by
increasing the frequency of molecular interactions, increasing the
chances of polymer nucleation. Another means of accelerating silk
gelation is through use of calcium ions which may interact with the
hydrophilic blocks at the ends of silk molecules in solution prior
to gelation. Decreasing pH and the addition of a hydrophilic
polymer have been shown to enhance gelation, possibly by decreasing
repulsion between individual silk molecules in solution and
subsequently competing with silk fibroin molecules in solution for
bound water, causing fibroin precipitation and aggregation.
[0008] Other silk fibroin gels have been produced by, for example,
mixing an aqueous silk fibroin solution with protein derived
biomaterials such as gelatin or chitosan. Recombinant proteins
materials based on silk fibroin's structure have also been used to
create self-assembling hydrogel structures. Another silk gel, a
silk fibroin-poly-(vinyl alcohol) gel was created by freeze- or
air-drying an aqueous solution, then reconstituting in water and
allowing to self-assemble. Silk hydrogels have also been generated
by either exposing the silk solution to temperature condition of
4.degree. C. (Thermgel) or by adding thirty percent (v/v) glycerol
(Glygel). Silk hydrogels created via a freeze-thaw process have not
only been generated but also used in vitro as a cell culture
scaffold.
[0009] The use of silk hydrogels as biomaterial matrices has also
been explored in a number of ways. General research on hydrogels as
platforms for drug delivery, specifically the release behavior of
benfotiamine (a synthetic variant of vitamin B.sub.1) coupled to
silk hydrogel was investigated. The study revealed both silk
concentration and addition of other compounds may factor in to the
eventual release profile of the material. Similarly, the release of
FITC-labeled dextran from a silk hydrogel could be manipulated by
altering the silk concentrations within the gel.
[0010] Further studies of silk hydrogels have been performed in
vivo as well. For example, the material has been used in vivo to
provide scaffolding for repair of broken bones in rabbits and
showed an accelerated healing rate relative to control animals. Of
particular interest, the in situ study also illustrated that the
particular formulation of silk hydrogel did not elicit an extensive
immune response from the host.
[0011] Despite early promise with silk hydrogel formulations in
vivo, sericin contamination remains a concern in their generation
and use just as with native fibroin for reasons of biocompatibility
as well as the potential for sericin to alter gelation kinetics.
The existence of sericin molecules in the silk solution
intermediate prior to gelation may also compromise final gel
structural quality, i.e., the distribution of .beta.-sheet
structure. For these reasons the removal of sericin from silk
fibroin material prior to hydrogel manufacture remains a concern.
The potential for disruption of gelation kinetics and structure by
contaminants also presents the need for development of a process
which consistently ensures structural uniformity and
biocompatibility.
SUMMARY
[0012] The present specification provides novel dermal fillers
useful for treating skin conditions.
[0013] Thus, aspects of the present specification disclose a
composition comprising a gel phase, wherein the gel phase includes
hydrogel particles comprising a silk fibroin. In other aspects of
the present specification disclose a composition comprising a gel
phase and a carrier phase, wherein the gel phase includes hydrogel
particles comprising a silk fibroin.
[0014] Other aspects of the present specification disclose a
composition comprising a gel phase, wherein the gel phase includes
a) hydrogel particles comprising a silk fibroin and b) hydrogel
particles comprising a matrix polymer. Matrix polymers useful to
make such compositions include, without limitation,
glycosaminoglycans (like chondroitin sulfate, dermatan sulfate,
keratan sulfate, heperan, heperan sulfate, and hyaluronan),
lubricins, and polysaccharides (like dextran, dextrin, starch,
hetastarch, glycogen, polyvinyl acetate, polyvinyl pyrrolidone,
polyethylene glycol, polyethylene imine, cellulose, methyl
cellulose, carboxymethyl cellulose, hydroxyethyl cellulose,
hydroxypropyl cellulose, hydroxyethyl methyl cellulose, and
hydroxypropyl methyl cellulose).
[0015] Yet other aspects of the present specification provide a
method of treating a skin condition in an individual in need
thereof, the method comprising the steps of administering a
composition disclosed herein into a dermal region of the
individual, wherein the administration improves the skin condition.
Skin conditions treated by the disclosed compositions include,
without limitation, augmentations, reconstructions, diseases,
disorders, defects, or imperfections of a body part, region or
area. In one aspect, a skin condition treated by the disclosed
compositions include, without limitation, a facial augmentation, a
facial reconstruction, a facial disease, a facial disorder, a
facial defect, or a facial imperfection. In one aspect, a skin
condition treated by the disclosed compositions include, without
limitation, skin dehydration, a lack of skin elasticity, skin
roughness, a lack of skin tautness, a skin stretch line or mark,
skin paleness, a dermal divot, a sunken check, a thin lip, a
retro-orbital defect, a facial fold, or a wrinkle.
DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 illustrates the impact of 23RGD on the gelation times
of silk hydrogels manufactured under various circumstances for
example without enhancers or with a water/23RGD enhancer (FIG. 1A),
or with an ethanol enhancer or combined ethanol-23RGD enhancers
(FIG. 1B). Depending upon the ratio of 23RGD to silk used and the
specific enhancer solvents, the peptide may function as either an
accelerant or decelerant of the process.
[0017] FIG. 2 is a graph of HPLC data illustrating the integration
of 23RGD and stability of its binding to 4% silk gel material made
with an enhancer solution consisting of a 3:1 molar ratio of
23RGD:silk dissolved in 90% ethanol, 10% water when rinsed multiple
times in ultra-purified water over several days. Data are shown for
both total peak area and calculated 23RGD:silk molar ratio based on
a 23RGD standard curve.
[0018] FIG. 3 is a graph comparing gel dry mass component at
different RGD concentrations for 2% silk gels (A), 4% silk gels
(B), and 6% gels (C). *Samples differ significantly, p<0.05;
.dagger. sample differs significantly from all others; .dagger-dbl.
all samples differ significantly.
[0019] FIG. 4 illustrates the impact upon silk hydrogel water
absorption and retention as identified in a gel drying assay. Data
are shown as the percentage of mass retained by a silk gel sample
(n=4 for each type) after being subjected to a 96-hour
lyophilization process. Increasing concentrations of 23RGD enhancer
caused increasing dry mass in the gel materials more substantial
than the mass of the peptide itself. This phenomenon is likely due
to structural differences in 23RGD-enhanced gels which do not
permit a level of water entrainment equal to those of gels enhanced
only with ethanol.
[0020] FIG. 5 shows a comparison of the percent mass loss over time
due to bioresorption of samples cast by PG and EEG methods (A),
cast from increasing silk concentrations (B), and cast using
increasing RGD concentrations (C). *Samples differ significantly,
p<0.05; .dagger. sample differs significantly from all others;
.dagger-dbl. all samples differ significantly.
[0021] FIG. 6 illustrates wet mass loss due to proteolytic
bioresorption of silk hydrogels enhanced by a combination of 23RGD
and ethanol at increasing concentrations of 23RGD. As a general
trend, gels enhanced with 23RGD tend to be bioresorbed more quickly
based upon this assay.
[0022] FIG. 7 is a second illustration of the bioresorption
behavior of 23RGD-enhanced and non-23RGD-enhanced silk hydrogels
when incubated in a protease solution. This bioresorption data
serves to reinforce the trend, illustrated in FIG. 5, of a slightly
more rapid rate of bioresorption of 23RGD-enhanced hydrogels in
comparison to non-23RGD-enhanced gels. The figure also supports the
more thorough removal of a-helix and random coil conformations from
23RGD-enhanced gels in FIG. 6 over four days of incubation in
protease.
[0023] FIG. 8 shows structural features observed by
Fourier-Transform Infrared (FTIR) spectroscopy of 4% silk fibroin
hydrogel devices which are enhanced by ethanol alone, and two
23RGD-ethanol enhancers. The full spectra (FIG. 9A) of the
materials are compared and the Amide I Band (1700-1600 cm.sup.-1)
highlighted for particular attention (FIG. 9B) because of its
relevance to secondary protein structure. Of specific interest is
the commonality between all gels in their rich .beta.-sheet
structure (1700 cm.sup.-1 and 1622 cm.sup.-1 respectively,
highlighted in FIGS. 9C and 9E) at all time points. These peaks
become more pronounced after bioresorption, and begin to
differentiate 23RGD-enhanced materials from materials enhanced with
ethanol alone. This is evidenced in 23RGD-enhanced gels by a peak
shift to lower wave numbers by the 1622 cm.sup.-1 peak and
dramatically increased prominence of the 1700 cm.sup.-1 peak.
Additional differences between bioresorbed and non-bioresorbed gels
may be seen in regions of the spectrum known to correlate to
.alpha.-helix and random coil conformations (1654 cm.sup.-1 and
1645 cm.sup.-1 respectively highlighted in FIG. 9D). These
conformations are extensively digested in all gel types, but most
completely in gels enhanced by 23RGD. This suggests that
23RGD-enhanced gels tend to bioresorb to a very .beta.-sheet rich
secondary structure in a more rapid fashion than non-23RGD-enhanced
gels. Spectra shown were collected on a Bruker Equinox 55 FTIR unit
using a compilation of 128 scans with a resolution of 4
cm.sup.-1.
[0024] FIG. 9 shows a comparative FTIR spectra illustrating the
effects of differing gelation techniques on gel protein structure
before (Day 0) and after (Day 4) proteolytic bioresorption. Groups
assessed included samples cast by PG and EEG methods (A), cast from
increasing silk concentrations (B), and cast using increasing RGD
concentrations (C).
[0025] FIG. 10 shows representative micrographs of H&E-stained
histological sections collected from silk gels implanted
subcutaneously in rats. Samples of 4% silk fibroin hydrogel formed
by passive gelation (4P), 4% silk fibroin hydrogel formed by
ethanol-enhanced gelation (4E), and 6% silk fibroin hydrogel formed
by ethanol-enhanced gelation (6E) were compared at 7 days (A, B,
and E respectively) with 4E and 6E samples compared again at days
28 (C and F) and 57 (D and G).
[0026] FIG. 11 shows representative gross photographs of 8% silk
fibroin hydrogel devices both unmodified (A) and 23RGD-enhanced (D)
after a two-week subcutaneous incubation in Lewis rats. Also shown
are micrographs resultant from H & E stains of the unmodified
(B and C) and 23RGD-coupled (E and F) samples at 10.times. and
20.times. magnification. These gross images coupled with the
histological micrographs provide evidence of a less extensive
inflammatory response during early device integration being
associated with 23RGD-enhanced gel than non-23RGD-enhanced gel.
[0027] FIG. 12 shows representative histology collected from a
thirteen-week study of 4% 3:1 23RGD-enhanced silk hydrogel blended
with 25% saline (left panels, H&E stain Trichrome stain) and
ZYPLAST.TM. (right panels H&E stain, Trichrome stain) and
injected into the intradermis of guinea pig. Each material type
exhibited some clear evidence of implanted device in 75% of their
respective implant sites. These micrographs indicate strong
similarities not only between the long-term bioresorption
characteristics but also long-term host tissue response between
collagen-derived biomaterials and this particular 23RGD-enhanced
silk hydrogel formulation.
[0028] FIG. 13 shows representative micrographs of H&E-stained
histological sections collected from Day 28 explants of 4% silk
fibroin, 10% saline (A); 4% silk fibroin, 1:1 23RGD, 10% saline
(B); 6% silk fibroin, 1:1 23RGD, 10% saline (C); ZYPLAST.TM. (D);
4% silk fibroin, 25% saline (E); 4% silk fibroin, 1:1 23RGD, 25%
saline (F); 6% silk fibroin, 10% saline (G); HYLAFORM.TM. (H); 6%
silk fibroin, 25% saline (I); 4% silk fibroin, 3:1 23RGD, 25%
saline (J); and 6% silk fibroin, 1:1 23RGD, 25% saline (K).
[0029] FIG. 14 shows representative micrographs of Day 92
histological sections of 4% silk fibroin, 3:1 23RGD, 25% saline
(A-D) and ZYPLAST.TM. samples (E-H) stained with H&E at
4.times. (A and E), 10.times. (B and F), stained with Masson's
Trichrome at 10.times. (C and G) and under polarized light at
10.times. (D and H).
[0030] FIG. 15 is a photograph of a custom-built testing jig used
in conjunction with an Instron 8511 (Instron Corporation, Canton
Mass.) in conjunction with Series IX software and a 100 N load cell
for characterizing the injection forces associated with forcing
silk gel through a 30 g needle.
[0031] FIG. 16 illustrates the average extrusion force data from
mechanical testing of various silk gel formulations illustrating
the effects of changing comminution method (A), saline
concentration (B), silk concentration (C), and RGD content (D).
Values are reported as an average of n=4 tests at each displacement
rate with standard deviation illustrated as error bars. *Samples
differ significantly, p<0.05; .dagger. sample differs
significantly from all others in group at same strain rate;
.dagger-dbl. all samples in group differ significantly from all
others in group at same strain rate.
[0032] FIG. 17 shows representative ESEM micrographs of selected
RGD/ethanol-induced silk precipitates generated from the previously
mentioned formulations. BASE (A), SCVLO (B), RHI (C), RLO (D), AVHI
(E), ECLO (F), AVLO (G), and 3R 6.7:1 (H) are shown at 200.times.
magnification.
[0033] FIG. 18 shows a comparison of the total dry mass of
precipitate recovered from each silk precipitate formulation (n=4
for each type) after being subjected to a 96-hour lyophilization
process. Data are grouped to compare the effects of changing volume
ratio of accelerant added (A), concentration of 23RGD in the
accelerant (B), changing the initial silk concentration (C), and
changing the concentration of ethanol in the accelerant (D). It was
shown that increasing any of these volumes or concentrations
resulted in greater quantities of precipitate, though none appear
to have substantially greater impact than another. This phenomenon
is likely due to basic kinetics of the assembly reaction, with each
reagent in turn appearing both as an excess and as limiting
dependent upon the specific formulation. *--significant difference,
p<0.05; .dagger.--Group differs significantly from all
others.
[0034] FIG. 19 shows a comparison of the percentage of dry mass in
each of precipitate recovered (n=4 for each type) after being
subjected to a 96-hour lyophilization process. Data are grouped to
compare the effects of changing volume ratio of accelerant added
(A), concentration of 23RGD in the accelerant (B), changing the
initial silk concentration (C), and changing the concentration of
ethanol in the accelerant (D). Increasing the concentration of
23RGD used increased the dry mass percentage of precipitates, while
increasing the ethanol percentage in the accelerant decreased dry
mass. These changes may stem from formation of altered gel network
structures caused by manipulation of these variables, likely more
crystalline in the case of 23RGD increases and less crystalline in
the case of ethanol concentration increases. *--significant
difference, p<0.05; .dagger.--Group differs significantly from
all others.
[0035] FIG. 20 shows representative FTIR spectra of the Amide I
band for 23RGD/ethanol-induced silk precipitates immediately after
processing (D0). Spectra are grouped to compare the effects of
changing volume ratio of accelerant added (A), concentration of
23RGD in the accelerant (B), changing the initial silk
concentration (C), and changing the concentration of ethanol in the
accelerant (D). These spectra illustrate that similarities exist
between all groups although changing 23RGD concentrations and
ethanol concentrations may substantially impact precipitate
structure. Increasing concentrations of decreased .beta.-sheet seen
in a peak shift from .about.1621 cm.sup.-1 in RVLO to .about.1624
cm.sup.-1 in RLO. A further increase in 23RGD concentration in both
BASE and RHI caused this weakened .beta.-sheet again along with
increased signal values in the 1654 cm.sup.-1 and 1645 cm.sup.-1
range, correlating to increased random coil and .alpha.-helical
content. An increased percentage of ethanol decreased the content
of .alpha.-helical and random coil shown by decreased signal
between 1670 cm.sup.-1 and 1630 cm.sup.-1 in both ECLO and BASE
samples relative to ECVLO. This decrease in a-helical and random
coil is accompanied by an increase in .beta.-sheet structure. The
findings relating to 23RGD and ethanol concentrations reinforce the
trends observed in the percent dry mass of the precipitates,
supposing that a-helical and random coil motifs entrain more water
than .beta.-sheet regions.
[0036] FIG. 21 is a representative micrograph of Congo red-stained
23RGD/ethanol-induced silk precipitates under polarized light at
20.times. magnification. A lack of emerald-green birefringence
indicates a negative result in testing for amyloid fibril
formation.
[0037] FIG. 22 shows comparison of 23RGD:silk molar ratio in each
of precipitate recovered. Data are grouped to compare the effects
of changing volume ratio of accelerant added (A), concentration of
23RGD in the accelerant (B), changing the initial silk
concentration (C), and changing the concentration of ethanol in the
accelerant (D). In examining the 23RGD bound to the precipitates,
all materials contained more 23RGD than predicted by initial
calculations aside of AVHI, RVLO, RHI, and SCVLO. In the cases of
AVHI and ECLO the 23RGD quantity was substantially more than was
expected. In the cases of BASE, RLO, SCVLO, and SCLO the 23RGD
quantities were approximately double that expected. This may be
indicative of the formation of a 23RGD dimer in the 90% ethanol
accelerant solution. The RVLO samples were made with a 23RGD
concentration of 0.49 mg/mL in the accelerant, the lowest used in
this study and potentially within the solubility range of 23RGD in
90% ethanol. RLO samples used 1.47 mg/mL and most other
formulations were made with a 23RGD accelerant concentration of
2.45 mg/mL, above the 23RGD concentration at which dimerization
became favorable in the solution. Further highlighting the
possibility of 23RGD dimerizing in the ethanol solution is the
behavior of ECLO precipitation. The 23RGD concentration remains
2.45 mg/mL as with BASE and AVLO but the water concentration in the
accelerant is increased to 20% and results in a binding of about
1.5-fold the expected total of 23RGD instead of 2-fold. This may be
due to dis-solution of a greater quantity of 23RGD, causing
coexistence between dimeric and monomeric 23RGD in solution
reflected in the subsequent binding ratios. *--significant
difference, p<0.05; .dagger.--Group differs significantly from
all others; .dagger-dbl.--All groups differ significantly.
[0038] FIG. 23 shows a representative FTIR spectra of the Amide I
band are shown for 23RGD/ethanol-induced silk precipitates
initially (D0) and after proteolytic bioresorption (D2). Spectra
are grouped to compare the effects of changing volume ratio of
accelerant added (A), concentration of 23RGD in the accelerant (B),
changing the initial silk concentration (C), and changing the
concentration of ethanol in the accelerant (D). Accelerant quantity
added did not substantially affect the bioresorption behavior of
the materials as BASE, AVHI and AVLO all featured decreased levels
of a-helix and random coil motifs. This decrease was slightly
larger in the case of AVLO which also featured a peak shift from
1624 cm.sup.-1 to 1622 cm.sup.-1, indicating a more stable
.beta.-sheet structure. 23RGD concentration did not appear to
affect bioresorption behavior of the materials either as RVLO, RLO,
BASE and RHI all showed decreased in .alpha.-helix and random coil
motifs, though a greater portion of a-helix and random coil
remained intact in RHI. Silk concentration did not substantially
affect the bioresorption behavior of the materials as BASE and SCLO
exhibited decreased levels of a-helix and random coil motifs and
featured slight peak shifts from 1624 cm.sup.-1 to 1623
cm.sup.-1.
[0039] FIG. 24 shows a H&E staining of tissue samples injected
with a dermal filler comprising 100% hyaluronan, a dermal filler
comprising 95% hyaluronan and 5% silk fibronin hydrogel, a dermal
filler comprising 75% hyaluronan and 25% silk fibronin hydrogel, a
dermal filler comprising 50% hyaluronan and 50% silk fibronin
hydrogel, a dermal filler comprising 25% hyaluronan and 75% silk
fibronin hydrogel, and a dermal filler comprising 100% silk
fibronin hydrogel.
[0040] FIG. 25 shows on the tope row, a H&E staining of tissue
samples injected with a dermal filler comprising 100% hyaluronan, a
dermal filler comprising 50% hyaluronan and 50% silk fibronin
hydrogel, and a dermal filler comprising 100% silk fibronin
hydrogel; and on the bottom row a CD-68 staining of tissue samples
injected with a dermal filler comprising 100% hyaluronan, a dermal
filler comprising 50% hyaluronan and 50% silk fibronin hydrogel,
and a dermal filler comprising 100% silk fibronin hydrogel.
DETAILED DESCRIPTION
[0041] Aspects of the present specification provide, in part, a
composition comprising a gel phase including a hydrogel comprising
a silk fibroin. As used herein, the term "silk fibroin" is
synonymous with "polymerized silk fibroin" and refers to silk
fibroin existing primarily as a polymer. A hydrogel comprising
polymerized silk fibroin or silk fibroin is made by, e.g., a
gelation process disclosed herein.
[0042] Aspects of the present specification provide, in part, a
depolymerized silk fibroin. As used herein, the term "depolymerized
silk fibroin" is synonymous with "dissolved silk" and "dissolved
silk fibroin" and refers to silk fibroin existing primarily as
monomers or other lower oligomeric units. Treatment of
naturally-occurring fibrous silk with a dissolution agent, such as,
e.g., a chaotropic agent results in depolymerized silk fibroin. The
depolymerized silk fibroin used for preparing silk fibroin hydrogel
is an intermediate in the silk hydrogel production process and a
direct precursor to the hydrogel material. The depolymerized silk
fibroin can be made from raw cocoons, previously degummed silk or
any other partially cleaned silk. This may also include material
commonly termed as "waste" from the reeling process, i.e. short
fragments of raw or degummed silk, the sole precaution being that
the silk must be substantially cleaned of sericin prior to making
fibroin solution and inducing gel formation. A particular source of
raw silk is from common domesticated silkworm B. marl, though
several other sources of silk may be appropriate. This includes
other strains of Bombycidae including Antheraea pernyi, Antheraea
yamamai, Antheraea mylitta, Antheraea assama, and Philosamia
cynthia ricini,as well as silk producing members of the families
Saturnidae, Thaumetopoeidae, and silk-producing members of the
order Araneae. The material may also be obtained from other spider,
caterpillar, or recombinant sources.
[0043] A hydrogel disclosed herein provide for a depolymerized silk
fibroin and/or silk fibroin that are substantially free of sericin.
Methods for performing sericin extraction have been described in
pending U.S. patent application Ser. No. 10/008,924, U.S.
Publication No. 2003/0100108, Matrix for the production of tissue
engineered ligaments, tendons and other tissue. That application
refers to cleaned fibroin fibers spun into yarns, used to create a
porous, elastic matrix suitable as a substrate for applications
requiring very high tensile strength, such as bioengineered
ligaments and tendons.
[0044] Extractants such as urea solution, hot water, enzyme
solutions including papain among others which are known in the art
to remove sericin from fibroin would also be acceptable for
generation of the silk. Mechanical methods may also be used for the
removal of sericin from silk fibroin. This includes but is not
limited to ultrasound, abrasive scrubbing and fluid flow. The rinse
post-extraction is conducted preferably with vigorous agitation to
remove substantially any ionic contaminants, soluble, and in
soluble debris present on the silk as monitored through microscopy
and solution electrochemical measurements. A criterion is that the
extractant predictably and repeatably remove the sericin coat of
the source silk without significantly compromising the molecular
structure of the fibroin. For example, an extraction may be
evaluated for sericin removal via mass loss, amino acid content
analysis, and scanning electron microscopy. Fibroin degradation may
in turn be monitored by FTIR analysis, standard protein gel
electrophoresis and scanning electron microscopy.
[0045] In certain cases, the silk utilized for generation of a silk
hydrogel has been substantially depleted of its native sericin
content (i.e., s 4% (w/w) residual sericin in the final extracted
silk). Alternatively, higher concentrations of residual sericin may
be left on the silk following extraction or the extraction step may
be omitted. In aspects of this embodiment, the sericin-depleted
silk fibroin has, e.g., about 1% (w/w) residual sericin, about 2%
(w/w) residual sericin, about 3% (w/w) residual sericin, or about
4% (w/w) residual sericin. In other aspects of this embodiment, the
sericin-depleted silk fibroin has, e.g., at most 1% (w/w) residual
sericin, at most 2% (w/w) residual sericin, at most 3% (w/w)
residual sericin, or at most 4% (w/w) residual sericin. In yet
other aspects of this embodiment, the sericin-depleted silk fibroin
has, e.g., about 1% (w/w) to about 2% (w/w) residual sericin, about
1% (w/w) to about 3% (w/w) residual sericin, or about 1% (w/w) to
about 4% (w/w) residual sericin.
[0046] In certain cases, the silk utilized for generation of a silk
hydrogel is entirely free of its native sericin content. As used
herein, the term "entirely free (i.e. "consisting of" terminology)
means that within the detection range of the instrument or process
being used, the substance cannot be detected or its presence cannot
be confirmed.
[0047] In certain cases, the silk utilized for generation of a silk
hydrogel is essentially free of its native sericin content. As used
herein, the term "essentially free" (or "consisting essentially
of") means that only trace amounts of the substance can be
detected.
[0048] Additionally, the possibility exists for deliberately
modifying hydrogel properties through controlled partial removal of
silk sericin or deliberate enrichment of source silk with sericin.
This may function to improve hydrogel hydrophilicity and eventual
host acceptance in particular biological settings despite sericin
antigen icily.
[0049] After initial degumming or sericin removal from fibrous silk
used for generation of a hydrogel, the silk is rinsed free of gross
particulate debris. It is of concern to remove such particles as
either solvent (i.e., specific solvent of interest for device
generation) soluble or insoluble compounds may profoundly affect
the outcome of the hydrogel generated from the intermediate
solution. Insoluble compounds may serve as nucleation points,
accelerating the gelation phenomenon and potentially altering
subsequent hydrogel protein structure. Soluble compounds may also
serve to interface with the protein network of the hydrogel,
altering the organizational state of the device. Either type of
compound could also compromise biocompatibility of the device.
[0050] Prior to dissolution, the prepared silk may be subjected to
association of various molecules. The binding between these
compounds and the silk molecules may be unaffected by the
dissolving agent used for preparation of silk solution
intermediate. The method for coupling the modifying compound to the
prepared silk may vary dependent upon the specific nature of the
bond desired between silk sequence and the modifier. Methods are
not limited to but may include hydrogen bonding through affinity
adsorption, covalent crosslinking of compounds or sequential
binding of inactive and active compounds. These molecules may
include, but would not be limited to, inorganic compounds,
peptides, proteins, glycoproteins, proteoglycans, ionic compounds,
natural, and synthetic polymers. Such peptides, proteins,
glycoproteins and proteoglycans may include classes of molecules
generally referred to as "growth factors", "cytokines",
"chemokines", and "extracellular matrix compounds". These compounds
might include such things as surface receptor binding motifs like
arginine-glycine-aspartic acid (RGD), growth factors like basic
fibroblast growth factor (bFGF), platelet derived growth factor
(PDGF), transforming growth factor (TGF), cytokines like tumor
necrosis factor (TNF), interferon (IFN), interleukins (IL), and
structural sequences including collagen, elastin, hyaluronic acid
and others. Additionally recombinant, synthetic, or non-native
polymeric compounds might be used as decoration including chitin,
poly-lactic acid (PLA), and poly-glycolic acid (PGA). Other
compounds linked to the material may include classes of molecules
generally referred to as tracers, contrasting agents, aptamers,
avimers, peptide nuclei acids and modified polysaccharide
coatings.
[0051] For example, the initially dissolved silk may be generated
by a 4 hour digestion at 60.degree. C. of pure silk fibroin at a
concentration of 200 g/L in a 9.3 M aqueous solution of lithium
bromide to a silk concentration of 20% (w/v). This process may be
conducted by other means provided that they deliver a similar
degree of dissociation to that provided by a 4 hour digestion at
60.degree. C. of pure silk fibroin at a concentration of 200 g/L in
a 9.3 M aqueous solution of lithium bromide. The primary goal of
this is to create uniformly and repeatably dissociated silk fibroin
molecules to ensure similar fibroin solution properties and,
subsequently, device properties. Less substantially dissociated
silk solution may have altered gelation kinetics resulting in
differing final gel properties. The degree of dissociation may be
indicated by Fourier-transform Infrared Spectroscopy (FTIR) or
x-ray diffraction (XRD) and other modalities that quantitatively
and qualitatively measure protein structure. Additionally, one may
confirm that heavy and light chain domains of the silk fibroin
dimer have remained intact following silk processing and
dissolution. This may be achieved by methods such as standard
protein sodium-dodecyl-sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) which assess molecular weight of the independent silk
fibroin domains.
[0052] System parameters which may be modified in the initial
dissolution of silk include but are not limited to solvent type,
silk concentration, temperature, pressure, and addition of
mechanical disruptive forces. Solvent types other than aqueous
lithium bromide may include but are not limited to aqueous
solutions, alcohol solutions, 1,1,1,3,3,3-hexafluoro-2-propanol,
and hexafluoroacetone, 1-butyl-3-methylimidazolium. These solvents
may be further enhanced by addition of urea or ionic species
including lithium bromide, calcium chloride, lithium thiocyanate,
zinc chloride, magnesium salts, sodium thiocyanate, and other
lithium and calcium halides would be useful for such an
application. These solvents may also be modified through adjustment
of pH either by addition of acidic of basic compounds.
[0053] Further tailoring of the solvent system may be achieved
through modification of the temperature and pressure of the
solution, as ideal dissolution conditions will vary by solvent
selected and enhancers added. Mechanical mixing methods employed
may also vary by solvent type and may vary from general agitation
and mixing to ultrasonic disruption of the protein aggregates.
Additionally, the resultant dissolved silk concentration may be
tailored to range from about 1% (w/v) to about 30% (w/v). It may be
possible to expand this range to include higher fractions of
dissolved silk depending upon the specific solvent system utilized.
In one example, following initial dissolution of the processed
silk, the silk protein may be left in a pure aqueous solution at 8%
(w/v) silk. This is accomplished by removal of the residual solvent
system while simultaneously ensuring that the aqueous component of
the silk solution is never fully removed nor compromised. In a
situation which involves an initial solution of 200 g/L silk in a
9.3 M aqueous solution of lithium bromide, this end is accomplished
by a dialysis step.
[0054] In aspects of this embodiment, the depolymerized silk
fibroin (dissolved silk fibroin) has a concentration of, e.g.,
about 1% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v),
about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v),
about 9% (w/v), about 10% (w/v), about 12% (w/v), about 15% (w/v),
about 18% (w/v), about 20% (w/v), about 25% (w/v), or about 30%
(w/v). In other aspects of this embodiment, the depolymerized silk
fibroin (dissolved silk fibroin) has a concentration of, e.g., at
least 1% (w/v), at least 2% (w/v), at least 3% (w/v), at least 4%
(w/v), at least 5% (w/v), at least 6% (w/v), at least 7% (w/v), at
least 8% (w/v), at least 9% (w/v), at least 10% (w/v), at least 12%
(w/v), at least 15% (w/v), at least 18% (w/v), at least 20% (w/v),
at least 25% (w/v), or at least 30% (w/v). In yet other aspects of
this embodiment, the depolymerized silk fibroin (dissolved silk
fibroin) has a concentration of, e.g., about 1% (w/v) to about 5%
(w/v), about 1% (w/v) to about 10% (w/v), about 1% (w/v) to about
15% (w/v), about 1% (w/v) to about 20% (w/v), about 1% (w/v) to
about 25% (w/v), about 1% (w/v) to about 30% (w/v), about 5% (w/v)
to about 10% (w/v), about 5% (w/v) to about 15% (w/v), about 5%
(w/v) to about 20% (w/v), about 5% (w/v) to about 25% (w/v), about
5% (w/v) to about 30% (w/v), about 10% (w/v) to about 15% (w/v),
about 10% (w/v) to about 20% (w/v), about 10% (w/v) to about 25%
(w/v), or about 10% (w/v) to about 30% (w/v).
[0055] Example dialysis conditions include a 3 mL-12 mL sample
volume dialysis cassettes with 3.5 kD molecular weight cutoff
cellulose membranes dialyzed for three days against ultra-pure
water with a series of six changes at regular intervals while
stirring constantly. Each cassette, 5 mL-12 mL cartridge size, may
be loaded (for example via 20-mL syringe) with 12 mL of a 20%
solution of silk dissolved in 9.3 M lithium bromide via an 18 gauge
needle. The resultant silk solution may be 8%.+-.0.5% (w/v). The
silk solution may be stored at a range of -80.degree. C. to
37.degree. C., such as 4.degree. C. prior to use. One method is to
dialyze the solution against water using a 3.5 kD molecular weight
cutoff cellulose membrane, for example, at one 12 mL cartridge per
1 L water in a 4 L beaker with stirring for 48 hours or 72 hours.
Water may be changed several times during the dialysis, for example
at 1 hour, 4 hours, 12 hours, 24 hours, and 36 hours (total of six
rinses). In other embodiments, this membrane may take the shape of
a cassette, tubing or any other semi-permeable membrane in a batch,
semi-continuous or continuous system. If desired, the concentration
of silk in solution may be raised following the original dialysis
step by inclusion of a second dialysis against a hygroscopic
polymer such as PEG, a poly(ethylene oxide) or amylase.
[0056] The parameters applied to the dialysis step may be altered
according to the specific needs or requirements of the particular
solution system involved. Although it may be undesirable to change
membrane composition or pore size in the interests of maintaining
efficiency of the process, it would be possible to change the
structuring of the dialysis barrier, as a dialysis tube or any
large semi-permeable membrane of similar construction should
suffice. Additionally it should be considered that any alteration
in the nature of the physical dialysis interface between solution
and buffer might alter rates of ion flux and thereby create
membrane-localized boundary conditions which could affect solution
dialysis and gelation rate kinetics. The duration and volume ratios
associated with this dialysis process must be tailored to any new
system as well, and removal of the solvent phase should be ensured
after purification before proceeding.
[0057] It is also possible to change the buffer phase in the
dialysis system, altering water purity or adding hygroscopic
polymers to simultaneously remove ions and water from the initial
silk solution. For example, if necessary, the silk solution can be
concentrated by dialysis against a hygroscopic polymer, for
example, PEG, a polyethylene oxide or amylase. The apparatus used
for dialysis can be cassettes, tubing, or any other semi-permeable
membrane.
[0058] Insoluble debris may be removed from the dialyzed silk
solution by centrifugation or filtration. For example, the dialyzed
silk may be removed from the cassette with a needle and syringe
(e.g., an 18 g needle at 20 mL syringe), and placed into a clean
centrifuge tube with sufficient volume (e.g., 40 mL). The
centrifuge may be run at 30,000 g relative centrifugal force (RCF)
for 30 minutes at 4.degree. C. The resulting supernatant may be
collected and centrifuged again under identical conditions, and the
remaining supernatant collected (e.g., in a 50 mL test tube) and
stored at 4.degree. C. The silk solution may also be evaluated via
X-ray photoelectron spectroscopy (to check for lithium bromide
residue) and dry mass (to check solution for dry protein mass,
concentration w/v).
[0059] Additionally, dependent upon the initial silk solvent, it
might be desirable to remove portions of either the silk phase or
solvent phase from the solution via an affinity column separation.
This could be useful in either selectively binding specific solvent
molecules or specific solute molecules to be eluted later in a new
solvent. The possibility also exists for a lyophilization of the
depolymerized silk fibroin (dissolved silk) followed by a
reconstitution step. This would be most useful in a case where
removing a solvent, is unlikely to leave residue behind. In the
case of a lyophilized solution, either used as a purification step
or as a procedure subsequent to purification, the type of solvent
used for reconstitution might be tailored for the process at hand.
Desirable solvents might include but are not limited to aqueous
alcohol solutions, aqueous solutions with altered pH, and various
organic solutions. These solvents may be selected based upon a
number of parameters which may include but are not limited to an
enhanced gelation rate, altered gel crystalline structure, altered
solution intermediate shelf-life, altered silk solubility, and
ability to interact with environmental milieu such as temperature
and humidity.
[0060] In certain embodiments, a silk hydrogel is prepared from
dissolved silk fibroin solution that uses an agent to enhance
gelation and an agent to improve the gel's biocompatibility. In
some instances, the same agent both enhances gelation and improves
biocompatibility. An example agent that both improves gel
biocompatibility and serves as a gelation enhancer is an
amphiphilic peptide which binds to silk molecules through
hydrophobic interactions, such as, e.g., a non-RDG integrin or a
RGD motif containing peptide like 23RGD. In other instances,
different agents serve these purposes. An example of an agent that
serves as a gelation enhancer is an alcohol, such as, e.g.,
ethanol, methanol, and isopropanol; glycerol; and acetone.
[0061] Regarding gelation enhancers, to accelerate the phenomenon
of silk gelation, a depolymerized silk fibroin solution (dissolved
silk solution) may be mixed with pure alcohol or aqueous alcohol
solution at varied volume ratios accompanied by mixing, either
through stirring, shaking or any other form of agitation. This
alcohol solution enhancer may then have a quantity of an
amphiphilic peptide added as a further enhancer of the final gel
outcome. The extent of acceleration may be heightened or lessened
by adding a larger or smaller enhancer component to the system.
[0062] In addition to organics, the gelation rate may be enhanced
by increasing the concentration of the depolymerized silk fibroin
(dissolved silk). This is done by methods including but not limited
to dialysis of intermediate silk solution against a buffer
incorporating a hygroscopic species such as polyethylene glycol, a
lyophilization step, and an evaporation step. Increased temperature
may also be used as an enhancer of the gelation process. In
addition to this, manipulation of intermediate silk solution pH by
methods including but not limited to direct titration and gas
exchange may be used to enhance the gelation process. Introduction
of select ionic species including calcium and potassium in
particular may also be used to accelerate gelation rate.
