U.S. patent application number 13/783485 was filed with the patent office on 2013-07-11 for silk-based drug delivery system.
This patent application is currently assigned to EIDGENOSSISCHES TECHNISCHE HOCHSCHULE (THE SWISS FEDERAL INSTITUTE OF TECHNOLOGY). The applicant listed for this patent is Eidgenossisches Techinsche Hochschule (The Swiss Federal Institute of Technology), TRUSTEES OF TUFTS COLLEGE. Invention is credited to David L. Kaplan, Lorenz Meinel.
Application Number | 20130177611 13/783485 |
Document ID | / |
Family ID | 35510265 |
Filed Date | 2013-07-11 |
United States Patent
Application |
20130177611 |
Kind Code |
A1 |
Kaplan; David L. ; et
al. |
July 11, 2013 |
SILK-BASED DRUG DELIVERY SYSTEM
Abstract
The present invention provides for novel sustained release
silk-based delivery systems. The invention further provides methods
for producing such formulations. In general, a silk fibroin
solution is combined with a therapeutic agent to form a silk
fibroin article. The article is then treated in such a way as to
alter its conformation. The change in conformation increases its
crystallinity or liquid crystallinity, thus controlling the release
of a therapeutic agent from the formulation. This can be
accomplished as single material carriers or in a layer-by-layer
fashion to load different therapeutic agents or different
concentrations of these agents in each layer.
Inventors: |
Kaplan; David L.; (Concord,
MA) ; Meinel; Lorenz; (Kassel, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TRUSTEES OF TUFTS COLLEGE;
Institute of Technology); Eidgenossisches Techinsche Hochschule
(The Swiss Federal |
Medford
Zurich |
MA |
US
CH |
|
|
Assignee: |
EIDGENOSSISCHES TECHNISCHE
HOCHSCHULE (THE SWISS FEDERAL INSTITUTE OF TECHNOLOGY)
Zurich
MA
TRUSTEES OF TUFTS COLLEGE
Medford
|
Family ID: |
35510265 |
Appl. No.: |
13/783485 |
Filed: |
March 4, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13443264 |
Apr 10, 2012 |
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13783485 |
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11628930 |
Oct 23, 2007 |
8178656 |
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PCT/US05/20844 |
Jun 13, 2005 |
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13443264 |
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60579065 |
Jun 11, 2004 |
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Current U.S.
Class: |
424/400 ;
424/130.1; 514/1.1; 514/44R; 514/773 |
Current CPC
Class: |
A61K 9/7007 20130101;
A61K 9/0002 20130101; A61K 47/46 20130101; A61K 9/209 20130101;
A61K 31/00 20130101; A61K 47/42 20130101 |
Class at
Publication: |
424/400 ;
514/773; 514/1.1; 514/44.R; 424/130.1 |
International
Class: |
A61K 47/42 20060101
A61K047/42; A61K 9/00 20060101 A61K009/00 |
Claims
1. A composition comprising at least one therapeutic agent
distributed in a silk fibroin article, wherein crystallinity
(beta-sheet) or liquid crystallinity of the silk fibroin article is
modified to release said at least one therapeutic agent over at
least about 12 hours.
2. The composition of claim 1, wherein the crystallinity
(beta-sheet) of the silk fibroin article is modified to release
said at least one therapeutic agent over at least about 24
hours.
3. The composition of claim 1, wherein the crystallinity
(beta-sheet) of the silk fibroin article is modified to release
said at least one therapeutic agent over a period of about 2 to 90
days.
4. The composition of claim 1, wherein the crystallinity
(beta-sheet) of the silk fibroin article is modified to release
said at least one therapeutic agent over a period of about 3 to 60
days.
5. The composition of claim 1, wherein the crystallinity
(beta-sheet) of the silk fibroin article is modified to release
said at least one therapeutic agent over a period of about 3 to 10
days.
6. The composition of claim 1, wherein the crystallinity
(beta-sheet) of the silk fibroin article is modified to reduce
initial burst of said at least one therapeutic agent.
7. The composition of claim 1, wherein the crystallinity
(beta-sheet) of the silk fibroin article is modified to release
said at least one therapeutic agent in an amount of about 1 ng/day
to about 1 mg/day.
8. The composition of claim 1, wherein the release of said at least
one therapeutic agent is sustained over the period of time.
9. The composition of claim 1, wherein said at least one
therapeutic agent is equal to or greater than 10 kilodaltons.
10. The composition of claim 9, wherein said at least one
therapeutic agent is selected from the group consisting of a
protein, a peptide, nucleic acid, PNA, aptamer, antibody, small
molecule, and any combinations thereof.
11. The composition of claim 1, wherein the silk fibroin article is
selected from the group consisting of a thread, fiber, foam, mesh,
hydrogel, three-dimensional scaffold, tablet filling material,
tablet coating, microsphere, and any combinations thereof.
12. The composition of claim 1, wherein the silk fibroin article is
biodegradable.
13. The composition of claim 1, wherein the silk fibroin article
further comprises a biocompatible polymer.
14. The composition of claim 1, wherein the silk fibroin article
comprises at least two layers, said at least one layer having
crystallinity or liquid crystallinity different from that of at
least one other layer.
15. The composition of claim 14, further comprising a targeting
agent that specifically targets the device to a specific cell or
tissue type.
16. A method comprising: i. forming a silk fibroin article
comprising a therapeutic agent; and ii. contacting said article
with water vapor at a pre-determined temperature, thereby inducing
crystallinity of the silk fibroin to modify release of said
therapeutic agent from said article.
17. The method of claim 16, wherein the crystallinity of the silk
fibroin is induced by water vapor to modify release of said
therapeutic agent from said article over at least about 12
hours.
18. The method of claim 16, wherein the crystallinity of the silk
fibroin is induced by water vapor to modify release of said
therapeutic agent from said article over at least about 24
hours.
19. The method of claim 16, wherein the crystallinity of the silk
fibroin is induced by water vapor to modify release of said
therapeutic agent from said article over a period of about 2 to 90
days.
20. The method of claim 16, wherein the pre-determined temperature
is about 25.degree. C.
21. The method of claim 16, further comprising subjecting said
article to a shear stress, an electric field, pressure, salt, or
any combinations thereof.
22. The method of claim 16, wherein the silk fibroin article
comprises a film.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 13/443,264 filed on Apr. 10, 2012,
which is a continuation application of U.S. patent application Ser.