[0063] Nucleating agents including organic and inorganic species,
both soluble and insoluble in an aqueous silk solution intermediate
may be used to enhance the gelation process. These may include but
are not limited to peptide sequences which bind silk molecules,
previously gelled silk, and poorly soluble p-sheet rich structures.
A further means of accelerating the gelation process is through the
introduction of mechanical excitation. This might be imparted
through a shearing device, ultrasound device, or mechanical mixer.
It should be borne in mind that any of these factors might
conceivably be used in concert with any other or group of others
and that the regime would need to be tailored to the desired
outcome.
[0064] The time necessary for complete silk solution gelation may
vary from seconds to hours or days, depending on the values of the
above mentioned parameters as well as the initial state of
aggregation and organization found in the silk solution (FIG. 1).
The volume fraction of added enhancer may vary from about 0% to
about 99% of the total system volume (i.e., either component may be
added to a large excess of the other or in any relative
concentration within the interval). The concentration of silk
solution used can range from about 1% (w/v) to about 20% (w/v). The
enhancer can be added to silk solution or the silk solution can be
added to enhancer. The formed silk hydrogel may be further
chemically or physically cross-linked to gain altered mechanical
properties.
[0065] In aspects of this embodiment, an enhancer solution is added
to a depolymerized silk fibroin (dissolved silk fibroin) solution,
the depolymerized silk fibroin solution having a concentration of
depolymerized silk fibroin of, e.g., about 1% (w/v), about 2%
(w/v), about 3% (w/v), about 4% (w/v), about 5% (w/v), about 6%
(w/v), about 7% (w/v), about 8% (w/v), about 9% (w/v), about 10%
(w/v), about 12% (w/v), about 15% (w/v), about 18% (w/v), about 20%
(w/v), about 25% (w/v), or about 30% (w/v). In other aspects of
this embodiment, an enhancer solution is added to a depolymerized
silk fibroin (dissolved silk fibroin) solution, the depolymerized
silk fibroin solution having a concentration of depolymerized silk
fibroin of, e.g., at least 1% (w/v), at least 2% (w/v), at least 3%
(w/v), at least 4% (w/v), at least 5% (w/v), at least 6% (w/v), at
least 7% (w/v), at least 8% (w/v), at least 9% (w/v), at least 10%
(w/v), at least 12% (w/v), at least 15% (w/v), at least 18% (w/v),
at least 20% (w/v), at least 25% (w/v), or at least 30% (w/v). In
yet other aspects of this embodiment, an enhancer solution is added
to a depolymerized silk fibroin (dissolved silk fibroin) solution,
the depolymerized silk fibroin solution having a concentration of
depolymerized silk fibroin of, e.g., about 1% (w/v) to about 5%
(w/v), about 1% (w/v) to about 10% (w/v), about 1% (w/v) to about
15% (w/v), about 1% (w/v) to about 20% (w/v), about 1% (w/v) to
about 25% (w/v), about 1% (w/v) to about 30% (w/v), about 5% (w/v)
to about 10% (w/v), about 5%(w/v) to about 15% (w/v), about 5%(w/v)
to about 20% (w/v), about 5% (w/v) to about 25% (w/v), about 5%
(w/v) to about 30% (w/v), about 10% (w/v) to about 15% (w/v), about
10% (w/v) to about 20% (w/v), about 10% (w/v) to about 25% (w/v),
or about 10% (w/v) to about 30% (w/v).
[0066] Aspects of the present specification provide, in part, a
hydrogel comprising an amphiphilic peptide. As used herein, the
term "amphiphilic peptide" refers to a peptide that includes both
hydrophobic and hydrophilic properties. Many other amphiphilic
molecules interact strongly with biological membranes by insertion
of the hydrophobic part into the lipid membrane, while exposing the
hydrophilic part to the aqueous environment. Particular embodiments
of hydrogels include silk fibroin, silk fibroin with 23RGD, silk
fibroin with alcohol and 23RGD, and silk fibroin with alcohol,
23RGD, and saline/PBS. The amount, relative ratio and sequence of
adding the components will change according to the specific
requirement for the device.
[0067] Additionally, an amphiphilic peptide may accelerate the
phenomenon of silk gelation under certain circumstances. Such gel
may be produced through combination of dissolved silk fibroin
solution and an enhancer solution of amphiphilic peptide in alcohol
across the silk concentration ranges from about 1% (w/v) to about
20% (w/v), amphiphilic peptide concentration ranges from about
1:100 to 100:1 moles 23RGD:moles silk, and alcohol concentration
ranges from about 1% (v/v) to about 99% (v/v) before removal. Thus,
for example, a particular silk gel is produced through direct
contact between an aqueous solution of depolymerized silk fibroin
and an enhancer solution comprising 23RGD in ethanol. For example,
the dissolved silk solution may be mixed with a 23RGD suspended in
pure ethanol or aqueous ethanol solution at varied volume ratios
accompanied by mixing, either through stirring, shaking or any
other form of agitation.
[0068] More specifically, as a non-limiting example, to infuse the
silk fibroin hydrogel with 23RGD, the 23RGD is first dissolved in a
solution of ethanol and water (e.g., 90% ethanol in purified water)
in an amount to generate the planned silk and 23RGD concentrations
of the final gel, and mixed (e.g., vortexed until there is no
visible 23RGD particulate). This solution is then mixed with
dissolved silk solution (e.g., by pipetting rapidly for 1-2
seconds). The gelling mixture may be allowed to stand covered under
ambient conditions for a suitable period, for example 24 hours (or
24 hours after the gel has solidified depending on enhancer
conditions).
[0069] The amount of time required for dissolved silk solutions to
gel may vary from seconds to hours or days, depending on the ratio
of silk solution volume and enhancer solution volume, dissolved
silk fibroin concentration, enhancer solution concentration,
enhancer type and amphiphilic peptide concentration. The
amphiphilic peptide may be mixed into the dissolved silk solution
in a variety of ways, for example water-dissolved amphiphilic
peptide can be added to a dissolved silk solution to form a gel; an
amphiphilic peptide can be added to water, blended with an alcohol,
then added to a dissolved silk solution; or an amphiphilic peptide
can be added to a silk fibroin hydrogel. The molar ratio of
amphiphilic peptide:silk fibroin can range from 100 to 0.01, the
dissolved silk solution concentration can be from about 1% to about
20%.
[0070] An example of an amphiphilic peptide is a 23RGD peptide
having the amino acid sequence:
HOOC-Gly-Arg-Gly-Asp-Ile-Pro-Ala-Ser-Ser-Lys-Gly-Gly-Gly-Gly-Ser-Arg-Leu--
Leu-Leu-Leu-Leu-Leu-Arg-NH.sub.2 (abbreviated
HOOC-GRGDIPASSKG.sub.4SRL.sub.6R--NH.sub.2) (SEQ ID NO: 1).
Optionally, each of the arginine residues may be of the D-form,
which may stabilize the RG bond to serine proteases. Additionally,
the COO-terminus may be acylated to block proteolysis. This example
23RGD has the amino acid sequence
Ac-GdRGDIPASSKG.sub.4SdRL.sub.6dR--NH.sub.2 (SEQ ID NO: 2). It may
be advantageous to include a spacer domain in the RGD peptide, for
example, a peptide such as SG.sub.4KSSAP (SEQ ID NO: 3) may present
the RGD on the surface of the silk biomaterial by optimally
separating the cell attachment domain from the bonding sequence at
the end of the peptide. The optional leucine tails of this example
may interact in a fashion analogous to a leucine zipper, and be
driven by entropy from an aqueous solution to form an approximation
of a Langmuir-Blodgett (LB), monomolecular film on the surface of
materials exposed to such solutions, thus presenting a `carpet` of
RGD attachment sites on those surfaces.
[0071] Other proteins or peptides may be used instead of 23RGD if
such proteins or peptides have the desired characteristics. Example
characteristics include hydrophilic domains that can
interfere/enhance/affect silk gelation, and/or cell integrin
binding domains that enhance cell adhesion, spreading, and
migration. Non-limiting examples of such non-RDG integrins include,
KQAGDV (SEQ ID NO: 4), PHSRN (SEQ ID NO: 5), YIGSR (SEQ ID NO: 6),
CDPGYIGSR (SEQ ID NO: 7), IKVAV (SEQ ID NO: 8), RNIAEIIKDI (SEQ ID
NO: 9), YFQRYLI (SEQ ID NO: 10), PDSGR (SEQ ID NO: 11), FHRRIKA
(SEQ ID NO: 12), PRRARV (SEQ ID NO: 13), and WQPPRARI (SEQ ID NO:
14). See also Hersel et al., 24 Biomaterials 4285-415 (2003).
[0072] In aspects of this embodiment, a hydrogel comprises a molar
ratio of amphiphilic peptide to silk fibroin of, e.g., about 100:1,
about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about
40:1, about 30:1, about 20:1, about 10:1, about 7:1, about 5:1,
about 3:1, about 1:1, about 1:3, about 1:5, about 1:7, about 1:10,
about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about
1:70, about 1:80, or about 1:90, or about 1:100. In other aspects
of this embodiment, a hydrogel comprises a molar ratio of
amphiphilic peptide to silk fibroin of, e.g., at least 100:1, at
least 90:1, at least 80:1, at least 70:1, at least 60:1, at least
50:1, at least 40:1, at least 30:1, at least 20:1, at least 10:1,
at least 7:1, at least 5:1, at least 3:1, at least 1:1, at least
1:3, at least 1:5, at least 1:7, at least 1:10, at least 1:20, at
least 1:30, at least 1:40, at least 1:50, at least 1:60, at least
1:70, at least 1:80, or at least 1:90, or at least 1:100. In yet
other aspects of this embodiment, a hydrogel comprises a molar
ratio of amphiphilic peptide to silk fibroin of, e.g., at most
100:1, at most 90:1, at most 80:1, at most 70:1, at most 60:1, at
most 50:1, at most 40:1, at most 30:1, at most 20:1, at most 10:1,
at most 7:1, at most 5:1, at most 3:1, at most 1:1, at most 1:3, at
most 1:5, at most 1:7, at most 1:10, at most 1:20, at most 1:30, at
most 1:40, at most 1:50, at most 1:60, at most 1:70, at most 1:80,
or at most 1:90, or at most 1:100. In still other aspects of this
embodiment, a hydrogel comprises a molar ratio of amphiphilic
peptide to silk fibroin of, e.g., about 100:1 to about 1:100; about
90:1 to about 1:90; about 80:1 to about 1:80; about 70:1 to about
1:70; about 60:1 to about 1:60; about 50:1 to about 1:50; about
40:1 to about 1:40; about 30:1 to about 1:30; about 20:1 to about
1:20; about 10:1 to about 1:10; about 7:1 to about 1:7; about 5:1
to about 1:5; or about 3:1 to about 1:3.
[0073] The use of an amphiphilic peptide not only alters the
protein structure characteristics of silk fibroin protein, but in
so doing alters its resistance to proteolytic bioresorption in
vitro. These alterations in proteolytic bioresorption resistance
stem from aspects of the protein structure alteration as
.alpha.-helix and random coil are typically thought to be less
stable and therefore more susceptible to proteolytic bioresorption
than .beta.-sheet regions of silk. .beta.-turn and .beta.-strand
regions of the hydrogel disclosed herein are most resistant to
proteolytic bioresorption as opposed to regions of .alpha.-helixes
and random coils. Through deliberate manipulation of this protein
structure by means of controlled solution concentration and
addition of enhancer factors (type, concentration, and driving
gradient), gelation kinetics and resultant gel properties might be
controlled to deliver optimal outcomes in terms of degradative and
resultant biological behaviors. The impact of amphiphilic peptide
addition to silk hydrogel in a silk hydrogel is evident upon
examination of data obtained through implantation studies conducted
in vivo, both subcutaneously in rats and intradermally in the
dermis of guinea pigs. See Example 10.
[0074] Aspects of the present specification provide, in part, a
hydrogel comprising a five-amino acid peptide "tail" capable of
linking or conjugating a molecule X to a silk molecule or fibroin
when the molecule X is attached to the tail. A molecule X is any
entity, natural or synthetic, that can be useful and can be use in
the context of silk hydrogels. As used herein, the term "linking"
or "conjugating" in the context of molecule X refers to an indirect
physical attachment of a molecule X to a silk fibroin via a third
entity, the five-amino acid peptide "tail" being that entity. In
one embodiment, the tail binds to silk fibroin by hydrophobic
interaction to the silk fibroin. Alternatively, the "tail" binds
the silk molecules by hydrogen bonding and/or covalen.sub.t
bonding. It is envisioned that the "tail" can bind silk fibroins by
a combination of hydrophobic interactions, hydrogen bonds, and
covalent bonds. By attaching a molecule X to a "tail" described
herein, it is possible to indirectly link the molecule X to silk
fibroin via the tail, and thus to the silk hydrogels described
herein.
[0075] In one embodiment, the molecule X is attached to a tail at
the carboxyl (COOH) end of the five-amino acid peptide. In another
embodiment, the molecule X is attached to a tail at the amino
(NH.sub.2) end of the five-amino acid peptide.
[0076] In one embodiment, the five-amino acid peptide "tail"
comprises hydrophobic and/or apolar (non polar) amino acid residues
such as valine, leucine, isoleucine, phenylalanine, tryptophan,
methionine, cysteine, alanine, tyrosine, serine, proline,
histidine, threonine and glycine. Various combinations of
hydrophobic and/or apolar amino acid residues are possible, for e.
g. LLLLL (SEQ ID NO: 15), LLFFL (SEQ ID NO: 16), LFLWL (SEQ ID NO:
17), FLWLL (SEQ ID NO: 18) and LALGL (SEQ ID NO: 19). In other
embodiments, the tail comprises any combination of the twenty
standard conventional amino acid residues. In other embodiments,
the tail comprises hydrophobic and/or apolar (non polar) and amino
acids residues with hydrophobic side chains, e.g. arginine and
lysine. As used herein, the term "comprising" or "comprises" means
that other elements can also be present in addition to the defined
elements presented. The use of "comprising" indicates inclusion
rather than limitation.
[0077] In one embodiment, the five-amino acid peptide "tail"
capable of linking or conjugating a molecule X to a silk molecule
or fibroin when the molecule X is attached to the tail comprise
more than five amino acid residues, e. g. six or seven hydrophobic
and/or apolar amino acid residues, such as LLLLLL (SEQ ID NO:
20).
[0078] In one embodiment, the five-amino acid peptide "tail"
comprises amino acid residues that are part hydrophobic (i.e. the
part of the side-chain nearest to the protein main-chain), for e.g.
arginine and lysine. in one embodiment, the part hydrophobic amino
acid residues flank the five-amino acid peptide "tail" such as in
RLLLLLR (SEQ ID NO: 21), KLLLLLR (SEQ ID NO: 22) and KLLLLLK (SEQ
ID NO: 23).
[0079] In one embodiment, the five-amino acid peptide "tail" is
separated from a molecule X by a spacer peptide. Spacer peptides
should generally have non-polar amino acid residues, such as,
glycine and proline. In one embodiment, the spacer comprises
unnatural amino acid residues such as nor amino acids and
keto-substituted amino acids. Such unnatural amino acid residues
are well known to one skilled in the art. In one embodiment, the
spacer peptide is attached to a tail at the carboxyl (COOH) end of
the five-amino acid peptide. In another embodiment, the spacer is
attached to a tail at the amino (NH.sub.2) end of the five-amino
acid peptide.
[0080] The length of the space peptide is variable. The spacer
serves to link the molecule X and tail together and also to provide
steric freedom to the molecule X, allowing for proper orientation
of a molecule X (e. g. cell binding domains such as the RGD domain)
and the correct interaction of the molecule X with cells in vivo. A
spacer which is too short can prevent the molecule X from being
properly functional (i.e., holding it too tight to the silk
molecules and away from cells), a spacer which is too long can
cause undesired effects as well (i.e., non-specific association of
peptides or shortened efficacy from peptide due to spacer
breakage). In one embodiment, the number of amino acid residues in
a spacer can range form 1 to 300. In one embodiment, the spacer
comprises a single amino acid residue, such as a G or a P. Examples
of spacers with more amino acid residues are GSPGISGGGGGILE (SEQ ID
NO: 24) and SGGGGKSSAPI (SEQ ID NO: 25).
[0081] In one embodiment, the molecule X is any biological molecule
or fragment thereof. Examples biological molecules include but are
not limited to growth factors, hormones, cytokines, chemokines,
extracellular matrix compounds, osteogenic protein (OP), bone
morphogenetic protein (BMP), growth and differentiation factor
(GDF), transforming growth factor (TGF), epidermal growth factor
(EGF), vascular endothelial growth factor (VEGF), interleukin (IL),
platelet derived growth factor (PDGF), fibroblast growth factor
(FGF), insulin-like growth factor (IGF), basic fibroblast growth
factor (BFGF), fibroblast activation protein (FAP), disintegrin,
metalloproteinase (ADAM), matrix metalloproteinase (MMP),
connective tissue growth factor (CTGF), stromal derived growth
factor (SDGF), keratinocyte growth factor (KGF), tumor necrosis
factor (TNF), interferon (IFN), erythropoietin (EPO), hepatocyte
growth factor (HGF), thrombopoietin (TPO), granulocyte colony
stimulating factor (GCSF), granulocyte macrophage colony
stimulating factor (GMCSF), myostatin (GDF-8), collagen, resilin,
elastin, laminin, hyaluronic acid, decorin, actin, and tubulin.
Examples fragments of biological molecules include but are not
limited to known cell integrin binding domains including but not
limited to RGD, KQAGDV (SEQ ID NO: 4), PHSRN (SEQ ID NO: 5), YIGSR
(SEQ ID NO: 6), CDPGYIGSR (SEQ ID NO: 7), IKVAV (SEQ ID NO: 8),
RNIAEIIKDI (SEQ ID NO: 9), YFQRYLI (SEQ ID NO: 10), PDSGR (SEQ ID
NO: 11), FHRRIKA (SEQ ID NO: 12), PRRARV (SEQ ID NO: 13), and
WQPPRARI (SEQ ID NO: 14).
[0082] In other embodiments, the molecule X is any recombinant,
synthetic, or non-native polymeric compounds. Examples include but
are not limited to chitin, poly-lactic acid (PLA), poly-glycolic
acid (PGA), as tracers (e. g. radioisotopes), contrasting agents
(e. g. imaging dyes), aptamers, avimers, peptides, nuclei acids,
modified polysaccharide coatings, drugs (chemotherapy drugs),and
recombinant antibodies or antibody-based moieties.
[0083] In one embodiment, the present specification provides a
synthetic molecule having the formula: (molecule X).sub.n-(spacer
peptide).sub.0-300-(tail)-NH.sub.2 for linking with silk molecule
or fibroin, wherein "n" is a whole integer ranging from 1-30, and
wherein the amino acid residues of the spacer ranges from 0-300.
Examples of such synthetic molecule capable for linking to silk
molecule or fibroin are: GRGDIPASSKG.sub.4SRL.sub.6R--NH.sub.2 (SEQ
ID NO: 1), Ac-GdRGDIPASSKG.sub.4SdRL.sub.6dR--NH.sub.2 (SEQ ID NO:
2), (VEGF)-(VEGF)-GSPGISGGGGGILEKLLLLLK--NH.sub.2 (SEQ ID NO: 26),
(HIV-C-peptide).sub.3-GSPGISGGGGGILEKLALWLLR-NH.sub.2 (SEQ ID NO:
27), (taxol).sub.2-GSPGISGGGGGILERLLLLR--NH.sub.2 (SEQ ID NO: 28),
and (EPO).sub.2-GSPGISGGGGGILERLLWLLR--NH.sub.2 (SEQ ID NO: 29).
When used in the context of the silk hydrogel described herein, the
synthetic molecule of SEQ ID NO: 1 enable better tissue attachment
of the hydrogel construct in vivo, the synthetic molecule of SEQ ID
NO: 26 can promote blood vessel generation (neo-angiogenesis) in
tissue engineered constructs, the synthetic molecule of SEQ ID NO:
28 can provide a slow release anti-HIV medication in the form of a
transdermal delivery patch, the synthetic molecule of SEQ ID. NO:
28 can provide sustained dosage of anti-cancer drug in vivo, and
the synthetic molecule of SEQ ID NO: 29 can provide a slow release
EPO during cancer chemotherapy treatment.
[0084] Aspects of the present specification disclose, in part, a
hydrogel comprising a synthetic molecule having the formula:
(molecule X).sub.n-(spacer peptide).sub.0-300-(tail)-NH.sub.2 or a
synthetic molecule having the formula: (molecule X).sub.n-(spacer
peptide).sub.0-300-(tail)-NH.sub.2 and an amphiphilic peptide. In
one embodiment, the amphiphilic peptide is 23RGD. In one
embodiment, the present specification provides a method of
conjugating a molecule X to a silk molecule or fibroin comprising
mixing a synthetic molecule having the formula: (molecule
X).sub.n-(spacer peptide).sub.0-300-(tail)-NH.sub.2 with a silk
molecule or fibroin or silk solution. Conjugation of individual
peptide can be effected by a linkage via the N-terminal or the
C-terminal of the peptide, resulting in an N-linked peptide
oligomer or a C-linked peptide oligomer, respectively.
[0085] Methods of peptide synthesis are known to one skilled in the
art, for example, the peptides described herein can be
synthetically constructed by suitable known peptide polymerization
techniques, such as exclusively solid phase techniques, partial
solid-phase techniques, fragment condensation or classical solution
couplings. For example, the disclosed peptides can be synthesized
by the solid phase method using standard methods based on either
t-butyloxycarbonyl (BOC) or 9-fluorenylmethoxy-carbonyl (FMOC)
protecting groups. This methodology is described by G. B. Fields et
al. in Synthetic Peptides: A User's Guide, W. M. Freeman &
Company, New York, N.Y., pp. 77-183 (1992) and in the textbook
"Solid-Phase Synthesis", Stewart & Young, Freemen &
Company, San Francisco, 1969, and is exemplified by the disclosure
of U.S. Pat. No. 4,105,603, issued Aug. 8, 1979. Classical solution
synthesis is described in detail in "Methoden der Organischen
Chemic (Houben-Weyl): Synthese von Peptiden", E. Wunsch (editor)
(1974) Georg Thieme Verlag, Stuttgart West Germany. The fragment
condensation method of synthesis is exemplified in U.S. Pat. No.
3,972,859. Other available syntheses are exemplified in U.S., Pat.
No. 3,842,067, U.S. Pat. No. 3,872,925, issued Jan. 28, 1975,
Merrifield B, Protein Science (1996), 5: 1947-1951; The chemical
synthesis of proteins; Mutter M, Int J Pept Protein Res 1979 March;
13 (3): 274-7 Studies on the coupling rates in liquid-phase peptide
synthesis using competition experiments; and Solid Phase Peptide
Synthesis in the series Methods in Enzymology (Fields, G. B. (1997)
Solid-Phase Peptide Synthesis. Academic Press, San Diego.
#9830).The foregoing disclosures are incorporated herein by
reference. Molecular DNA methods can also be used. The coding
sequence of the short spacer can be constructed be annealing a
complementary pair of primers. One of skill in the art can design
and synthesize oligonucleotides that will code for the selected
spacer.
[0086] Methods of linking peptides are also known in the art. The
physical linking of the individual isolated peptides into
oligomeric peptides as set forth herein, can be effected by
chemical conjugation procedures well known in the art, such as by
creating peptide linkages, use of condensation agents, and by
employing well known bifunctional cross-linking reagents. The
conjugation may be direct, which includes linkages not involving
any intervening group, e.g., direct peptide linkages, or indirect,
wherein the linkage contains an intervening moiety, such as a
protein or peptide, e.g., plasma albumin, or other spacer molecule.
For example, the linkage may be via a heterobifunctional or
homobifunctional cross-linker, e.g., carbodiimide, glutaraldehyde,
N-succinimidyl 3-(2-pyridydithio) propionate (SPDP) and
derivatives, bis-maleimide,
4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and the like.
[0087] Cross-linking can also be accomplished without exogenous
cross-linkers by utilizing reactive groups on the molecules being
conjugated. Methods for chemically cross-linking peptide molecules
are generally known in the art, and a number of hetero- and
homobifunctional agents are described in, e.g., U.S. Pat. Nos.
4,355,023, 4,657,853, 4,676,980, 4,925,921, and 4,970,156, and
Immuno Technology Catalogue and Handbook, Pierce Chemical Co.
(1989), each of which is incorporated herein by reference. Such
conjugation, including cross-linking, should be performed so as not
to substantially affect the desired function of the peptide
oligomer or entity conjugated thereto, including therapeutic
agents, and moieties capable of binding substances of interest.
[0088] It will be apparent to one skilled in the art that
alternative linkers can be used to link peptides, for example the
use of chemical protein crosslinkers. For example homobifunctional
crosslinker such as disuccinimidyl-suberimidate-dihydrochloride;
dimethyl-adipimidate-dihydrochloride; 1,5,-2, 4dinitrobenezene or
heterobifunctional crosslinkers such as N-hydroxysuccinimidyl
2,3-dibromopropionate; 1ethyl-3-[3-dimethylaminopropyl]carbodiimide
hydrochloride; and succinim
idyl4-[n-maleimidomethyl]-cyclohexane-1-carboxylate.
[0089] A composition disclosed herein is typically a biodegradable,
bioerodible, and/or bioresorbable. In an embodiment, a silk fibroin
hydrogel disclosed herein has a protein structure that makes the
hydrogel resist biodegradation. In aspects of this embodiment, a
hydrogel is resistant to biodegradation for, e.g., about 10 days,
about 20 days, about 30 days, about 40 days, about 50 days, about
60 days, about 70 days, about 80 days, or about 90 days. In other
aspects of this embodiment, a hydrogel is resistant to
biodegradation for, e.g., at least 10 days, at least 20 days, at
least 30 days, at least 40 days, at least 50 days, at least 60
days, at least 70 days, at least 80 days, or at least 90 days. In
yet other aspects of this embodiment, a hydrogel is resistant to
biodegradation for, e.g., about 10 days to about 30 days, about 20
days to about 50 days, about 40 days to about 60 days, about 50
days to about 80 days, or about 60 days to about 90 days.
[0090] In an embodiment, a silk fibroin hydrogel disclosed herein
has a protein structure that makes the hydrogel resist bioerosion.
In aspects of this embodiment, a hydrogel is resistant to
bioerosion for, e.g., about 10 days, about 20 days, about 30 days,
about 40 days, about 50 days, about 60 days, about 70 days, about
80 days, or about 90 days. In other aspects of this embodiment, a
hydrogel is resistant to bioerosion for, e.g., at least 10 days, at
least 20 days, at least 30 days, at least 40 days, at least 50
days, at least 60 days, at least 70 days, at least 80 days, or at
least 90 days. In yet other aspects of this embodiment, a hydrogel
is resistant to bioerosion for, e.g., about 10 days to about 30
days, about 20 days to about 50 days, about 40 days to about 60
days, about 50 days to about 80 days, or about 60 days to about 90
days.
[0091] In an embodiment, a silk fibroin hydrogel disclosed herein
has a protein structure that makes the hydrogel resist
bioresorption. In aspects of this embodiment, a hydrogel is
resistant to bioresorption for, e.g., about 10 days, about 20 days,
about 30 days, about 40 days, about 50 days, about 60 days, about
70 days, about 80 days, or about 90 days. In other aspects of this
embodiment, a hydrogel is resistant to bioresorption for, e.g., at
least 10 days, at least 20 days, at least 30 days, at least 40
days, at least 50 days, at least 60 days, at least 70 days, at
least 80 days, or at least 90 days. In yet other aspects of this
embodiment, a hydrogel is resistant to bioresorption for, e.g.,
about 10 days to about 30 days, about 20 days to about 50 days,
about 40 days to about 60 days, about 50 days to about 80 days, or
about 60 days to about 90 days.
[0092] In yet another embodiment, a silk fibroin hydrogel disclosed
herein has a protein structure that substantially includes
.beta.-turn and .beta.-strand regions. In aspects of this
embodiment, a hydrogel has a protein structure including, e.g.,
about 10% .beta.-turn and .beta.-strand regions, about 20%
.beta.-turn and .beta.-strand regions, about 30% .beta.-turn and
.beta.-strand regions, about 40% .beta.-turn and 3-strand regions,
about 50% .beta.-turn and .beta.-strand regions, about 60%
.beta.-turn and .beta.-strand regions, about 70% .beta.-turn and
.beta.-strand regions, about 80% .beta.-turn and .beta.-strand
regions, about 90% .beta.-turn and .beta.-strand regions, or about
100% .beta.-turn and .beta.-strand regions. In other aspects of
this embodiment, a hydrogel has a protein structure including,
e.g., at least 10% .beta.-turn and .beta.-strand regions, at least
20% .beta.-turn and .beta.-strand regions, at least 30% .beta.-turn
and .beta.-strand regions, at least 40% .beta.-turn and
.beta.-strand regions, at least 50% .beta.-turn and .beta.-strand
regions, at least 60% .beta.-turn and .beta.-strand regions, at
least 70% .beta.-turn and .beta.-strand regions, at least 80%
.beta.-turn and .beta.-strand regions, at least 90% .beta.-turn and
.beta.-strand regions, or at least 95% 3-turn and .beta.-strand
regions. In yet other aspects of this embodiment, a hydrogel has a
protein structure including, e.g., about 10% to about 30%
.beta.-turn and .beta.-strand regions, about 20% to about 40%
.beta.-turn and .beta.-strand regions, about 30% to about 50%
.beta.-turn and .beta.-strand regions, about 40% to about 60%
.beta.-turn and .beta.-strand regions, about 50% to about 70%
.beta.-turn and .beta.-strand regions, about 60% to about 80%
.beta.-turn and .beta.-strand regions, about 70% to about 90%
.beta.-turn and .beta.-strand regions, about 80% to about 100%
.beta.-turn and .beta.-strand regions, about 10% to about 40%
.beta.-turn and .beta.-strand regions, about 30% to about 60%
.beta.-turn and .beta.-strand regions, about 50% to about 80%
.beta.-turn and .beta.-strand regions, about 70% to about 100%
.beta.-turn and .beta.-strand regions, about 40% to about 80%
.beta.-turn and .beta.-strand regions, about 50% to about 90%
.beta.-turn and .beta.-strand regions, about 60% to about 100%
.beta.-turn and .beta.-strand regions, or about 50% to about 100%
.beta.-turn and .beta.-strand regions.
[0093] In yet another embodiment, a silk fibroin hydrogel disclosed
herein has a protein structure that is substantially-free of
.alpha.-helix and random coil regions. In aspects of this
embodiment, a hydrogel has a protein structure including, e.g.,
about 5% .alpha.-helix and random coil regions, about 10%
.alpha.-helix and random coil regions, about 15% .alpha.-helix and
random coil regions, about 20% .alpha.-helix and random coil
regions, about 25% .alpha.-helix and random coil regions, about 30%
.alpha.-helix and random coil regions, about 35% .alpha.-helix and
random coil regions, about 40% .alpha.-helix and random coil
regions, about 45% .alpha.-helix and random coil regions, or about
50% .alpha.-helix and random coil regions. In other aspects of this
embodiment, a hydrogel has a protein structure including, e.g., at
most 5% .alpha.-helix and random coil regions, at most 10%
.alpha.-helix and random coil regions, at most 15% .alpha.-helix
and random coil regions, at most 20% .alpha.-helix and random coil
regions, at most 25% .alpha.-helix and random coil regions, at most
30% .alpha.-helix and random coil regions, at most 35%
.alpha.-helix and random coil regions, at most 40% .alpha.-helix
and random coil regions, at most 45% .alpha.-helix and random coil
regions, or at most 50% .alpha.-helix and random coil regions. In
yet other aspects of this embodiment, a hydrogel has a protein
structure including, e.g., about 5% to about 10% .alpha.-helix and
random coil regions, about 5% to about 15% .alpha.-helix and random
coil regions, about 5% to about 20% .alpha.-helix and random coil
regions, about 5% to about 25% .alpha.-helix and random coil
regions, about 5% to about 30% .alpha.-helix and random coil
regions, about 5% to about 40% .alpha.-helix and random coil
regions, about 5% to about 50% .alpha.-helix and random coil
regions, about 10% to about 20% .alpha.-helix and random coil
regions, about 10% to about 30% .alpha.-helix and random coil
regions, about 15% to about 25% .alpha.-helix and random coil
regions, about 15% to about 30% .alpha.-helix and random coil
regions, or about 15% to about 35% .alpha.-helix and random coil
regions.
[0094] Aspects of the present specification provide, in part, a
silk fibroin hydrogel having hardness. Hardness refers to various
properties of an object in the solid phase that gives it high
resistance to various kinds of shape change when force is applied.
Hardness is measured using a durometer and is a unitless value that
ranges from zero to 100. The ability or inability of a hydrogel to
be easily compressed will affect its suitability for application in
different tissue replacement roles, i.e., mechanical compliance as
bone, fat, connective tissue. Hardness will also affect the ability
of a hydrogel to be effectively comminuted, the reason being that a
hard material may be more easily and consistently comminuted.
Hardness will also affect extrudability, as a soft material may be
more readily able to be slightly compressed during injection to
pack with other particles or change shape to pass through a syringe
barrel or needle.
[0095] In an embodiment, a silk fibroin hydrogel exhibits low
hardness. In aspects of this embodiment, a silk fibroin hydrogel
exhibits a hardness of, e.g., about 5, about 10, about 15, about
20, about 25, about 30, or about 35. In other aspects of this
embodiment, a silk fibroin hydrogel exhibits a hardness of, e.g.,
at most 5, at most 10, at most 15, at most 20, at most 25, at most
30, or at most 35. In yet other aspects of this embodiment, a silk
fibroin hydrogel exhibits a hardness of, e.g., about 5 to about 35,
about 10 to about 35, about 15 to about 35, about 20 to about 35,
or about 25 to about 35, about 5 to about 40, about 10 to about 40,
about 15 to about 40, about 20 to about 40, about 25 to about 40,
or about 30 to about 40
[0096] In an embodiment, a silk fibroin hydrogel exhibits medium
hardness. In aspects of this embodiment, a silk fibroin hydrogel
exhibits a hardness of, e.g., about 40, about 45, about 50, about
55, or about 60. In other aspects of this embodiment, a silk
fibroin hydrogel exhibits a hardness of, e.g., at least 40, at
least 45, at least 50, at least 55, or at least 60. In yet other
aspects of this embodiment, a silk fibroin hydrogel exhibits a
hardness of, e.g., at most 40, at most 45, at most 50, at most 55,
or at most 60. In still other aspects of this embodiment, a silk
fibroin hydrogel exhibits a hardness of, e.g., about 35 to about
60, about 35 to about 55, about 35 to about 50, about 35 to about
45, about 40 to about 60, about 45 to about 60, about 50 to about
60, about 55 to about 60, about 40 to about 65, about 45 to about
65, about 50 to about 65, about 55 to about 65.
[0097] In another embodiment, a silk fibroin hydrogel exhibits high
hardness. In aspects of this embodiment, a silk fibroin hydrogel
exhibits a hardness of, e.g., about 65, about 70, about 75, about
80, about 85, about 90, about 95, or about 100. In other aspects of
this embodiment, a silk fibroin hydrogel exhibits a hardness of,
e.g., at least 65, at least 70, at least 75, at least 80, at least
85, at least 90, at least 95, or at least 100. In yet other aspects
of this embodiment, a silk fibroin hydrogel exhibits a hardness of,
e.g., about 65 to about 100, about 70 to about 100, about 75 to
about 100,about 80 to about 100, about 85 to about 100, about 90 to
about 100, about 65 to about 75, about 65 to about 80, about 65 to
about 85, about 65 to about 90, about 65 to about 95, about 60 to
about 75, about 60 to about 80, about 60 to about 85, about 60 to
about 90, or about 60 to about 95.
[0098] In an embodiment, a silk fibroin hydrogel exhibits high
resistant to deformation. In aspects of this embodiment, a silk
fibroin hydrogel exhibits resistant to deformation of, e.g., about
100%, about 99%, about 98%, about 97%, about 96%, about 95%, about
94%, about 93%, about 92%, about 91%, about 90%, about 89%, about
88%, about 87%, about 86%, or about 85%. In other aspects of this
embodiment, a silk fibroin hydrogel exhibits resistant to
deformation of, e.g., at least 99%, at least 98%, at least 97%, at
least 96%, at least 95%, at least 94%, at least 93%, at least 92%,
at least 91%, at least 90%, at least 89%, at least 88%, at least
87%, at least 86%, or at least 85%. In yet other aspects of this
embodiment, a silk fibroin hydrogel exhibits resistant to
deformation of, e.g., at most 99%, at most 98%, at most 97%, at
most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at
most 91%, at most 90%, at most 89%, at most 88%, at most 87%, at
most 86%, or at most 85%. In still aspects of this embodiment, a
silk fibroin hydrogel exhibits resistant to deformation of, e.g.,
about 85% to about 100%, about 87% to about 100%, about 90% to
about 100%, about 93% to about 100%, about 95% to about 100%, or
about 97% to about 100%.
[0099] A silk fibroin hydrogel exhibits an elastic modulus. Elastic
modulus, or modulus of elasticity, refers to the ability of a
hydrogel material to resists deformation, or, conversely, an
object's tendency to be non-permanently deformed when a force is
applied to it. The elastic modulus of an object is defined as the
slope of its stress-strain curve in the elastic deformation region:
.lamda.=stress/strain, where .lamda. is the elastic modulus in
Pascal's; stress is the force causing the deformation divided by
the area to which the force is applied; and strain is the ratio of
the change caused by the stress to the original state of the
object. Specifying how stresses are to be measured, including
directions, allows for many types of elastic moduli to be defined.
The three primary elastic moduli are tensile modulus, shear
modulus, and bulk modulus.
[0100] Tensile modulus (E) or Young's modulus is an objects
response to linear strain, or the tendency of an object to deform
along an axis when opposing forces are applied along that axis. It
is defined as the ratio of tensile stress to tensile strain. it is
often referred to simply as the elastic modulus. The shear modulus
or modulus of rigidity refers to an object's tendency to shear (the
deformation of shape at constant volume) when acted upon by
opposing forces. It is defined as shear stress over shear strain.
The shear modulus is part of the derivation of viscosity. The shear
modulus is concerned with the deformation of a solid when it
experiences a force parallel to one of its surfaces while its
opposite face experiences an opposing force (such as friction). The
bulk modulus (K) describes volumetric elasticity or an object's
resistance to uniform compression, and is the tendency of an object
to deform in all directions when uniformly loaded in all
directions. it is defined as volumetric stress over volumetric
strain, and is the inverse of compressibility. The bulk modulus is
an extension of Young's modulus to three dimensions.
[0101] In another embodiment, a silk fibroin hydrogel exhibits a
tensile modulus. in aspects of this embodiment, a silk fibroin
hydrogel exhibits a tensile modulus of, e.g., about 1 MPa, about 10
MPa, about 20 MPa, about 30 MPa, about 40 MPa, about 50 MPa, about
60 MPa, about 70 MPa, about 80 MPa, about 90 MPa, about 100 MPa,
about 200 MPa, about 300 MPa, about 400 MPa, about 500 MPa, about
750 MPa, about 1 GPa, about 5 GPa, about 10 GPa, about 15 GPa,
about 20 GPa, about 25 GPa,or about 30 GPa. In other aspects of
this embodiment, a silk fibroin hydrogel exhibits a tensile modulus
of, e.g., at least 1 MPa, at least 10 MPa, at least 20 MPa, at
least 30 MPa, at least 40 MPa, at least 50 MPa, at least 60 MPa, at
least 70 MPa, at least 80 MPa, at least 90 MPa, at least 100 MPa,
at least 200 MPa, at least 300 MPa, at least 400 MPa, at least 500
MPa, at least 750 MPa, at least 1 GPa, at least 5 GPa, at least 10
GPa, at least 15 GPa, at least 20 GPa, at least 25 GPa,or at least
30 GPa In yet other aspects of this embodiment, a silk fibroin
hydrogel exhibits a tensile modulus of, e.g., about 1 MPa to about
30 MPa, about 10 MPa to about 50 MPa, about 25 MPa to about 75 MPa,
about 50 MPa to about 100 MPa, about 100 MPa to about 300 MPa,
about 200 MPa to about 400 MPa, about 300 MPa to about 500 MPa,
about 100 MPa to about 500 MPa, about 250 MPa to about 750 MPa,
about 500 MPa to about 1 GPa, about 1GPa to about 30 GPa, about 10
GPa to about 30 GPa.
[0102] In another embodiment, a silk fibroin hydrogel exhibits
shear modulus. In aspects of this embodiment, a silk fibroin
hydrogel exhibits a shear modulus of, e.g., about 1 MPa, about 10
MPa, about 20 MPa, about 30 MPa, about 40 MPa, about 50 MPa, about
60 MPa, about 70 MPa, about 80 MPa, about 90 MPa, about 100 MPa,
about 200 MPa, about 300 MPa, about 400 MPa, about 500 MPa, about
750 MPa, about 1 GPa, about 5 GPa, about 10 GPa, about 15 GPa,
about 20 GPa, about 25 GPa, or about 30 GPa. In other aspects of
this embodiment, a silk fibroin hydrogel exhibits a shear modulus
of, e.g., at least 1 MPa, at least 10 MPa, at least 20 MPa, at
least 30 MPa, at least 40 MPa, at least 50 MPa, at least 60 MPa, at
least 70 MPa, at least 80 MPa, at least 90 MPa, at least 100 MPa,
at least 200 MPa, at least 300 MPa, at least 400 MPa, at least 500
MPa, at least 750 MPa, at least 1 GPa, at least 5 GPa, at least 10
GPa, at least 15 GPa, at least 20 GPa, at least 25 GPa,or at least
30 GPa In yet other aspects of this embodiment, a silk fibroin
hydrogel exhibits a shear modulus of, e.g., about 1 MPa to about 30
MPa, about 10 MPa to about 50 MPa, about 25 MPa to about 75 MPa,
about 50 MPa to about 100 MPa, about 100 MPa to about 300 MPa,
about 200 MPa to about 400 MPa, about 300 MPa to about 500 MPa,
about 100 MPa to about 500 MPa, about 250 MPa to about 750 MPa,
about 500 MPa to about 1 GPa, about 1GPa to about 30 GPa, about 10
GPa to about 30 GPa.
[0103] In another embodiment, a silk fibroin hydrogel exhibits a
bulk modulus. In aspects of this embodiment, a silk fibroin
hydrogel exhibits a bulk modulus of, e.g., about 5 GPa, about 6
GPa, about 7 GPa, about 8 GPa, about 9 GPa, about 10 GPa, about 15
GPa, about 20 GPa, about 25 GPa, about 30 GPa, about 35 GPa, about
40 GPa, about 45 GPa, about 50 GPa, about 60 GPa, about 70 GPa,
about 80 GPa, about 90 GPa, about 100 GPa. In other aspects of this
embodiment, a silk fibroin hydrogel exhibits a bulk modulus of,
e.g., at least 5 GPa, at least 6 GPa, at least 7 GPa, at least 8
GPa, at least 9 GPa, at least 10 GPa, at least 15 GPa, at least 20
GPa, at least 25 GPa, at least 30 GPa, at least 35 GPa, at least 40
GPa, at least 45 GPa, at least 50 GPa, at least 60 GPa, at least 70
GPa, at least 80 GPa, at least 90 GPa, at least 100 GPa. In yet
other aspects of this embodiment, a silk fibroin hydrogel exhibits
a bulk modulus of, e.g., about 5 GPa to about 50 GPa, about 5 GPa
to about 100 GPa, about 10 GPa to about 50 GPa, about 10 GPa to
about 100 GPa, or about 50 GPa to about 100 GPa.
[0104] A silk fibroin hydrogel exhibits high tensile strength.
Tensile strength has three different definitional points of stress
maxima. Yield strength refers to the stress at which material
strain changes from elastic deformation to plastic deformation,
causing it to deform permanently. Ultimate strength refers to the
maximum stress a material can withstand when subjected to tension,
compression or shearing. It is the maximum stress on the
stress-strain curve. Breaking strength refers to the stress
coordinate on the stress-strain curve at the point of rupture, or
when the material pulls apart.
[0105] In another embodiment, a silk fibroin hydrogel exhibits high
yield strength relative to other polymer classes. In aspects of
this embodiment, a silk fibroin hydrogel exhibits a yield strength
of, e.g., about 0.1 MPa, about 0.5 MPa, about 1 MPa, about 5 MPa,
about 10 MPa, about 20 MPa, about 30 MPa, about 40 MPa, about 50
MPa, about 60 MPa, about 70 MPa, about 80 MPa, about 90 MPa, about
100 MPa, about 200 MPa, about 300 MPa, about 400 MPa, about 500
MPa. In other aspects of this embodiment, a silk fibroin hydrogel
exhibits a yield strength of, e.g., at least 0.1 MPa, at least 0.5
MPa, at least 1 MPa, at least 5 MPa, at least 10 MPa, at least 20
MPa, at least 30 MPa, at least 40 MPa, at least 50 MPa, at least 60
MPa, at least 70 MPa, at least 80 MPa, at least 90 MPa, at least
100 MPa, at least 200 MPa, at least 300 MPa, at least 400 MPa, at
least 500 MPa. In yet other aspects of this embodiment, a silk
fibroin hydrogel exhibits a yield strength of, e.g., at most 1 MPa,
at most 5 MPa, at most 10 MPa, at most 20 MPa, at most 30 MPa, at
most 40 MPa, at most 50 MPa, at most 60 MPa, at most 70 MPa, at
most 80 MPa, at most 90 MPa, at most 100 MPa, at most 200 MPa, at
most 300 MPa, at most 400 MPa, at most 500 MPa, at most 600 MPa, at
most 700 MPa, at most 800 MPa, at most 900 MPa, at most 1000 MPa,
at most 1500 MPa, or at most 2000 MPa. In still other aspects of
this embodiment, a silk fibroin hydrogel exhibits a yield strength
of, e.g., about 1 MPa to about 50 MPa, about 1 MPa to about 60 MPa,
about 1 MPa to about 70 MPa, about 1 MPa to about 80 MPa, about 1
MPa to about 90 MPa, about 1 MPa to about 100 MPa, about 10 MPa to
about 50 MPa, about 10 MPa to about 60 MPa, about 10 MPa to about
70 MPa, about 10 MPa to about 80 MPa, about 10 MPa to about 90 MPa,
about 10 MPa to about 100 MPa, about 10 MPa to about 200 MPa, about
10 MPa to about 300 MPa, or about 100 MPa to about 300 MPa.
[0106] In another embodiment, a silk fibroin hydrogel exhibits high
ultimate strength. In aspects of this embodiment, a silk fibroin
hydrogel exhibits an ultimate strength of, e.g., about 0.1 MPa,
about 0.5 MPa, about 1 MPa, about 5 MPa, about 10 MPa, about 20
MPa, about 30 MPa, about 40 MPa, about 50 MPa, about 60 MPa, about
70 MPa, about 80 MPa, about 90 MPa, about 100 MPa, about 200 MPa,
about 300 MPa, about 400 MPa, about 500 MPa. In other aspects of
this embodiment, a silk fibroin hydrogel exhibits an ultimate
strength of, e.g., at least 0.1 MPa, at least 0.5 MPa, at least 1
MPa, at least 5 MPa, at least 10 MPa, at least 20 MPa, at least 30
MPa, at least 40 MPa, at least 50 MPa, at least 60 MPa, at least 70
MPa, at least 80 MPa, at least 90 MPa, at least 100 MPa, at least
200 MPa, at least 300 MPa, at least 400 MPa, at least 500 MPa. In
yet other aspects of this embodiment, a silk fibroin hydrogel
exhibits an ultimate strength of, e.g., at most 1 MPa, at most 5
MPa, at most 10 MPa, at most 20 MPa, at most 30 MPa, at most 40
MPa, at most 50 MPa, at most 60 MPa, at most 70 MPa, at most 80
MPa, at most 90 MPa, at most 100 MPa, at most 200 MPa, at most 300
MPa, at most 400 MPa, at most 500 MPa, at most 600 MPa, at most 700
MPa, at most 800 MPa, at most 900 MPa, at most 1000 MPa, at most
1500 MPa, or at most 2000 MPa. In still other aspects of this
embodiment, a silk fibroin hydrogel exhibits an ultimate strength
of, e.g., about 1 MPa to about 50 MPa, about 1 MPa to about 60 MPa,
about 1 MPa to about 70 MPa, about 1 MPa to about 80 MPa, about 1
MPa to about 90 MPa, about 1 MPa to about 100 MPa, about 10 MPa to
about 50 MPa, about 10 MPa to about 60 MPa, about 10 MPa to about
70 MPa, about 10 MPa to about 80 MPa, about 10 MPa to about 90 MPa,
about 10 MPa to about 100 MPa, about 10 MPa to about 200 MPa, about
10 MPa to about 300 MPa, or about 100 MPa to about 300 MPa.
[0107] In another embodiment, a silk fibroin hydrogel exhibits high
breaking strength. In aspects of this embodiment, a silk fibroin
hydrogel exhibits a breaking strength of, e.g., about 0.1 MPa,
about 0.5 MPa, about 1 MPa, about 5 MPa, about 10 MPa, about 20
MPa, about 30 MPa, about 40 MPa, about 50 MPa, about 60 MPa, about
70 MPa, about 80 MPa, about 90 MPa, about 100 MPa, about 200 MPa,
about 300 MPa, about 400 MPa, about 500 MPa. In other aspects of
this embodiment, a silk fibroin hydrogel exhibits a breaking
strength of, e.g., at least 0.1 MPa, at least 0.5 MPa, at least 1
MPa, at least 5 MPa, at least 10 MPa, at least 20 MPa, at least 30
MPa, at least 40 MPa, at least 50 MPa, at least 60 MPa, at least 70
MPa, at least 80 MPa, at least 90 MPa, at least 100 MPa, at least
200 MPa, at least 300 MPa, at least 400 MPa, at least 500 MPa. In
yet other aspects of this embodiment, a silk fibroin hydrogel
exhibits a breaking strength of, e.g., at most 1 MPa, at most 5
MPa, at most 10 MPa, at most 20 MPa, at most 30 MPa, at most 40
MPa, at most 50 MPa, at most 60 MPa, at most 70 MPa, at most 80
MPa, at most 90 MPa, at most 100 MPa, at most 200 MPa, at most 300
MPa, at most 400 MPa, at most 500 MPa, at most 600 MPa, at most 700
MPa, at most 800 MPa, at most 900 MPa, at most 1000 MPa, at most
1500 MPa, or at most 2000 MPa. In still other aspects of this
embodiment, a silk fibroin hydrogel exhibits a breaking strength
of, e.g., about 1 MPa to about 50 MPa, about 1 MPa to about 60 MPa,
about 1 MPa to about 70 MPa, about 1 MPa to about 80 MPa, about 1
MPa to about 90 MPa, about 1 MPa to about 100 MPa, about 10 MPa to
about 50 MPa, about 10 MPa to about 60 MPa, about 10 MPa to about
70 MPa, about 10 MPa to about 80 MPa, about 10 MPa to about 90 MPa,
about 10 MPa to about 100 MPa, about 10 MPa to about 200 MPa, about
10 MPa to about 300 MPa, or about 100 MPa to about 300 MPa.
[0108] Aspects of the present specification provide, in part, a
silk fibroin hydrogel having a transparency and/or translucency.
Transparency (also called pellucidity or diaphaneity) is the
physical property of allowing light to pass through a material,
whereas translucency (also called translucence or translucidity)
only allows light to pass through diffusely. The opposite property
is opacity. Transparent materials are clear, while translucent ones
cannot be seen through clearly. The silk fibroin hydrogels
disclosed herein may, or may not, exhibit optical properties such
as transparency and translucency. In certain cases, e.g.,
superficial line filling, it would be an advantage to have an
opaque hydrogel. In other cases such as development of a lens or a
"humor" for filling the eye, it would be an advantage to have a
translucent hydrogel. These properties could be modified by
affecting the structural distribution of the hydrogel material.
Factors used to control a hydrogel's optical properties include,
without limitation, silk fibroin concentration, gel crystallinity,
and hydrogel homogeneity.
[0109] When light encounters a material, it can interact with it in
several different ways. These interactions depend on the nature of
the light (its wavelength, frequency, energy, etc.) and the nature
of the material. Light waves interact with an object by some
combination of reflection, and transmittance with refraction. As
such, an optically transparent material allows much of the light
that falls on it to be transmitted, with little light being
reflected. Materials which do not allow the transmission of light
are called optically opaque or simply opaque.
[0110] In an embodiment, a silk fibroin hydrogel is optically
transparent. In aspects of this embodiment, a silk fibroin hydrogel
transmits, e.g., about 75% of the light, about 80% of the light,
about 85% of the light, about 90% of the light, about 95% of the
light, or about 100% of the light. In other aspects of this
embodiment, a silk fibroin hydrogel transmits, e.g., at least 75%
of the light, at least 80% of the light, at least 85% of the light,
at least 90% of the light, or at least 95% of the light. In yet
other aspects of this embodiment, a silk fibroin hydrogel
transmits, e.g., about 75% to about 100% of the light, about 80% to
about 100% of the light, about 85% to about 100% of the light,
about 90% to about 100% of the light, or about 95% to about 100% of
the light.
[0111] In another embodiment, a silk fibroin hydrogel is optically
opaque. In aspects of this embodiment, a silk fibroin hydrogel
transmits, e.g., about 5% of the light, about 10% of the light,
about 15% of the light, about 20% of the light, about 25% of the
light, about 30% of the light, about 35% of the light, about 40% of
the light, about 45% of the light, about 50% of the light, about
55% of the light, about 60% of the light, about 65% of the light,
or about 70% of the light. In other aspects of this embodiment, a
silk fibroin hydrogel transmits, e.g., at most 5% of the light, at
most 10% of the light, at most 15% of the light, at most 20% of the
light, at most 25% of the light, at most 30% of the light, at most
35% of the light, at most 40% of the light, at most 45% of the
light, at most 50% of the light, at most 55% of the light, at most
60% of the light, at most 65% of the light, at most 70% of the
light, or at most 75% of the light. In other aspects of this
embodiment, a silk fibroin hydrogel transmits, e.g., about 5% to
about 15%, about 5% to about 20%, about 5% to about 25%, about 5%
to about 30%, about 5% to about 35%, about 5% to about 40%, about
5% to about 45%, about 5% to about 50%, about 5% to about 55%,
about 5% to about 60%, about 5% to about 65%, about 5% to about
70%, about 5% to about 75%, about 15% to about 20%, about 15% to
about 25%, about 15% to about 30%, about 15% to about 35%, about
15% to about 40%, about 15% to about 45%, about 15% to about 50%,
about 15% to about 55%, about 15% to about 60%, about 15% to about
65%, about 15% to about 70%, about 15% to about 75%, about 25% to
about 35%, about 25% to about 40%, about 25% to about 45%, about
25% to about 50%, about 25% to about 55%, about 25% to about 60%,
about 25% to about 65%, about 25% to about 70%, or about 25% to
about 75%, of the light.
[0112] In an embodiment, a silk fibroin hydrogel is optically
translucent. In aspects of this embodiment, a silk fibroin hydrogel
diffusely transmits, e.g., about 75% of the light, about 80% of the
light, about 85% of the light, about 90% of the light, about 95% of
the light, or about 100% of the light. In other aspects of this
embodiment, a silk fibroin hydrogel diffusely transmits, e.g., at
least 75% of the light, at least 80% of the light, at least 85% of
the light, at least 90% of the light, or at least 95% of the light.
In yet other aspects of this embodiment, a silk fibroin hydrogel
diffusely transmits, e.g., about 75% to about 100% of the light,
about 80% to about 100% of the light, about 85% to about 100% of
the light, about 90% to about 100% of the light, or about 95% to
about 100% of the light.
[0113] After formation of a hydrogel described herein, the hydrogel
can further processed. For example, to remove enhancer species and
become a more complete, the formed hydrogel may be leeched against
a solvent, such as, e.g., water, under ambient temperature and
pressure conditions for three days with five changes of water. The
hydrogel may be leeched against ultra-pure water of a volume at
least 100-times that of the gel. More specifically, for example,
the gels may be placed in a bulk of purified water and the rinse
changed at hours 12, 24 and 48 with 15 mL gel per 1.5 L water. The
number of rinses and volume ratios involved may be altered so long
as the resultant hydrogel is substantially free of residual
gelation enhancer.
[0114] A hydrogel may be further processed by pulverizing the
hydrogel into particles and mixed with a carrier phase such as,
e.g., water or a saline solution to form an injectable or topical
substance like a solution, oil, lotion, gel, ointment, cream,
slurry, salve, or paste. A hydrogel may be milled to a particle
size from about 10 .mu.m to about 1000 .mu.m in diameter, such as
15 .mu.m to 30 .mu.m. Saline is then added as a carrier phase by
first determining the bulk volume of a hydrogel, then vigorously
pulverizing the hydrogel into particles while incorporating an
appropriate volume of saline to achieve a desired carrier to
hydrogel particle ratio. For example, hydrogel milling may be
accomplished by means of a forced sieving of bulk hydrogel through
a series of stainless steel cloth sieves of decreasing pore sizes.
In another example, a hydrogel may be loaded into a syringe and
pulverized with a spatula to a fine paste with saline.
[0115] A composition disclosed herein may be formulated using
material processing constraints such as silk concentration and
saline concentration to tailor material longevity in vivo. In one
example, a silk hydrogel might be tailored for a persistence of
five weeks to six weeks in vivo by using a 1%-3% (w/v) silk gel
with 25%-50% (v/v) saline carrier. In another example, a silk
hydrogel might be tailored for a persistence of two months to three
months in vivo by using a 3%-5% (w/v) silk gel with 20%-40% (v/v)
saline. In another example, a silk hydrogel might be tailored for a
persistence of 5-6 months by using 4-6% (w/v) silk gel with 20-40%
(v/v) saline. In another example, a silk hydrogel might be tailored
for a persistence of 7-10 months by using a 6-8% (w/v) silk gel
with 20-30% (v/v) saline. The persistence of these materials might
also be increased or decreased by increasing or decreasing particle
size respectively.
[0116] Gel emulsion saline content and gel silk concentration could
be used to modify the mechanical profile of the silk gel materials
for particular applications. For example, a gel emulsion of about
1% (w/v) to about 5% (w/v) silk gel concentration with 5%-95%
lubricant (e.g., 5%-95% (w/v) saline/PBS) may be useful as a dermal
filler, bulking agent, camouflage agent, intramuscular or sub-Q
filler, or pharmaceutical delivery vector. A gel emulsion of, for
example, about 5% (w/v) to about 8% (w/v) silk gel concentration
with 0% to about 30% lubricant fluid may be useful in bone defects
or cartilage defects.
[0117] Aspects of the present specification provide, in part, a
composition comprising a gel phase including a hydrogel comprising
a matrix polymer. The compositions disclosed herein can further
comprise a hydrogel comprising one or more matrix polymers in
addition to hydrogel particles comprising silk fibroin, or a
hydrogel comprising one or more matrix polymers and silk fibroin.
As used herein, the term "matrix polymer" refers to a polymer that
can become part of and/or function as an extracellular matrix
polymer and pharmaceutically acceptable salts thereof. Non-limiting
examples of a matrix polymer include a glycosaminoglycan like
chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronan;
a lubricin; a polysaccharide, and an elastic protein (like silk
protein, resilin, resilin-like polypeptides (RLPs), elastin
(including tropoelastin, fibrillin and fibullin), elastin-like
polypeptides (ELPs), gluten (including gliadin and glutenin),
abductin, byssus, and collagen). Non-limiting examples of a
pharmaceutically acceptable salt of a matrix polymer includes
sodium salts, potassium salts, magnesium salts, calcium salts, and
combinations thereof. Matrix polymers useful in the compositions
and methods disclosed herein are described in, e.g., Piron and
Tholin, Polysaccharide Crosslinking, Hydrogel Preparation,
Resulting Polysaccharides(s) and Hydrogel(s), uses Thereof, U.S.
Patent Publication 2003/0148995; Lebreton, Cross-Linking of Low and
High Molecular Weight Polysaccharides Preparation of Injectable
Monophase Hydrogels; Lebreton, Viscoelastic Solutions Containing
Sodium Hyaluronate and Hydroxypropyl Methyl Cellulose, Preparation
and Uses, U.S. Patent Publication 2008/0089918; Lebreton,
Hyaluronic Acid-Based Gels Including Lidocaine, U.S. Patent
Publication 2010/0028438; and Polysaccharides and Hydrogels thus
Obtained, U.S. Patent Publication 2006/0194758; and Di Napoli,
Composition and Method for Intradermal Soft Tissue Augmentation,
International Patent Publication WO 2004/073759, each of which is
hereby incorporated by reference in its entirety.
[0118] Aspects of the present specification provide, in part, a
composition comprising a glycosaminoglycan. As used herein, the
term "glycosaminoglycan" is synonymous with "GAG" and
"mucopolysaccharide" and refers to long unbranched polysaccharides
consisting of a repeating disaccharide units. The repeating unit
consists of a hexose (six-carbon sugar) or a hexuronic acid, linked
to a hexosamine (six-carbon sugar containing nitrogen) and
pharmaceutically acceptable salts thereof. Members of the GAG
family vary in the type of hexosamine, hexose or hexuronic acid
unit they contain, such as, e.g., glucuronic acid, iduronic acid,
galactose, galactosamine, glucosamine) and may also vary in the
geometry of the glycosidic linkage. Any glycosaminoglycan is useful
in the compositions disclosed herein with the proviso that the
glycosaminoglycan improves a condition of the skin, such as, e.g.,
hydration or elasticity. GAGs useful in the compositions and
methods disclosed herein are commercially available. Table 1 lists
representative GAGs.
TABLE-US-00001 TABLE 1 Examples of GAGs Glycosidic Hexuronic
linkage Name acid/Hexose Hexosamine geometry Unique features
Chondroitin GlcUA or GalNAc or -4GlcUA.beta.1- Most prevalent GAG
sulfate GlcUA(2S) GalNAc(4S) or 3GalNAc.beta.1- GalNAc(6S) or
GalNAc(4S,6S) Dermatan GlcUA or GalNAc or -4IdoUA.beta.1-
Distinguished from chondroitin sulfate IdoUA or GalNAc(4S) or
3GalNAc.beta.1- sulfate by the presence of IdoUA(2S) GalNAc(6S) or
iduronic acid, although some GalNAc(4S,6S) hexuronic acid
monosaccharides may be glucuronic acid. Keratan Gal or GlcNAc or
-3Gal(6S).beta.1- Keratan sulfate type II may be sulfate Gal(6S)
GlcNAc(6S) 4GlcNAc(6S).beta.1- fucosylated. Heparin GlcUA or GlcNAc
or -4IdoUA(2S).alpha.1- Highest negative charge IdoUA(2S) GlcNS or
4GlcNS(6S).alpha.1- density of any known GlcNAc(6S) or biological
molecule GlcNS(6S) Heparan GlcUA or GlcNAc or -4GlcUA.beta.1-
Highly similar in structure to sulfate IdoUA or GlcNS or
4GlcNAc.alpha.1- heparin, however heparan IdoUA(2S) GlcNAc(6S) or
sulfates disaccharide units are GlcNS(6S) organised into distinct
sulfated and non-sulfated domains. Hyaluronan GlcUA GlcNAc
-4GlcUA.beta.1- The only GAG that is 3GlcNAc.beta.1- exclusively
non-sulfated GlcUA = .beta.-D-glucuronic acid GlcUA(2S) =
2-O-sulfo-.beta.-D-glucuronic acid IdoUA = .alpha.-L-iduronic acid
IdoUA(2S) = 2-O-sulfo-.alpha.-L-iduronic acid Gal =
.beta.-D-galactose Gal(6S) = 6-O-sulfo-.beta.-D-galactose GalNAc =
.beta.-D-N-acetylgalactosamine GalNAc(4S) =
.beta.-D-N-acetylgalactosamine-4-O-sulfate GalNAc(6S) =
.beta.-D-N-acetylgalactosamine-6-O-sulfate GalNAc(4S,6S) =
.beta.-D-N-acetylgalactosamine-4-O, 6-O-sulfate GlcNAc =
.alpha.-D-N-acetylglucosamine GlcNS = .alpha.-D-N-sulfoglucosamine
GlcNS(6S) = .alpha.-D-N-sulfoglucosamine-6-O-sulfate
[0119] Aspects of the present specification provide, in part, a
composition comprising a chondroitin sulfate. As used herein, the
term "chondroitin sulfate" refers to an unbranched, sulfated GAG of
variable length comprising disaccharides of two alternating
monosaccharides of D-glucuronic acid (GlcA) and
N-acetyl-D-galactosamine (GalNAc) and pharmaceutically acceptable
salts thereof. A chondroitin sulfate may also include D-glucuronic
acid residues that are epimerized into L-iduronic acid (IdoA), in
which case the resulting disaccharide is referred to as dermatan
sulfate. A chondroitin sulfate polymer can have a chain of over 100
individual sugars, each of which can be sulfated in variable
positions and quantities. Chondroitin sulfate is an important
structural component of cartilage and provides much of its
resistance to compression. Any chondroitin sulfate is useful in the
compositions disclosed herein with the proviso that the chondroitin
sulfate improves a condition of the skin, such as, e.g., hydration
or elasticity. Non-limiting examples of pharmaceutically acceptable
salts of chondroitin sulfate include sodium chondroitin sulfate,
potassium chondroitin sulfate, magnesium chondroitin sulfate,
calcium chondroitin sulfate, and combinations thereof.
[0120] Aspects of the present specification provide, in part, a
composition comprising a keratan sulfate. As used herein, the term
"keratan sulfate" refers to a GAG of variable length comprising
disaccharide units, which themselves include .beta.-D-galactose and
N-acetyl-D-gafactosamine (GaINAc) and pharmaceutically acceptable
salts thereof. Disaccharides within the repeating region of keratan
sulfate may be fucosylated and N-Acetylneuraminic acid caps the end
of the chains. Any keratan sulfate is useful in the compositions
disclosed herein with the proviso that the keratan sulfate improves
a condition of the skin, such as, e.g., hydration or elasticity.
Non-limiting examples of pharmaceutically acceptable salts of
keratan sulfate include sodium keratan sulfate, potassium keratan
sulfate, magnesium keratan sulfate, calcium keratan sulfate, and
combinations thereof.
[0121] Aspects of the present specification provide, in part, a
composition comprising a hyaluronan. As used herein, the term
"hyaluronic acid" is synonymous with "HA", "hyaluronic acid", and
"hyaluronate" refers to an anionic, non-sulfated glycosaminoglycan
polymer comprising disaccharide units, which themselves include
D-glucuronic acid and D-N-acetylglucosamine monomers, linked
together via alternating .beta.-1,4 and .beta.-1,3 glycosidic bonds
and pharmaceutically acceptable salts thereof. Hyaluronan can be
purified from animal and non-animal sources. Polymers of hyaluronan
can range in size from about 5,000 Da to about 20,000,000 Da. Any
hyaluronan is useful in the compositions disclosed herein with the
proviso that the hyaluronan improves a condition of the skin, such
as, e.g., hydration or elasticity. Non-limiting examples of
pharmaceutically acceptable salts of hyaluronan include sodium
hyaluronan, potassium hyaluronan, magnesium hyaluronan, calcium
hyaluronan, and combinations thereof.
[0122] Aspects of the present specification provide, in part, a
composition comprising a lubricin. As used herein, the term
"lubricin" is synonymous with "proteoglycan 4" or superficial zone
protein" and refers to a large, water soluble glycoprotein encoded
by the PRG4 gene and pharmaceutically acceptable salts thereof.
Lubricin is about 345 kDa approximately equal proportions of
protein and the GAGs chondroitin sulfate and keratan sulfate. The
structure of lubricin molecule is that of a partially extended
flexible rod and, in solution, occupies a smaller spatial domain
than would be expected from structural predictions. This
characteristic may aid in the molecule's boundary lubricating
ability. Lubricin is present in synovial fluid and on the surface
(superficial layer) of articular cartilage and therefore plays an
important role in joint lubrication and synovial homeostasis. The
expression of lubricin has also been detected and the protein
localized in tendon, meniscus, lung, liver, heart, bone, ligament,
muscle and skin. Any lubricin is useful in the compositions
disclosed herein with the proviso that the lubricin improves a
condition of the skin, such as, e.g., hydration or elasticity.
Non-limiting examples of pharmaceutically acceptable salts of
lubricin include sodium lubricin, potassium lubricin, magnesium
lubricin, calcium lubricin, and combinations thereof.
[0123] Aspects of the present pharmaceutical compositions provide,
in part, a polysaccharide. As used herein, the term
"polysaccharide" refers to high molecular weight compounds
comprising at least eleven monosaccharide units. Polysaccharides
consisting of only one kind of repeating unit are called homo
polysaccharide polymers, whereas polysaccharides formed from two or
more different repeating units and called co polysaccharide
polymers. A polysaccharide can be natural or synthetic.
Non-limiting examples of polysaccharide include, e.g., dextrans
(like dextran 1K, dextran 4K, dextran 40K, dextran 60K, and dextran
70K), dextrin, glycogen, inulin, starch, starch derivatives (like
hydroxymethyl starch, hydroxyethyl starch, hydroxypropyl starch,
hydroxybutyl starch, and hydroxypentyl starch), hetastarch,
cellulose, MOLL, methyl cellulose (MC), carboxymethyl cellulose
(CMC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC),
hydroxyethyl methyl cellulose (HEMC), hydroxypropyl methyl
cellulose (HPMC); polyvinyl acetates (PVA); polyvinyl pyrrolidones
(PVP), also known as povidones, having a K-value of less than or
equal to 18, a K-value greater than 18 or less than or equal to 95,
or a K-value greater than 95, like PVP 12 (KOLLIDON.RTM. 12), PVP
17 (KOLLIDON.RTM. 17), PVP 25 (KOLLIDON.RTM. 25), PVP 30
(KOLLIDON.RTM. 30), PVP 90 (KOLLIDON.RTM. 90); polyethylene glycols
like PEG 100, PEG 200, PEG 300, PEG 400, PEG 500, PEG 600, PEG 700,
PEG, 800, PEG 900, PEG 1000, PEG 1100, PEG 1200, PEG 1300, PEG
1400, PEG 1500, PEG 1600, PEG 1700, PEG 1800, PEG 1900, PEG 2000,
PEG 2100, PEG 2200, PEG 2300, PEG 2400, PEG 2500, PEG 2600, PEG
2700, PEG 2800, PEG 2900, PEG 3000, PEG 3250, PEG 3350, PEG 3500,
PEG 3750, PEG 4000, PEG 4250, PEG 4500, PEG 4750, PEG 5000, PEG
5500, PEG 6000, PEG 6500, PEG 75000, PEG 7500, or PEG 8000; and
polyethylene imines (PEI).
[0124] Aspects of the present specification provide, in part, a
composition comprising an elastic protein. As used herein, the term
"elastic protein" refers to a structural polypeptide with a wide
range of physical properties including, without limitation, high
resilience in that the polypeptide can be deformed reversibly
without loss of energy, the ability to be deformed to large strains
with little force, and/or with low stiffness in that the
polypeptide can be stretched. For example, elastic proteins like
elastin and resilin have a combination of high resilience, large
strains and low stiffness is characteristic of rubber-like proteins
that function in the storage of elastic-strain energy. Other
elastic proteins, like collagen, provides exceptional energy
storage capacity but are not very stretchy. Mussel byssus threads
and spider dragline silks are also elastic proteins because they
are remarkably stretchy, in spite of their considerable strength,
low resilience, and stiffness. The silk fibroin disclosed herein is
another elastic protein. Non-limiting examples of elastic proteins
include silk protein, resilin, resilin-like polypeptides (RLPs),
elastin (including tropoelastin, fibrillin and fibullin),
elastin-like polypeptides (ELPs), gluten (including gliadin and
glutenin), abductin, byssus, and collagen. In general, elastic
proteins have at least one domain containing elastic repeat motifs
and another non-elastic domain where crosslinks can be formed. See,
e.g., Tatham and Shewry, Comparative Structures and Properties of
Elastic Proteins, Phil. Trans. R. Soc. Lond. B 357: 229-234 (2002),
which is hereby incorporated by reference in its entirety. However,
both resilin and abductin are exceptions since crosslinking can
occur within the elastic repeat motif.
[0125] Aspects of the present specification provide, in part, a
composition comprising a crosslinked matrix polymer. As used
herein, the term "crosslinked" refers to the intermolecular bonds
joining the individual polymer molecules, or monomer chains, into a
more stable structure like a gel. As such, a crosslinked matrix
polymer has at least one intermolecular bond joining at least one
individual polymer molecule to another one. Matrix polymers
disclosed herein may be crosslinked using dialdehydes and disuf
ides crosslinking agents including, without limitation,
multifunctional PEG-based crosslinking agents, divinyl sulfones,
diglycidyl ethers, and bis-epoxides. Non-limiting examples of
hyaluronan crosslinking agents include divinyl sulfone (DVS),
1,4-butanediol diglycidyl ether (BDDE),
1,2-bis(2,3-epoxypropoxy)ethylene (EG DG E), 1,2,7,8-diepoxyoctane
(DEO), biscarbodiimide (BCDI), pentaerythritol tetraglycidyl ether
(PETGE), adipic dihydrazide (ADH), bis(sulfosuccinimidyl)suberate
(BS), hexamethylenediamine (HMDA),
1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, or combinations
thereof.
[0126] Aspects of the present specification provide, in part, a
composition comprising a crosslinked matrix polymer having a degree
of crosslinking. As used herein, the term "degree of crosslinking"
refers to the percentage of matrix polymer monomeric units that are
bound to a cross-linking agent, such as, e.g., the disaccharide
monomer units of hyaluronan. Thus, a composition that that has a
crosslinked matrix polymer with a 4% degree of crosslinking means
that on average there are four crosslinking molecules for every 100
monomeric units. Every other parameter being equal, the greater the
degree of crosslinking, the harder the gel becomes. Non-limiting
examples of a degree of crosslinking include about 1% to about
15%.
[0127] In an embodiment, a composition comprises a crosslinked
matrix polymer. In other aspects of this embodiment, a composition
comprises a crosslinked matrix polymer where the partially
crosslinked matrix polymer represents, e.g., about 1% by weight,
about 2% by weight, about 3% by weight, about 4% by weight, about
5% by weight, about 6% by weight, about 7% by weight, about 8% by
weight, or about 9%, or about 10% by weight, of the total matrix
polymer present in the composition. In yet other aspects of this
embodiment, a composition comprises a crosslinked matrix polymer
where the partially crosslinked matrix polymer represents, e.g., at
most 1% by weight, at most 2% by weight, at most 3% by weight, at
most 4% by weight, at most 5% by weight, at most 6% by weight, at
most 7% by weight, at most 8% by weight, at most 9% by weight, or
at most 10% by weight, of the total matrix polymer present in the
composition. In still other aspects of this embodiment, a
composition comprises a crosslinked matrix polymer where the
partially crosslinked matrix polymer represents, e.g., about 0% to
about 10% by weight, about 1% to about 10% by weight, about 3% to
about 10% by weight, or about 5% to about 10% by weight, of the
total matrix polymer present in the composition.