No. 11/628,930 filed on Oct. 23, 2007 and issued as U.S. Pat. No.
8,178,656 on May 15, 2012, which is a 35 U.S.C. .sctn.371 U.S.
National Stage Entry of International Application No.
PCT/US2005/020844 filed on Jun. 13, 2005, which designated the
U.S., and which claims the benefit under 35 U.S.C. .sctn.119(e) of
U.S. Provisional Application No. 60/579,065 filed on Jun. 11, 2004,
the contents of each of which are hereby incorporated by reference
in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates generally to a silk-based drug
delivery system. In particular, the system is capable of releasing
a therapeutic agent from the device at a sustained and controllable
rate.
BACKGROUND OF THE INVENTION
[0003] Silk, as the term is generally known in the art, means a
filamentous fiber product secreted by an organism such as a
silkworm or spider. Silks produced from insects, namely (i) Bombyx
mori silkworms, and (ii) the glands of spiders, typically Nephilia
clavipes, are the most often studied forms of the material;
however, hundreds to thousands of natural variants of silk exist in
nature. Fibroin is produced and secreted by a silkworm's two silk
glands.
[0004] Silkworm silk has been used in biomedical applications for
over 1,000 years. The Bombyx mori specie of silkworm produces a
silk fiber (known as a "bave") and uses the fiber to build its
cocoon. The bave, as produced, includes two fibroin filaments or
"broins", which are surrounded with a coating of gum, known as
sericin--the silk fibroin filament possesses significant mechanical
integrity. When silk fibers are harvested for producing yarns or
textiles, including sutures, a plurality of fibers can be aligned
together, and the sericin is partially dissolved and then
resolidified to create a larger silk fiber structure having more
than two broins mutually embedded in a sericin coating.
[0005] The unique mechanical properties of reprocessed silk such as
fibroin and its biocompatibility make the silk fibers especially
attractive for use in biotechnological materials and medical
applications. Silk provides an important set of material options
for biomaterials and tissue engineering because of the impressive
mechanical properties, biocompatibility and biodegradability
(Altman, G. H., et al., Biomaterials 2003, 24, 401-416; Cappello,
J., et al., J. Control. Release 1998, 53, 105-117; Foo, C. W. P.,
et al., Adv. Drug Deliver. Rev. 2002, 54, 1131-1143; Dinerman, A.
A., et al., J. Control. Release 2002, 82, 277-287; Megeed, Z., et
al., Adv. Drug Deliver. Rev. 2002, 54, 1075-1091; Petrini, P., et
al., J. Mater. Sci-Mater. M. 2001, 12, 849-853; Altman, G. H., et
al., Biomaterials 2002, 23, 4131-4141; Panilaitis, B., et al.,
Biomaterials 2003, 24, 3079-3085). For example, 3-dimensional
porous silk scaffolds have been described for use in tissue
engineering (Meinel et al., Ann Biomed Eng. 2004 January;
32(1):112-22; Nazarov, R., et al., Biomacromolecules in press).
Further, regenerated silk fibroin films have been explored as
oxygen- and drug-permeable membranes, supports for enzyme
immobilization, and substrates for cell culture (Minoura, N., et
al., Polymer 1990, 31, 265-269; Chen, J., et al., Minoura, N.,
Tanioka, A. 1994, 35, 2853-2856; Tsukada, M., et al., Polym. Sci.
Part B Polym. Physics 1994, 32, 961-968).
[0006] The desirability of sustained release has long been
recognized in the pharmaceutical field. Sustained-release
drug-delivery systems can provide many benefits over conventional
dosage forms. Generally, sustained-release preparations provide a
longer period of therapeutic or prophylactic response compared to
conventional rapid release dosage forms. For example, in treatment
of pain, sustained-release formulations are useful to maintain
relatively constant analgesic drug release rates over a period of
time, for example 12-24 hours, so that blood serum concentration of
the drug remains at a therapeutically effective level for a longer
duration than is possible with a conventional dosage form of the
drug. In addition, whereas standard dosage forms typically exhibit
high initial drug release rates that can result in unnecessarily
elevated blood serum levels of the drug, sustained-release
formulations can help maintain blood serum levels of the drug at or
slightly above the therapeutically effective threshold. Such
reduced fluctuation in blood serum concentration of the drug can
also help prevent excess dosing.
[0007] Furthermore, sustained-release compositions, by optimizing
the kinetics of delivery, also increase patient compliance as
patients are less likely to miss a dose with less frequent
administration, particularly when a once-a-day dosage regimen is
possible; less frequent administration also increases patient
convenience. Sustained-release formulations may also reduce overall
healthcare costs. Although the initial cost of sustained-release
delivery systems may be greater than the costs associated with
conventional delivery systems, average costs of extended treatment
over time can be lower due to less frequent dosing, enhanced
therapeutic benefit, reduced side-effects, and a reduction in the
time required to dispense and administer the drug and monitor
patient compliance.
[0008] Many polymer-based systems have been proposed to accomplish
the goal of sustained release. These systems generally have relied
upon either degradation of the polymer or diffusion through the
polymer as a means to control release.
[0009] Polymer-based attempts to develop sustained-release
formulations have included the use of a variety of biodegradable
and non-biodegradable polymer (e.g. poly(lactide-co-glycolide))
microparticles containing the active ingredient (see e.g., Wise et
al., Contracgption, 1:227-234 (1973); and Hutchinson et al.,
Biochem. Soc. Trans., 13:520-523 (1985)), and a variety of
techniques are known by which active agents, e.g. proteins, can be
incorporated into polymeric microspheres (see e.g., U.S. Pat. No.
4,675,189 and references cited therein). In addition, various
microcapsules, microparticles, and larger sustained-release
implants have been used to deliver pharmaceuticals to patients over
an extended period of time. For example, polyesters such as
poly-DL-lactic acid, polyglycolic acid, polylactide, and other
copolymers, have been used to release biologically active molecules
such as progesterone and luteinizing hormone-releasing hormone
(LH-RH) analogs, e.g., as described in Kent et al., U.S. Pat. No.
4,675,189, and Hutchinson et al., U.S. Pat. No. 4,767,628.