[0128] In other aspects of this embodiment, a composition comprises
a crosslinked matrix polymer where the degree of crosslinking is
about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about
7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,
about 14%, or about 15%. In yet other aspects of this embodiment, a
composition comprises a crosslinked matrix polymer where the degree
of crosslinking is at most 1%, at most 2%, at most 3%, at most 4%,
at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, at most
10%, at most 11%, at most 12%, at most 13%, at most 14%, or at most
15%. In still other aspects of this embodiment, a composition
comprises a crosslinked matrix polymer where the degree of
crosslinking is about 1% to about 15%, about 2% to about 11%, about
3% to about 10%, about 1% to about 5%, about 10% to about 15%,
about 11% to about 15%, about 6% to about 10%, or about 6% to about
8%.
[0129] In another embodiment, a composition comprises a crosslinked
glycosaminoglycan. In aspect of this embodiment, a composition
comprises a crosslinked chondroitin sulfate polymer, a crosslinked
dermatan sulfate polymer, a crosslinked keratan sulfate polymer, a
crosslinked heparan polymer, a crosslinked heparan sulfate polymer,
or a crosslinked hyaluronan polymer. In other aspects of this
embodiment, a composition comprises a crosslinked glycosaminoglycan
where the crosslinked glycosaminoglycan represents, e.g., about 1%
by weight, about 2% by weight, about 3% by weight, about 4% by
weight, about 5% by weight, about 6% by weight, about 7% by weight,
about 8% by weight, or about 9%, or about 10% by weight, of the
total glycosaminoglycan present in the composition. In yet other
aspects of this embodiment, a fluid composition comprises a
crosslinked glycosaminoglycan where the crosslinked
glycosaminoglycan represents, e.g., at most 1% by weight, at most
2% by weight, at most 3% by weight, at most 4% by weight, at most
5% by weight, at most 6% by weight, at most 7% by weight, at most
8% by weight, at most 9% by weight, or at most 10% by weight, of
the total glycosaminoglycan present in the composition. In still
other aspects of this embodiment, a composition comprises a
crosslinked glycosaminoglycan where the crosslinked
glycosaminoglycan represents, e.g., about 0% to about 10% by
weight, about 1% to about 10% by weight, about 3% to about 10% by
weight, or about 5% to about 10% by weight, of the total
glycosaminoglycan present in the composition.
[0130] In other aspects of this embodiment, a composition comprises
a crosslinked glycosaminoglycan where the degree of crosslinking is
about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about
7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,
about 14%, or about 15%. In yet other aspects of this embodiment, a
composition comprises a crosslinked glycosaminoglycan where the
degree of crosslinking is at most 1%, at most 2%, at most 3%, at
most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most
9%, at most 10%, at most 11%, at most 12%, at most 13%, at most
14%, or at most 15%. In still other aspects of this embodiment, a
composition comprises a crosslinked glycosaminoglycan where the
degree of crosslinking is about 1% to about 15%, about 2% to about
11%, about 3% to about 10%, about 1% to about 5%, about 10% to
about 15%, about 11% to about 15%, about 6% to about 10%, or about
6% to about 8%.
[0131] In yet another embodiment, a composition comprises a
crosslinked lubricin. In aspects of this embodiment, a composition
comprises a crosslinked lubricin where the crosslinked lubricin
represents, e.g., about 1% by weight, about 2% by weight, about 3%
by weight, about 4% by weight, about 5% by weight, about 6% by
weight, about 7% by weight, about 8% by weight, or about 9%, or
about 10% by weight, of the total lubricin present in the
composition. In other aspects of this embodiment, a composition
comprises a crosslinked lubricin where the crosslinked lubricin
represents, e.g., at most 1% by weight, at most 2% by weight, at
most 3% by weight, at most 4% by weight, at most 5% by weight, at
most 6% by weight, at most 7% by weight, at most 8% by weight, at
most 9% by weight, or at most 10% by weight, of the total lubricin
present in the composition. In yet other aspects of this
embodiment, a composition comprises a crosslinked lubricin where
the crosslinked lubricin represents, e.g., about 0% to about 10% by
weight, about 1% to about 10% by weight, about 3% to about 10% by
weight, or about 5% to about 10% by weight, of the total lubricin
present in the composition.
[0132] In other aspects of this embodiment, a composition comprises
a crosslinked lubricin where the degree of crosslinking is about
1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,
about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,
about 14%, or about 15%. In yet other aspects of this embodiment, a
composition comprises a crosslinked lubricin where the degree of
crosslinking is at most 1%, at most 2%, at most 3%, at most 4%, at
most 5%, at most 6%, at most 7%, at most 8%, at most 9%, at most
10%, at most 11%, at most 12%, at most 13%, at most 14%, or at most
15%. in still other aspects of this embodiment, a composition
comprises a crosslinked lubricin where the degree of crosslinking
is about 1% to about 15%, about 2% to about 11%, about 3% to about
10%, about 1% to about 5%, about 10% to about 15%, about 11% to
about 15%, about 6% to about 10%, or about 6% to about 8%.
[0133] In another embodiment, a composition comprises a crosslinked
polysaccharide. In aspect of this embodiment, a composition
comprises a crosslinked dextran, a crosslinked dextrin, a
crosslinked starch, a crosslinked hetastarch, a crosslinked
glycogen, a crosslinked polyvinyl acetate, a crosslinked polyvinyl
pyrrolidone, a crosslinked polyethylene glycol, a crosslinked
polyethylene imine, a crosslinked cellulose, a crosslinked methyl
cellulose, a crosslinked carboxymethyl cellulose, a crosslinked
hydroxyethyl cellulose, a crosslinked hydroxypropyl cellulose, a
crosslinked hydroxyethyl methyl cellulose, or a crosslinked
hydroxypropyl methyl cellulose. In other aspects of this
embodiment, a composition comprises a crosslinked polysaccharide
where the crosslinked polysaccharide represents, e.g., about 1% by
weight, about 2% by weight, about 3% by weight, about 4% by weight,
about 5% by weight, about 6% by weight, about 7% by weight, about
8% by weight, or about 9%, or about 10% by weight, of the total
polysaccharide present in the composition. In yet other aspects of
this embodiment, a composition comprises a crosslinked
polysaccharide where the crosslinked polysaccharide represents,
e.g., at most 1% by weight, at most 2% by weight, at most 3% by
weight, at most 4% by weight, at most 5% by weight, at most 6% by
weight, at most 7% by weight, at most 8% by weight, at most 9% by
weight, or at most 10% by weight, of the total polysaccharide
present in the composition. In still other aspects of this
embodiment, a composition comprises a crosslinked polysaccharide
where the crosslinked polysaccharide represents, e.g., about 0% to
about 10% by weight, about 1% to about 10% by weight, about 3% to
about 10% by weight, or about 5% to about 10% by weight, of the
total polysaccharide present in the composition.
[0134] In other aspects of this embodiment, a composition comprises
a crosslinked polysaccharide where the degree of crosslinking is
about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about
7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,
about 14%, or about 15%. In yet other aspects of this embodiment, a
composition comprises a crosslinked polysaccharide where the degree
of crosslinking is at most 1%, at most 2%, at most 3%, at most 4%,
at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, at most
10%, at most 11%, at most 12%, at most 13%, at most 14%, or at most
15%. In still other aspects of this embodiment, a composition
comprises a crosslinked polysaccharide where the degree of
crosslinking is about 1% to about 15%, about 2% to about 11%, about
3% to about 10%, about 1% to about 5%, about 10% to about 15%,
about 11% to about 15%, about 6% to about 10%, or about 6% to about
8%.
[0135] Aspects of the present specification provide, in part, a
composition comprising an uncrosslinked matrix polymer. As used
herein, the term "uncrosslinked" refers to a lack of intermolecular
bonds joining the individual matrix polymer molecules, or monomer
chains. As such, an uncrosslinked matrix polymer is not linked to
any other matrix polymer by an intermolecular bond.
[0136] Aspects of the present specification provide, in part, a
composition comprising a substantially uncrosslinked matrix
polymer. As sued herein, the term "substantially uncrosslinked"
refers to the presence of uncrosslinked matrix polymers in a
composition disclosed herein at a level of at least 90% by weight
of the composition, with the remaining at most 10% by weight of the
composition being comprised of other components including
crosslinked matrix polymers.
[0137] In an embodiment, a composition comprises a substantially
uncrosslinked matrix polymer. In other aspects of this embodiment,
a composition comprises an uncrosslinked matrix polymer where the
uncrosslinked matrix polymer represents, e.g., about 90% by weight,
about 91% by weight, about 92% by weight, about 93% by weight,
about 94% by weight, about 95% by weight, about 96% by weight,
about 97% by weight, about 98% by weight, or about 99%, or about
100% by weight, of the total matrix polymer present in the
composition. In yet other aspects of this embodiment, a composition
comprises an uncrosslinked matrix polymer where the uncrosslinked
matrix polymer represents, e.g., at least 90% by weight, at least
91% by weight, at least 92% by weight, at least 93% by weight, at
least 94% by weight, at least 95% by weight, at least 96% by
weight, at least 97% by weight, at least 98% by weight, or at least
99% by weight, of the total matrix polymer present in the
composition. In still other aspects of this embodiment, a
composition comprises an uncrosslinked matrix polymer where the
uncrosslinked matrix polymer represents, e.g., about 90% to about
100% by weight, about 93% to about 100% by weight, about 95% to
about 100% by weight, or about 97% to about 100% by weight, of the
total matrix polymer present in the composition.
[0138] In another embodiment, a composition comprises a
substantially uncrosslinked glycosaminoglycan. In aspects of this
embodiment, a composition comprises a substantially uncrosslinked
chondroitin sulfate polymer, a substantially uncrosslinked dermatan
sulfate polymer, a substantially uncrosslinked keratan sulfate
polymer, a substantially uncrosslinked heparan polymer, a
substantially uncrosslinked heparan sulfate polymer, or a
substantially uncrosslinked hyaluronan polymer. In other aspects of
this embodiment, a fluid composition comprises an uncrosslinked
glycosaminoglycan where the uncrosslinked glycosaminoglycan
represents, e.g., about 90% or more by weight, about 91% or more by
weight, about 92% or more by weight, about 93% or more by weight,
about 94% or more by weight, about 95% or more by weight, about 96%
or more by weight, about 97% or more by weight, about 98% or more
by weight, or about 99% or more, or about 100% by weight, of the
total glycosaminoglycan present in the composition. In yet other
aspects of this embodiment, a fluid composition comprises an
uncrosslinked glycosaminoglycan where the uncrosslinked
glycosaminoglycan represents, e.g., about 90% to about 100% by
weight, about 93% to about 100% by weight, about 95% to about 100%
by weight, or about 97% to about 100% by weight, of the total
glycosaminoglycan present in the composition.
[0139] In yet another embodiment, a fluid composition comprises a
substantially uncrosslinked lubricin. In aspects of this
embodiment, a fluid composition comprises an uncrosslinked lubricin
where the uncrosslinked lubricin represents, e_g., about 90% or
more by weight, about 91% or more by weight, about 92% or more by
weight, about 93% or more by weight, about 94% or more by weight,
about 95% or more by weight, about 96% or more by weight, about 97%
or more by weight, about 98% or more by weight, or about 99% or
more, or about 100% by weight, of the total lubricin present in the
composition. In other aspects of this embodiment, a fluid
composition comprises an uncrosslinked lubricin where the
uncrosslinked lubricin represents, e.g., about 90% to about 100% by
weight, about 93% to about 100% by weight, about 95% to about 100%
by weight, or about 97% to about 100% by weight, of the total
lubricin present in the composition.
[0140] In another embodiment, a composition comprises a
substantially uncrosslinked polysaccharide. In aspects of this
embodiment, a composition comprises a substantially uncrosslinked
dextran, a substantially uncrosslinked dextrin, a substantially
uncrosslinked starch, a substantially uncrosslinked hetastarch, a
substantially uncrosslinked glycogen, a substantially uncrosslinked
polyvinyl acetate, a substantially uncrosslinked polyvinyl
pyrrolidone, a substantially uncrosslinked polyethylene glycol, a
substantially uncrosslinked polyethylene imine, a substantially
uncrosslinked cellulose, a substantially uncrosslinked methyl
cellulose, a substantially uncrosslinked carboxymethyl cellulose, a
substantially uncrosslinked hydroxyethyl cellulose, a substantially
uncrosslinked hydroxypropyl cellulose, a substantially
uncrosslinked hydroxyethyl methyl cellulose, or a substantially
uncrosslinked hydroxypropyl methyl cellulose. In other aspects of
this embodiment, a composition comprises an uncrosslinked
polysaccharide where the uncrosslinked polysaccharide represents,
e.g., about 90% or more by weight, about 91% or more by weight,
about 92% or more by weight, about 93% or more by weight, about 94%
or more by weight, about 95% or more by weight, about 96% or more
by weight, about 97% or more by weight, about 98% or more by
weight, or about 99% or more, or about 100% by weight, of the total
polysaccharide present in the composition. in yet other aspects of
this embodiment, a composition comprises an uncrosslinked
polysaccharide where the uncrosslinked polysaccharide represents,
e.g., about 90% to about 100% by weight, about 93% to about 100% by
weight, about 95% to about 100% by weight, or about 97% to about
100% by weight, of the total polysaccharide present in the
composition.
[0141] Aspects of the present specification provide, in part, a
composition that is essentially free of a crosslinked matrix
polymer. As used herein, the term "essentially free" (or
"consisting essentially of") refers to a composition where only
trace amounts of cross-linked matrix polymers can be detected.
[0142] In an embodiment, a composition comprises a
glycosaminoglycan that is essentially free of a crosslinked
glycosaminoglycan. In an aspect of this embodiment, a composition
comprises a chondroitin sulfate that is essentially free of a
crosslinked chondroitin sulfate polymer, a dermatan sulfate
essentially free of a crosslinked dermatan sulfate polymer, a
keratan sulfate essentially free of a crosslinked keratan sulfate
polymer, a heparan essentially free of a crosslinked heparan
polymer, a heparan sulfate essentially free of a crosslinked
heparan sulfate polymer, or a hyaluronan sulfate essentially free
of a crosslinked hyaluronan polymer.
[0143] In yet another embodiment, a fluid composition comprises a
lubricin that is essentially free of a crosslinked lubricin.
[0144] In an embodiment, a composition comprises a polysaccharide
that is essentially free of a crosslinked polysaccharide. In an
aspect of this embodiment, a composition comprises a dextran that
is essentially free of a crosslinked dextran, dextrin that is
essentially free of a crosslinked dextrin, dextran that is
essentially free of a crosslinked starch, hetastarch that is
essentially free of a crosslinked hetastarch, glycogen that is
essentially free of a crosslinked glycogen, polyvinyl acetate that
is essentially free of a crosslinked polyvinyl acetate, polyvinyl
pyrrolidone that is essentially free of a crosslinked polyvinyl
pyrrolidone, polyethylene glycothat is essentially free of a
crosslinked polyethylene glycol, polyethylene imine that is
essentially free of a crosslinked polyethylene imine, cellulose
that is essentially free of a crosslinked cellulose, methyl
cellulose that is essentially free of a crosslinked methyl
cellulose, carboxymethyl cellulose that is essentially free of a
crosslinked carboxymethyl cellulose, hydroxyethyl cellulose that is
essentially free of a crosslinked hydroxyethyl cellulose,
hydroxypropyl cellulose that is essentially free of a crosslinked
hydroxypropyl cellulose, hydroxyethyl methyl cellulosthat is
essentially free of a crosslinked hydroxyethyl methyl cellulose, or
hydroxypropyl methyl cellulose that is essentially free of a
crosslinked hydroxypropyl methyl cellulose.
[0145] Aspects of the present specification provide, in part, a
composition that is entirely free of a crosslinked matrix polymer.
As used herein, the term "entirely free" refers to a fluid
composition that within the detection range of the instrument or
process being used, crosslinked matrix polymers cannot be detected
or its presence cannot be confirmed.
[0146] In an embodiment, a composition comprises a
glycosaminoglycan that is entirely free of a crosslinked
glycosaminoglycan. In an aspect of this embodiment, a composition
comprises a chondroitin sulfate that is entirely free of a
crosslinked chondroitin sulfate polymer, a dermatan sulfate
entirely free of a crosslinked dermatan sulfate polymer, a keratan
sulfate entirely free of a crosslinked keratan sulfate polymer, a
heparan entirely free of a crosslinked heparan polymer, a heparan
sulfate entirely free of a crosslinked heparan sulfate polymer, or
a hyaluronan sulfate entirely free of a crosslinked hyaluronan
polymer.
[0147] In yet another embodiment, a fluid composition comprises a
lubricin that is entirely free of a crosslinked lubricin.
[0148] In an embodiment, a composition comprises a polysaccharide
that is entirely free of a crosslinked polysaccharide. In an aspect
of this embodiment, a composition comprises a dextran that is
entirely free of a crosslinked dextran, dextrin that is entirely
free of a crosslinked dextrin, dextran that is entirely free of a
crosslinked starch, hetastarch that is entirely free of a
crosslinked hetastarch, glycogen that is entirely free of a
crosslinked glycogen, polyvinyl acetate that is entirely free of a
crosslinked polyvinyl acetate, polyvinyl pyrrolidone that is
entirely free of a crosslinked polyvinyl pyrrolidone, polyethylene
glycothat is entirely free of a crosslinked polyethylene glycol,
polyethylene imine that is entirely free of a crosslinked
polyethylene imine, cellulose that is entirely free of a
crosslinked cellulose, methyl cellulose that is entirely free of a
crosslinked methyl cellulose, carboxymethyl cellulose that is
entirely free of a crosslinked carboxymethyl cellulose,
hydroxyethyl cellulose that is entirely free of a crosslinked
hydroxyethyl cellulose, hydroxypropyl cellulose that is entirely
free of a crosslinked hydroxypropyl cellulose, hydroxyethyl methyl
cellulosthat is entirely free of a crosslinked hydroxyethyl methyl
cellulose, or hydroxypropyl methyl cellulose that is entirely free
of a crosslinked hydroxypropyl methyl cellulose.
[0149] Aspects of the present specification provide, in part, a
composition comprising a ratio of crosslinked matrix polymer and
uncrosslinked polymer. This ratio of crosslinked and uncrosslinked
matrix polymer is also known as the gel:fluid ratio. Any gel:fluid
ratio is useful in making the compositions disclosed herein with
the proviso that such ratio produces a composition disclosed herein
that improves a skin condition as disclosed herein. Non-limiting
examples of gel:fluid ratios include 100:0, 98:2, 90:10, 75:25,
70:30, 60:40, 50:50, 40:60, 30:70, 25:75, 10:90; 2:98, and
0:100.
[0150] In an embodiment, a composition comprises a crosslinked
matrix polymer and an uncrosslinked matrix polymer. In another
aspect of this embodiment, a composition comprises a crosslinked
matrix polymer and an uncrosslinked matrix polymer where the
gel:fluid ratio is sufficient to form a fluid. In other aspects of
this embodiment, a composition comprises a crosslinked matrix
polymer and an uncrosslinked matrix polymer where the gel:fluid
ratio is, e.g., about 0:100, about 1:99, about 2:98, about 3:97,
about 4:96, about 5:95, about 6:94, about 7:93, about 8:92, about
9:91, or about 10:90. In yet other aspects of this embodiment, a
composition comprises a crosslinked matrix polymer and an
uncrosslinked matrix polymer where the gel:fluid ratio is, e.g., at
most 1:99, at most 2:98, at most 3:97, at most 4:96, at most 5:95,
at most 6:94, at most 7.:93, at most 8:92, at most 9:91, or at most
10:90. In still other aspects of this embodiment, a composition
comprises a crosslinked matrix polymer and an uncrosslinked matrix
polymer where the gel:fluid ratio is, e.g., about 0:100 to about
3:97, about 0:100 to about 5:95, or about 0:100 to about 10:90.
[0151] In other aspects of this embodiment, a composition comprises
a crosslinked matrix polymer and an uncrosslinked matrix polymer
where the gel:fluid ratio is, e.g., about 15:85, about 20:80, about
25:75, about 30:70, about 35:65, about 40:60, about 45:55, about
50:50, about 55:45, about 60:40, about 65:35, about 70:30, about
75:25, about 80:20, about 85:15, about 90:10, about 95:5, about
98:2, or about 100:0. In yet other aspects of this embodiment, a
composition comprises a crosslinked matrix polymer and an
uncrosslinked matrix polymer where the gel:fluid ratio is, e.g., at
most 15:85, at most 20:80, at most 25:75, at most 30:70, at most
35:65, at most 40:60, at most 45:55, at most 50:50, at most 55:45,
at most 60:40, at most 65:35, at most 70:30, at most 75:25, at most
80:20, at most 85:15, at most 90:10, at most 95:5, at most 98:2, or
at most 100:0. In still other aspects of this embodiment, a
composition comprises a crosslinked matrix polymer and an
uncrosslinked matrix polymer where the gel:fluid ratio is, e.g.,
about 10:90 to about 70:30, about 15:85 to about 70:30, about 10:90
to about 55:45, about 80:20 to about 95:5, about 90:10 to about
100:0, about 75:25 to about 100:0, or about 60:40 to about
100:0.
[0152] In still another embodiment, a composition comprises an
uncrosslinked matrix polymer where the uncrosslinked matrix polymer
is present in an amount sufficient to improve a condition of the
skin, such as, e.g., hydration or elasticity. In aspects of this
embodiment, a composition comprises an uncrosslinked matrix polymer
where the uncrosslinked matrix polymer is present at a
concentration of, e.g., about 5 mg/mL, about 6 mg/mL, about 7
mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 11
mg/mL, about 12 mg/mL, about 13 mg/mL, about 13.5 mglmL, about 14
mg/mL, about 15 mg/mL, about 16 mg/mL, about 17 mg/mL, about 18
mg/mL, about 19 mg/mL, or about 20 mg/mL. In other aspects of this
embodiment, a composition comprises an uncrosslinked matrix polymer
where the uncrosslinked matrix polymer is present at a
concentration of, e.g., at least 1 mg/mL, at least 5 mg/mL, at
least 10 mg/mL, at least 15 mg/mL, at least 20 mg/mL, or at least
25 mg/mL. In yet other aspects of this embodiment, a composition
comprises an uncrosslinked matrix polymer where the uncrosslinked
matrix polymer is present at a concentration of, e.g., at most 1
mg/mL, at most 5 mg/mL, at most 10 mg/mL, at most 15 mg/mL, at most
20 mg/mL, or at most 25 mg/mL. In still other aspects of this
embodiment, a composition comprises an uncrosslinked matrix polymer
where the uncrosslinked matrix polymer is present at a
concentration of, e.g., about 7.5 mg/mL to about 19.5 mg/m L, about
8.5 mg/mL to about 18.5 mg/mL, about 9.5 mg/mL to about 17.5 mg/mL,
about 10.5 mg/mL to about 16.5 mglmL, about 11.5 mg/mL to about
15.5 mg/mL, or about 12.5 mg/mL to about 14.5 mg/mL.
[0153] In aspects of this embodiment, a composition comprises an
uncrosslinked glycosaminoglycan where the uncrosslinked
glycosaminoglycan is present at a concentration of, e.g., about 2
mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL,
about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about
11 mg/mL, about 12 mg/mL, about 13 mg/mL, about 13.5 mg/mL, about
14 mg/mL, about 15 mg/mL, about 16 mg/mL, about 17 mg/mL, about 18
mg/mL, about 19 mg/mL, or about 20 mg/mL. In other aspects of this
embodiment, a composition comprises an uncrosslinked
glycosaminoglycan where the uncrosslinked glycosaminoglycan is
present at a concentration of, e.g., at least 1 mg/mL, at least 2
mg/mL, at least 3 mg/mL, at least 4 mg/mL, at least 5 mg/mL, at
least 10 mg/mL, at least 15 mg/m L, at least 20 mg/mL, or at least
25 mg/mL. In yet other aspects of this embodiment, a composition
comprises an uncrosslinked glycosaminoglycan where the
uncrosslinked glycosaminoglycan is present at a concentration of,
e.g., at most 1 mg/mL, at most 2 mg/mL, at most 3 mg/m L, at most 4
mg/mL, at most 5 mg/mL, at most 10 mg/mL, at most 15 mg/mL, at most
20 mg/mL, or at most 25 mg/mL. In still other aspects of this
embodiment, a composition comprises an uncrosslinked
glycosaminoglycan where the uncrosslinked glycosaminoglycan is
present at a concentration of, e.g., about 7.5 mg/mL to about 19.5
mg/mL, about 8.5 mg/mL to about 18.5 mg/mL, about 9.5 mg/mL to
about 17.5 mg/mL, about 10.5 mg/mL to about 16.5 mg/mL, about 11.5
mg/mL to about 15.5 mg/mL, or about 12.5 mg/mL to about 14.5
mg/mL.
[0154] In aspects of this embodiment, a composition comprises an
uncrosslinked lubricin where the uncrosslinked lubricin is present
at a concentration of, e.g., about 5 mg/mL, about 6 mg/mL, about 7
mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 11
mg/mL, about 12 mg/mL, about 13 mg/mL, about 13.5 mg/mL, about 14
mg/mL, about 15 mg/mL, about 16 mg/mL, about 17 mg/mL, about 18
mg/mL, about 19 mg/mL, or about 20 mg/mL. In other aspects of this
embodiment, a composition comprises an uncrosslinked lubricin where
the uncrosslinked lubricin is present at a concentration of, e.g.,
at least 1 mg/mL, at least 5 mg/mL, at least 10 mg/mL, at least 15
mg/mL, at least 20 mg/mL, or at least 25 mg/mL. In yet other
aspects of this embodiment, a composition comprises an
uncrosslinked lubricin where the uncrosslinked lubricin is present
at a concentration of, e.g., at most 1 mg/mL, at most 5 mg/mL, at
most 10 mg/mL, at most 15 mg/mL, at most 20 mg/mL, or at most 25
mg/mL. In still other aspects of this embodiment, a composition
comprises an uncrosslinked lubricin where the uncrosslinked
lubricin is present at a concentration of, e.g., about 7.5 mg/mL to
about 19.5 mg/mL, about 8.5 mg/mL to about 18.5 mg/mL, about 9.5
mg/mL to about 17.5 mg/mL, about 10.5 mg/mL to about 16.5 mg/mL,
about 11.5 mg/mL to about 15.5 mg/mL, or about 12.5 mg/mL to about
14.5 mg/mL.
[0155] In aspects of this embodiment, a composition comprises an
uncrosslinked polysaccharide where the uncrosslinked polysaccharide
is present at a concentration of, e.g., about 2 mg/mL, about 3
mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL,
about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 11 mg/mL, about
12 mg/mL, about 13 mg/mL, about 13.5 mg/mL, about 14 mg/mL, about
15 mg/mL, about 16 mg/mL, about 17 mg/mL, about 18 mg/mL, about 19
mg/mL, or about 20 mg/mL. In other aspects of this embodiment, a
composition comprises an uncrosslinked polysaccharide where the
uncrosslinked polysaccharide is present at a concentration of,
e.g., at least 1 mg/mL, at least 2 mg/mL, at least 3 mg/mL, at
least 4 mg/mL, at least 5 mg/mL, at least 10 mg/mL, at least 15
mg/mL, at least 20 mg/mL, or at least 25 mg/mL. In yet other
aspects of this embodiment, a composition comprises an
uncrosslinked polysaccharide where the uncrosslinked polysaccharide
is present at a concentration of, e.g., at most 1 mg/mL, at most 2
mg/mL, at most 3 mg/mL, at most 4 mg/mL, at most 5 mg/mL, at most
10 mg/mL, at most 15 mg/mL, at most 20 mg/mL, or at most 25 mg/mL.
In still other aspects of this embodiment, a composition comprises
an uncrosslinked polysaccharide where the uncrosslinked
polysaccharide is present at a concentration of, e.g., about 7.5
mg/mL to about 19.5 mg/mL, about 8.5 mg/mL to about 18.5 mg/mL,
about 9.5 mg/mL to about 17.5 mg/mL, about 10.5 mg/mL to about 16.5
mg/mL, about 11.5 mg/mL to about 15.5 mg/mL, or about 12.5 mg/mL to
about 14.5 mg/mL.
[0156] In an embodiment, a composition comprises an uncrosslinked
hyaluronan where the uncrosslinked hyaluronan comprises a
combination of both high molecular weight hyaluronan and low
molecular weight hyaluronan in a ratio of about 20:1, about 15:1,
about 10:1, about 5:1, about 1:1, about 1:5 about 1:10, about 1:15,
or about 1:20.
[0157] In another embodiment, a composition comprises an
uncrosslinked hyaluronan where the uncrosslinked hyaluronan
comprises a combination of both high molecular weight hyaluronan
and low molecular weight hyaluronan, in various ratios. As used
herein, the term "high molecular weight hyaluronan" refers to a
hyaluronan polymer that has a molecular weight of 1,000,000 Da or
greater. Non-limiting examples of a high molecular weight
hyaluronan include a hyaluronan of about 1,500,000 Da, a hyaluronan
of about 2,000,000 Da, a hyaluronan of about 2,500,000 Da, a
hyaluronan of about 3,000,000 Da, a hyaluronan of about 3,500,000
Da, a hyaluronan of about 4,000,000 Da, a hyaluronan of about
4,500,000 Da, and a hyaluronan of about 5,000,000 Da. As used
herein, the term "low molecular weight hyaluronan" refers to a
hyaluronan polymer that has a molecular weight of less than
1,000,000 Da. Non-limiting examples of a low molecular weight
hyaluronan include a hyaluronan of about 200,000 Da, a hyaluronan
of about 300,000 Da, a hyaluronan of about 400,000 Da, a hyaluronan
of about 500,000 Da, a hyaluronan of about 600,000 Da, a hyaluronan
of about 700,000 Da, a hyaluronan of about 800,000 Da, and a
hyaluronan of about 900,000 Da.
[0158] In other aspects of this embodiment, a composition comprises
a crosslinked hyaluronan where the crosslinked hyaluronan has a
mean molecular weight of, e.g., about 1,000,000 Da, about 1,500,000
Da, about 2,000,000 Da, about 2,500,000 Da, about 3,000,000 Da,
about 3,500,000 Da, about 4,000,000 Da, about 4,500,000 Da, or
about 5,000,000 Da. In yet other aspects of this embodiment, a
composition comprises a crosslinked hyaluronan where the
crosslinked hyaluronan has a mean molecular weight of, e.g., at
least 1,000,000 Da, at least 1,500,000 Da, at least 2,000,000 Da,
at least 2,500,000 Da, at least 3,000,000 Da, at least 3,500,000
Da, at least 4,000,000 Da, at least 4,500,000 Da, or at least
5,000,000 Da. In still other aspects of this embodiment, a
composition comprises a crosslinked hyaluronan where the
crosslinked hyaluronan has a mean molecular weight of, e.g., about
1,000,000 Da to about 5,000,000 Da, about 1,500,000 Da to about
5,000,000 Da, about 2,000,000 Da to about 5,000,000 Da, about
2,500,000 Da to about 5,000,000 Da, about 2,000,000 Da to about
3,000,000 Da, about 2,500,000 Da to about 3,500,000 Da, or about
2,000,000 Da to about 4,000,000 Da.
[0159] In other aspects of this embodiment, a composition comprises
an uncrosslinked hyaluronan where the uncrosslinked hyaluronan has
a mean molecular weight of, e.g., about 1,000,000 Da, about
1,500,000 Da, about 2,000,000 Da, about 2,500,000 Da, about
3,000,000 Da, about 3,500,000 Da, about 4,000,000 Da, about
4,500,000 Da, or about 5,000,000 Da. In yet other aspects of this
embodiment, a composition comprises an uncrosslinked hyaluronan
where the uncrosslinked hyaluronan has a mean molecular weight of,
e.g., at least 1,000,000 Da, at least 1,500,000 Da, at least
2,000,000 Da, at least 2,500,000 Da, at least 3,000,000 Da, at
least 3,500,000 Da, at least 4,000,000 Da, at least 4,500,000 Da,
or at least 5,000,000 Da. In still other aspects of this
embodiment, a composition comprises an uncrosslinked hyaluronan
where the uncrosslinked hyaluronan has a mean molecular weight of,
e.g., about 1,000,000 Da to about 5,000,000 Da, about 1,500,000 Da
to about 5,000,000 Da, about 2,000,000 Da to about 5,000,000 Da,
about 2,500,000 Da to about 5,000,000 Da, about 2,000,000 Da to
about 3,000,000 Da, about 2,500,000 Da to about 3,500,000 Da, or
about 2,000,000 Da to about 4,000,000 Da. In further aspects, a
composition comprises an uncrosslinked hyaluronan where the
uncrosslinked hyaluronan has a mean molecular weight of, e.g.,
greater than 2,000,000 Da and less than about 3,000,000 Da, greater
than 2,000,000 Da and less than about 3,500,000 Da, greater than
2,000,000 Da and less than about 4,000,000 Da, greater than
2,000,000 Da and less than about 4,500,000 Da, greater than
2,000,000 Da and less than about 5,000,000 Da.
[0160] A composition disclosed herein comprises a gel phase
including a silk fibroin hydrogel component or particle and matrix
polymer hydrogel component or particle. In aspects of this
embodiment, the percent amount of silk fibroin hydrogel present in
a composition relative to matrix polymer hydrogel is from about
0.1% (v/v) to about 25% (v/v). In aspects of this embodiment, the
percent amount of matrix polymer hydrogel present in a composition
relative to silk fibroin hydrogel is from about 99.9% (v/v) to
about 75% (v/v). In aspects of this embodiment, the ratio of silk
fibroin hydrogel to matrix polymer hydrogel in the gel phase of a
composition comprises, e.g., about 0.1% (v/v) silk fibroin hydrogel
and about 99.9% (v/v) matrix polymer hydrogel, about 1% (v/v) silk
fibroin hydrogel and about 99% (v/v) matrix polymer hydrogel, about
5% (v/v) silk fibroin hydrogel and about 95% (v/v) matrix polymer
hydrogel, about 10% (v/v) silk fibroin hydrogel and about 90% (v/v)
matrix polymer hydrogel, about 15% (v/v) silk fibroin hydrogel and
about 85% (v/v) matrix polymer hydrogel, about 20% (v/v) silk
fibroin hydrogel and about 80% (v/v) matrix polymer hydrogel, or
about 25% (v/v) silk fibroin hydrogel and about 75% (v/v) matrix
polymer hydrogel.
[0161] A composition disclosed herein may comprise a gel phase
where the silk fibroin hydrogel component and matrix polymer
hydrogel component are processed separately. The resulting
processed hydrogel materials, e.g., hydrogel particles of both
types, are then mixed together, such as, e.g., after a milling step
and/or after re-homogenization in a carrier phase, to form the
final composition. In addition, a matrix polymer may be initially
mixed with depolymerized silk fibroin solution, with subsequent
polymerization occurring only after the completion of the mixing
step to form an integrated matrix polymer/silk fibroin composite
hydrogel. Similarly, the silk fibroin and matrix polymers may be
linked together to form a hydrogel composite that is then
subsequently processed into the gel phase of the composition. Such
linkage can occur by a typical crosslinking method or by linking
the matrix polymer to the silk fibroin hydrogel via a peptide
linker disclosed herein, such as, e.g., a five-amino acid peptide
"tail" and synthetic molecule. As disclosed herein, a composition
may comprise a gel phase that comprises both separately processed
hydrogel components as well as particles of hydrogel
composites.
[0162] As a non-limiting example, a solution comprising about 1% to
about 30% depolymerized silk fibroin may be mixed with about 6 mg/g
to about 30 mg/g of hyaluronan having a degree of crosslinking of
from 0 to about 17% where the percent weight of the silk fibroin
component is from about 1% to about 75%. As another non-limiting
example, hydrogel particles comprising from about 1% to about 8%
silk fibroin are mixed with hydrogel particles comprising about 6
mg/g to about 30 mg/g of hyaluronan having a degree of crosslinking
of from 0 to about 17% where the percent weight of the silk fibroin
component is from about 1% to about 75%. As yet another
non-limiting example, a hydrogel composition comprising hydrogel
particles comprising from about 1% to about 8% silk fibroin mixed
together with a carrier phase (about 20% (v/v) to about 50% (v/v))
is mixed with a hydrogel composition comprising hydrogel particles
comprising about 6mg/g to about 30 mg/g of hyaluronan having a
degree of crosslinking of from 0 to about 17% where the percent
weight of the silk fibroin component is from about 1% to about
75%.
[0163] Aspects of the present specification provide, in part, a
composition comprising a silk fibroin hydrogel component or
particle and matrix polymer hydrogel component or particle having
an opacity. Opacity is the measure of impenetrability to
electromagnetic or other kinds of radiation, especially visible
light. An opaque object is neither transparent (allowing all light
to pass through) nor translucent (allowing some light to pass
through). In certain applications, it would be an advantage to have
an opaque composition. For example, in applications where a
composition disclosed herein is administered to a superficial
region, an opaque composition provides coloration and appearance of
the overlying skin.