[0010] Unfortunately, the successes of current polymer-based
sustained delivery systems have been limited. This is due, in large
part, to their necessity on using organic solvents during
preparation. Even solvents which are well tolerated in vivo, i.e.
ethylacetate, may cause immunological reactions or anaphylactic
shock. In addition, all organic solvents are volatile and require
expensive production processes.
[0011] There is, therefore, a need for a biocompatible,
biodegradable, sustained-release drug-delivery system. Such
products should have the desired mechanical properties of tensile
strength, elasticity, formability, and the like, provide for
controlled resorption, and be physiologically acceptable. Moreover,
such products should allow for ease of administration for a variety
of in vivo indications and in best-case scenarios be inexpensive to
manufacture.
SUMMARY OF THE INVENTION
[0012] The present invention provides a novel sustained release
silk-based drug delivery system. The invention further provides
methods for producing such devices.
[0013] In one embodiment, a method for producing a pharmaceutical
formulation for controlled release of a therapeutic agent is
provided. The method comprises contacting a silk fibroin solution
with the therapeutic agent. Therapeutic agents include, for
example, proteins, peptides and small molecules. In a preferred
embodiment, an aqueous silk fibroin solution is utilized.
[0014] Next, a silk fibroin article that contains the therapeutic
agent is formed. The silk fibroin article may be a thread, fiber,
film, foam, mesh, hydrogel, three-dimensional scaffold, tablet
filling material, tablet coating, or microsphere.
[0015] The conformation of the article is then altered in order to
increase its crystallinity or liquid crystlallinity, thus providing
controlled release of the therapeutic agent from the silk fibroin
article.
[0016] In one embodiment of the present invention, the conformation
of the article is altered by contacting the fibroin article with
methanol. The methanol concentration is at least 50%, at least 70%,
at least 90% or at least 100%.
[0017] In an alternative embodiment, alteration in the conformation
of the fibroin article is induced by treating the article with
sheer stress. The sheer stress may be applied by passing the
article through a needle.
[0018] The conformation of the fibroin article may also be altered
by contacting the article with an electric field, by applying
pressure, or by contacting the article with salt.
[0019] Preferably, the therapeutic agent is equal to or greater
than about 10 kilodaltons (kDa). More preferably the therapeutic
agent is greater than about 20 kDa.
[0020] In a further embodiment, a pharmaceutical formulation with a
plurality of silk fibroin articles (i.e. layers) is provided. In
this embodiment, at least one layer has an induced conformational
change that differs from at least one other layer. The silk fibroin
article layers may each contain different therapeutic agents, each
layer having the same or different induced conformational
changes.
[0021] The pharmaceutical formulation is biodegradable and may
comprise a targeting agent that specifically targets the device to
a specific cell or tissue type. The targeting agent may be, for
example, a sugar, peptide, or fatty acid.
[0022] In one embodiment, the silk fibroin solution is obtained
from a solution containing a dissolved silkworm silk, such as, for
example, from Bombyx mori. Alternatively, the silk fibroin solution
is obtained from a solution containing a dissolved spider silk,
such as, for example, from Nephila clavipes. The silk fibroin
solution may also be obtained from a solution containing a
genetically engineered silk. In one embodiment, the genetically
engineered silk comprises a therapeutic agent. This may be a fusion
protein with a cytokine, an enzyme, or any number of hormones or
peptide-based drugs, antimicrobials and related substrates.
[0023] Also encompassed in the present invention is the
pharmaceutical formulation for controlled release of a therapeutic
agent, produced by the above methods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate embodiments of
the invention and, together with the description, serve to explain
the objects, advantages, and principles of the invention.
[0025] FIG. 1 shows the drug release of FD4 after 24, 48, and 130
hours.
[0026] FIG. 2 shows the influence of methanol concentration on drug
release. Treatment with methanol concentrations up to 50% results
in a high burst release within the first 12 hours with minute
amounts of FD4 released at later time points. In contrast,
treatment with 90% or 100% methanol solutions results in a
sustained and faster release for about 200 hours.
[0027] FIG. 3 shows coating of core with silk fibroin solution
treated with different concentrations of methanol (I) core without
coating; (d) core with coating treated with 90% methanol solution;
(c) core with two layers of fibroin; and (b) core with three layers
of fibroin. The results demonstrate that the release of a
therapeutic can be controlled through the thickness of the coating
around the core.
[0028] FIG. 4 shows release of FITC-dextrans with different
molecular weights and from silk films treated with H2O (4A) or
methanol (4B).
[0029] FIG. 5 shows cumulative release and adsorption of
horseradish peroxidase (HRP; 5A, 5C) and Lysozyme (Lys, 5B, 5D)
from silk films treated with methanol or H2O, respectively.
[0030] FIG. 6 shows AFM images of native silk films (6A, 6C) or
films treated with methanol (6B, 6D). Bar length 2.5 .mu.m (6A, 6B)
and 0.5 .mu.m (6B, 6D).
[0031] FIG. 7 shows physicochemical characterization of native silk
films or films treated with methanol. (7A) FTIR analysis and X-ray
diffractogramm of methanol treated (7B) and untreated (7C) films
Contact angle measurements of a water drop on methanol treated (7D)
and untreated (7E) films over time.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Methods for preparation of silk-based drug delivery systems
are described. In particular, the drug delivery system allows for
the controlled and sustained release of therapeutic agents in vivo.
In general, a silk fibroin solution is combined with a therapeutic
agent to form a silk fibroin article. The article is then treated
in such a way as to alter its conformation. The change in
conformation increases its crytallinity, thus controlling the
release of a therapeutic agent from the formulation.
[0033] As used herein, the term "fibroin" includes silkworm fibroin
and insect or spider silk protein (Lucas et al., Adv. Protein Chem
13: 107-242 (1958)). Preferably, fibroin is obtained from a
solution containing a dissolved silkworm silk or spider silk. The
silkworm silk protein is obtained, for example, from Bombyx mori,
and the spider silk is obtained from Nephila clavipes. In the
alternative, the silk proteins suitable for use in the present
invention can be obtained from a solution containing a genetically
engineered silk, such as from bacteria, yeast, mammalian cells,
transgenic animals or transgenic plants. See, for example, WO
97/08315 and U.S. Pat. No. 5,245,012.
[0034] The silk fibroin solution can be prepared by any
conventional method known to one skilled in the art. For example,
B. mori cocoons are boiled for about 30 minutes in an aqueous
solution. Preferably, the aqueous solution is about 0.02M
Na.sub.2CO.sub.3. The cocoons are rinsed, for example, with water
to extract the sericin proteins and the extracted silk is dissolved
in an aqueous salt solution. Salts useful for this purpose include
lithium bromide, lithium thiocyanate, calcium nitrate or other
chemicals capable of solubilizing silk. Preferably, the extracted
silk is dissolved in about 9-12 M LiBr solution. The salt is
consequently removed using, for example, dialysis.
[0035] If necessary, the solution can then be concentrated using,
for example, dialysis against a hygroscopic polymer, for example,
PEG, a polyethylene oxide, amylose or sericin.
[0036] Preferably, the PEG is of a molecular weight of 8,000-10,000
g/mol and has a concentration of 25-50%. A slide-a-lyzer dialysis
cassette (Pierce, MW CO 3500) is preferably used. However, any
dialysis system may be used. The dialysis is for a time period
sufficient to result in a final concentration of aqueous silk
solution between 10-30%. In most cases dialysis for 2-12 hours is
sufficient. See, for example, PCT application PCT/US/04/11199.
[0037] Alternatively, the silk fibroin solution can be produced
using organic solvents. Such methods have been described, for
example, in Li, M., et al., J. Appl. Poly Sci. 2001, 79, 2192-2199;
Min, S., et al. Sen'I Gakkaishi 1997, 54, 85-92; Nazarov, R. et
al., Biomacromolecules 2004 May-June; 5(3):718-26.
[0038] In accordance with the present invention, the silk fibroin
solutions contain at least one therapeutic agent. The silk fibroin
solution is contacted with a therapeutic agent prior to forming the
fibroin article, e.g. a fiber, mesh, scaffold, or loaded into the
article after it is formed. For loading after formation, silk
assembly is used to control hydrophilic/hydrophobic partitioning
(see, for example, Jin et al., Nature. 2003 Aug. 28;
424(6952):1057-61) and the adsorption of phase separation of the
therapeutic agent. The material can also be loaded by entrapping
the therapeutic agent in the silk by inducing the transition to the
beta sheet (e.g. methanol, shear, salts, electric) and adding
layers on this with each layer entrapping the next therapeutic.
This layer-by-layer approach would allow onion like structures with
selective loading in each layer.
[0039] The variety of different therapeutic agents that can be used
in conjunction with the formulations of the present invention is
vast and includes small molecules, proteins, peptides and nucleic
acids. In general, therapeutic agents which may be administered via
the invention include, without limitation: anti-infectives such as
antibiotics and antiviral agents; chemotherapeutic agents (i.e.
anticancer agents); anti-rejection agents; analgesics and analgesic
combinations; anti-inflammatory agents; hormones such as steroids;
growth factors (bone morphogenic proteins (i.e. BMP's 1-7), bone
morphogenic-like proteins (i.e. GFD-5, GFD-7 and GFD-8), epidermal
growth factor (EGF), fibroblast growth factor (i.e. FGF 1-9),
platelet derived growth factor (PDGF), insulin like growth factor
(IGF-I and IGF-II), transforming growth factors (i.e.
TGF-.beta.-III), vascular endothelial growth factor (VEGF));
anti-angiogenic proteins such as endostatin, and other naturally
derived or genetically engineered proteins, polysaccharides,
glycoproteins, or lipoproteins. Growth factors are described in The
Cellular and Molecular Basis of Bone Formation and Repair by Vicki
Rosen and R. Scott Thies, published by R. G. Landes Company, hereby
incorporated herein by reference.
[0040] Additionally, the silk based devices of the present
invention can be used to deliver any type of molecular compound,
such as, pharmacological materials, vitamins, sedatives, steroids,
hypnotics, antibiotics, chemotherapeutic agents, prostaglandins,
and radiopharmaceuticals. The delivery system of the present
invention is suitable for delivery of the above materials and
others including but not limited to proteins, peptides,
nucleotides, carbohydrates, simple sugars, cells, genes,
anti-thrombotics, anti-metabolics, growth factor inhibitor, growth
promoters, anticoagulants, antimitotics, fibrinolytics,
anti-inflammatory steroids, and monoclonal antibodies.
[0041] Additionally, the pharmaceutical formulation of the present
invention may also have a targeting ligand. Targeting ligand refers
to any material or substance which may promote targeting of the
pharmaceutical formulation to tissues and/or receptors in vivo
and/or in vitro with the formulations of the present invention. The
targeting ligand may be synthetic, semi-synthetic, or
naturally-occurring. Materials or substances which may serve as
targeting ligands include, for example, proteins, including
antibodies, antibody fragments, hormones, hormone analogues,
glycoproteins and lectins, peptides, polypeptides, amino acids,
sugars, saccharides, including monosaccharides and polysaccharides,
carbohydrates, vitamins, steroids, steroid analogs, hormones,
cofactors, and genetic material, including nucleosides,
nucleotides, nucleotide acid constructs, petptide nucleic acids
(PNA), aptamers, and polynucleotides. Other targeting ligands in
the present invention include cell adhesion molecules (CAM), among
which are, for example, cytokines, integrins, cadherins,
immunoglobulins and selectin. The pharmaceutical formulations of
the present invention may also encompass precursor targeting
ligands. A precursor to a targeting ligand refers to any material
or substance which may be converted to a targeting ligand. Such
conversion may involve, for example, anchoring a precursor to a
targeting ligand. Exemplary targeting precursor moieties include
maleimide groups, disulfide groups, such as ortho-pyridyl
disulfide, vinylsulfone groups, azide groups, and [agr]-iodo acetyl
groups.
[0042] Silk formulations containing bioactive materials may be
formulated by mixing one or more therapeutic agents with the silk
solution used to make the article. Alternatively, a therapeutic
agent can be coated onto the pre-formed silk fibroin article,
preferably with a pharmaceutically acceptable carrier. Any
pharmaceutical carrier can be used that does not dissolve the silk
material. The therapeutic agents may be present as a liquid, a
finely divided solid, or any other appropriate physical form.
[0043] The above described silk fibroin solution, which contains at
least one therapeutic agent, is next processed into a thread,
fiber, film, mesh, hydrogel, three-dimensional scaffold, tablet
filling material, tablet coating, or microsphere. Methods for
generating such are well known in the art. See, e.g. Altman, et
al., Biomaterials 24:401, 2003; PCT Publications, WO 2004/000915
and WO 2004/001103; and PCT Application No's PCT/US/04/11199 and
PCT/US04/00255, which are herein incorporated by reference.