[0164] In an embodiment, a composition comprising a silk fibroin
hydrogel and a polymer matrix is optically opaque. In aspects of
this embodiment, a composition comprising a silk fibroin hydrogel
and a polymer matrix transmits, e.g., about 5% of the light, about
10% of the light, about 15% of the light, about 20% of the light,
about 25% of the fight, about 30% of the light, about 35% of the
light, about 40% of the light, about 45% of the light, about 50% of
the light, about 55% of the light, about 60% of the light, about
65% of the light, or about 70% of the light. In other aspects of
this embodiment, a composition comprising a silk fibroin hydrogel
and a polymer matrix transmits, e.g., at most 5% of the light, at
most 10% of the light, at most 15% of the light, at most 20% of the
light, at most 25% of the light, at most 30% of the light, at most
35% of the light, at most 40% of the light, at most 45% of the
light, at most 50% of the light, at most 55% of the light, at most
60% of the light, at most 65% of the light, at most 70% of the
light, or at most 75% of the light. In other aspects of this
embodiment, a composition comprising a silk fibroin hydrogel and a
polymer matrix transmits, e.g., about 5% to about 15%, about 5% to
about 20%, about 5% to about 25%, about 5% to about 30%, about 5%
to about 35%, about 5% to about 40%, about 5% to about 45%, about
5% to about 50%, about 5% to about 55%, about 5% to about 60%,
about 5% to about 65%, about 5% to about 70%, about 5% to about
75%, about 15% to about 20%, about 15% to about 25%, about 15% to
about 30%, about 15% to about 35%, about 15% to about 40%, about
15% to about 45%, about 15% to about 50%, about 15% to about 55%,
about 15% to about 60%, about 15% to about 65%, about 15% to about
70%, about 15% to about 75%, about 25% to about 35%, about 25% to
about 40%, about 25% to about 45%, about 25% to about 50%, about
25% to about 55%, about 25% to about 60%, about 25% to about 65%,
about 25% to about 70%, or about 25% to about 75%, of the
light.
[0165] In aspects of this embodiment, a composition comprising a
silk fibroin hydrogel and a polymer matrix exhibits, e.g., about
5%, about 10%, about 15%, about 20%, about 25%, about 30%, about
35%, about 40%, about 45%, about 50%, about 55%, about 60%, about
65%, about 70%, about 75%, about 80%, about 85%, about 90%, about
95%, about 100% reduction in tyndalling. In other aspects of this
embodiment, a composition comprising a silk fibroin hydrogel and a
polymer matrix, e.g., at least 5%, at least 10%, at least 15%, at
least 20%, at least 25%, at least 30%, at least 35%, at least 40%,
at least 45%, at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, or at least 100%, reduction in tyndalling.
In other aspects of this embodiment, a composition comprising a
silk fibroin hydrogel and a polymer matrix exhibits, e.g., about
20% to about 100%, about 50% to about 100%, about 70% to about
100%, about 15% to about 35%, about 20% to about 40%, about 25% to
about 45%, about 30% to about 50%, about 35% to about 55%, about
40% to about 60%, about 45% to about 65%, about 50% to about 70%,
about 55% to about 75%, about 60% to about 80%, about 65% to about
85%, about 70% to about 90%, about 75% to about 95%, or about 80%
to about 100%, reduction in tyndalling.
[0166] Aspects of the present specification provide, in part, a
composition comprising a carrier phase. A composition disclosed
herein may, but may not, include a carrier phase. As such, the
disclosed compositions can be monophasic or multiphasic
compositions. As used herein, the term "carrier phase" is
synonymous with "carrier" and refers to a material used to increase
fluidity of a hydrogel. A carrier is advantageously a
physiologically-acceptable carrier and may include one or more
conventional excipients useful in pharmaceutical compositions. As
used herein, the term "a physiologically-acceptable carrier" refers
to a carrier in accord with, or characteristic of, the normal
functioning of a living organism. As such, administration of a
composition comprising a hydrogel and a carrier has substantially
no long term or permanent detrimental effect when administered to a
mammal. The present compositions include a carrier where a major of
the volume is water or saline. However, other useful carriers
include any physiologically tolerable material which improves upon
extrudability or intrudability of the hydrogel through a needle or
into a target host environment. Potential carriers could include
but are not limited to physiological buffer solutions, serum, other
protein solutions, gels composed of polymers including proteins,
glycoproteins, proteoglycans, or polysaccharides. Any of the
indicated potential carriers may be either naturally derived,
wholly synthetic, or combinations of both.
[0167] The volume of carrier per volume of hydrogel may be
increased or decreased in a range between 0% to about 100%
depending upon the desired physical properties of the resultant
composition including dose delivery, viscosity, injectability, and
desired in vivo behavioral characteristics. This carrier is then
mixed with the hydrogel until achieving a "uniform" consistency
which may be termed an emulsion or suspension. More specifically,
for example, a hydrogel may be passed through an 18 g needle
several times to create hydrogel particles, injecting back and
forth between a pair of syringes, then this procedure repeated with
22 g needles affixed to 1 mL syringes. Advantages derived from
adding a carrier to a hydrogel or hydrogel particles include
decreased viscosity in the extracellular in vivo microenvironment;
release of focal mechanical stress loading after drug delivery
platform administration; and improved ionic composition resulting
in improved biocompatibility.
[0168] Aspects of the present specification provide, in part, a
composition disclosed herein exhibiting a dynamic viscosity.
Viscosity is resistance of a fluid to shear or flow caused by
either shear stress or tensile stress. Viscosity describes a
fluid's internal resistance to flow caused by intermolecular
friction exerted when layers of fluids attempt to slide by one
another and may be thought of as a measure of fluid friction. The
less viscous the fluid, the greater its ease of movement
(fluidity).
[0169] Viscosity can be defined in two ways; dynamic viscosity
(.mu., although .eta. is sometimes used) or kinematic viscosity
(v). Dynamic viscosity, also known as absolute or complex
viscosity, is the tangential force per unit area required to move
one horizontal plane with respect to the other at unit velocity
when maintained a unit distance apart by the fluid. The SI physical
unit of dynamic viscosity is the Pascal-second (Pas), which is
identical to Nm-2s. Dynamic viscosity can be expressed as
.tau.=.mu.dvx/dz, where .tau.=shearing stress, .mu.=dynamic
viscosity, and dvx/dz is the velocity gradient over time. For
example, if a fluid with a viscosity of one Pas is placed between
two plates, and one plate is pushed sideways with a shear stress of
one Pascal, it moves a distance equal to the thickness of the layer
between the plates in one second. Dynamic viscosity symbolize by is
also used, is measured with various types of rheometers, devices
used to measure the way in which a liquid, suspension or slurry
flows in response to applied forces.
[0170] Kinematic viscosity (v) is the ratio of dynamic viscosity to
density, a quantity in which no force is involved and is defined as
follows: v=.mu./.rho., where .mu. is the dynamic viscosity .rho. is
density with the SI unit of kg/m.sup.3. Kinematic viscosity is
usually measured by a glass capillary viscometer as has an SI unit
of m.sup.2/s.
[0171] The viscosity of a fluid is highly temperature dependent and
for either dynamic or kinematic viscosity to be meaningful, the
reference temperature must be quoted. For the viscosity values
disclosed herein, a dynamic viscosity is measured at 1 Pa with a
cone/plane geometry 2.degree./40 cm and a temperature of 20.degree.
C. Examples of the dynamic viscosity of various fluids at
20.degree. C. is as follows: water is about 1.0.times.10.sup.-3
Pas, blood is about 3-4.times.10.sup.'Pas, vegetable oil is about
60-85.times.10.sup.-3 Pas, motor oil SE 30 is about 0.2 Pas,
glycerin is about 1.4 Pas, maple syrup is about 2-3 Pas, honey is
about 10 Pas, chocolate syrup is about 10-25 Pas, peanut butter is
about 150-250 Pas, lard is about 1,000 Pas, vegetable shortening is
about 1,200 Pas, and tar is about 30,000 Pas.
[0172] In aspects of this embodiment, a composition disclosed
herein exhibits a dynamic viscosity of, e.g., about 10 Pas, about
20 Pas, about 30 Pas, about 40 Pas, about 50 Pas, about 60 Pas,
about 70 Pas, about 80 Pas, about 90 Pas, about 100 Pas, about 125
Pas, about 150 Pas, about 175 Pas, about 200 Pas, about 225 Pas,
about 250 Pas, about 275 Pas, about 300 Pas, about 400 Pas, about
500 Pas, about 600 Pas, about 700 Pas, about 750 Pas, about 800
Pas, about 900 Pas, about 1,000 Pas, about 1,100 Pas, or about
1,200 Pas. In other aspects of this embodiment, a composition
disclosed herein exhibits a dynamic viscosity of, e.g., at most 10
Pas, at most 20 Pas, at most 30 Pas, at most 40 Pas, at most 50
Pas, at most 60 Pas, at most 70 Pas, at most 80 Pas, at most 90
Pas, at most 100 Pas, at most 125 Pas, at most 150 Pas, at most 175
Pas, at most 200 Pas, at most 225 Pas, at most 250 Pas, at most 275
Pas, at most 300 Pas, at most 400 Pas, at most 500 Pas, at most 600
Pas, at most 700 Pas, at most 750 Pas, at most 800 Pas, at most 900
Pas, or at most 1000 Pas. In yet other aspects of this embodiment,
a composition disclosed herein exhibits a dynamic viscosity of,
e.g., about 10 Pas to about 100 Pas, about 10 Pas to about 150 Pas,
about 10 Pas to about 250 Pas, about 50 Pas to about 100 Pas, about
50 Pas to about 150 Pas, about 50 Pas to about 250 Pas, about 100
Pas to about 500 Pas, about 100 Pas to about 750 Pas, about 100 Pas
to about 1,000 Pas, about 100 Pas to about 1,200 Pas, about 300 Pas
to about 500 Pas, about 300 Pas to about 750 Pas, about 300 Pas to
about 1,000 Pas, or about 300 Pas to about 1,200 Pas.
[0173] Aspects of the present specification provide, in part, a
composition disclosed herein is injectable. As used herein, the
term "injectable" refers to a material having the properties
necessary to administer the composition into a skin region of an
individual using an injection device with a fine needle. As used
herein, the term "fine needle" refers to a needle that is 27 gauge
or smaller. Injectability of a composition disclosed herein can be
accomplished by sizing the hydrogel particles as discussed
above.
[0174] In aspect of this embodiment, a composition disclosed herein
is injectable through a fine needle. In other aspects of this
embodiment, a composition disclosed herein is injectable through a
needle of, e.g., about 27 gauge, about 30 gauge, or about 32 gauge.
In yet other aspects of this embodiment, a composition disclosed
herein is injectable through a needle of, e.g., 27 gauge or
smaller, 30 gauge or smaller, or 32 gauge or smaller. In still
other aspects of this embodiment, a composition disclosed herein is
injectable through a needle of, e.g., about 27 gauge to about 32
gauge.
[0175] In aspects of this embodiment, a composition disclosed
herein can be injected with an extrusion force of about 60 N, about
55 N, about 50 N, about 45 N, about 40 N, about 35 N, about 30 N,
about 25 N, about 20 N, or about 15 N. In other aspects of this
embodiment, a composition disclosed herein can be injected through
a 27 gauge needle with an extrusion force of about 60 N or less,
about 55 N or less, about 50 N or less, about 45 N or less, about
40 N or less, about 35 N or less, about 30 N or less, about 25 N or
less, about 20 N or less, about 15 N or less, about 10 N or less,
or about 5 N or less. In yet other aspects of this embodiment, a
composition disclosed herein can be injected through a 30 gauge
needle with an extrusion force of about 60 N or less, about 55 N or
less, about 50 N or less, about 45 N or less, about 40 N or less,
about 35 N or less, about 30 N or less, about 25 N or less, about
20 N or less, about 15 N or less, about 10 N or less, or about 5 N
or less. In still other aspects of this embodiment, a composition
disclosed herein can be injected through a 32 gauge needle with an
extrusion force of about 60 N or less, about 55 N or less, about 50
N or less, about 45 N or less, about 40 N or less, about 35 N or
less, about 30 N or less, about 25 N or less, about 20 N or less,
about 15 N or less, about 10 N or less, or about 5 N or less.
[0176] Aspects of the present specification provide, in part, a
composition disclosed herein exhibits cohesiveness. Cohesion or
cohesive attraction, cohesive force, or compression force is a
physical property of a material, caused by the intermolecular
attraction between like-molecules within the material that acts to
unite the molecules. A composition should be sufficiently cohesive
as to remain localized to a site of administration. Additionally,
in certain applications, a sufficient cohesiveness is important for
a composition to retain its shape, and thus functionality, in the
event of mechanical load cycling. As such, in one embodiment, a
composition exhibits strong cohesive attraction, on par with water.
In another embodiment, a composition exhibits low cohesive
attraction. In yet another embodiment, a composition exhibits
sufficient cohesive attraction to remain localized to a site of
administration. In still another embodiment, a composition exhibits
sufficient cohesive attraction to retain its shape. In a further
embodiment, a composition exhibits sufficient cohesive attraction
to retain its shape and functionality.
[0177] In aspects of this embodiment, a composition disclosed
herein has a compression force of about 10 grams-force, about 20
grams-force, about 30 grams-force, about 40 grams-force, about 50
grams-force, about 60 grams-force, about 70 grams-force, about 80
grams-force, about 90 grams-force, about 100 grams-force, about 200
grams-force, about 300 grams-force, about 400 grams-force, about
500 grams-force, about 600 grams-force, about 700 grams-force, or
about 800 grams-force. In other aspects of this embodiment, a
composition disclosed herein has a compression force of at least
500 grams-force, at least 600 grams-force, at least 700
grams-force, at least 800 grams-force, at least 900 grams-force, at
least 1000 grams-force, at least 1250 grams-force, at least 1500
grams-force, at least 1750 grams-force, at least 2000 grams-force,
at least 2250 grams-force, at least 2500 grams-force, at least 2750
grams-force, or at least 3000 grams-force. In other aspects of this
embodiment, a composition disclosed herein has a compression force
of at most 10 grams-force, at most 20 grams-force, at most 30
grams-force, at most 40 grams-force, at most 50 grams-force, at
most 60 grams-force, at most 70 grams-force, at most 80
grams-force, at most 90 grams-force, at most 100 grams-force, at
most 200 grams-force, at most 300 grams-force, at most 400
grams-force, at most 500 grams-force, at most 600 grams-force, at
most 700 grams-force, or at most 800 grams-force.
[0178] In yet other aspects of this embodiment, a composition
disclosed herein has a compression force of about 10 grams-force to
about 50 grams-force, about 25 grams-force to about 75 grams-force,
about 50 grams-force to about 150 grams-force, about 100
grams-force to about 200 grams-force, about 100 grams-force to
about 300 grams-force, about 100 grams-force to about 400
grams-force, about 100 grams-force to about 500 grams-force, about
200 grams-force to about 300 grams-force, about 200 grams-force to
about 400 grams-force, about 200 grams-force to about 500
grams-force, about 200 grams-force to about 600 grams-force, about
200 grams-force to about 700 grams-force, about 300 grams-force to
about 400 grams-force, about 300 grams-force to about 500
grams-force, about 300 grams-force to about 600 grams-force, about
300 grams-force to about 700 grams-force, about 300 grams-force to
about 800 grams-force, about 400 grams-force to about 500, about
400 grams-force to about 600, about 400 grams-force to about 700,
about 400 grams-force to about 800, about 500 grams-force to about
600 grams-force, about 500 grams-force to about 700 grams-force,
about 500 grams-force to about 800 grams-force, about 600
grams-force to about 700 grams-force, about 600 grams-force to
about 800 grams-force, about 700 grams-force to about 800
grams-force, about 1000 grams-force to about 2000 grams-force,
about 1000 grams-force to about 3000 grams-force, or about 2000
grams-force to about 3000 grams-force.
[0179] Aspects of the present hydrogel formulations provide, in
part, a surfactant. As used herein, the term "surfactant" refers to
a natural or synthetic amphiphilic compound. A surfactant can be
non-ionic, zwitterionic, or ionic. It is envisioned that any
surfactant is useful in making a hydrogel formulation disclosed in
the present specification, with the proviso that a therapeutically
effective amount of the hydrogel formulation is recovered using
this surfactant amount. Non-limiting examples of surfactants
include polysorbates like polysorbate 20 (TWEEN.RTM. 20),
polysorbate 40 (TWEEN.RTM. 40), polysorbate 60 (TWEEN.RTM. 60),
polysorbate 61 (TWEEN.RTM. 61), polysorbate 65 (TWEEN.RTM. 65),
polysorbate 80 (TWEEN.RTM. 80), and polysorbate 81 (TWEEN.RTM. 81);
poloxamers (polyethylene-polypropylene copolymers), like Poloxamer
124 (PLURONIC.RTM. L44), Poloxamer 181 (PLURONIC.RTM. L61),
Poloxamer 182 (PLURONIC.RTM. L62), Poloxamer 184 (PLURONIC.RTM.
L64), Poloxamer 188 (PLURONIC.RTM. F68), Poloxamer 237
(PLURONIC.RTM. F87), Poloxamer 338 (PLURONIC.RTM. L108), Poloxamer
407 (PLURONIC.RTM. F127), polyoxyethyleneglycol dodecyl ethers,
like BRIJ.RTM. 30, and BRIJ.RTM. 35; 2-dodecoxyethanol
(LUBROL.RTM.-PX); polyoxyethylene octyl phenyl ether (TRITON.RTM.
X-100); sodium dodecyl sulfate (S DS);
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS);
3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate
(CHAPSO); sucrose monolaurate; and sodium cholate. Other
non-limiting examples of surfactant excipients can be found in,
e.g., Pharmaceutical Dosage Forms and Drug Delivery Systems (Howard
C. Ansel et al., eds., Lippincott Williams & Wilkins
Publishers, 7.sup.th ed. 1999); Remington: The Science and Practice
of Pharmacy (Alfonso R. Gennaro ed., Lippincott, Williams &
Wilkins, 20.sup.th ed. 2000); Goodman & Gilman's The
Pharmacological Basis of Therapeutics (Joel G. Hardman et al.,
eds., McGraw-Hill Professional, 10.sup.th ed. 2001); and Handbook
of Pharmaceutical Excipients (Raymond C. Rowe et al., APhA
Publications, 4.sup.th edition 2003), each of which is hereby
incorporated by reference in its entirety.
[0180] In aspects of this embodiment, a hydrogel formulation
comprises a polysorbate, a poloxamer, a polyoxyethyleneglycol
dodecyl ether, 2-dodecoxyethanol, polyoxyethylene octyl phenyl
ether, sodium dodecyl sulfate,
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate,
3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate,
sucrose monolaurate; or sodium cholate.
[0181] Aspects of the present specification provide, in part, a
method of treating a soft tissue condition of an individual by
administering a composition disclosed herein. As used herein, the
term "treating," refers to reducing or eliminating in an individual
a cosmetic or clinical symptom of a soft tissue condition
characterized by a soft tissue imperfection, defect, disease,
and/or disorder; or delaying or preventing in an individual the
onset of a cosmetic or clinical symptom of a condition
characterized by a soft tissue imperfection, defect, disease,
and/or disorder. For example, the term "treating" can mean reducing
a symptom of a condition characterized by a soft tissue defect,
disease, and/or disorder by, e.g., at least 20%, at least 30%, at
least 40%, at least 50%, at least 60%, at least 70%, at least 80%,
at least 90% or at least 100%. The effectiveness of a compound
disclosed herein in treating a condition characterized by a soft
tissue defect, disease, and/or disorder can be determined by
observing one or more cosmetic, clinical symptoms, and/or
physiological indicators associated with the condition. An
improvement in a soft tissue defect, disease, and/or disorder also
can be indicated by a reduced need for a concurrent therapy. Those
of skill in the art will know the appropriate symptoms or
indicators associated with specific soft tissue defect, disease,
and/or disorder and will know how to determine if an individual is
a candidate for treatment with a compound or composition disclosed
herein.
[0182] A composition or compound is administered to an individual.
An individual is typically a human being. Typically, any individual
who is a candidate for a conventional procedure to treat a soft
tissue condition is a candidate for a method disclosed herein. In
addition, the presently disclosed compositions and methods may
apply to individuals seeking a small/moderate enlargement, shape
change or contour alteration of a body part or region, which may
not be technically possible or aesthetically acceptable with
existing soft tissue implant technology. Pre-operative evaluation
typically includes routine history and physical examination in
addition to thorough informed consent disclosing all relevant risks
and benefits of the procedure.
[0183] The composition and methods disclosed herein are useful in
treating a soft tissue condition. A soft tissue condition includes,
without limitation, a soft tissue imperfection, defect, disease,
and/or disorder. Non-limiting examples of a soft tissue condition
include breast imperfection, defect, disease and/or disorder, such
as, e.g., a breast augmentation, a breast reconstruction,
mastopexy, micromastia, thoracic hypoplasia, Poland's syndrome,
defects due to implant complications like capsular contraction
and/or rupture; a facial imperfection, defect, disease or disorder,
such as, e.g., a facial augmentation, a facial reconstruction,
Parry-Romberg syndrome, lupus erythematosus profundus, dermal
divots, sunken checks, thin lips, nasal imperfections or defects,
retro-orbital imperfections or defects, a facial fold, line and/or
wrinkle like a glabellar line, a nasolabial line, a perioral line,
and/or a marionette line, and/or other contour deformities or
imperfections of the face; a neck imperfection, defect, disease or
disorder; a skin imperfection, defect, disease and/or disorder;
other soft tissue imperfections, defects, diseases and/or
disorders, such as, e.g., an augmentation or a reconstruction of
the upper arm, lower arm, hand, shoulder, back, torso including
abdomen, buttocks, upper leg, lower leg including calves, foot
including plantar fat pad, eye, genitals, or other body part,
region or area, or a disease or disorder affecting these body
parts, regions or areas; urinary incontinence, fecal incontinence,
other forms of incontinence; and gastroesophageal reflux disease
(GERD).
[0184] The amount of a composition used with any of the methods as
disclosed herein will typically be determined based on the
alteration and/or improvement desired, the reduction and/or
elimination of a soft tissue condition symptom desired, the
clinical and/or cosmetic effect desired by the individual and/or
physician, and the body part or region being treated. The
effectiveness of composition administration may be manifested by
one or more of the following clinical and/or cosmetic measures:
altered and/or improved soft tissue shape, altered and/or improved
soft tissue size, altered and/or improved soft tissue contour,
altered and/or improved tissue function, tissue ingrowth support
and/or new collagen deposition, sustained engraftment of
composition, improved patient satisfaction and/or quality of life,
and decreased use of implantable foreign material.
[0185] For example, for breast augmentation procedures,
effectiveness of the compositions and methods may be manifested by
one or more of the following clinical and/or cosmetic measures:
increased breast size, altered breast shape, altered breast
contour, sustained engraftment, reduction in the risk of capsular
contraction, decreased rate of liponecrotic cyst formation,
improved patient satisfaction and/or quality of life, and decreased
use of breast implant.
[0186] As another example, effectiveness of the compositions and
methods in treating a facial soft tissue may be manifested by one
or more of the following clinical and/or cosmetic measures:
increased size, shape, and/or contour of facial feature like
increased size, shape, and/or contour of lip, cheek or eye region;
altered size, shape, and/or contour of facial feature like altered
size, shape, and/or contour of lip, cheek or eye region shape;
reduction or elimination of a wrinkle, fold or line in the skin;
resistance to a wrinkle, fold or line in the skin; rehydration of
the skin; increased elasticity to the skin; reduction or
elimination of skin roughness; increased and/or improved skin
tautness; reduction or elimination of stretch lines or marks;
increased and/or improved skin tone, shine, brightness and/or
radiance; increased and/or improved skin color, reduction or
elimination of skin paleness; sustained engraftment of composition;
decreased side effects; improved patient satisfaction and/or
quality of life.
[0187] As yet another example, for urinary incontinence procedures,
effectiveness of the compositions and methods for sphincter support
may be manifested by one or more of the following clinical
measures: decreased frequency of incontinence, sustained
engraftment, improved patient satisfaction and/or quality of life,
and decreased use of implantable foreign filler.
[0188] The amount of a composition used with any of the methods
disclosed herein will typically be a therapeutically effective
amount. As used herein, the term "therapeutically effective amount"
is synonymous with "effective amount", "therapeutically effective
dose", and/or " effective dose" and refers to the amount of
compound that will elicit the biological, cosmetic or clinical
response being sought by the practitioner in an individual in need
thereof. As a non-limiting example, an effective amount is an
amount sufficient to achieve one or more of the clinical and/or
cosmetic measures disclosed herein. The appropriate effective
amount to be administered for a particular application of the
disclosed methods can be determined by those skilled in the art,
using the guidance provided herein. For example, an effective
amount can be extrapolated from in vitro and in vivo assays as
described in the present specification. One skilled in the art will
recognize that the condition of the individual can be monitored
throughout the course of therapy and that the effective amount of a
composition disclosed herein that is administered can be adjusted
accordingly.
[0189] In aspects of this embodiment, the amount of a composition
administered is, e.g., 0.01 g, 0.05 g, 0.1 g, 0.5 g, 1 g, 5 g, 10
g, 20 g, 30 g, 40 g, 50 g, 60 g, 70 g, 80 g, 90 g, 100 g, 150 g, or
200 g. In other aspects of this embodiment, the amount of a
composition administered is, e.g., about 0.01 g to about 0.1 g,
about 0.1 g to about 1 g, about 1 g to about 10 g, about 10 g to
about 100 g, or about 50 g to about 200 g. In yet other aspects of
this embodiment, the amount of a composition administered is, e.g.,
0.01 mL, 0.05 mL, 0.1 mL, 0.5 mL, 1 mL, 5 mL, 10 mL, 20 mL, 30 mL,
40 mL, 50 mL, 60 mL, 70 g, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL.
In other aspects of this embodiment, the amount of a composition
administered is, e.g., about 0.01 mL to about 0.1 mL, about 0.1 mL
to about 1 mL, about 1 mL to about 10 mL, about 10 mL to about 100
mL, or about 50 mL to about 200 mL.
[0190] Aspects of the present invention provide, in part,
administering a composition disclosed herein. As used herein, the
term "administering" means any delivery mechanism that provides a
composition disclosed herein to an individual that potentially
results in a clinically, therapeutically, or experimentally
beneficial result. The actual delivery mechanism used to administer
a composition to an individual can be determined by a person of
ordinary skill in the art by taking into account factors,
including, without limitation, the type of skin condition, the
location of the skin condition, the cause of the skin condition,
the severity of the skin condition, the degree of relief desired,
the duration of relief desired, the particular composition used,
the rate of excretion of the particular composition used, the
pharmacodynamics of the particular composition used, the nature of
the other compounds included in the particular composition used,
the particular route of administration, the particular
characteristics, history and risk factors of the individual, such
as, e.g., age, weight, general health and the like, or any
combination thereof. In an aspect of this embodiment, a composition
disclosed herein is administered to a skin region of an individual
by injection.
[0191] The route of administration of composition administered to
an individual patient will typically be determined based on the
cosmetic and/or clinical effect desired by the individual and/or
physician and the body part or region being treated. A composition
disclosed herein may be administered by any means known to persons
of ordinary skill in the art including, without limitation, syringe
with needle, catheter, topically, or by direct surgical
implantation. The composition disclosed herein can be administered
into a skin region such as, e.g., a dermal region or a hypodermal
region. In addition, a composition disclosed herein can be
administered once, or over a plurality of times. Ultimately, the
timing used will follow quality care standards.
[0192] For a breast soft tissue replacement procedure, the route of
administration may include axillary, periareolar, and/or
inframammary routes. Alternatively or in addition, a composition
may be delivered through a transaxillary endoscopic subpectoral
approach. For a facial soft tissue replacement procedure, the route
of administration can be frontal, temporal, zygomatic, periocular,
amdibula, perioral or chin routes. In urinary incontinence
procedures, the route of administration may include transurethral
or periurethral routes. Alternatively or in addition,
administration may be delivered via an antegrade route. The routes
discussed herein do not exclude the use of multiple routes to
achieve the desired clinical effect.
[0193] Aspects of the present invention provide, in part, a dermal
region. As used herein, the term "dermal region" refers to the
region of skin comprising the epidermal-dermal junction and the
dermis including the superficial dermis (papillary region) and the
deep dermis (reticular region). The skin is composed of three
primary layers: the epidermis, which provides waterproofing and
serves as a barrier to infection; the dermis, which serves as a
location for the appendages of skin; and the hypodermis
(subcutaneous adipose layer). The epidermis contains no blood
vessels, and is nourished by diffusion from the dermis. The main
type of cells which make up the epidermis are keratinocytes,
melanocytes, Langerhans cells and Merkels cells.
[0194] The dermis is the layer of skin beneath the epidermis that
consists of connective tissue and cushions the body from stress and
strain. The dermis is tightly connected to the epidermis by a
basement membrane. It also harbors many Mechanoreceptor/nerve
endings that provide the sense of touch and heat. It contains the
hair follicles, sweat glands, sebaceous glands, apocrine glands,
lymphatic vessels and blood vessels. The blood vessels in the
dermis provide nourishment and waste removal from its own cells as
well as from the Stratum basale of the epidermis. The dermis is
structurally divided into two areas: a superficial area adjacent to
the epidermis, called the papillary region, and a deep thicker area
known as the reticular region.
[0195] The papillary region is composed of loose areolar connective
tissue. It is named for its fingerlike projections called papillae
that extend toward the epidermis. The papillae provide the dermis
with a "bumpy" surface that interdigitates with the epidermis,
strengthening the connection between the two layers of skin. The
reticular region lies deep in the papillary region and is usually
much thicker. It is composed of dense irregular connective tissue,
and receives its name from the dense concentration of collagenous,
elastic, and reticular fibers that weave throughout it. These
protein fibers give the dermis its properties of strength,
extensibility, and elasticity. Also located within the reticular
region are the roots of the hair, sebaceous glands, sweat glands,
receptors, nails, and blood vessels. Tattoo ink is held in the
dermis. Stretch marks from pregnancy are also located in the
dermis.
[0196] The hypodermis lies below the dermis. Its purpose is to
attach the dermal region of the skin to underlying bone and muscle
as well as supplying it with blood vessels and nerves. It consists
of loose connective tissue and elastin. The main cell types are
fibroblasts, macrophages and adipocytes (the hypodermis contains
50% of body fat). Fat serves as padding and insulation for the
body.
[0197] In an aspect of this embodiment, a composition disclosed
herein is administered to a skin region of an individual by
injection into a dermal region or a hypodermal region. In aspects
of this embodiment, a composition disclosed herein is administered
to a dermal region of an individual by injection into, e.g., an
epidermal-dermal junction region, a papillary region, a reticular
region, or any combination thereof.
[0198] Aspects of the present specification disclose, in part, a
method of treating a soft tissue condition of an individual, the
method comprising the steps of administering a composition
disclosed herein to a site of the soft tissue condition of the
individual, wherein the administration of the composition improves
the soft tissue condition, thereby treating the soft tissue
condition. In aspects of this embodiment, a soft tissue condition
is a breast tissue condition, a facial tissue condition, a neck
condition, a skin condition, an upper arm condition, a lower arm
condition, a hand condition, a shoulder condition, a back
condition, a torso including abdominal condition, a buttock
condition, an upper leg condition, a lower leg condition including
calf condition, a foot condition including plantar fat pad
condition, an eye condition, a genital condition, or a condition
effecting another body part, region or area.
[0199] Other aspects of the present specification disclose, in
part, a method of treating a skin condition comprises the step of
administering to an individual suffering from a skin condition a
composition disclosed herein, wherein the administration of the
composition improves the skin condition, thereby treating the skin
condition. In an aspect of this embodiment, a skin condition is a
method of treating skin dehydration comprises the step of
administering to an individual suffering from skin dehydration a
composition disclosed herein, wherein the administration of the
composition rehydrates the skin, thereby treating skin dehydration.
In another aspect of this embodiment, a method of treating a lack
of skin elasticity comprises the step of administering to an
individual suffering from a lack of skin elasticity a composition
disclosed herein, wherein the administration of the composition
increases the elasticity of the skin, thereby treating a lack of
skin elasticity. In yet another aspect of this embodiment, a method
of treating skin roughness comprises the step of administering to
an individual suffering from skin roughness a composition disclosed
herein, wherein the administration of the composition decreases
skin roughness, thereby treating skin roughness. In still another
aspect of this embodiment, a method of treating a lack of skin
tautness comprises the step of administering to an individual
suffering from a lack of skin tautness a composition disclosed
herein, wherein the administration of the composition makes the
skin tauter, thereby treating a lack of skin tautness.
[0200] In a further aspect of this embodiment, a method of treating
a skin stretch line or mark comprises the step of administering to
an individual suffering from a skin stretch line or mark a
composition disclosed herein, wherein the administration of the
composition reduces or eliminates the skin stretch line or mark,
thereby treating a skin stretch line or mark. In another aspect of
this embodiment, a method of treating skin paleness comprises the
step of administering to an individual suffering from skin paleness
a composition disclosed herein, wherein the administration of the
composition increases skin tone or radiance, thereby treating skin
paleness. In another aspect of this embodiment, a method of
treating skin wrinkles comprises the step of administering to an
individual suffering from skin wrinkles a composition disclosed
herein, wherein the administration of the composition reduces or
eliminates skin wrinkles, thereby treating skin wrinkles. In yet
another aspect of this embodiment, a method of treating skin
wrinkles comprises the step of administering to an individual a
composition disclosed herein, wherein the administration of the
composition makes the skin resistant to skin wrinkles, thereby
treating skin wrinkles.
[0201] Aspects of the present specification provide, in part,
administration of a composition disclosed herein wherein such
administration promotes new collagen deposition. The compositions
comprising a silk fibroin hydrogel component or particle and matrix
polymer hydrogel component or particle support tissue ingrowth and
new deposition of collagen (Example 21).
[0202] In an embodiment, administration of a composition comprising
a silk fibroin hydrogel component and a matrix polymer hydrogel
component as disclosed herein increases new collagen deposition. In
aspects of this embodiment, administration of a composition
comprising a silk fibroin hydrogel component and a matrix polymer
hydrogel component as disclosed herein increases new collagen
deposition by about 10%, about 20%, about 30%, about 40%, about
50%, about 60%, about 70%, about 80%, about 90%, or about 100%,
relative to a the same or similar composition comprising the matrix
polymer hydrogel component, but lacking the silk fibroin hydrogel
component. In other aspects of this embodiment, administration of a
composition comprising a silk fibroin hydrogel component and a
matrix polymer hydrogel component as disclosed herein increases new
collagen deposition by at least 25%, at least 50%, at least 75%, at
least 100%, at least 125%, at least 150%, at least 175%, at least
200%, at least 225%, at least 250%, at least 275%, or at least
300%, relative to a the same or similar composition comprising the
matrix polymer hydrogel component, but lacking the silk fibroin
hydrogel component. In yet other aspects of this embodiment,
administration of a composition comprising a silk fibroin hydrogel
component and a matrix polymer hydrogel component as disclosed
herein increases new collagen deposition by about 10% to about
100%, about 50% to about 150%, about 100% to about 200%, about 150%
to about 250%, about 200% to about 300%, about 350% to about 450%,
about 400% to about 500%, about 550% to about 650%, about 600% to
about 700%, relative to a the same or similar composition
comprising the matrix polymer hydrogel component, but lacking the
silk fibroin hydrogel component.
EXAMPLES
[0203] The following examples illustrate representative embodiments
now contemplated, but should not be construed to limit the
disclosed purified silk fibroin and method for purifying such silk
fibroins, hydrogels comprising such silk fibroin with or without an
amphiphilic peptide and methods for making hydrogels comprising
such silk fibroin and the use of silk fibroin hydrogels in a
variety of medical uses, including, without limitation fillers for
tissue space, templates for tissue reconstruction or regeneration,
scaffolds for cells in tissue engineering applications and for
disease models, a surface coating to improve medical device
function, or as a platform for drug delivery.
Example 1
Silk Sericin Extraction
[0204] Silk fibroin for generation of the hydrogel was obtained in
the form of degummed B. mori silk at a size of 20 denier -22 denier
(38 .mu.m.+-.5.6 .mu.m diameter). This degummed silk was further
processed in order to remove the inherently present and potentially
antigenic protein glue, sericin that conjoins independent fibroin
filaments. This was done as described previously herein. Following
removal of sericin, the pure fibroin was dried carefully to ambient
humidity levels using a laminar flow hood.
Example 2
Generation of Silk Fibroin Solution
[0205] Silk fibroin filaments, cleaned of their sericin and rinsed
free of insoluble debris and ionic contaminants were used for the
generation of an aqueous silk solution. These silk fibers were
added to a solution of 9.3M LiBr and purified water (e.g.,
MILLI-Q.RTM. Ultrapure Water Purification Systems) (Millipore,
Billerica, Mass.) to make a solution consisting of 20% pure silk (%
w/v). This mixture was then heated to a temperature of 60.degree.
C. and digested for a period of four hours. A total of 12 mL of the
resultant solution was then loaded into a 3 mL-12 mL Slide-A-Lyzer
dialysis cassette (Pierce Biotechnology, Inc., Rockford, Ill.)
(molecular weight cutoff of 3.5 kD) and placed into a beaker
containing purified water as a dialysis buffer at a volume of 1 L
water per 12 mL cassette of silk solution. The beakers were placed
on stir plates and stirred continuously for the duration of the
dialysis. Changes of dialysis buffer occurred at 1, 4, 12, 24, and
36 hours of processing time.
[0206] Following dialysis, the solution was removed from the
cassettes by means of a syringe and needle and centrifuged at
30,000 g relative centrifugal force (RCF) at 4.degree. C. for 30
minutes, decanting the supernatant (silk solution) into a clean
centrifuge tube, then repeating the centrifugation for a further 30
minutes. This process of centrifugation is beneficial for removal
of insoluble particulate debris associated with the silk solution
both prior to and following after dialysis. It is believed that
such insoluble debris could serve as antigens in vivo or perhaps
nucleation points about which gelation of the silk could occur,
shortening storage life of the solution and compromising the
uniformity of the gelation system. After completion of the second
centrifugation, the supernatant was again collected and stored at
4.degree. C. until needed. To confirm uniformity of the dialysis
product, known volumes of the solution were collected, massed, and
then dried completely through lyophilization. These lyophilized
samples were then massed and the dry mass of solution compared to
initial solution volume to determine percent silk present per unit
volume of solution. Additionally, the solution was assessed via
X-ray Photoelectron Spectroscopy (XPS) analysis to ensure that no
detectable quantities of Li.sup.+ or Br.sup.- ions were present in
the solution.
Example 3
Induction of Gelation
[0207] A variety of different methods were employed in the course
of hydrogel development for the purposes of contrasting and
comparing certain relevant properties of various formulae.
Regardless of the nature in which the gelation process was carried
out, the final determination that a "gel" state had been reached
was applied uniformly to all groups. A solution or composite of
solutions (i.e., silk solution blended with an enhancer or enhancer
solution) was considered a gel after observing formation of a
uniform solid phase throughout the entire volume, generally opaque
and white in appearance.
[0208] Samples to be produced by passive gelation were not exposed
to any enhancer additives. These gels were produced by measuring a
volume of silk solution into a casting vessel, for the purposes of
these experiments, polypropylene tubes sealed against air
penetration and water loss, and the sample allowed to stand under
ambient room conditions (nominally 20-24.degree. C., 1 atm, 40%
relative humidity) until fully gelled. Care was taken to ensure
uniformity of casting vessels material of construction across
groups so as to avoid potential influence from surface effects.
These effects may serve to enhance or inhibit gelation and may be
caused by factors including but not limited to siliconization,
surface roughness, surface charge, debris contamination, surface
hydrophobicity/hydrophilicity, and altered mass transfer
dynamics.
[0209] Samples produced by means of a 23RGD-induced process were
made in one of two ways, the first being direct addition of 23RGD
in a pre-determined ratio to the silk solution without any sort of
reconstitution. The 23RGD (obtained as a desiccated fine powder
form) was blended into a measured volume of 8% silk solution within
the casting vessel by pipetting using a 1000 .mu.L pipette. These
gels were then cast in polypropylene tubes, sealed against air
penetration and water loss, and the sample was allowed to stand
under ambient room conditions (nominally 20-24.degree. C., 1 atm,
40% relative humidity) until fully gelled.
[0210] The 23RGD-induced gels were also produced by first
dissolving the 23RGD powder in purified water. The concentration of
this solution was determined based upon the amount of 23RGD to be
introduced into a gel and the final concentration of silk desired
in the gel. In the case of 4% silk gels enhanced with 23RGD,
quantities of water equal to the amount of 8% silk solution to be
used in the gel were used for the dissolution of appropriate
quantities of 23RGD. In the case of gels induced by addition of
23RGD to be generated at a molar ratio of 3:1 23RGD:silk, a
quantity of 23RGD was dissolved in 1 mL of water per 1 mL of 8%
silk solution to be gelled. This mixing was performed in the
casting vessel as well, being accomplished by means of rapid
pipetting with a 1000 .mu.L pipette when appropriate. These gels
were then cast in polypropylene tubes, sealed against air
penetration and water loss, and the sample was allowed to stand
under ambient room conditions (nominally 20-24.degree. C., 1 atm,
40% relative humidity) until fully gelled.
[0211] Samples produced by means of ethanol-enhanced gelation (EEG)
were generated by means of directly adding ethanol to a measured
volume of 8% silk solution in the casting vessel. The ethanol is
added in a quantity such that the volume added should yield a
volumetric dilution of the 8% silk solution resulting in the final
required concentration of silk within the gel, assuming minimal
volume loss due to miscibility of the organic added. The mixture of
ethanol and silk solution is then mixed by means of pipetting with
a 1000 .mu.L pipette when appropriate. These gels were then cast in
polypropylene tubes, sealed against air penetration and water loss,
and the sample was allowed to stand under ambient room conditions
(nominally 20-24.degree. C., 1 atm, 40% relative humidity) until
fully gelled.
[0212] Samples produced by a combined 23RGD-ethanol effect (RGDEEG)
were generated using a solution of 90% ethanol, 10% purified water
and appropriate quantities of 23RGD dissolved in this solvent. It
was not possible to readily dissolve 23RGD in pure ethanol and it
was believed that undissolved 23RGD might cause poor distribution
of the peptide throughout the gel phase. As a result, it was
determined that since a solution of ethanol and water offering
similar gelation acceleration characteristics to a pure ethanol
solution and reasonable 23RGD solubility would be an acceptable
alternative. A solution of 90% ethanol and 10% water met both of
these criteria and as a result was used for generation of these
gels. The 23RGD concentration of this ethanol solution was
determined based upon the amount of 23RGD to be introduced into a
gel and the final concentration of silk desired in the gel. In the
case of 4% silk gels enhanced with 23RGD, quantities of 90% ethanol
equal to the amount of 8% silk solution to be used in the gel were
used for the dissolution of appropriate quantities of 23RGD. In the
case of gels induced by addition of 23RGD to be generated at a
molar ratio of 3:1 23RGD:silk, a quantity of 23RGD was dissolved in
1 mL of 90& ethanol per 1 mL of 8% silk solution to be gelled.
This mixing was performed in the casting vessel as well, being
accomplished by means of rapid pipetting with a 1000 .mu.L pipette
when appropriate. These gels were then cast in polypropylene tubes,
sealed against air penetration and water loss, and the sample was
allowed to stand under ambient room conditions (nominally
20-24.degree. C., 1 atm, 40% relative humidity) until fully
gelled.
[0213] Silk gelation times were determined by casting gels
according to the methods above, the exception being that gels were
mixed not through pipetting, but through vigorous mechanical
shaking. These studies were conducted using 1.5 mL microcentrifuge
tubes as casting vessels with sample groups of N=6 used for each
gel formulation (FIG. 1). The determination that a "gel" state had
been reached was made in the method as described above, based upon
observation of a uniform solid phase throughout the entire volume,
generally opaque and white in appearance.
[0214] Gelation time varied widely depending on specific
formulation. The 8P silk samples took 21 days until gelation while
the 4P samples required 31.+-.1 day (data not shown). EEG samples
gelled significantly faster than PG samples with a 4E sample
requiring 27.+-.5.4 seconds for gelation (p.ltoreq.0.05). EEG
samples gelled more rapidly as the concentration of ethanol added
increased with time required gelation times of 1770.+-.600 s,
670.3.+-.101.0 s, 29.8.+-.5.2 s, 9.7.+-.2.0 s, and 4.2.+-.0.8 s for
6.4E, 6E, 4.8E, 4E, and 3.2E respectively. There were significant
differences between all times except 4.8E and 4E, 4E and 3.2E, and
4.8E and 3.2E. RGDEEG gels generated a tightly localized white
fibrous precipitate instantaneously upon addition of the ethanol
solution to the silk and gelled more quickly than PG samples,
though they were slower than EEG gels. 4RL, 4RM and 4RH samples
took 22.7.+-.2.5 seconds, 38.8.+-.4.5 seconds, and 154.5.+-.5
seconds to gel with 4RH differing significantly from the other
RGDEEG formulations.
[0215] Gelation timing experiments revealed the time constraints
posed by the PG method. Results indicated that, while increased
silk concentration decreased gelation time, the total time to gel
was decreased only from 31 days for 4P to 21 days for 8P. This may
result from the increased frequency of collisions between silk
molecules in solution and resultant gel network assembly. Using
ethanol directly added to silk solution as an accelerant proved to
dramatically decrease the gelation time of the silk by increasing
the volume of ethanol added in a fashion well-modeled by a power
function. This increasingly rapid gelation is likely caused by
greater competition for hydrating water molecules between silk and
ethanol coupled with altered electronegativity of the solution,
both favoring forced aggregation of the silk molecules. Studies
conducted on RGDEEG samples revealed that addition of greater
concentrations of RGD led to increasing gelation times modeled by
an exponential function. This appears counter-intuitive as it was
expected that RGD should function in some capacity to accelerate
gelation.
[0216] The slowing of gelation in RGDEEG samples may result from
difficulties in silk molecular binding to the RGD-coated silk
precipitates, perhaps due to stearic interference with hydrophobic
regions of silk chains. Upon RGD-ethanol accelerant addition to the
silk solution, a large quantity of silk-RGD complexes was
precipitated from the solution. It was noted during the gelation of
RGDEEG samples that a fibrillar, white, opaque precipitate was
consistently formed within the solution mixture immediately upon
mixing. This precipitation from solution may be evidence of this
rapid assembly of high concentration silk-RGD precipitates. This
formation may be caused by association between silk micelles and
peptide molecules in solution, disruption of the silk micelles, and
rapid assembly of them into a tightly-localized fibrillar
structure. This rapid assembly may progress until driving gradients
generated by the differing solvent chemistries provided by the
ethanol and water reach an equilibrium state. At this point, silk
molecules are able to remain stably in solution with further silk
network assembly occurring only by slow lengthening of the
initially formed precipitates. While this precipitation provided a
high number of nucleation points to initiate completion of a gel
network, these nucleation points may be of limited utility based
upon availability of binding sites. The remaining silk molecules
were much slower to assemble as a result. These precipitates also
tended to initiate assembly of a peripheral network comprised
largely of loose .alpha.-helix and random coil motifs, possibly due
to interference in silk packing due to the interference of these
particles.
[0217] The hydrogels produced by the methods described above derive
substantial benefit from the ability to more precisely control the
time course for its gelation in comparison to that of a
conventionally designed and cast gel. It is evident from monitoring
the time between casting and gelation of the device and similarly
cast, non-enhanced or exclusively ethanol modified gels that 23RGD
under certain circumstances may be manipulated to have an
additional accelerant effect upon the process of gelation. This
observed enhancer effect both mitigates the time constraints and
controllability associated with non-modified gels and additionally
alters the manner in which the protein aggregate network is formed
relative to solely ethanol enhanced gels
Example 4
Determination of Residual Ethanol by Colorimetric Analysis
[0218] Following gelation of a sample produced with either an
ethanol or 23RGD component, the gel was removed from the casting
vessel and immersed in a bulk of purified water as a rinse buffer.
This bulk comprised a volume such that the volumetric ratio of
water to gel was .gtoreq.100:1. The gel was permitted to lay static
in the rinse buffer for a period of 72 hours, changing the water
every 12 hours.
[0219] Samples of silk gel were evaluated to determine the total
residual content of ethanol in a series of 23RGD-ethanol- and
ethanol-enhanced gels. Briefly, samples of gel (N=4 of each type)
generated as described above were processed and analyzed using an
Ethanol Assay Kit (kit #K620-100 from BioVision Research Prods,
Mountain View, Calif.). Samples of gel were cut to a size of
approximately 0.3 cm in height by 0.5 cm in diameter (approximately
250 mg). These samples were massed to the nearest 0.1 mg using an
APX-60 (Denver Instrument, Denver Colo.) balance as per the
manufacturer's instructions. These gel samples were individually
ground using a metal spatula and placed into 250 .mu.L of Milli-Q
water in microcentrifuge tubes. These gels were incubated at
37.degree. C. for a period of 24 hours. After incubation, the gels
were centrifuged on an Eppendorf 5415 microcentrifuge with an HA
45-18-11 rotor (Hamburg, Germany) at 18,000 rpm for 30 minutes. At
the conclusion of this centrifugation step, the supernatant was
used as the sample of interest according to the instructions
provided by the kit manufacturer. Colorimetric analyses of the
sample was performed at an absorbance of 570 nm using a
spectrophotometer, and in conjunction with a standard curve,
residual percentages of ethanol in the gel were calculated (Table
1, FIG. 2). It was shown in this process that the leeching step is
capable of substantially removing residual ethanol from the silk
gels, as none of these materials exhibited a residual ethanol
component of greater than 5% ethanol by mass.
TABLE-US-00002 TABLE 1 Determination of Residual Ethanol by
Colorimetric Analysis Silk Enhancer Enhancer Initial Ethanol Final
Ethanol Concentration Concentration Solvent Solute Concentration
Mean Stdev 2% 90% None 68% 2.49% 0.06% 3:1 23RDG:Silk 4.44% 0.13%
10:1 23RDG:Silk 4.77% 0.29% 4% None 45% 2.55% 0.07% 3:1 23RDG:Silk
2.86% 0.08% 10:1 23RDG:Silk 2.97% 0.07% 6% None 22.5%.sup. 3.12%
0.05% 3:1 23RDG:Silk 3.16% 0.04% 10:1 23RDG:Silk 2.99% 0.10%
Example 5
23RGD Quantification by HPLC
[0220] 23RGD-infused gels were studied to quantify the amount of
23RGD bound to the silk-hydrogel device as well as the quantity of
free 23RGD which might be rinsed free of the device under relevant
conditions. Briefly, samples of 23RGD-infused gel were cast and
rinsed according to the methods above, with samples of rinse buffer
being collected from each rinse for subsequent analysis by HPLC.
Additionally, subsequent to the last rinse, the gel samples were
mechanically pulverized by means of a stainless steel stirring rod
and the adsorbed 23RGD removed by incubation for 4 hours in a
dissolving buffer. This mixture of gel and solvent was then
centrifuged on an Eppendorf 5415C at 16,000 g RCF for 30 minutes.
The supernatant was collected and centrifuged another 30 minutes at
16,000 g RCF after which time the supernatant was collected in a
sample vial for HPLC analysis. Samples of rinse buffer from the
first and last rinse were centrifuged in the same fashion after
being diluted with the same solvent the gel was extracted with in a
volumetric ratio of 1 part rinse buffer to 4 parts solvent. To
ensure 23RGD-hydrogel device rinse-exposed surface area was not a
limiting factor, the same rinse and extraction process was
performed upon devices pulverized after gelation and before
rinsing. The peak area consistent with 23RGD for each HPLC sample
was taken and these data compared against a standard curve
generated for 23RGD on the same HPLC unit under identical handling
and run conditions.
[0221] The resultant data indicated levels of signal from 23RGD in
samples collected from rinse buffer were just slightly higher than
values for 23RGD solvent alone and were immeasurable by the
standard curve, expected to resolve a relative 23RGD:silk ratio of
0.05:1. By comparison, the assay was able to detect a ratio of
3.35:1 in the final rinsed and extracted 23RGD-enhanced gel.
[0222] HPLC data confirmed complete retention of RGD on the silk
hydrogel material after the rinse process. This provides not only a
functional RGD component to this specific series of hydrogel
formulations, but indication for use of amphiphilic peptides as
candidates for introduction of other components into silk gels.
This knowledge might be applied to a number of other biologically
active peptide sequences, though additional work must be done to
understand how these specific peptides might influence gelation and
how gelation in turn impacts the functionality of these
peptides.
Example 6
Silk Gel Dry Massing
[0223] Silk gel samples of various 23RGD-ethanol- and
ethanol-enhanced formulations were cut into sample cylinders (N=4
of each type) of approximately 0.7 cm in height by 0.5 cm in
diameter (approximately 500 mg). These samples were massed to the
nearest 0.1 mg using an APX-60 (Denver Instrument, Denver Colo.)
balance as per the manufacturer's instructions and placed into
massed microcentrifuge tubes. After this, the samples were frozen
to -80.degree. C. for 24 hours. At the conclusion of this time, the
samples were placed into a lyophilizer unit for a period of 96
hours to remove all water content. Following the completion of this
96 hour drying, the remaining protein components of the silk gel
samples were massed again and the mass fraction of water in the
samples determined.
[0224] Gel dry massing showed an increasing percentage of dry mass
as RGD component increased in each silk concentration group (FIG.
3). The dry mass of 2E was significantly less than 2RL and 2RM
(p.ltoreq.0.05) at 1.63.+-.0.30%, 3.85.+-.1.23% and 4.03.+-.0.53%
respectively (FIG. 3A). The dry masses of 4E, 4RL and 4RM all
differed significantly from each other at 4.05.+-.0.10%,
4.56.+-.0.12%, and 5.19.+-.0.18% respectively (FIG. 3B). The dry
mass of 6E was significantly less than both 6RL and 6RM at
5.84.+-.0.15%, 6.53.+-.0.28%, and 6.95.+-.0.40% respectively (FIG.
3C).
[0225] The gels, regardless of the silk concentration, showed a
statistically significant trend toward decreasing percentage of
water mass in each gel material as 23RGD component increased as
determined by analysis of each silk concentration group with ANOVA
(FIG. 4, Tukey post hoc, p<0.05). This phenomenon might be
explained by the possibility that the 23RGD causes formation of a
different secondary structure within the silk hydrogels and that
this structure might be less hydrophilic than non-23RGD-enhanced
material. It is possible that this may be manifested in a different
ratio of .beta.-sheet structure, .alpha.-helix structure, and
unordered random coil for 23RGD-treated materials than their
counterparts, tending to favor the more hydrophobic .beta.-sheet
conformation.
[0226] Silk gel dry mass data revealed that increasing
concentrations of both silk and RGD in the silk gels increased the
percentage of dry mass in these materials, though the increase from
ROD was too large to attribute solely to additional peptide mass.
This phenomenon might be explained by the hypothesized structure of
the RGDEEG gels mentioned previously relative to PG and EEG gels.
It is likely that the large regions of poorly-associated 13-sheet
structure in the RGDEEG gels do a poor job at integrating water
into the structure. The inter-connecting regions of .alpha.-helix
structures and unordered random coil are able to entrain water, but
do so with less success than in the case of the more homogenous EEG
gels. It may also be possible that the hydrophilic ROD sequence
interfered with the dry massing procedure, causing rapid gain of
water mass upon exposure of the samples to atmospheric
conditions.
Example 7
Enzymatic Bioresorption
[0227] Gels specified were subjected to in vitro digestion by a
solution consisting of non-specific protease mixture. Briefly, gel
samples were cast to generate uniform, cylindrical samples of
approximately 1 gram total weight (about 1 mL of gel). These
samples were digested with a protease obtained from the bacteria
Streptomyces griseus (Sigma catalog No. P-5147) suspended in
phosphate buffered saline at a concentration of 1 mg/ml. A ratio of
3 mL of protease solution per 1 ml of initial gel was used for the
purposes of this study. The protease solution was added to a sealed
tube containing the gel and incubated for 24 hours at 37.degree. C.
with no mechanical mixing. After 24 hours, the solution was drained
through a piece of 316 stainless steel woven wire cloth. This
permitted retention of all gel particles greater than 50 .mu.m in
diameter (gap size was 43 .mu.m by 43 .mu.m), those smaller than
that were considered to be "bioresorbed" for the purposes of this
assay. After thorough draining of the solution, the mass of the gel
was measured wet, but devoid of excess entrained moisture. The
protease solution was then replaced and the sample incubated a
further 24 hours at 37.degree. C. This process was repeated until
the samples were bioresorbed for a total of four days, changing
solutions and massing each day.
[0228] PG samples and EEG samples bioresorbed similarly, differing
significantly only at D4 where 4P samples retained 62.89.+-.4.26%
of the original mass and 4E samples retained 53.27.+-.5.45%
(p.ltoreq.0.05) (FIG. 5A). 6E gels incubated in PBS showed no
significant mass loss over the course of the 4 day incubation (FIG.
5B). EEG silk gels with high concentrations of fibroin exhibited
higher mass retention than lower concentrations at all days. At Day
1 there were significant differences between 2E and all other gel
types with 2E, 4E and 6E gels retaining 57.04.+-.10.03%,
93.21.+-.9.47%, and 103.98.+-.3.65%, respectively while 6E in PBS
retained 101.18%.+-.12.01%. At Day 2, there were significant
differences again between 2E and all other gel types with 2E, 4E
and 6E gels retaining 36.59.+-.7.07%, 90.60.+-.9.24%, and
103.24.+-.6.38% of the original mass while 6E in PBS retained
98.28%.+-.12.38%. At Day 3 there were significant differences
between all gel types in protease, with 2E, 4E and 6E gels
retaining 32.36.+-.10.48%, 67.85.+-.8.82%, and 95.51.+-.8.97% of
the original mass. 6E samples incubated in PBS did not differ from
those incubated in protease, retaining 100.39%.+-.12.73% of the
original mass. At Day 4 there were significant differences between
all gel types with 2E, 4E, and 6E gels retaining 28.14.+-.4.75%,
53.27.+-.5.45%, and 81.76%.+-.3.35% of the original mass while 6E
in PBS retained 102.45.+-.12.50%. Addition of RGD to silk gels
appeared to slightly decrease the mass retention of these materials
when subjected to proteolytic bioresorption (FIG. 5C). 4E samples
retained significantly more mass than 4RM and 4RH at Day 2 as they
retained 90.6.+-.9.24%, 74.47.+-.4.55%, and 71.23.+-.6.06% of the
initial masses respectively. There were no further significant
differences in 4E samples relative to 4RM and 4RH samples over the
course of the bioresorption assay.
[0229] Gel samples treated with 23RGD exhibit a trend toward more
rapid bioresorption within the constraints of this particular
assay. This was illustrated at the 4% silk concentration (FIG. 6)
and then confirmed at a concentration of 6% silk fibroin in the gel
materials (FIG. 7). Significant differences in the bioresorption
rates of 23RGD enhanced samples recorded by two-way ANOVA using a
Bonferroni post test (p<0.05), particularly with 6% silk,
reinforced the trend. The unique behavior attributed to
23RGD-enhanced materials may be due in part to its unique protein
structure, as the bioresorption method considers particles below a
size of 50 .mu.m to be bioresorbed, regardless of their stability.
It may be possible for a rich beta sheet structure to exist within
23RGD gels which is broken up into small, discrete regions by
interfering regions of .alpha.-helix structure and random coil
which bioresorb more quickly, creating a plethora of tiny,
non-resorbed fragments in solution.
[0230] In vitro bioresorption of 4P and 4E samples showed both
materials had a similar resistance to proteolysis (FIG. 5A). This
is indicative that the basic process of ethanol-enhanced gelation
is capable of generating a gel structure rapidly without
sacrificing important material properties. It was also shown that
increasing the concentration of silk in EEG gels from 2% to 4% to
6% in 2E, 4E, and 6E respectively, substantially decreased sample
bioresorption mass loss (FIG. 5B). This may correlate to a more
homogeneous, stable and resilient gel structure, or simply to a
greater quantity of silk molecules to be cleaved by the proteases
in order to bioresorb the samples. In either case, these data
clearly indicate a potential for tailoring of bioresorption time
scale of a silk gel material through alteration of the silk protein
content of gels. It was also illustrated that a 4 day exposure to
PBS did not appreciably alter the mass of 6E samples, providing a
preliminary indication that EEG samples are not substantially
degraded by hydrolysis. This is a further reinforcement of the
stability and bulk integrity of these silk gels as many gel
materials suffer from limited resilience in vivo due to high
susceptibility to hydrolysis. Addition of increasing quantities of
RGD to silk gels was shown to slightly increase the rates of
bioresorption mass loss in comparing 4E, 4RM and 4RH (FIG. 5C).
This behavior indicates that there may be some structural
differences between RGDEEG and EEG gels which cause less mass loss
in EEG gels as compared to RGDEEG in this bioresorption assay. This
may relate directly to the previously proposed idea that RGDEEG
materials consist of many small regions of robust .beta.-sheet
structure loosely bound together by a weak inter-connecting matrix
of .alpha.-helix and unordered random coil structures. This stands
in contrast to EEG materials, which are thought to assemble from
similar, though less prominent and numerous, precipitates into a
more homogeneous structure than RGDEEG gels as a result. The
inter-connecting matrix of the RGDEEG gels is therefore more
susceptible to rapid bioresorption through this proteolytic assay
than that of EEG gels. While .beta.-sheet regions may remain intact
in RGDEEG gels, bulk material integrity is lost as the
inter-connecting network is resorbed as are the residual
.beta.-sheet particles due to the sieving method used as a cutoff
for degradation product particle size. This is indicative that it
may be possible to use varying levels of RGD in order to further
manipulate the structure and bioresorption profile of a silk
gel.
Example 8
Fourier-Transform Infrared Spectrum Capture
[0231] Silk hydrogels, 23RGD-ethanol-enhanced 4% silk, 3:1 and
10:1, were cast as described above and subjected to proteolytic
bioresorption as described above. Additionally, non-bioresorbed
control samples were obtained for sake of analysis via FTIR in
quantities of 0.5ml each. Using a Bruker Equinox 55
spectrophotometer (Bruker Optics, Inc., Billerica, Mass.) coupled
with a Pike MIRACLE.TM. germanium crystal (PIKE Technologies,
Madison, Wis.), sample absorbance spectra were obtained. Samples
were imaged by pressing them upon the crystal via a pressure arm
until single sample scans indicated viable signal from the material
then performing a 128-scan integration. Resolution was set to 4
cm.sup.-1 with a 1 cm.sup.-1 interval from a range of 4000
cm.sup.-1 to 400 cm.sup.-1.
[0232] Resultant spectra were subjected to analysis via OPUS 4.2
software (Bruker Optics, Inc). A peak-find feature was used to
identify peaks between 4000 cm.sup.-1 and 600 cm.sup.-1, with the
search criteria being automatic selection of local inflection
points of a second-derivative, nine-point smoothing function.
Program sensitivity was set to 3.5% for all spectra based upon
operator discretion regarding magnitude of peaks identified and
likely relevance to compound identification and
"fingerprinting".
[0233] Each of the samples subjected to FTIR analysis exhibited a
spectrum with very pronounced peaks at the Amide I band (1600-1700
cm.sup.-1) (FIG. 8). Additionally, the specific wave numbers of
these peaks are consistent between the 23RGD-infused silk fibroin
hydrogel and other silk gel groups. All samples exhibit major peaks
at .about.1622 cm.sup.-1 and a minor peak/toe region at .about.1700
cm.sup.-1, a pattern associated with a high degree of .beta.-sheet
structure within a sample (FIG. 8). There are also similarities
across all samples types at the Amide II band with a major peak at
.about.1514 cm.sup.-1.
[0234] Use of the EEG process to produce silk gels did not
dramatically impact gel secondary structure but did slightly
increase the resistance of the gel formulation to proteolytic
bioresorption (FIG. 9A). Evaluation of characteristic FTIR spectra
of 4P and 4E gels at Day 0 revealed few distinguishing
characteristics as both formulations exhibited a characteristic
.beta.-sheet peak around 1622 cm-1 and toe region of .beta.-turn at
1700 cm-1. Each sample also had additional portions of
.beta.-sheet, .beta.-turn, .alpha.-helix, and unordered random coil
at 1677 cm.sup.-1, 1663 cm.sup.-1, 1654 cm.sup.-1, and 1645
cm.sup.-1 respectively with higher relative quantities of
.alpha.-helix and random coil appearing in 4P than 4E at Day 0. At
Day 4, both samples showed pronounced decreases in 1677 cm.sup.-1
.beta.-sheet, .beta.-turn, .alpha.-helix and random coil signal,
though this 4P exhibited this to a greater extent than 4E,
indicating preferential resorption of these motifs and greater
resistance to this in 4E gels.
[0235] Increasing the final silk concentration of EEG gels had
little impact on initial gel secondary structure, though there was
a pronounced increase in .beta.-sheet structures at Day 4 with
greater silk concentrations (FIG. 98). At Day 0, 2E, 4E, and 6E
gels all showed strong signal for 1622 cm.sup.-1 .beta.-sheet and
1700 cm.sup.-1 .beta.-turn strong, with 6E having particularly
prominent peaks in these regions. Each sample also had additional
portions of 1677 cm.sup.-1 .beta.-sheet, 1663 cm.sup.-1
.beta.-turn, .alpha.-helix, and unordered random coil. At Day 4 all
gels showed decreases in 1677cm-1 .beta.-sheet, 1663 cm.sup.-1
.beta.-turn, .alpha.-helix and random coil peaks relative to 1622
cm.sup.-1 .beta.-sheet and .beta.-turn peaks with this behavior
being more marked in 4E and 6E than 2E. The Day 4 6E sample also
showed a more stable .beta.-sheet structure indicated by a peak
shift to lower wave number at .about.1620 cm.sup.-1.
[0236] Pronounced differences in the 23RGD-ethanol-enhanced and
ethanol-enhanced spectra only became evident after a four-day
period of bioresorption in protease. The day 4 samples exhibited
differences primarily in the order of magnitude of certain
secondary structure modalities seen through slight differences in
FTIR Amide I band shape. At day 4, the 23RGD-ethanol-enhanced
samples exhibit higher levels of .beta.-turn structure evidenced by
far more pronounced and distinct peaks at .about.1700 cm.sup.-1
while also showing considerably lower levels of .alpha.-helix
structure (1654 cm.sup.-1) and unordered random coil (1645
cm.sup.-1) structures. For example, FTIR spectra from 4E, 4RM and
4RH all show similar structures featuring 1622 cm.sup.-1
.beta.-sheet and 1700 cm.sup.-1 .beta.-turn prominently with
indications of 1677 cm.sup.-1 .beta.-sheet, 1663 cm.sup.-1
.beta.-turn, .alpha.-helix, and unordered random coil secondary
structures (FIG. 9C). At Day 4, 4RM and 4RH both show a less
pronounced 1677 cm.sup.-1 .beta.-sheet, 1663 cm-1 .beta.-turn,
.alpha.-helix, and random coil component than the 4E sample with
4RH also showing a more stable .beta.-sheet structure, indicated by
a peak shift to lower wave number at .about.1620 cm.sup.-1.
Additionally, a peak shift occurred in both the 10:1
23RGD-ethanol-enhanced and ethanol-enhanced samples in the
.beta.-strand peak at 1622 cm.sup.-1, indicative of increased
.beta.-sheet stability. Considered as a whole, the collective peak
shifts and peak magnitudes observed in the spectra at day 4
compared to day 0, all gel types experienced substantial
strengthening of .beta.-sheet component, likely due to removal of
less-stable .alpha.-helix and random coil. These effects were most
pronounced in 23RGD-enhanced gel materials, likely due to intrinsic
differences in the initial organization of the structural network
of the gel materials.
[0237] FTIR analysis and comparison of PG, EEG and RGDEEG showed
strong behavioral similarities across all gel groups. Each material
exhibited .beta.-sheet-dominated secondary protein structures,
featuring elements of .alpha.-helical and random coil structures
and each resorbed in such a fashion that the quantities of
.beta.-sheet-rich structure increased relative to .alpha.-helical
and random coil structures. The selective bioresorption of
.alpha.-helical and random coil structures indicates that they are
likely favorably degraded by proteolysis relative to .beta.-sheet
structures, thus the bioresorption profile of a gel might be
influenced by altering the balance between .beta.-sheet motifs and
the combination of .alpha.-helical and random coil structures. An
evaluation of ethanol as an accelerant revealed a minimal effect on
silk gel structure at Day 0 as both 4P and 4E had high .beta.-sheet
contents with .alpha.-helical and random coil structures (FIG. 9A).
At Day 4 though, there was a slightly greater relative .beta.-sheet
content in 4E than 4P samples. This may be caused by structural
differences in 4E and 4P formulations that were imperceptible at
Day 0 by ATR-FTIR, possibly in the uniformity and homogeneity of
the silk gels. It is possible that the same differences
hypothesized between EEG and RGDEEG gels derived from their
different extents of precipitate/nucleation point formation in
early-phase gelation causes differences between PG and EEG
materials as well. As PG samples are not accelerated, it is likely
that very few nucleation points will form quickly and as a result,
the gelation process occurs in a very slow but homogeneous fashion,
allowing for an optimal stearic packing of silk molecules
throughout the solution volume. This results in a consistent
protein structure throughout the final gel volume, corresponding to
good bulk material integrity. This would contrast with EEG gels, as
the previously postulated nucleation phenomenon associated with
RGDEEG materials likely occurs with EEG materials as well, though
in a less prominent fashion. This results in a non-uniform
distribution of highly organized regions of .beta.-sheet held
together by .alpha.-helical and random coil structures in the EEG
materials relative to the PG materials, with .alpha.-helical and
random coil degraded more rapidly than .beta.-sheet. This is in
keeping with previous studies which have shown that more poorly
packed .beta.-sheet structures and .alpha.-helix structures are
more susceptible to degradation. Increasing silk concentration in
EEG gels from 2E to 4E to 6E revealed the most prominent
.beta.-sheet structures in 6E at both Day 0 and Day 4 while 2E had
considerably more .alpha.-helix and random coil at both days than
2E and 4E (FIG. 9B). This would seem to indicate that dilute
concentrations of silk in the final hydrogel result in a less
densely packed secondary structure, possibly due to stearic freedom
within the gel volume relative to 4% and 6% states. This indicates
that silk concentration may be used to manipulate the secondary
structure of silk gel to influence bioresorption. A study of the
effect of increasing RGD concentration indicated that while gels
were virtually identical at Day 0, the .alpha.-helix structure and
unordered random coil in 4RM and 4RH gels were less resilient to
bioresorption than in 4E as seen at Day 4 (FIG. 9C). This might
also be explained by inhomogeneities within the 4RM and 4RH gels
relative to 4E as mentioned previously. This may be particularly
likely in light of the formation of precipitates observed in RGDEEG
samples. This data may be indicative that RGD or a similar peptide
could be used to further tailor the nature of the bioresorption
profile of silk gels.
[0238] These results indicate that silk gels produced through PG,
EEG, and RGDEEG result from a two-phase assembly process consisting
of nucleation and aggregation. Silk gels contain predominantly
.beta.-sheet structure which is more resistant to in vitro
bioresorption than .alpha.-helix and random coil. EEG gels form
more quickly than PG, likely due to a more rapid precipitation and
nucleation event mediated by the effects of ethanol on the solution
solvent phase. EEG gels form a non-homogeneous structure likely
consisting of localized, initially-precipitated .beta.-sheet
regions inter-connected by .alpha.-helix and random coil assembled
subsequently. RGDEEG gels form a non-homogeneous structure likely
consisting of localized, initially-precipitated .beta.-sheet
regions inter-connected by .alpha.-helix and random coil assembled
subsequently. RGDEEG gels reach completion more slowly than EEG
gels due to stearic RGD-mediated interference encountered in gel
assembly following nucleation. RGDEEG gels are less homogeneous
than EEG gels due to these difficulties associated with late-phase
assembly.
Example 9
Injectable Gel Processing
[0239] Silk hydrogels were prepared as described above in Examples
1-4. Gels were then comminuted by grinding the silk gel to a paste
using a stainless steel spatula. Gel formulations including PBS
were massed with an SI-215 balance (Denver Instrument, Denver
Colo.) and the correct volume percentage of PBS (Invitrogen
Corporation, Carlsbad, Calif.) was blended in with the assumption
that both the gel and PBS had a density of 1 g/ml. Silk hydrogels
to be used for in vivo assessment were subjected to vigorous
mechanical pulverization by means of a stainless steel stir rod.
When specified as containing a saline component, gels were blended
with saline at volumetric ratios based upon the original volume of
gel (i.e., prior to mechanical disruption) following pulverizing by
the stainless steel bar. This addition of phosphate buffered saline
serves to regulate tonicity of the gel as well as improve
injectability. Following this initial pulverizing, the gel was
further disrupted by means of repeated injection through a 26-gauge
needle in order to decrease overall particle size within the gel
and improve injectability characteristics. In some samples, gel was
further disrupted by means of repeated injection first through an
18 g needle repeatedly until the gel flowed readily, and then the
material was then cycled in like fashion through a 23 g needle and
26 g needle.
Example 10
In Vivo Investigation of Silk Hydrogel in Rodent Models
[0240] Samples of silk gel which had been processed for
implantation or injection in vivo as described in Example 9 were
double-bagged with appropriate sterilization bags for gamma
irradiation and sterilized by exposure to a dose of 25 kGy of gamma
radiation.
[0241] In one trial silk hydrogel samples, both 23RGD-enhanced and
native were implanted subcutaneously in male Lewis rats having an
average weight of 400 g. This was done according to protocol#86-04
on file with New England Medical Center's Department of Laboratory
Animal Medicine (DLAM) and approved by the Institutional Animal
Care and Use Committee (IACUC). On the day of surgery, animals were
anesthetized via a ketamine/xylazine solution injected IM in the
animals' hind legs. Following administration of anesthesia, the
skin of the rats was shaved closely and swabbed with alcohol,
allowed to dry, swabbed with BETADINE.RTM. microbicide (Purdue
Pharma, Stamford, Conn.) then draped with sterile towels. In the
case of implanted devices, two dorsal midline incisions were made
directly over the spine, the first 0.5 cm below the shoulders and
the second 2.5 cm above the pelvic crest, each 1 cm long each. The
incisions were expanded into 1 cm deep pockets using a blunt
dissection technique beneath the panniculus carnosus at each side
yielding 4 potential implant sites. Implants, 3 per animal; each 1
cm.times.1 cm.times.0.3 cm in size were inserted into the pockets
without fixation with the final site undergoing the same dissection
but replacing the implant with 0.5 mL of sterile saline solution.
The skin was closed with interrupted absorbable sutures. Depending
on study, samples were harvested at 7 days, 14 days, 28 days,
and/or 57 days after implantation surgery. Gross observations were
collected semi-weekly regarding implant site appearance. After
sample harvest, gross observations of the implants were conducted
and samples were processed for histological evaluation. Analysis of
histology slides was provided by a trained veterinary
pathologist.