[0044] Silk films can be produced by preparing the concentrated
aqueous silk fibroin solution and casting the solution. See, for
example PCT application PCT/US/04/11199. The film can be contacted
with water or water vapor, in the absence of alcohol. The film can
then be drawn or stretched mono-axially or biaxially. The
stretching of a silk blend film induces molecular alignment of the
film and thereby improves the mechanical properties of the
film.
[0045] If desired, the film comprises from about 50 to about 99.99
part by volume aqueous silk protein solution and from about 0.01 to
about 50 part by volume biocompatible polymer e.g., polyethylene
oxide (PEO). Preferably, the resulting silk blend film is from
about 60 to about 240 .mu.m thick, however, thicker samples can
easily be formed by using larger volumes or by depositing multiple
layers.
[0046] Foams may be made from methods known in the art, including,
for example, freeze--drying and gas foaming in which water is the
solvent or nitrogen or other gas is the blowing agent,
respectively. Alternately, the foam is made by contacting the silk
fibroin solution with granular salt. The pore size of foams can be
controlled, for example by adjusting the concentration of silk
fibroin and the particle size of a granular salt (for example, the
preferred diameter of the salt particle is between about 50 microns
and about 1000 microns). The salts can be monovalent or divalent.
Preferred salts are monovalent, such as NaCl and KCl. Divalent
salts, such as CaCl.sub.2 can also be used. Contacting the
concentrated silk fibroin solution with salt is sufficient to
induce a conformational change of the amorphous silk to a-sheet
structure that is insoluble in the solution. After formation of the
foam, the excess salt is then extracted, for example, by immersing
in water. The resultant porous foam can then be dried and the foam
can be used, for example, as a cell scaffold in biomedical
application. See, for example PCT application PCT/US/04/11199.
[0047] In one embodiment, the foam is micropatterned foam.
Micropatterned foams can be prepared using, for example, the method
set forth in U.S. Pat. No. 6,423,252, the disclosure of which is
incorporated herein by reference. The method comprises contacting
the concentrated silk solution with a surface of a mold, the mold
comprising on at least one surface thereof a three-dimensional
negative configuration of a predetermined micropattern to be
disposed on and integral with at least one surface of the foam,
lyophilizing the solution while in contact with the micropatterned
surface of the mold, thereby providing a lyophilized,
micropatterned foam, and removing the lyophilized, micropatterned
foam from the mold. Foams prepared according to this method
comprise a predetermined and designed micropattern on at least one
surface, which pattern is effective to facilitate tissue repair,
ingrowth or regeneration.
[0048] Fibers may be produced using, for example, wet spinning or
electrospinning Alternatively, as the concentrated solution has a
gel-like consistency, a fiber can be pulled directly from the
solution.
[0049] Electrospinning can be performed by any means known in the
art (see, for example, U.S. Pat. No. 6,110,590). Preferably, a
steel capillary tube with a 1.0 mm internal diameter tip is mounted
on an adjustable, electrically insulated stand. Preferably, the
capillary tube is maintained at a high electric potential and
mounted in the parallel plate geometry. The capillary tube is
preferably connected to a syringe filled with silk solution.
Preferably, a constant volume flow rate is maintained using a
syringe pump, set to keep the solution at the tip of the tube
without dripping. The electric potential, solution flow rate, and
the distance between the capillary tip and the collection screen
are adjusted so that a stable jet is obtained. Dry or wet fibers
are collected by varying the distance between the capillary tip and
the collection screen.
[0050] A collection screen suitable for collecting silk fibers can
be a wire mesh, a polymeric mesh, or a water bath. Alternatively
and preferably, the collection screen is an aluminum foil. The
aluminum foil can be coated with Teflon fluid to make peeling off
the silk fibers easier. One skilled in the art will be able to
readily select other means of collecting the fiber solution as it
travels through the electric field. The electric potential
difference between the capillary tip and the aluminum foil counter
electrode is, preferably, gradually increased to about 12 kV,
however, one skilled in the art should be able to adjust the
electric potential to achieve suitable jet stream.
[0051] The present invention additionally provides a non-woven
network of fibers comprising a pharmaceutical formulation of the
present invention. The fiber may also be formed into yarns and
fabrics including for example, woven or weaved fabrics.
[0052] The fibroin silk article of the present invention may also
be coated onto various shaped articles including biomedical devices
(e.g. stents), and silk or other fibers, including fragments of
such fibers.
[0053] Silk hydrogels can be prepared by methods known in the art,
see for example PCT application PCT/US/04/11199. The sol-gel
transition of the concentrated silk fibroin solution can be
modified by changes in silk fibroin concentration, temperature,
salt concentrations (e.g. CaCl.sub.2, NaCl, and KCl), pH,
hydrophilic polymers, and the like. Before the sol-gel transition,
the concentrated aqueous silk solution can be placed in a mold or
form. The resulting hydrogel can then be cut into any shape, using,
for example a laser.
[0054] The silk fibroin articles described herein can be further
modified after fabrication. For example, the scaffolds can be
coated with additives, such as bioactive substances that function
as receptors or chemoattractors for a desired population of cells.
The coating can be applied through absorption or chemical
bonding.
[0055] Additives suitable for use with the present invention
include biologically or pharmaceutically active compounds. Examples
of biologically active compounds include, but are not limited to:
cell attachment mediators, such as collagen, elastin, fibronectin,
vitronectin, laminin, proteoglycans, or peptides containing known
integrin binding domains e.g. "RGD" integrin binding sequence, or
variations thereof, that are known to affect cellular attachment
(Schaffner P & Dard 2003 Cell Mol Life Sci. January;
60(1):119-32; Hersel U. et al. 2003 Biomaterials. November;
24(24):4385-415); biologically active ligands; and substances that
enhance or exclude particular varieties of cellular or tissue
ingrowth. Other examples of additive agents that enhance
proliferation or differentiation include, but are not limited to,
osteoinductive substances, such as bone morphogenic proteins (BMP);
cytokines, growth factors such as epidermal growth factor (EGF),
platelet-derived growth factor (PDGF), insulin-like growth factor
(IGF-I and II) TGF- and the like. As used herein, the term additive
also encompasses antibodies, DNA, RNA, modified RNA/protein
composites, glycogens or other sugars, and alcohols.