[0242] Sections were scored for presence (0=none, 1=present) of
implant mineralization, cyst formation, fibrosis, sebaceous cell
hyperplasia, and focal follicular atrophy. Additionally, the
density of inflammatory response (0=none . . . 5=extensive) and
extent epidermal hyperplasia (0=none . . . 3=extensive) were
graded. These data were reported as percentages of the highest
score possible for the group of slides. Sections were also examined
for presence of any particular characteristic cell types including
lymphocytes, neutrophils, eosinophils, mononuclear giant cells,
macrophages, and fibroblasts. Additional commentary relevant to the
host response was included at the discretion of the reviewing
pathologist. Prism 4.03 (GraphPad Software Inc., San Diego, Calif.)
was used to perform analysis of variance (ANOVA) with a
significance threshold set at p.ltoreq.0.05. One-way ANOVA was used
to compare differences average extrusion forces for comminuted
gels. For all tests, Tukey's post-hoc test was also performed for
multiple comparisons.
[0243] Table 2 lists the formulations of silk gel, both
23RGD-ethanol-enhanced and ethanol-enhanced developed and assessed
intradermally in a rat model. Silk gels explanted from rats at Day
7 were visibly well-defined and easily identifiable with no gross
indications of edema, erythema, or transdermal elimination of
material. It was not possible to differentiate sites of PBS control
implantation from surrounding tissue. H&E sections of 4% silk
fibroin hydrogels formed by passive gelation (4P), 4% silk fibroin
hydrogels formed by ethanol-enhanced gelation (4E) and 6% silk
fibroin hydrogels formed by ethanol-enhanced gelation (6E) all
appeared similar, with mild inflammation in all cases characterized
by lymphocytes, macrophages, some neutrophils and fibroblasts (FIG.
10). Cellular infiltration was observed in all sample types with
complete penetration in 4P and peripheral ingrowth to a depth of
about 100 .mu.m in both EEG gels with no evidence of cyst formation
observed. In all gels, early bioresorption was indicated by implant
edge erosion with residual implant material remaining localized
into large lakes. Host integration of implanted gel had progressed
in Day 28 samples of 4E and 6E evidenced by greater cellular
ingrowth into the material with complete implant penetration in 4E
samples and robust peripheral ingrowth in 6E samples. The cellular
response at this time point was characterized by fibroblasts,
lymphocytes and macrophages with the addition of a few
multi-nucleated giant cells.
TABLE-US-00003 TABLE 2 Silk Hydrogel Formulations Group Silk Saline
Name Concentration Enhancer Component 4E10 4% 90% Ethanol 10% 4R10
90% Ethanol, 1:1 23RGD 4RH10 90% Ethanol, 3:1 23RGD 4E25 90%
Ethanol 25% 4R25 90% Ethanol, 1:1 23RGD 4RH25 90% Ethanol, 3:1
23RGD 6E10 6% 90% Ethanol 10% 6R10 90% Ethanol, 1:1 23RGD 6E25 90%
Ethanol 25% 6R25 90% Ethanol, 1:1 23RGD 6RH25 90% Ethanol, 3:1
23RGD
[0244] Day 57 samples of 4E and 6E showed continued host
bioresorption of the gel material as there was little residual 4E
and while 6E remained visible in large, intact lakes, the gel had
been completely penetrated with host tissue. The host response to
4E had dramatically decreased in cellularity between Day 28 and Day
57 with very little evidence of hypercellularity at Day 57 with
some scattered macrophages and fibroblasts around the implant site.
The pathology of the host response of 6E was similar to the Day 28
response to 4E, with fibroblasts as the predominant cell type and
scattered lymphocytes, macrophages and multi-nucleated giant cells.
This was viewed as a low-grade, persistent, fibrotic-type
inflammatory response to the material.
[0245] Samples of 23RGD-enhanced gel exhibited a less robust
inflammatory response at the 14 day time point in comparison to
non-23RGD-enhanced gel (FIG. 11). This is observed through an
appreciable decrease in hyper-cellularity proximal to the gel
implant and an accompanying decrease in the fragmentation of the
implant material. It is possible that this improvement in implant
integrity is due to a less robust foreign body response by the host
animal and it may also be evidence that there is less mechanical
contraction of the implant site, a commonly observed phenomenon
with biomaterials including the "RGD" motif. These effects indicate
that 23RGD-enhancement of silk gels leads to a more biocompatible
material with better implant outcomes.
[0246] In a second trial, intraderm ally-injected samples of silk
hydrogel, both ethanol enhanced and 23RGD-ethanol enhanced and
relevant control materials were investigated using male Hartley
guinea pigs. This was done according to protocol#29-05 on file with
New England Medical Center's Department of Laboratory Animal
Medicine (DLAM) and approved by the Institutional Animal Care and
Use Committee (IACUC). Briefly, male Hartley guinea pigs weighing
300-350 g were anesthetized via a ketamine/xylazine cocktail
injected intramuscularly into the animals' hind legs. The dorsal
skin of the guinea pigs was then shaved closely and swabbed with
alcohol, allowed to dry, swabbed with BETADINE.RTM. microbicide or
Chloraprep (Enturia, Inc., Leawood, Kans.), then draped with
sterile towels. A 50 .mu.L volume of the desired material was
injected through a 26 g needle at six different sites along the
left side of the animal's back. Further injections of an
appropriate silk gel control were made at the six contralateral
sites. Explanation of the silk gels was performed at 28 days after
implantation. Gross observations were collected semi-weekly
regarding implant site appearance. After sample harvest, gross
observations of the implants were conducted and samples were
processed for histological evaluation. Analysis of histology slides
was provided by a trained veterinary pathologist. Scoring and
statistical analysis was performed as described above.
[0247] Table 3 lists the formulations of silk gel, both
23RGD-ethanol-enhanced and ethanol-enhanced developed and assessed
intradermally in a guinea pig model in a twenty-eight day screen.
Although no statistically significant differences were identified,
the data for both gross observations and histology (Tables 4 and 5)
indicate a general trend supporting the previous data that
23RGD-enhancement of gel improves material biocompatibility. Among
sites implanted with silk gel, gross outcomes varied. Ulceration
and hair loss rates were lower in groups with 25% PBS compared to
10% saline, 6% silk compared to 4% silk and RGDEEG casting as
compared to just EEG casting (Table 4). Site redness rates followed
a similar pattern with the exception that RGDEEG samples induced
more site redness than EEG samples. All silk gels showed evidence
of epidermal cyst formation, fibrosis, epidermal hyperplasia and
pronounced inflammation with traces of follicular atrophy in all
EEG samples. Sebaceous cell hyperplasia was present to a limited
extent in all formulations with the exception of 6% silk, 10%
saline, 1:1 23RGD (Table 5). This is particularly evident in the
case of silk gels of 4% silk with 25% saline added and either
enhanced with an ethanol-based enhancer or an 23RGD-ethanol-based
enhancer, and more specifically, in the case of site ulcerations
(Table 5). This material indicated strong improvements with
increasing 23RGD concentration in the number of sites ulcerating
throughout the course of the trial. These results are indicative
that use of 23RGD in conjunction with an ethanol enhancer provides
an improved outcome when compared to an ethanol enhancer alone.
TABLE-US-00004 TABLE 3 Silk Hydrogel Formulations Group Silk Saline
Name Concentration Enhancer Component 4E10 4% 90% Ethanol 10% 4R10
90% Ethanol, 1:1 23RGD 4E25 90% Ethanol 25% 4R25 90% Ethanol, 1:1
23RGD 4RH25 90% Ethanol, 3:1 23RGD 6E10 6% 90% Ethanol 10% 6R10 90%
Ethanol, 1:1 23RGD 6E25 90% Ethanol 25% 6R25 90% Ethanol, 1:1
23RGD
TABLE-US-00005 TABLE 4 Gross Evaluation of Guinea Pigs Group Site
Hair Name Redness Loss Palpability Ulceration 4E10 38% 58% 65% 33%
4R10 57% 49% 67% 33% 4E25 28% 34% 49% 28% 4R25 44% 34% 64% 17%
4RH25 50% 23% 66% 6% 6E10 63% 52% 68% 33% 6R10 78% 51% 68% 22% 6E25
33% 31% 69% 11% 6R25 56% 30% 68% 13% HYLAFORM .TM. 6% 12% 63% 0%
ZYPLAST .TM. 17% 10% 52% 0%
TABLE-US-00006 TABLE 5 Histological Evaluation of Guinea Pigs
Epidermal Cyst Epidermal Follicular Sebaceous Group Name Formation
Fibrosis Inflammation Hyperplasia Atrophy Hyperplasia 4E10 22% 100%
70% 59% 11% 22% 4R10 74% 100% 62% 67% 0% 14% 4E25 50% 100% 69% 67%
13% 13% 4R25 29% 100% 39% 62% 0% 14% 4RH25 14% 100% 64% 50% 0% 43%
6E10 44% 100% 70% 56% 11% 33% 6R10 25% 100% 63% 38% 0% 0% 6E25 30%
100% 60% 40% 10% 20% 6R25 29% 100% 64% 33% 0% 14% HYLAFORM .TM. 0%
0% 3% 6% 0% 0% ZYPLAST .TM. 0% 25% 28% 31% 0% 0%
[0248] A third trial also used male Hartley guinea pigs to
investigate intradermally injected samples of silk hydrogel as
described above, comparing samples of 4% and 6% silk, 25% saline
3:1 23RGD-ethanol enhanced silk gels with a collagen-based control
material, ZYPLAST.TM. (Allergan Inc., Irvine Calif.) and
HYLAFORM.TM. (Allergan Inc., Irvine Calif.). Explanation of the
silk gels was performed at 92 days after implantation. Gross
observations were collected semi-weekly regarding implant site
appearance. After sample harvest, gross observations of the
implants were conducted and samples were processed for histological
evaluation. During the course of the 92 day trial, none of the 24
implant sites, either 23RGD-ethanol-enhanced hydrogel or
ZYPLAST.TM., ulcerated. Histology revealed that 75% of all
ZYPLAST.TM. sites had residual material as did 75% of all
23RGD-ethanol-enhanced silk gel sites (both 4% and 6%). Both
materials exhibited very similar chronic phase cellular responses,
as the sites were characterized by a mild fibrotic reaction with
abundant deposition of collagen in and around the implant site
(FIG. 12). The collagen appears less ordered than does that in the
surrounding dermal reticulum based upon the color density when
viewed with Trichrome staining and also when viewed under polarized
light. Silk gel sites had similar palpability scores to both
control materials but exhibited higher rates of site redness, hair
loss and ulceration than did ZYPLAST.TM. and HYLAFORM.TM.. These
results not only reinforce that 23RGD-ethanol-enhanced silk gel is
biocompatible, but also indicate that it is comparable to collagen
biomaterials in terms of its persistence and long-term behavior in
vivo.
[0249] ZYPLAST.TM. exhibited no epidermal cysts, follicular
atrophy, or sebaceous cell hyperplasia, though it did show small
levels of fibrosis, inflammation and epidermal hyperplasia.
Examination of histological sections showed residual silk gel
material which stained in a mildly eosinophilic fashion and
appeared as large lakes of material at a central location with
smaller masses of material distributed more widely throughout the
reticular dermis (FIG. 13). These smaller masses were typically
surrounded by fibroblasts and macrophages with occasional
multi-nucleated giant cells present. Eosinophils were located
proximal to these smaller masses of implant as well. In general,
host response to the silk fibroin gels was characterized as mildly
fibrotic and included populations of fibroblasts, lymphocytes,
macrophages, multi-nucleated giant cells and eosinophils. Little
difference was evident between silk gel types except in terms of
the extent of eosinophilia. Larger eosinophil populations were
observed for 6% as compared to 4% silk gels and were also observed
to increase with RGD concentration in the silk gel samples in both
4% and 6% groups. ZYPLAST.TM. exhibited strong eosinophilic
staining and was distributed as large lakes in the reticular dermis
with smaller masses throughout the area. Hypercellularity near the
injection site was lessened in ZYPLAST.TM. samples when compared to
silk gel. Fibroblasts, lymphocytes, macrophages, multi-nucleated
giant cells and eosinophils were present with less tendency to
localize at the implant periphery. HYLAFORM.TM. samples examined
showed many very small masses of material throughout the reticular
dermis. HYLAFORM.TM. exhibited no epidermal cysts, fibrosis,
follicular atrophy, or sebaceous cell hyperplasia with extremely
limited instances of inflammation and epidermal hyperplasia. There
was no observable hypercellularity near the implanted material or
other evidence of inflammation at the implant sites.
[0250] At day 92, histological evaluation of 4% silk fibroin
hydrogel, 3:1 23RGD, 25% saline (4RH25) samples and ZYPLAST.TM.
samples showed similar material persistence and host response (FIG.
14). Very little implant material remained visible in the dermis of
the animals with no hypercellularity present at this time point,
evidence of hyperplasia or cellular inflammation. The eosinophils
found at day 28 in the ZYPLAST.TM. and silk gel samples were not
observed at day 92. Of particular interest, 4RH25 also exhibited
residual disruption to the reticular dermis in the form of an
irregular collagen pattern near the implant material. The
disorganization of the collagen was seen as a region of stained
collagen seen to be devoid of the typical cross-hatch pattern of
normal reticular dermis (FIG. 14C). This disorganization was
confirmed when viewing the histological sections under polarized
light with the disorganized collagen appearing as an interruption
in the birefringence associated with the surrounding reticular
dermis (FIG. 14D).
Example 11
Enhanced Injectable Gel Formulation
[0251] Silk hydrogels were prepared as described above in Examples
1-4. Once processed, the gels were sized into coarse or fine
particles using a sieving step (Table 6). Gel materials were
pressed through a 316SS stainless steel wire cloth sieve with a
stainless steel spatula and into clean polystyrene Petri dishes.
Sieves with gap sizes of 711 .mu.m.times.711 .mu.m, 295
.mu.m.times.295 .mu.m, 104 .mu.m.times.104 .mu.m and 74
.mu.m.times.74 .mu.m were used. After passing through the 74
.mu.m.times.74 .mu.m gap sieve, the material was considered
processed to a "coarse" state. Samples to be processed to a "fine"
state were further forced through a 43 .mu.m.times.43 .mu.m sieve
in the same fashion. This sieving was conducted four separate times
for each sample type, each sieving using an approximate quantity of
0.5 mL of gel material.
TABLE-US-00007 TABLE 6 Particle sizing Nominal Silk 23RGD Molar
Ratio Group Mass Percentage with Silk Fineness Name 2% 1:1 Fine 2RF
4% 0 4F 1:1 Coarse 4RC Fine 4RF 3:1 RHRF 10:1 4VHRF 8% 1:1 Coarse
8RC Fine 8RF
[0252] Samples of silk gel material (N=4 of each type) were
evaluated under light microscopy. Briefly, a 100 mg portion of silk
gel or control device was massed using an SI-215 Summit series
balance. This material was loaded into the open back end of a 3 mL
syringe using a stainless steel spatula. The plunger was replaced
in the syringe, an 18 g needle was attached to the end of the
syringe and approximately 900 .mu.L of ultra-pure water was drawn
up. This mixture of water and silk gel was mixed through gentle
shaking. After mixing to suspend evenly, a sample of approximately
30 .mu.L of dilute silk gel was placed on a 75 mm.times.25 mm
single frosted, pre-cleaned micro slide (Fisher Scientific Co.,
Waltham, Mass.) and covered with a 22 mm.times.40 mm premium cover
glass (Corning Inc., Corning, N.Y.). This sample slide was then be
imaged with a microscope. Sample slides were imaged using a System
Microscope Model BX41 (Olympus, Melville, N.Y.) in conjunction with
a Microscope PC MACROFIRE.TM. Model S99831 Camera (Optronics,
Goleta, Calif.) and PICTUREFRAME.TM. 2.1 software (Optronics,
Goleta, Calif.). Briefly, slides were scanned for clearly separated
gel particles using the 4.times. objective lens and locations
determined for a series of 3 representative images of the sample
slide. Each of these locations was imaged after first switching the
microscope objective lens to 10.times.. Micrograph image files were
subjected to analysis with IMAGE-PRO.RTM. Plus 5.1 software (Media
Cybernetics, Inc., Silver Spring, Md.). Image files were checked
for particle size distribution, average particle size, average
aspect ratio, maximum particle size, minimum particle size and
standard particle size deviation. A compilation of the data is
presented in Table 7.
TABLE-US-00008 TABLE 7 Particle Comminution Data Group Min to Max
Object Mean Object Name Area (.mu.m.sup.2) Area (.mu.m.sup.2) 2RF
5.33 to 1.32 .times. 10.sup.4 52.43 .+-. 261.82 4F 5.33 to 8.07
.times. 10.sup.3 27.82 .+-. 129.34 4RC 5.33 to 8.52 .times.
10.sup.3 38.41 .+-. 196.67 4RF 5.33 to 5.29 .times. 10.sup.3 34.12
.+-. 135.31 4HRF 5.33 to 7.51 .times. 10.sup.3 40.62 .+-. 166.61
4VHRF 5.33 to 3.14 .times. 10.sup.3 35.4 .+-. 105.43 8RC 5.33 to
8.04 .times. 10.sup.3 46.57 .+-. 225.43 8RF 5.33 to 2.85 .times.
10.sup.3 35.26 .+-. 129.63 ZYPLAST .TM. 5.33 to 1.95 .times.
10.sup.3 22.08 .+-. 41.71
[0253] Examination of the particles under light microscopy revealed
some clumped gel particles which were removed from particle sizing
data manually. Particle sizes ranged from 5.3 to 1.3.times.10.sup.4
.mu.m.sup.2, comparable in range to commercially available
ZYPLAST.TM. which ranged from 5.3 to 1.95.times.10.sup.3
.mu.m.sup.2. The data also revealed mean particle sizes ranging
from 27.8 .mu.m.sup.2 to 52.4 .mu.m.sup.2, again, comparable to
ZYPLAST.TM. with a mean particle size of 22.1 .mu.m.sup.2. These
data illustrate that silk gel may be successfully comminuted to
small and functionally useful particle sizes in a fashion similar
to presently utilized injectable gel materials. The basic
forced-sieving method could easily be replaced with more
sophisticated, reproducible methods for purposes of scale-up.
[0254] After comminution and blending, samples of silk gel
emulsions were subjected to extrusion force testing. Gel materials
prepared as described in Examples 1-4 were blended with appropriate
ratios of saline in order to evaluate injection (extrusion) force
profiles relative to a control material, ZYPLAST.TM. (Table 8).
This was accomplished by massing 5 g of gel material in a large
weighing boat using an SI-215 balance (Denver Instrument, Denver,
Colo.). An appropriate quantity of saline will be added to
constitute the correct volume percentage making the assumption that
both the gel material and saline have a density of 1 g/mL. This
material was then blended to an even consistency using a stainless
steel spatula and loaded into the back end of a 10 mL syringe with
an 18 g needle attached for subsequent use.
TABLE-US-00009 TABLE 8 Silk Gel Injection Force Profile Generation
Nominal Silk 23RGD Molar Ratio Saline Group Mass with Silk Fineness
Content Name 2% 1:1 Fine 25% 2RF25 4% 0 25% 4F25 1:1 Coarse 25%
4RC25 Fine 0% 4RFO 25% 4RF25 50% 4RF50 3:1 25% 4HRF25 10:1 25%
4VHRF25 8% Fine 25% 6RF25
[0255] These samples were tested using an Instron 8511 (Instron
Corp., Canton, Mass.) in conjunction with Series IX software and a
custom-designed aluminum frame attached to a 100 N load cell (FIG.
15). For the material testing, 1 mL of the sample material of
interest was loaded into a 1mL gas-tight glass syringe. The sample
syringe was mounted in the custom-designed aluminum frame mounted
on the Instron unit and the material extruded. The sample was then
checked for the force required to extrude the gel at each of 3
strain rates, 10 mm/minute, 50 mm/minute, and 200 mm/minute with
total actuator displacement set at 7 mm. A series of four tests
were run on each material type at each piston displacement rate.
Load-displacement data was collected at a frequency of 100 Hz and
are presented as the mean .+-. the standard deviation of the 4
average extrusion forces experienced of each gel type at each
strain rate. The average extrusion force was defined as the average
load measured in the plateau region of the load-displacement curve
resultant from each extrusion test. The data were reported as the
average amount of force required for extrusion of the sample
material and are compiled in Table 9 and FIG. 16.
TABLE-US-00010 TABLE 9 Average Force (N) to Extrude Silk Gel from
30 g Needle Plunger Displacement Rate 10 mm/min 50 mm/min 200
mm/min Group Name Ave Stdev Ave Stdev Ave Stdev 2RF25 0.6 0.0 2.9
0.6 7.3 0.7 4RF25 3.7 2.0 4.5 1.3 22.4 6.7 4RD25 7.1 3.7 6.7 0.5
25.1 3.9 4RF0 9.5 1.0 28.5 3.1 66.2 10.0 4RF26 3.2 0.9 7.4 0.6 30.4
5.0 4RF50 1.2 0.2 2.7 0.1 10.1 0.3 4HRF25 2.2 0.4 8.9 1.0 22.0 0.6
4VHRF25 2.8 1.6 5.2 1.4 14.6 2.1 8RF25 3.6 0.7 10.1 1.3 29.2 2.4
ZYPLAST .TM. 1.6 0.5 18.7 0.7 29.1 1.4
[0256] A comparison between milling techniques revealed that there
were no significant differences between 4RC25 and 4RF25, having
average extrusion forces of 7.1.+-.3.7N and 3.2.+-.0.9N at 10
mm/min, 6.7.+-.0.5N and 7.4.+-.0.6N at 50 mm/min, and 25.1.+-.3.9N
and 30.4.+-.5.0N at 200 mm/min respectively (Table 6, FIG. 16A).
Both of these formulations differed significantly (p.ltoreq.0.05)
from ZYPLAST.TM. at strain rates of 10 and 50 mm/min, which had
extrusion forces of 1.6.+-.0.5 N, 18.7.+-.0.7 N, and 29.1.+-.1.4 N
at 10, 50, and 200 mm/min strain rates.
[0257] Data regarding the extrudability of silk gel formulations
clearly illustrated that the addition of saline as a carrier fluid
to the comminuted silk particles offers an improved degree of
extrudability, substantially reducing the force necessary to
extrude silk gel at all strain rates. Adding increasing
concentrations of saline to the comminuted silk gels significantly
decreased the extrusion force required for silk gels at each strain
rate, with gels again exhibiting shear-thickening behavior (Table
9, FIG. 16B). At all strain rates, 4RF0 required significantly more
force to extrude than 4RF25, which in turn required significantly
more than 4RF50. At a strain rate of 10 mm/min, 4R0, 4R10, and 4R25
showed a significant decrease (p.ltoreq.0.05) in extrusion force
with increasing PBS concentration, having average forces of
9.5.+-.3.1 N, 6.1.+-.0.5 N, and 4.7.+-.0.7 N respectively (Table
9). At 50mm/min, these relationships were more pronounced with
average extrusion forces of 14.0.+-.0.9 N, 5.4.+-.0.7 N, and
3.9.+-.0.2 N respectively and all differed significantly (Table 6,
FIG. 16). At 200 mm/min, the trend remained as average extrusion
forces were 26.4.+-.4.5 N, 10.6.+-.1.6 N, and 6.4.+-.0.5 N
respectively with 0% PBS differing significantly from the other two
groups. Samples of 6R25 had an average extrusion force of
29.3.+-.4.8 N at 10 mm/min, significantly higher than 4R25 (Table
9). At 50 mm/min and 200 mm/min, the force to extrude the 6R25 was
greater than 80 N, causing the test to abort in order prevent
damage to the load cell.
[0258] The data also illustrate that use of very low concentrations
of silk may improve the extrudability of gel relative to higher
concentrations as in the case of 2RF25 as compared to 4RF25 and
8RF25. Increasing the concentration of silk in the comminuted silk
gels increased the extrusion force required for silk gels at each
strain rate, with significant increases between 2RF25 and both
4RF25 and 8RF25 at 10 mm/min and 200 mm/min (Table 9, FIG. 16C).
All groups differed significantly at the 50 mm/min strain rate and
gels continued to exhibit shear-thickening behavior, seen in the
increased extrusion forces associated with increased strain rates.
At 10 mm/min 2RF25 and 8RF25 required 0.6.+-.0.0 N and 3.6.+-.0.7 N
respectively, at 50 mm/min they required 2.9.+-.0.6 N and
10.1.+-.1.3 N, and at 200 mm/min 7.3.+-.0.6 N and 29.2.+-.2.4
N.
[0259] The data further indicated that use of 23RGD to enhance the
silk gel material did not appreciably impact the force necessary to
extrude silk gel formulations. Adding increasing concentrations of
RGD did not have a consistent effect upon the extrusion force
necessary for the gel materials (Table 9, FIG. 16D). At a 10 mm/min
strain rate there were no significant differences between 4F25 at
3.7.+-.2.0 N, 4R25, 4HR25 at 2.2.+-.0.4 N, and 4VHR25 at 2.8.+-.1.6
N. At a 50 mm/min strain rate 4HR25 was significantly higher than
all other extrusion forces at 8.9.+-.1.0 N as compared to 4F25 at
4.5.+-.1.3 N, 4R25, and 4VHR25 at 5.2.+-.1.4N. At a 200 mm/min
strain rate 4HR25 at 22.0.+-.0.6 N was significantly higher than
only 4VHR25 at 14.6.+-.2.1 N as compared to 4F25 at 22.4.+-.6.7 N
and 4R25.
[0260] Lastly, the data showed that silk gels blended with saline
had very similar extrudability to ZYPLAST.TM., a material already
proven to be readily handled as an injectable material. Based upon
this data it is believed that through careful manipulation of the
carrier species associated with the silk gel, modulation of silk
concentration, and control of particle size, silk gel materials may
be made to behave as a readily injectable material.
[0261] These results indicate that silk gels may be comminuted to a
particle range of about 25-50 .mu.m.sup.2 in cross-sectional area.
Silk gels may be comminuted to a size similar to ZYPLAST.TM.. Silk
gel particle size can be decreased by increasing silk concentration
or by changing the method of comminution. Increasing concentrations
of RGD did not develop a clear trend in silk particle size. Silk
gels may be extruded at a relevant strain rate of 50 mm/min at a
force comparable to or less than ZYPLAST.TM.. Silk gel extrusion
force may be decreased by adding increased quantities of saline
carrier or decreased concentrations of silk in the original gel.
Changes of comminution method attempted in this study did not
substantially affect the amount of force necessary for silk
extrusion. Increasing concentrations of RGD did not develop a clear
trend in silk gel extrusion force.
Example 12
Silk Gel Precipitates
[0262] The silk gel precipitate materials outlined in Table 10 were
generated for analysis. Silk solution of the specified
concentration was generated using the stock solution of 8% (w/v)
aqueous silk and diluting with purified water (Milli-Q purified).
23RGD/ethanol accelerant was prepared by generating a solution of
ethanol and purified water, then dissolving the specified 23RGD
quantity by vortexing. Silk precipitates were generated by directly
adding the specified volume of accelerant solution to that of silk
solution in 50 mL centrifuge tubes, shaking once to mix and
allowing the mixture to stand for 5 additional seconds before
adding about 45 mL purified water to halt the gelation process.
This material stood for 24 hours under ambient conditions and was
then strained through stainless steel cloth with 150
.mu.m.times.150 .mu.m pores to recover precipitates. These
precipitates were rinsed twice for 24 hours in 50 mL of purified
(Milli-Q) water at room conditions, strained a final time and used
for evaluation.
TABLE-US-00011 TABLE 10 Silk Gel Precipitate Types Generated
Initial Silk Solution 23RGD/ethanol Accelerant Silk Accelerant
Final Precipitate Silk Solution Ethanol 23RGD Solution Final Silk
RGD:Silk Group Concentration Volume Concentration Concentration
Volume Concentration Molar Name (mg/mL) (mL) (%) (mg/mL) (mL)
(mg/mL) Ratio BASE 80 1 90 2.45 1 40 5.0 RHI 80 1 90 4.90 1 40 10.0
RVLO 80 1 90 0.49 1 40 1.0 RLO 80 1 90 1.47 1 40 3.0 SCLO 80 1 90
2.45 1 30 6.7 SCVLO 80 1 90 2.45 1 20 10.0 ECLO 80 1 80 2.45 1 40
5.0 ECVLO 80 1 70 2.45 1 40 5.0 AVHI 80 0.67 90 2.45 1.33 27 10.0
AVLO 80 1.33 90 2.45 0.67 53 2.5
[0263] Samples of gel were examined under low-vacuum conditions
(.about.1 Torr) on a Quanta 200 (FEI Co., Hillsboro, Oreg.)
environmental scanning electron microscope with images collected at
magnifications of 200.times.. Representative images were taken to
illustrate surface topography characteristics of silk precipitate
samples (FIG. 17). All silk precipitate types appeared similar
under ESEM analysis. Each sample exhibited a mixture of both
granular and filamentous regions with occasional appearance of
large, contiguous masses of smooth material.
Example 13
Silk Gel Precipitate Massing
[0264] Silk precipitate samples, as described in Example 12, were
isolated after rinsing by straining through stainless steel wire
cloth with a pore size of 104 .mu.m.times.104 .mu.m and gently
blotted with a clean, lint-free wipe. Samples were massed to the
nearest 0.01 mg using an S-215 balance (Denver Instrument, Denver,
Colo.). These samples were frozen to -80.degree. C. for 24 hours
and placed into a Labconco lyophilizer unit (Labconco Corp., Kansas
City, Mo.) for 96 hours to remove all water content. The
precipitate residual solids were massed again and the dry mass
fraction in the samples determined. One-Way analysis of variance
(ANOVA) was used to test for significant differences caused by
changing silk concentration, 23RGD concentration and accelerant
volume. A Student's t-test was used to test the significance of
differences resulting from altered ethanol concentrations.
[0265] Increasing silk fibroin concentration increased precipitate
dry mass with Increasing the percentage of ethanol in the
accelerant solution also increased dry mass of the precipitates
with ECVLO produced only trace quantities of precipitate (visible,
but not recoverable in measurable quantities).
[0266] Increasing accelerant volume significantly increased
precipitate dry mass as AVHI was significantly greater than both
AVLO and BASE (p.ltoreq.0.05, FIG. 18A). For example, AVHI
(18.02.+-.3.9 mg) was significantly greater than both AVLO
(7.37.+-.1.33 mg) and BASE (11.07.+-.2.86 mg). Increasing
concentrations of 23RGD in the accelerant also increased the dry
mass of precipitate with BASE and RHI both significantly higher
than RVLO at (FIG. 18B). Fore example, BASE at 11.07.+-.2.86 mg,
RHI at 15.61.+-.3.62 mg, and RMED at 10.2.+-.1.42 mg were all
significantly higher than RLO at 1.9.+-.0.6 mg. Increasing silk
fibroin concentration increased precipitate dry mass with BASE
being greater than SCLO and significantly greater than SCVLO (FIG.
18C). For example, BASE was greater than SOLO at 7.84.+-.1.49 mg
and significantly greater than SCVLO at 4.15.+-.1.0 mg. Increasing
the percentage of ethanol in the accelerant solution also increased
dry mass of the precipitates with BASE producing significantly more
than ECLO (FIG. 18D). For example, BASE produced significantly more
than ECLO at 2.8.+-.0.91 mg. ECVLO produced only trace quantities
of precipitate (visible, but not recoverable in measurable
quantities). These results indicate that greater concentrations of
reactants (i.e., accelerant solution, RGD, silk and ethanol) all
increased the quantity of precipitant resultant.
[0267] The percent water in silk precipitates was determined as the
percentage of mass lost after silk precipitates of each formulation
types were subjected to a lyophilization step. Increasing the
volumetric fraction of accelerant added to make silk precipitates
did not significantly (p.ltoreq.0.05) affect the dry mass fraction
of the resultant precipitates (FIG. 19A). For example, AVLO at
(85.57.+-.2.32%, BASE at 88.99.+-.0.8%, and AVHI was
86.83.+-.1.95%. Increasing concentrations of 23RGD in the
accelerant showed a significant increase in dry mass percentage
with RVLO significantly less than RLO, RHI, and BASE (FIG. 19B).
For example, RLO at 95.01.+-.1.76% retained significantly more
water than RMED at 86.52.+-.2.67%, RHI at 88.39.+-.0.98%, and BASE.
Increasing concentrations of silk fibroin did not result in a clear
trend although SCLO was significantly greater than both SCVLO and
BASE (FIG. 19C). For example, SCLO at 80.77.+-.1.97% was
significantly less than both SCVLO at 86.94.+-.1.98% and BASE.
Increasing the percentage of ethanol in the accelerant solution
significantly decreased the dry mass percentage with ECLO compared
to BASE (FIG. 19D). For example, ECLO at 86.97.+-.1.16% compared to
BASE. In summary, greater concentrations of reactants (i.e.,
accelerant solution, 23RGD, silk and ethanol) increased the
quantity of resultant precipitate. It is also of interest that
there were significant differences between the dry mass fractions
of BASE and both RVLO and ECLO, possibly indicating different
protein structures. These differing protein structures might be
more hydrophobic than BASE in the case of ECLO and more hydrophilic
in the case of RVLO. These properties might used to affect the
stability of the gels in an in vivo environment with more
hydrophilic materials being more readily bioresorbed by the host
while more hydrophobic materials prove more resistant.
[0268] In examining the percent of water in the precipitates it is
of particular interest that there were significant differences
between BASE and both RLO and ECLO. This may result from structural
motifs different than other precipitate types generated by RLO and
ECLO. With respect to ECLO, it has a greater proportion of
.beta.-sheet structure than BASE and would be expected to entrain
less water. However, the difference observed between RLO and base
is difficult to explain. RLO has a greater extent of .beta.-sheet
structure with less .alpha.-helix and random coil motifs than BASE,
yet it entrains a greater quantity of water. In fact, this same
trend is seen when comparing RLO to RMED, BASE, and RHI. The
situation is further confounded in examining the relationship
between the initial secondary structures of RMED, BASE and RHI, as
all initially exhibit greater quantities of .alpha.-helix and
random coil than RLO, yet all entrain significantly less water.
SCLO samples also had a significantly higher dry mass percentage as
compared to BASE and SCVLO sample with no clear trend or reason for
this occurrence. These data indicate that there may be a structural
difference in these precipitates not apparent in the secondary
structure of the materials which is affecting the manner in which
the precipitates associate with water. It may be the case that the
RGD bound to these precipitates has altered in some fashion the
manner in which the silk molecules are presented to water,
enhancing their ability to associate with it.
Example 14
Gel Precipitate FTIR Spectrum Capture
[0269] Gel precipitates of each type, as described in Example 12,
were analyzed by attenuated total reflectance Fourier-transform
infrared (ATR-FTIR) spectroscopy using a Bruker Equinox 55
spectrophotometer (Bruker Optics, Inc., Billerica, Mass.) coupled
with a Pike MIRACLE.TM. germanium crystal (PIKE Technologies,
Madison, Wis.). Sample ATR signal spectra were obtained by
performing a 128-scan integration. Resolution was set to 4
cm.sup.-1 with a 1 cm.sup.-1 interval from a range of 4000 to 400
cm.sup.-1. FTIR spectra of pure water were also collected and
subtracted manually from the gel spectra to remove confounding
water signal at a ratio conducive to flattening the region between
1800 cm.sup.-1 and 1700 cm.sup.-1 on the spectrum. After
subtraction, the Amide I bands (1700-1600 cm.sup.-1) of
representative spectra were evaluated against characteristic peaks
commonly accepted to be associated with secondary protein
structures.
[0270] Examination of the silk precipitates under FTIR revealed
that increasing the volumetric ratio of accelerant added to the
silk solution had little effect on their protein secondary
structure (FIG. 20A). AVLO, BASE, and AVHI all exhibited similar
characteristics with characteristic peaks around 1624 cm.sup.-1 and
a toe region at 1698 cm.sup.-1 indicating a predominance of
.beta.-sheet and .beta.-turn structure respectively. Each sample
also exhibited additional structures at 1677 cm.sup.-1, 1663
cm.sup.-1, 1654 cm.sup.-1 and 1645 cm.sup.-1 denoting additional
interspersed .beta.-sheet, .beta.-turn, .alpha.-helical and random
coil conformations respectively. Increasing concentrations of 23RGD
in the accelerant decreased .beta.-sheet stability indicated by a
peak shift from .about.1621 cm.sup.-1 in RVLO to .about.1624
cm.sup.-1 in RLO (FIG. 20B). Further increasing the concentration
of 23RGD in BASE and RHI caused this weakened .beta.-sheet again
accompanied by an increase in higher signal values in the 1654
cm.sup.-1 and 1645 cm.sup.-ranges, indicating increased random coil
and .alpha.-helical constituents. Otherwise, RVLO, RLO, BASE, and
RHI revealed similar structures with dominant peaks in the 1620
cm.sup.-1 range and a toe region at 1698 cm.sup.-1 with additional
structures at 1654 cm.sup.-1 and 1645 cm.sup.-1. Increasing
concentrations of silk fibroin had little perceptible effect on
protein secondary structure (FIG. 20C). The spectra for SCVLO,
SCLO, and BASE each exhibited similar characteristic peaks around
1624 cm.sup.-1 with toe regions at 1698 cm.sup.-1 indicating a
predominant .beta.-sheet structure with additional .alpha.-helical
and random coil conformations interspersed. Increasing the
percentage of ethanol in the accelerant solution resulted in less
evidence of .alpha.-helical and random coil conformations indicated
by a decrease in the signal between 1670 cm.sup.-1 and 1630
cm.sup.-1 in both ECLO and BASE samples relative to ECVLO (FIG.
20D). This decrease in .alpha.-helical and random coil is
accompanied by an increase in .beta.-sheet structure.
[0271] Substantial similarity existed between all groups except for
RVLO and ECVLO, which each differ from BASE formulation. Each of
these material types exhibited a different secondary structure from
both each other and from BASE, reinforcing the trend observed
previously in the percent dry mass of the precipitates. Higher
concentrations of 23RGD yielded less organized .beta.-sheet
structures and lower concentrations of ethanol yielded greater
quantities of .alpha.-helix and random coil motifs. It is possible
that used in conjunction with one another, these two phenomena
could be adjusted to develop silk structures resulting from silk
solutions in any of a variety of different protein conformations.
These conformations could, in turn, be tailored based upon the
desired ultimate bulk properties of the silk material.
[0272] It is expected that higher .beta.-sheet components might
provide the gel with greater resistance to bioresorption and
compressive loading, while at the same time, making the material
more rigid.
Example 15
Congo Red Staining of Gel Precipitates
[0273] Silk precipitate samples were stained with 100 .mu.M Congo
red in purified water. Silk precipitate samples weighing 5-10 mg
were vortexed with 500 .mu.L of this solution for 15 seconds,
allowed to stand at room temperature (.about.20-24.degree. C.) for
10 minutes, then centrifuged at 16,000 g (RCF) for 10 minutes. The
supernatant was discarded and the pellet re-suspended by vortexing
for 30 seconds in 1 mL of purified water. The process of soaking,
centrifugation, aspirating and rinsing was repeated 3 times. The
final pellet was removed, smeared on a glass microscope slide, and
imaged under white and polarized light using a Microscope PC
MACROFIRE.TM. Model S99831 Camera (Optronics, Goleta, Calif.) and
PICTUREFRAME.TM. 2.1 software (Optronics, Goleta, Calif.) and a
System Microscope Model BX41 (Olympus, Melville, N.Y.).
[0274] None of the silk precipitate types exhibited the emerald
luminescence typically associated with amyloid fibrillar structures
(FIG. 21). All precipitate types did exhibit bright white
luminescence, indicative of a robust crystalline structure. The
extent of this brightness does not appear to vary substantially by
formulation, but only by sample quantity on the slide. Based on
these results, it is unlikely that any of these precipitate types
is amyloid in nature, a positive sign, as amyloid fibrils are
associated with a number of negative pathologies in humans.
Example 16
23RGD Quantification in Gel Precipitates by HPLC
[0275] The amount of 23RGD bound to silk precipitates was
quantified by analyzing lyophilized samples. The 23RGD was removed
by incubating the samples for 4 hours in a dissolving buffer, then
centrifuging on an Eppendorf 5415C (Eppendorf North America Inc.,
Westbury, N.Y.) at 16,000 g (RCF) for 30 minutes and the
supernatant collected. This supernatant was then centrifuged in
identical fashion and the final supernatant collected for HPLC
analysis using a PerkinElmer Series 200 (PerkinElmer, Waltham,
Mass.). The 23RGD peak areas from each curve were compared against
a standard curve. 1-Way ANOVA was used to test for significant
differences caused by changing silk concentration, 23RGD
concentration, and accelerant volume. A Student's t-test was used
to test the significance of differences resulting from altered
ethanol concentrations.
[0276] Increasing the quantity of 23RGD/ethanol accelerant added
resulted in a significant increase (p.ltoreq.0.05) in 23RGD:silk
ratio for both BASE and AVHI as compared to AVLO (FIG. 22A). For
example, BASE at 8.7.+-.0.6 and AVHI at 10.5.+-.1.2 were
significantly increased as compared to AVLO at 5.2.+-.1.8.
Increasing the quantity of 23RGD in the accelerant solution
resulted in significant increases in 23RGD:silk ratio for each of
RVLO, RLO, BASE, and RHI relative to each other (FIG. 22B). For
example, RLO at 1.1.+-.0.2, RMED at 6.95.+-.0.49, BASE and RHI at
10.7.+-.0.8 relative to each other. Changing the starting
concentration of silk in solution prior to precipitation did not
affect 23RGD:silk ratio as those in SCVLO, SCLO, and BASE did not
differ significantly (FIG. 22C). For example, SCVLO at 11.0.+-.0.4,
SCLO at 9.9.+-.1.8, and BASE did not differ significantly.
Decreasing the ethanol content in the accelerant did not produce a
significant effect as observed by comparing ECLO and BASE (FIG.
22D).
[0277] Reviewing this data in light of the precipitate dry massing
data, none of the conditions explored resulted in isolation of silk
(.about.10-35% precipitated) nor 23RGD (.about.5-30% precipitated)
as limiting reagents in the reaction. Precipitate samples generated
at a calculated 10:1 23RGD:silk ratio consistently generated a
"correct" molecular binding ratio. In the case of AVHI, this runs
contrary to the trend of bound 23RGD concentrations being
approximately double the projected values as indicated by AVLO and
BASE (about 5:1 and about 9:1, respectively). This might be
explained by saturation of the silk with 23RGD in the case of 10:1
23RGD precipitates. This is further reinforced by the behavior of
SCVLO and 0.6S 3R 10:1, both of which were produced using 2.45
mg/mL 23RGD in 90% ethanol as the AVHI was. Both materials
projected to have greater than 10:1 ratios of bound 23RGD (20:1 and
13.4:1, respectively) based on the behavior of AVLO and BASE, but
which both reached only about 10:1 ratios. RHI, generated using a
4.5 mg/mL 23RGD concentration in the accelerant which conceivably
should have been high enough to induce the postulated dimeric 23RGD
reached only the expected 23RGD ratio of about 10:1 not the
postulated 20:1.
[0278] Few of the silk precipitates entrained a molar ratio similar
to what was initially calculated (FIG. 23). Four groups, SCVLO,
AVHI, RHI, and RLO contained ratios similar to their calculated
values of RGD per mole of silk. The six remaining groups contained
ratios substantially greater than their calculated values. In the
cases of AVLO, BASE, RMED, and SCLO, the RGD quantities were about
2-fold greater than expected. Although not wishing to be limited by
theory, this greater observed molar ratio may be indicative of the
formation of a RGD bi-layer. It may be the case that either
micelles or lamellar structures of RGD existed in the 90% ethanol
solution prior to addition to the silk, upon contacting the aqueous
phase, micellar stability was disrupted. As a result, a bi-layer of
RGD was formed at the solution interface, where these molecules
began to interact with the silk molecules. The RLO samples were
made with a RGD concentration of 0.49 mg/mL in the accelerant, the
lowest used in this study and potentially within the solubility
range of RGD in 90% ethanol. RMED samples used 1.47 mg/mL and most
other formulations were made with a RGD accelerant concentration of
2.45 mg/mL, above the RGD concentration at which dimerization
became favorable in the solution. Further highlighting the
possibility of RGD dimerizing in the ethanol solution is the
behavior of ECLO precipitation. The RGD concentration remains 2.45
mg/mL as with BASE and AVLO but the water concentration in the
accelerant is increased to 20% and results in a binding of about
1.5-fold the expected total of RGD. This may be due to a decreased
driving force for RGD bi-layer formation at the solution interface
caused by the lower ethanol content. This might in turn cause
disruption to fewer micellar structures in the initial accelerant
solution. It could also be explained by altered micellar structure,
varying between a single peptide layer and a multi-lamellar
structure depending upon the concentrations of water and ethanol in
the accelerant phase.
[0279] Precipitate samples generated at a calculated 10:1 RGD:silk
ratio consistently generated a "correct" molecular binding ratio.
In the case of AVHI, this runs contrary to the trend of bound RGD
concentrations being approximately double the projected values as
indicated by AVLO and BASE (about 5:1 and about 9:1 respectively).
It is possible that this might be explained by saturation of the
silk with RGD in the case of 10:1 RGD precipitates. This is further
reinforced by the behavior of SCVLO and 0.6S 3R 10:1, both of which
were produced using 2.45 mg/mL RGD in 90% ethanol as was AVHI. Both
materials projected to have greater than 10:1 ratios of bound RGD
(20:1 and 13.4:1 respectively) based on the behavior of AVLO and
BASE, but which both reached only about 10:1 ratios. RHI, generated
using a 4.5 mg/mL RGD concentration in the accelerant which
conceivably should have been high enough to induce the postulated
dimeric RGD, reached only the expected RGD ratio of about 10:1 not
the postulated 20:1. This may be attributed to the mode of binding
between the silk molecules and the RGD molecules. It is expected
that RGD will bind through a hydrophobic association mechanism and
despite the largely hydrophobic sequence of silk, it may be
possible that there are approximately 5 sites which offer
preferable RGD binding stability. This presumption stems from the
apparent saturation at 10:1 RGD molecules per molecule of silk.
Dependent upon the nature of RGD self-association at the solution
boundary, it may be a case where single RGD molecules or RGD dimers
bind to these sites.
[0280] There are a series of properties further indicating the
possibility of a specific molecular assembly interaction between
the silk and 23RGD accelerant. Conspicuously, that 23RGD does
localize to the precipitates in a greater-than-calculated ratio but
that it binds at intuitive concentrations which can be related
quickly to the initially calculated molar ratios. The fact that
this occurs without fully depleting either the 23RGD or the silk
fibroin molecules is of further interest. The FTIR data also
indicated that use of 0.49 mg/mL 23RGD in RVLO precipitates induced
formation of distinctly different structures than use of 2.45 mg/mL
in BASE or 4.9 mg/mL in RHI which appeared similar to each other.
RMED precipitates generated with 1.47 mg/mL of 23RGD contained
characteristics of both RVLO and BASE/RHI material spectra. FTIR
indicated a different structure from a 2.45 mg/mL of 23RGD in 70%
ethanol accelerant in the case of ECVLO. These outcomes were both
reinforced in examining the percentage of dry mass from the
resultant precipitates (though ECLO is used to illustrate the trend
in 23RGD solubility in ethanol solution instead of ECVLO). Both of
these assays indicate the formation of different precipitate
structures based upon the extent of 23RGD saturation in the ethanol
solution, conceivably resulting from dimeric 23RGD binding or
monomeric 23RGD binding.
[0281] This phenomenon likely results from the amphiphilic nature
of 23RGD and the varied chemistry of the solution phase between
heavily ethanolic and heavily aqueous. It is possible that the
hydrophilic ends of two 23RGD molecules associate in the 90%
ethanol solution, exposing the two hydrophobic ends to solution.
Addition of this accelerant solution with dimeric 23RGD causes
rapid association of the exposed hydrophobic ends of the 23RGD with
hydrophobic domains of the silk molecules, rapidly precipitating
them. This process occurs until the 90% ethanol accelerant solution
is sufficiently diluted with the aqueous silk solution to cause the
dimeric assembly of the 23RGD molecules to no longer be favorable,
as a result stopping precipitation. Based upon the apparent
saturation at about 10 for 23RGD:silk ratio, there may also be a
maximum of 5 binding sites for the 23RGD dimer per molecule of
silk. This knowledge may be used to bind specific quantities of
23RGD to silk, while at the same time dictating silk gel structure
and resultant behavior. Additionally, this method may also
potentially be applied to other amphiphilic peptides of interest
during their integration into a silk gel material.
[0282] These results indicate that silk precipitate quantity may be
increased by increasing the quantity of any reactant in the RGDEEG
system. Silk precipitates occurring during RGDEEG gelation are
unlikely to be amyloid. Silk precipitate .beta.-sheet structure may
be increased by higher concentrations of ethanol accelerant or
lower concentrations of RGD. RGD molecules may self-associate into
micelles, lamellar structures, or dimers when placed into a
strongly ethanolic solution, in turn, assembling with silk in a
dimeric fashion during RGDEEG gelation. Silk molecules may become
saturated with RGD once they have bound about 10 molecules. Silk
precipitate structures may be altered by changing RGD
concentrations added, though the extent and nature of these changes
remains unclear, as they are not perceptible in material secondary
structure. These altered structures may account for otherwise
unexplained increased appearance of .alpha.-helix and random coil
motifs at high RGD concentrations in precipitates. These altered
structures may account for otherwise unexplained increased
resistance to proteolytic bioresorption of .alpha.-helix and random
coil motifs at high RGD concentrations in precipitates
Example 17
Enzymatic Bioresorption of Gel Precipitates
[0283] A single sample of precipitate types selected for distinctly
different behaviors from BASE in the previously listed assays
including RVLO, RLO, BASE, RHI, ECLO, 0.65 3R 5:1 weighing
approximately 60 mg were massed using an S-215 balance. These
samples were placed in a solution of Protease Type XIV from
Streptomyces griseus (Sigma catalog no. P-5147) in phosphate
buffered saline (PBS) was generated at a concentration of 0.3 mg/mL
(activity was 4.5 U/mg) at a ratio of 1 mL of protease solution per
100 mg of silk precipitate. The gel and protease solution were
incubated for 24 hours at 37.degree. C. with no mechanical mixing.
After 24 hours, the residual precipitate was isolated by straining
through stainless steel cloth as before and the specimens analyzed
by FTIR as described.
[0284] Accelerant quantity added did not substantially affect the
bioresorption behavior of the materials as BASE, AVHI and AVLO all
featured decreased levels of .alpha.-helix and random coil motifs
(FIG. 23A). This decrease was slightly larger in the case of AVLO
which also featured a peak shift from 1624 cm.sup.-1 to 1622
cm.sup.-1, indicating a more stable .beta.-sheet structure. The
23RGD concentration did not appear to affect bioresorption behavior
of the materials either as RVLO, RLO, BASE and RHI all showed
decreased in .alpha.-helix and random coil motifs, though a greater
portion of .alpha.-helix and random coil remained intact in RHI
(FIG. 23B). However, a greater portion of .alpha.-helix and random
coil remained intact in RHI at Day 2 relative to the other samples.
Silk concentration did not substantially affect the bioresorption
behavior of the materials as BASE and SCLO exhibited decreased
levels of .alpha.-helix and random coil motifs and featured slight
peak shifts from 1624 cm.sup.-1 to 1623 cm.sup.-1 (FIG. 23C).
[0285] Despite differences in initial structures, all precipitate
types bioresorbed in a similar fashion with .alpha.-helix and
random coil motifs degraded preferentially to .beta.-sheet. Only
increasing the concentration of 23RGD, as in the case of RHI,
appeared to have any appreciable effect on the final secondary
structure of the precipitates. This appears to be a case where
there is simply more .alpha.-helix and random coil structure upon
initial formation of these materials and they take more time to
degrade to a similar extent of .beta.-sheet structure as the other
formulations. Use of this knowledge in conjunction with an ability
to manipulate the secondary protein structures of these materials
could lead to biomaterials with very specific lifetimes in
vivo.
Example 18
Composition Comprising Silk Fibroin Hydrogel Particles and Matrix
Polymer
[0286] Silk fibroin hydrogels were cast according to the methods
described above in Examples 1-4. A silk hydrogel consisting of 6%
silk fibroin by dry mass percent with a 23RGD molar ratio of 1:1
with silk molecules was generated for particle comminution. This
material was subjected to the forced sieving method described above
to generate silk gel particles ranging nominally between 0.1
.mu.m.sup.2 and 5 .mu.m.sup.2. These materials were blended with
crosslinked hyaluronan at various volumetric ratios for evaluation
as a potential filler material. The blends were made at volumetric
percentages of 5% silk fibroin hydrogel with 95% hyaluronan, 25%
silk fibroin hydrogel with 75% hyaluronan, 50% silk fibroin
hydrogel with 50% hyaluronan.
Example 19
Composition Comprising Silk Fibroin Hydrogel Particles and Matrix
Polymer
[0287] The composition described above in Example 18 are modified
in terms of the 23RGD component (0 to 3:1), silk fibroin
concentration (1% to 8%), particle size (0.1 .mu.m.sup.2 to 500
.mu.m.sup.2), and silk format (may be silk solution intermediate
mentioned above added directly to hyaluronan). Materials also vary
in the percent composition of silk hydrogel in hyaluronan between
0.1% and 99.9% depending upon application and desired material
properties.
Example 20
Extrudability Characteristics of Composition Comprising Silk
Fibroin Hydrogel Particles and Matrix Polymer
[0288] To assess the extrusion force necessary to inject a
composition disclosed herein through a needle, the dermal fillers
comprising silk fibroin hydrogel particles in Table 11 were
compared with JUVEDERM.RTM. Ultra Plus (Allergan, Inc., Irvine
Calif.), a crosslinked hyaluronan dermal filler sample lacking silk
fibroin hydrogel particles (6PUR00). The extrusion force test was
performed by measuring the force necessary to extrude a hydrogel
through a 27 gauge or 30 gauge needle using a 0.8 mL syringe.
[0289] To prepare the compositions listed in Table 11, a 6% silk
fibroin hydrogel was prepared according to Examples 1-4. Some of it
was used as component for the filler formulation, and another batch
was mixed with 25% (v/v) PBS (saline buffer) according to Example
9, for a final silk fibroin content of around 4.5%. These hydrogels
were mixed with a commercial crosslinked hyaluronan in various
proportions using a mechanic mixer. This hyaluronan hydrogel had
the following characteristics: 24 mg/g sodium hyaluronate (with an
average molecular weight of 3,000,000 Da before crosslinking), a
degree of crosslinking of 5%-6% (crosslinker: 1,4-butanediol
diglycidyl ether). Table 11 lists the formulations prepared by
mixing silk fibroin hydrogels with hyaluronan gels in 100 g pots by
using several homogenization cycles of 1 minute at 3500rpm in the
mixer. The pH was adjusted to about 7.0 by addition of small
volumes of a diluted sodium hydroxide solution between
homogenization cycles.
TABLE-US-00012 TABLE 11 Examples of filler formulations containing
silk fibroin and crosslinked hyaluronan Composition Silk Component
Final Silk Fibroin Final HA Name Silk Fibroin Hydrogel Weight %
Concentration Concentration 6PUR00 no silk (blank) 0% 0 mg/g 24.0
mg/g 6PUR05 6% silk fibroin gel 5% 3.0 mg/g 22.8 mg/g 6PUR25 25%
15.0 mg/g 18.0 mg/g 6PUR50 50% 30.0 mg/g 12 mg/g 6PBS05 4.5% silk
fibroin gel 5% 2.25 mg/g 22.8 mg/g 6PBS25 (made from 6% gel + 25%
11.25 mg/g 18.0 mg/g 6PBS50 25% (v/v) saline buffer) 50% 22.5 mg/g
12 mg/g 6PBS75 75% 33.75 mg/g 6 mg/g
[0290] Analysis of these compositions indicate that compositions
comprising about 5% to about 50% silk fibroin hydrogel particles
exhibited extrudability characteristics similar to a composition
comprising 0% silk fibroin hydrogel particles. For example, at a
plunger displacement rate of about 13 mm/min, compositions
comprising 0% to about 50% silk fibroin hydrogel particles all
exhibited an extrusion force of about 10N. Similarly, at a plunger
displacement rate of about 50 mm/min, compositions comprising 0%,
about 5%, and about 50% silk fibroin hydrogel particles all
exhibited an extrusion force of about 17N, with compositions
comprising 25% silk fibroin hydrogel particles exhibiting an
extrusion force of about 20N.
Example 21
In Vivo Evaluation of Composition Comprising Silk Fibroin Hydrogel
Particles and Matrix Polymer
[0291] To examine the in vivo effects of the compositions disclosed
herein, the compositions disclosed in Table 12 were subcutaneously
injected into Sprague Dawley rats.
[0292] To prepare the compositions listed in Table 12, a 8% silk
fibroin hydrogel was prepared according to Examples 1-4. The silk
fibroin hydrogel was milled into particles of a mean
cross-sectional area of about 1 .mu.m and blended with the saline
component indicated in Table 12. Saline was added by first mixing
by spatula into a bulk of silk fibroin hydrogel, then shearing 60
times through a 1.5 mm orifice. After saline blending, the material
was sterilized by gamma irradiation at a dose of 25-40 kgy. The
sterilized silk fibroin hydrogel particles were blended with
JUVEDERM.RTM. Ultra Plus (Allergan, Inc., Irvine Calif.), a
hyaluronan dermal filler, in ratios according to Table 12
immediately prior to surgery. Blending was conducted by means of
shearing back and forth between a pair of syringes connected by a
stopcock until combined gel appearance was uniform.
TABLE-US-00013 TABLE 12 Examples of filler formulations containing
silk fibroin and crosslinked hyaluronan Silk Fibroin Hyaluronan
Saline Silk Fibroin SST:Silk Group Sample Name Component Component
Component Percent Silk Ratio 1 JUVEDERM .RTM. Ultra Plus N/A 100%
N/A N/A N/A 2 5% Base Silk Fibroin 3.75% 95% 1.25% 6% 1:1 molar 3
25% Base Silk Fibroin 18.75% 75% 6.25% 6% 1:1 molar 4 50% Base Silk
Fibroin 37.5% 50% 12.5% 6% 1:1 molar 5 75% Base Silk Fibroin 56.25%
5% 18.75% 6% 1:1 molar 6 Base Silk Fibroin 75% 0% 25% 6% 1:1
molar
[0293] Eight male Sprague Dawley rats weighing 250-275 grams,
acclimated for one week at the animal facility prior to surgery,
were anesthetized with 4% isoflurane and maintained at 1-2%
isoflurane on a heated pad. The back of the animal was shaved and
cleaned with alcohol. The animals were injected at four different
sites on the back with a volume of 50 .mu.L/injection. Compositions
were distributed across multiple animals in a successive and
cyclical pattern. Material Group 1 will be injected into Animal 1,
Site 1; Material Group 2 into Animal 1, Site 2; Material Group 3
into Animal 1, Site 3; Material Group 4 into Animal 1, Site 4;
Material Group 5 will be injected into Animal 2, Site 1, Material
Group 6 will be injected into Animal 2, Site 2; Material Group 1
will be injected into Animal 2, Site 3; Material Group 2 will be
injected into Animal 2, Site 4, etc. A total of 24 sites will be
injected in 6 rats. On Day 42, animals were euthanized via carbon
dioxide asphyxiation and all sample sites were identified,
harvested, and prepared for subsequent sectioning and staining.
[0294] Material cross-sections mounted on slides were stained with
H&E and CD-68 according to standard methods (FIG. 24 and FIG.
25). It was observed that all sample types infiltrated the animal
dermis and appeared as lakes of material. Pure JUVEDERM.RTM. Ultra
Plus control elicited a minimal extent of cellular response, very
consistent with ambient cellularity in the surrounding tissue (FIG.
24). The extent of cellular infiltrate and total presence was
increased by adding silk fibroin hydrogel particles to the HA
dermal filler (FIG. 24). Base silk fibroin hydrogel particle
material by comparison, exhibited significant cellular response
which tended to occur circumferentially around smaller agglomerated
lakes of material (FIG. 24). Staining with CD-68 revealed the
presence of small populations of CD-68+ cells in all silk fibroin
hydrogel particle containing samples but in none of the pure
JUVEDERM.RTM. Ultra Plus control samples (FIG. 25). This suggests
increased macrophage activity in the silk fibroin hydrogel
particle-containing samples as compared to the pure JUVEDERM.RTM.
Ultra Plus control.
[0295] The results reveal the potential for increasing the extent
of cellular interaction with a pure crosslinked hyaluronan material
through introduction of a silk fibroin hydrogel component. This
increased cellularity at the implant site could ultimately
correlate to an alternative host response to the hyaluronan
including a wound-healing-type response involving neo-collagen
deposition during implant biorseorption. Taken together, the data
here suggest that the combination hyaluronan/silk fibroin hydrogel
material not only acts as a dermal filler for intradermal defects,
but also encourages neo-collagen deposition through a native
healing response.
Example 22
Use of Dermal Filler Composition for Treating a Facial Defect of
the Cheek
[0296] This example illustrates the use of compositions and methods
disclosed herein for treating a facial defect of the cheek.
[0297] A 28-year-old woman presents with a lean face. She felt her
face looked old, sad and bitter because of the less fullness of her
check contour. Pre-operative evaluation of the person includes
routine history and physical examination in addition to thorough
informed consent disclosing all relevant risks and benefits of the
procedure. The physician evaluating the individual determines that
she is a candidate for soft tissue treatment using the compositions
and methods disclosed herein. A composition comprising silk fibroin
hydrogel component and a hyaluronan component is administered
subcutaneously and under superficial musculoaponeurotix system into
the checks regions; about 15 mL of composition into the left and
right cheeks. The individual is monitored for approximately 7 days.
The physician evaluates the cheeks tissue and determines that the
treatment was successful. Both the woman and her physician are
satisfied with the results of the procedure because she looked
younger. Approximately one month after the procedure, the woman
indicates that his quality of life has improved.
Example 23
Use of Dermal Filler Composition for Treating Facial Imperfection
of Eyelids
[0298] This example illustrates the use of compositions and methods
disclosed herein for treating a facial imperfection of the
eyelids.
[0299] A 37-year-old woman presents with sunken eyes and this
appearance made her look old and fierce. Pre-operative evaluation
of the person includes routine history and physical examination in
addition to thorough informed consent disclosing all relevant risks
and benefits of the procedure. The physician evaluating the
individual determines that she is a candidate for soft tissue
treatment using the compositions and methods disclosed herein. A
composition comprising silk fibroin hydrogel component and a
hyaluronan component is administered subcutaneously and under
superficial musculoaponeurotix system into the upper eyelid
regions; about 2.5 mL of composition into the left and right eyelid
regions. The individual is monitored for approximately 7 days. The
physician evaluates the eyelid regions and determines that the
treatment was successful. Both the woman and her physician are
satisfied with the results of the procedure because she looked
younger. Approximately one month after the procedure, the woman
indicates that his quality of life has improved.
Example 24
Use of Dermal Filler Composition for Treating Wrinkles
[0300] This example illustrates the use of compositions and methods
disclosed herein for treating wrinkles.
[0301] A 55-year-old woman presents with wrinkles around the eyes
and cheek areas. Pre-operative evaluation of the person includes
routine history and physical examination in addition to thorough
informed consent disclosing all relevant risks and benefits of the
procedure. The physician evaluating the individual determines that
she is a candidate for soft tissue treatment using the compositions
and methods disclosed herein. A composition comprising silk fibroin
hydrogel component and a hyaluronan component is administered
subcutaneously and under superficial musculoaponeurotix system into
the upper eyelid and cheek regions; about 1.5 mL of composition
into the left and right eyelid and cheek regions. The individual is
monitored for approximately 7 days. The physician evaluates the
facial regions and determines that the treatment was successful.
Both the woman and her physician are satisfied with the results of
the procedure because she looked younger. Approximately one month
after the procedure, the woman indicates that his quality of life
has improved.
Example 25
Use of Dermal Filler Composition for Treating a Breast Defect
[0302] This example illustrates the use of compositions and methods
disclosed herein for treating a breast defect.
[0303] A 32-year-old woman presents with complaints that the medial
portions of her breast implants are visible, which accentuated the
"bony" appearance of her sternum. In addition she felt her breast
are too far apart. Pre-operative evaluation of the person includes
routine history and physical examination in addition to thorough
informed consent disclosing all relevant risks and benefits of the
procedure. The physician evaluating the individual determines that
she is a candidate for soft tissue treatment using the compositions
and methods disclosed herein. A composition comprising silk fibroin
hydrogel component and a hyaluronan component is administered
subcutaneously over the lateral sternum and medial breast
bilaterally, 15 mL on the right and 10 mL on the left. The
composition is administered in a tear like fashion to increase the
surface area to volume ratio. The individual is monitored for
approximately 7 days. The physician evaluates the breasts and
determines that the treatment was successful. Both the woman and
her physician are satisfied with the results of the procedure.
Approximately one month after the procedure, the woman indicates
that his quality of life has improved.
Example 26
Use of Dermal Filler Composition for Breast Augmentation
[0304] This example illustrates the use of compositions and methods
disclosed herein for breast augmentation.
[0305] A 28-year-old woman presents micromastia or breast
hypoplasia. Pre-operative evaluation of the person includes routine
history and physical examination in addition to thorough informed
consent disclosing all relevant risks and benefits of the
procedure. The physician evaluating the individual determines that
she is a candidate for soft tissue treatment using the compositions
and methods disclosed herein. A composition comprising silk fibroin
hydrogel component and a hyaluronan component is administered
subcutaneously using axillary, periareolar, and inframammary routes
bilaterally, 90 mL on the right and 145 mL on the left. The
composition is administered in a tear like fashion to increase the
surface area to volume ratio. The individual is monitored for
approximately 7 days. The physician evaluates the breasts and
determines that the treatment was successful. Both the woman and
her physician are satisfied with the results of the procedure.
Approximately one month after the procedure, the woman indicates
that his quality of life has improved.
Example 27
Adipose Tissue Transplant for Breast Disorder
[0306] This example illustrates the use of compositions and methods
disclosed herein for treating a breast disorder.
[0307] A 29-year-old woman presents with bilaterial tiberous breast
deformity. Pre-operative evaluation of the person includes routine
history and physical examination in addition to thorough informed
consent disclosing all relevant risks and benefits of the
procedure. The physician evaluating the individual determines that
she is a candidate for soft tissue treatment using the compositions
and methods disclosed herein. A composition comprising silk fibroin
hydrogel component and a hyaluronan component is administered
subcutaneously in multiple planes axillary, periareolar, and
inframammary routes bilaterally, 180 mL on the right and 170 mL on
the left. The composition is administered in a tear like fashion to
increase the surface area to volume ratio. The individual is
monitored for approximately 7 days. The physician evaluates the
breasts and determines that the treatment was successful. Both the
woman and her physician are satisfied with the results of the
procedure. Approximately one month after the procedure, the woman
indicates that his quality of life has improved.
[0308] In closing, it is to be understood that although aspects of
the present specification have been described with reference to the
various embodiments, one skilled in the art will readily appreciate
that the specific examples disclosed are only illustrative of the
principles of the subject matter disclosed herein. Therefore, it
should be understood that the disclosed subject matter is in no way
limited to a particular methodology, protocol, and/or reagent,
etc., described herein. As such, various modifications or changes
to or alternative configurations of the disclosed subject matter
can be made in accordance with the teachings herein without
departing from the spirit of the present specification. Lastly, 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.
Accordingly, the present invention is not limited to that precisely
as shown and described.
[0309] Certain embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Of course, variations on these described embodiments
will become apparent to those of ordinary skill in the art upon
reading the foregoing description. The inventor expects skilled
artisans to employ such variations as appropriate, and the
inventors intend for the invention to be practiced otherwise than
specifically described herein. Accordingly, this invention includes
all modifications and equivalents of the subject matter recited in
the claims appended hereto as permitted by applicable law.
Moreover, any combination of the above-described elements in all
possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by
context.
[0310] Groupings of alternative elements or embodiments of the
invention disclosed herein are not to be construed as limitations.
Each group member may be referred to and claimed individually or in
any combination with other members of the group or other elements
found herein. It is anticipated that one or more members of a group
may be included in, or deleted from, a group for reasons of
convenience and/or patentability. When any such inclusion or
deletion occurs, the specification is deemed to contain the group
as modified thus fulfilling the written description of all Markush
groups used in the appended claims.
[0311] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as molecular weight,
reaction conditions, and so forth used in the specification and
claims are to be understood as being modified in all instances by
the term "about." As used herein, the term "about" means that the
item, parameter or term so qualified encompasses a range of plus or
minus ten percent above and below the value of the stated item,
parameter or term. Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the specification and
attached claims are approximations that may vary depending upon the
desired properties sought to be obtained by the present invention.
At the very least, and not as an attempt to limit the application
of the doctrine of equivalents to the scope of the claims, each
numerical parameter should at least be construed in light of the
number of reported significant digits and by applying ordinary
rounding techniques. Notwithstanding that the numerical ranges and
parameters setting forth the broad scope of the invention are
approximations, the numerical values set forth in the specific
examples are reported as precisely as possible. Any numerical
value, however, inherently contains certain errors necessarily
resulting from the standard deviation found in their respective
testing measurements
[0312] The terms "a," "an," "the" and similar referents used in the
context of describing the invention (especially in the context of
the following claims) are to be construed to cover both the
singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. Recitation of ranges of values
herein is merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range. Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein is intended
merely to better illuminate the invention and does not pose a
limitation on the scope of the invention otherwise claimed. No
language in the specification should be construed as indicating any
non-claimed element essential to the practice of the invention.
[0313] Specific embodiments disclosed herein may be further limited
in the claims using consisting of or consisting essentially of
language. When used in the claims, whether as filed or added per
amendment, the transition term "consisting of" excludes any
element, step, or ingredient not specified in the claims. The
transition term "consisting essentially of" limits the scope of a
claim to the specified materials or steps and those that do not
materially affect the basic and novel characteristic(s).
Embodiments of the invention so claimed are inherently or expressly
described and enabled herein.
[0314] All patents, patent publications, and other publications
referenced and identified in the present specification are
individually and expressly incorporated herein by reference in
their entirety for the purpose of describing and disclosing, for
example, the compositions and 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.
Sequence CWU 1
1
29123PRTArtificial SequenceChemically synthesized RGD peptide 1Gly
Arg Gly Asp Ile Pro Ala Ser Ser Lys Gly Gly Gly Gly Ser Arg1 5 10
15Leu Leu Leu Leu Leu Leu Arg 20223PRTArtificial SequenceChemically
synthesized acylated RGD peptide 2Gly Arg Gly Asp Ile Pro Ala Ser
Ser Lys Gly Gly Gly Gly Ser Arg1 5 10 15Leu Leu Leu Leu Leu Leu Arg
20310PRTArtificial SequenceChemically synthesized spacer peptide
SGGGGKSSAP 3Ser Gly Gly Gly Gly Lys Ser Ser Ala Pro1 5
1046PRTArtificial SequenceChemically synthesized hydrophilic domain
4Lys Gln Ala Gly Asp Val1 555PRTArtificial SequenceChemically
synthesized hydrophilic domain 5Pro His Ser Arg Asn1
565PRTArtificial SequenceChemically synthesized hydrophilic domain
YIGSR 6Tyr Ile Gly Ser Arg1 579PRTArtificial SequenceChemically
synthesized hydrophilic domain CDPGYIGSR 7Cys Asp Pro Gly Tyr Ile
Gly Ser Arg1 585PRTArtificial SequenceChemically synthesized
hydrophilic domain IKVAV 8Ile Lys Val Ala Val1 5910PRTArtificial
SequenceChemically synthesized hydrophilic domain RNIAEIIKDI 9Arg
Asn Ile Ala Glu Ile Ile Lys Asp Ile1 5 10107PRTArtificial
SequenceChemically synthesized hydrophilic domain YFQRYLI 10Tyr Phe
Gln Arg Tyr Leu Ile1 5115PRTArtificial SequenceChemically
synthesized hydrophilic domain PDSGR 11Pro Asp Ser Gly Arg1
5127PRTArtificial SequenceChemically synthesized hydrophilic domain
FHRRIKA 12Phe His Arg Arg Ile Lys Ala1 5136PRTArtificial
SequenceChemically synthesized hydrophilic domain PRRARV 13Pro Arg
Arg Ala Arg Val1 5148PRTArtificial SequenceChemically synthesized
hydrophilic domain WQPPRARI 14Trp Gln Pro Pro Arg Ala Arg Ile1
5155PRTArtificial SequenceChemically synthesized hydrophobic/apolar
tail LLLLL 15Leu Leu Leu Leu Leu1 5165PRTArtificial
SequenceChemically synthesized hydrophobic/apolar tail LLFFL 16Leu
Leu Phe Phe Leu1 5175PRTArtificial SequenceChemically synthesized
hydrophobic/apolar tail LFLWL 17Leu Phe Leu Trp Leu1
5185PRTArtificial SequenceChemically synthesized hydrophobic/apolar
tail FLWLL 18Phe Leu Trp Leu Leu1 5195PRTArtificial
SequenceChemically synthesized hydrophobic/apolar tail LALGL 19Leu
Ala Leu Gly Leu1 5206PRTArtificial SequenceChemically synthesized
hydrophobic/apolar tail LLLLLL 20Leu Leu Leu Leu Leu Leu1
5217PRTArtificial SequenceChemically synthesized hydrophobic/apolar
tail RLLLLLR 21Arg Leu Leu Leu Leu Leu Arg1 5227PRTArtificial
SequenceChemically synthesized hydrophobic/apolar tail KLLLLLR
22Lys Leu Leu Leu Leu Leu Arg1 5237PRTArtificial SequenceChemically
synthesized hydrophobic/apolar tail KLLLLLK 23Lys Leu Leu Leu Leu
Leu Lys1 52414PRTArtificial SequenceChemically synthesized spacer
GSPGISGGGGGILE 24Gly Ser Pro Gly Ile Ser Gly Gly Gly Gly Gly Ile
Leu Glu1 5 102511PRTArtificial SequenceChemically synthesized
spacer SGGGGKSSAPI 25Ser Gly Gly Gly Gly Lys Ser Ser Ala Pro Ile1 5
102621PRTArtificial SequenceChemically synthesized spacer-tail
peptide GSPGISGGGGGILEKLLLLLK 26Gly Ser Pro Gly Ile Ser Gly Gly Gly
Gly Gly Ile Leu Glu Lys Leu1 5 10 15Leu Leu Leu Leu Lys
202722PRTArtificial SequenceChemically synthesized spacer-tail
peptide GSPGISGGGGGILEKLALWLLR 27Gly Ser Pro Gly Ile Ser Gly Gly
Gly Gly Gly Ile Leu Glu Lys Leu1 5 10 15Ala Leu Trp Leu Leu Arg
202820PRTArtificial SequenceChemically synthesized spacer-tail
peptide GSPGISGGGGGILERLLLLR 28Gly Ser Pro Gly Ile Ser Gly Gly Gly
Gly Gly Ile Leu Glu Arg Leu1 5 10 15Leu Leu Leu Arg
202921PRTArtificial SequenceChemically synthesized spacer-tail
peptide GSPGISGGGGGILERLLWLLR 29Gly Ser Pro Gly Ile Ser Gly Gly Gly
Gly Gly Ile Leu Glu Arg Leu1 5 10 15Leu Trp Leu Leu Arg 20
* * * * *