[0056] Biocompatible polymers can be added to the silk article to
generate composite matrices in the process of the present
invention.
[0057] Biocompatible polymers useful in the present invention
include, for example, polyethylene oxide (PEO) (U.S. Pat. No.
6,302,848), polyethylene glycol (PEG) (U.S. Pat. No. 6,395,734),
collagen (U.S. Pat. No. 6,127,143), fibronectin (U.S. Pat. No.
5,263,992), keratin (U.S. Pat. No. 6,379,690), polyaspartic acid
(U.S. Pat. No. 5,015,476), polylysine (U.S. Pat. No. 4,806,355),
alginate (U.S. Pat. No. 6,372,244), chitosan (U.S. Pat. No.
6,310,188), chitin (U.S. Pat. No. 5,093,489), hyaluronic acid (U.S.
Pat. No. 387,413), pectin (U.S. Pat. No. 6,325,810),
polycaprolactone (U.S. Pat. No. 6,337,198), polylactic acid (U.S.
Pat. No. 6,267,776), polyglycolic acid (U.S. Pat. No. 5,576,881),
polyhydroxyalkanoates (U.S. Pat. No. 6,245,537), dextrans (U.S.
Pat. No. 5,902,800), and polyanhydrides (U.S. Pat. No. 5,270,419).
Two or more biocompatible polymers can be used.
[0058] As a next step in the method for producing pharmaceutical
formulations for controlled release of therapeutic agents, the
conformation of the silk fibroin article is altered. The induced
conformational change alters the crystallinity of the article, thus
altering the rate of release of the therapeutic agent from the silk
fibroin article. The conformational change may be induced by
treating the fibroin article with methanol. The methanol
concentration is at least 50%, at least 70%, at least 90% or at
least 100%.
[0059] Alternatively, the alteration in the conformation of the
fibroin article may be induced by treating the article with sheer
stress. The sheer stress may be applied, for example, by passing
the article through a needle. Other methods of inducing
conformational changes include contacting the article with an
electric field, salt or by applying pressure.
[0060] The silk-based drug delivery system of the present invention
may comprise a plurality (i.e. layers) of silk fibroin articles,
where at least one silk fibroin article may have an induced
conformational change that differs from at least one alternative
silk fibroin article. For example, each layer may have different
solutions of fibroin (concentrations, drugs) and have different
conformational changes. These can be combined in various sequences
to create `onion-like` structures such that the delivery vehicle
will offer changing rates of release of each layer depending on
crystallinity, thickness, concentration of drug, type of drug, etc.
This approach is very amenable to scale up and combinatorial or
related approaches to formulation to create multiple control points
in release profiles and drug combinations.
[0061] Additionally, the release of the therapeutic agent from the
pharmaceutical formulations of the present invention can be
controlled through the thickness of the silk fibroin article. As
shown in FIG. 3, with increasing article thickness, the initial
burst and amount of drug released within the first 100 hours was
reduced. However, the sustained release of drug over time was
significantly higher with increasing film numbers.
[0062] Drug delivery composites are also encompassed. A family of
such structures are prepared as above and then dispersed in various
amounts into the fibroin hydrogels. These composite systems would
then be used in various modes of delivery, such as, for example,
the "onion-like" vehicles described above.
[0063] The materials produced using the present invention, e.g.,
hydrogels, fibers, films, foams, or meshes, may be used in a
variety of medical applications such as a drug (e.g, small
molecule, protein, or nucleic acid) delivery device, including
controlled release systems.
[0064] Controlled release permits dosages to be administered over
time, with controlled release kinetics. In some instances, delivery
of the therapeutic agent is continuous to the site where treatment
is needed, for example, over several weeks. Controlled release over
time, for example, over several days or weeks, or longer, permits
continuous delivery of the therapeutic agent to obtain optimal
treatment. The controlled delivery vehicle is advantageous because
it protects the therapeutic agent from degradation in vivo in body
fluids and tissue, for example, by proteases.
[0065] Controlled release from the pharmaceutical formulation may
be designed to occur over time, for example, for greater than about
12 or 24 hours. The time of release may be selected, for example,
to occur over a time period of about 12 hours to 24 hours; about 12
hours to 42 hours; or, e.g., about 12 to 72 hours. In another
embodiment, release may occur for example on the order of about 2
to 90 days, for example, about 3 to 60 days. In one embodiment, the
therapeutic agent is delivered locally over a time period of about
7-21 days, or about 3 to 10 days. In other instances, the
therapeutic agent is administered over 1,2,3 or more weeks in a
controlled dosage. The controlled release time may be selected
based on the condition treated. For example, longer times may be
more effective for wound healing, whereas shorter delivery times
may be more useful for some cardiovascular applications.
[0066] Controlled release of the therapeutic agent from the fibroin
article in vivo may occur, for example, in the amount of about 1 ng
to 1 mg/day, for example, about 50 ng to 500 pg/day, or, in one
embodiment, about 100 ng/day. Delivery systems comprising
therapeutic agent and a carrier may be formulated that include, for
example, 10 ng to 1 mg therapeutic agent, or in another embodiment,
about 1 ug to 500 ug, or, for example, about 10 ug to 100 ug,
depending on the therapeutic application.
[0067] The silk-based drug delivery vehicle may be administered by
a variety of routes known in the art including topical, oral,
parenteral (including intravenous, intraperitoneal, intramuscular
and subcutaneous injection as well as intranasal or inhalation
administration) and implantation. The delivery may be systemic,
regional, or local. Additionally, the delivery may be intrathecal,
e.g., for CNS delivery. For example, administration of the
pharmaceutical formulation for the treatment of wounds may be by
topical application, systemic administration by enteral or
parenteral routes, or local or regional injection or implantation.
The silk-based vehicle may be formulated into appropriate forms for
different routes of administration as described in the art, for
example, in"Remington: The Science and Practice of Pharmacy", Mack
Publishing Company, Pennsylvania, 1995, the disclosure of which is
incorporated herein by reference.
[0068] The controlled release vehicle may include excipients
available in the art, such as diluents, solvents, buffers,
solubilizers, suspending agents, viscosity controlling agents,
binders, lubricants, surfactants, preservatives and stabilizers.
The formulations may include bulking agents, chelating agents, and
antioxidants. Where parenteral formulations are used, the
formulation may additionally or alternately include sugars, amino
acids, or electrolytes.
[0069] Excipients include polyols, for example of a molecular
weight less than about 70,000 kD, such as trehalose, mannitol, and
polyethylene glycol. See for example, U.S. Pat. No. 5,589,167, the
disclosure of which is incorporated herein. Exemplary surfactants
include nonionic surfactants, such as Tweeng surfactants,
polysorbates, such as polysorbate 20 or 80, etc., and the
poloxamers, such as poloxamer 184 or 188, Pluronic (r) polyols, and
other ethylene/polypropylene block polymers, etc. Buffers include
Tris, citrate, succinate, acetate, or histidine buffers.
Preservatives include phenol, benzyl alcohol, metacresol, methyl
paraben, propyl paraben, benzalconium chloride, and benzethonium
chloride. Other additives include carboxymethylcellulose, dextran,
and gelatin. Stabilizing agents include heparin, pentosan
polysulfate and other heparinoids, and divalent cations such as
magnesium and zinc.
[0070] The pharmaceutical formulation of the present invention may
be sterilized using conventional sterilization process such as
radiation based sterilization (i.e. gamma-ray), chemical based
sterilization (ethylene oxide), autoclaving, or other appropriate
procedures. Preferably the sterilization process will be with
ethylene oxide at a temperature between 52-55.degree. C. for a time
of 8 or less hours. After sterilization the formulation may be
packaged in an appropriate sterilize moisture resistant package for
shipment.
[0071] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. Although methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of the invention, the preferred methods and materials
are described below. All publications, patent applications, patents
and other references mentioned herein are incorporated by
reference. In addition, the materials, methods and examples are
illustrative only and not intended to be limiting. In case of
conflict, the present specification, including definitions,
controls.
[0072] The invention will be further characterized by the following
examples which are intended to be exemplary of the invention.
EXAMPLES
Example I
Materials and Methods: Preparation of Silk
[0073] Silk cocoons were cut in quarters and washed in
Na.sub.2CO.sub.3 solution for 1 hour. The silk was washed 2 times
with hot water and 20 times with cold water. Silk was dried over
night and dissolved in 9M LiBr to become a 10% silk-fibroin
solution. The solution was centrifuged at 27,000 g for 30 minutes
and the supernatant was transferred in a dialysis cassette (MWCO
3,500) and dialyzed for 2 days. Silk-fibroin concentration was
adjusted to 5% (m/V in water) by evaporation at 250 mbar and
45.degree. C. The resulting silk solution was either mixed with a
solution of FITC coupled to dextran (molecular weight of 4 kDa;
FD4; concentration 10 mg/ml, Sigma) in a ratio of 150 ul
silk-fibroin solution to 20 ul of FD4 solution, or the FD-4 was
added later for control groups (see table 1). As a further group,
parts of this solution were sonicated with 50 Hz, 2A, 10 sec
(Hielscher, UP200H). 170 ul of the solution (FD-4/silk mixture) was
added into each well of a 96 well plate. For control, wells were
filled with 150 ul of the silk-fibroin solution. Water was
evaporated over night at room temperature and 250 mbar. 20 ul of
the FD4 solution was added to the wells, which received the
silk-fibroin solution only (to analyze the effect of mixing the
drug with the solved silk-fibroin vs. later incubation of the solid
films). Further wells were not incubated with silk (to analyze a
possible fluorescence of the silk-fibroin films themselves). The
films were either treated with water, methanol 20% or methanol 90%
(V/V) for 3 hours. The solutions were aspirated and replaced by 300
ul PBS for the drug release study. Total release medium was
replaced with fresh PBS after 24, 48, 130 hours and fluorescence
was read with a Lumicounter (Packard, 480V, Gain Level, 1, ex. 485
nm, em. 530 nm).
Results & Discussion:
[0074] Plain silk-fibroin films do not show fluorescence. Without
MeOH treatment or treatment with 20% MeOH, the release was
characterized by a high initial burst and only minute release after
24 hours (Groups I+0 and I+20), although more FD4 was released when
treated with 20% MeOH. This is probably due to the reduced
solubility of FD4 in presence of MeOH. When treated with 90% MeOH,
significantly more FD4 was released after 48 and after 130 hours,
demonstrating the feasibility to get a sustained release from silk
polymers by inducing a transformational change (I+90). Similar to
the observation with the groups I+0 and I+20, the increase in MeOH
concentration to 90% results in a higher encapsulation of total
FD4. An incubation of the prepared film with FD4 for 3 hours (I-90)
results in a high initial burst, similar to the I+0 and I+20. The
absence of a substantial sustained release is probably due to a
hindrance of drug diffusion in the (amorphous) silk-fibroin films.
Therefore, conformational changes do not substantially affect FD4
absorbed to the surface of the films Essentially the same results
were obtained for the groups, which were also treated with
ultrasonication. However, a treatment with ultrasonication does not
have an influence on drug release as compared to the non sonicated
group.
[0075] In conclusion, a sustained release of drugs (FD4) can be
obtained with silk-fibroin polymers, when a conformational change
is induced with 90% MeOH. Ultrasonication does not have an impact
on drug release.
Example 2
[0076] We evaluated the feasibility to formulate drug delivery
systems based on silk-fibroin. The experiments started from an
aqueous fibroin-drug solution, followed by a slow evaporation of
the water resulting in a solid fibroin film with suspended drug
molecules. A conformational change of the fibroin was induced
through methanol treatment, resulting in an increase of
crystallinity. The degree of crystallinity governed the release of
the drug.
Results & Discussion
[0077] Dextrans coupled to a fluorescent dye were chosen as a model
drug. They allow a straightforward assessment of the influence of
drug molecular weight on swelling gels. FD4--a term used in this
summary describes FITC (F) coupled to a dextran (D) with a
molecular weight of 4,000 g/mol. A first set of experiments
compared FD4 release from fibroin gels treated with water and
ascending methanol concentrations (FIG. 2).
[0078] FIG. 2 shows the release of FD4 over time. The differences
in maximum FD4 concentrations stems from different solubilities of
the drug in methanol solutions of different concentrations or
water. However, the release pattern was different. Treatment with
methanol concentrations up to 50% resulted in a high burst release
within the first 12 hours with minute amounts of FD4 released at
later time points. In contrast, treatment with 90% or 100% methanol
solutions resulted in a sustained and faster release for about 200
hours. At later time-points, the amount of drug released per time
(slope) significantly decreased, with the release continuing
throughout the observation period (exceeding 1,000 hours).
Treatment with shear stress alone (syringe treated, FIG. 2) also
induced gel formation through an increase of crystallinity.
[0079] To control the high initial burst-more than 30% of the total
FD4 used-, identical films were prepared and treated with 90%
methanol (core). This core was coated with additional layers of
fibroin and again treated with 90% methanol solution or water (FIG.
3).
[0080] The results demonstrated in FIG. 3 show that the release can
be controlled through the thickness of the coating around the core.
With increasing film thickness (d-b-c in FIG. 3), the initial burst
was reduced, the amount of drug released within the first 50 hours
significantly less as compared to the core (I, FIG. 3), and the
drug release (slope) was significantly higher for the coated cores
than for the uncoated one. In particular the cores coated with 2
(c) or 3 (b) layers of fibroin had a nearly linear release for the
first 300 hours (about 12.5 days), following zero order kinetics.
The coating had a minimal effect on drug release, when treated with
water instead of methanol solution, demonstrating the importance of
inducing the conformational change (data not shown).
[0081] These findings allow for at least 2 conclusions: (i) silk
fibroin when treated with methanol solution can be used to
fabricate a controlled drug delivery system; and (ii) silk-fibroin
coatings, treated with methanol solution can modify the release of
drugs from a drug containing core.
[0082] This allows for numerous applications, including the
preparation of microspheres or the coating of tablets to modify the
release.
Example 3
[0083] The exposure of silk films to methanol suggested an increase
in crystallinity, as determined by FTIR analysis (FIG. 7A). This
finding was based on a Amide II bond shift from 1540 cm-1 to 1535
cm-1, a finding typical for the increase in .beta.-crystalline
structures. Similarly, an additional shoulder appeared in response
to methanol treatment at 1630 cm-1 (Amide I) and 1265 cm-1 (Amide
III).
[0084] This data was corroborated by X-ray diffractometry (FIGS. 7B
and 7C). The hydrophobicity of the film surfaces were significantly
influenced by the crystallinity change of the films in response to
methanol treatment, as determined by contact angle measurements
(FIGS. 7D and 7E). For methanol treated films no change of the
contact angle was observed over time, indicating the
water-insolubility of these films as opposed to water treated
films, in which a rapid decrease in contact angles resulted after 3
minutes of exposure to a water droplet.
[0085] The topology of silk films before and after methanol
treatment was assessed by atomic force microscopy (FIG. 6).
Exposure to methanol as opposed to untreated films resulted in a
rougher surface (FIGS. 6A and 6B) and the formation of globular
structures (FIGS. 6C and 6D).
[0086] Conclusion: Methanol treatment of silk films resulted in an
increase in crystallinity (.beta.-sheet), an increase in
hydrophobicity, a decrease in water solubility, and a change in
surface topology.
Example 4
[0087] The release of fluorescently marked dextrans with different
molecular weights was evaluated as a function of methanol
treatment.
[0088] The release of dextrans with size ranges from 4 to 20 kDa
was not apparently sustained, whereas the release of larger
molecules (40 kDa) was retarded (FIG. 4A). In contrast, the
methanol treatment of the silk films resulted in a strong
retardation of release for all dextrans, particularly for molecular
weights equal to or exceeding 10 kDa (FIG. 4B).
[0089] The efficacy of silk films as drug delivery systems for
protein drugs was evaluated using horseradish peroxidase (HRP) and
Lysozyme (Lys; FIG. 5), and analyzed by biological potency tests. A
discontinuous release from native silk films was observed for HRP,
characterized by an initial burst of 5% of the total loading,
followed by a lag phase of two days and a continuous release from
days 3 to 8 (FIG. 5A). HRP release was significantly changed after
exposure of the HRP loaded films to methanol. No initial burst was
observed and the HRP release started at day 5, from which on it was
continuously released until day 23 (FIG. 5A). Lysozyme release from
native silk films was similar to HRP, with an initial burst of
about 30%, a lag phase of 1 day and a continuous release between
days 3 and 8 (FIG. 5B). In contrast to HRP, Lys loaded and methanol
treated films did not release substantial amounts over time (FIG.
5B).
[0090] The adsorption of HRP and Lys to native film surfaces was
similar for both proteins, but apparent and statistically
insignificant (p=0.08) differences were observed for methanol
treated films for Lys loaded films but not for HRP loaded films
(FIGS. 5C and 5D).
[0091] Conclusion: Drug release from silk films was a function of
the drugs molecular weight and film treatment with methanol.
Sustained release profiles with a linear release of bioactive
protein were observed for HRP and Lys, resulting in nearly zero
order kinetics from days 3 to 8 (water treated films), whereas
substantially less protein-drug activity was observed upon methanol
treatment. This decrease of activity was correlated to methanol
sensitivity of Lys (and to a lesser extent for HRP). Alternatively,
crystallinity can be induced by water vapor treatment (data not
shown) of drug loaded films at 25.degree. C. over a saturated
Na.sub.2SO.sub.4 solution for 24 hours. No loss of protein activity
(Lys or HRP) is expected under these vapor conditions an assumption
corroborated by preliminary findings.
TABLE-US-00001 TABLE 1 Groups and treatments I 0 + I + 20 I + 90 I
- 90 II + 0 II + 20 II + 90 II - 90 FD4 FD4 FD4 FD4 FD4 FD4 FD4 FD4
preincubated preincubated preincubated added to preincubated
preincubated preincubated added to with silk- with silk- with silk-
silk- with silk- with silk- with silk- silk- fibroin fibroin
fibroin fibroin fibroin fibroin fibroin fibroin solution, no
solution, solution, film, 90% solution, US solution, US solution,
US film, US MeOH 20% MeOH 90% MeOH MeOH treatment of treatment of
treatment of treatment treatment treatment treatment treatment
mixture, no mixture, 20% mixture, 90% of (n = 3) (n = 3) (n = 15)
(n = 3) MeOH MeOH MeOH mixture, treatment treatment treatment 90%
(n = 3) (n = 3) (n = 9) MeOH treatment (n = 3)
* * * * *