U.S. patent application number 15/746141 was filed with the patent office on 2020-03-12 for biodegradable silk ear tubes.
The applicant listed for this patent is The General Hospital Corporation - dba Mass General Hospital, Massachusetts Eye and Ear Infirmary, Tufts University. Invention is credited to Christopher Hartnick, David Kaplan, Michael Whalen.
Application Number | 20200078495 15/746141 |
Document ID | / |
Family ID | 57835176 |
Filed Date | 2020-03-12 |
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United States Patent
Application |
20200078495 |
Kind Code |
A1 |
Kaplan; David ; et
al. |
March 12, 2020 |
BIODEGRADABLE SILK EAR TUBES
Abstract
In some embodiments, the present invention provides methods for
making resorbable ear tubes including the steps of providing a silk
fibroin solution, and forming a silk ear tube from the silk fibroin
solution, wherein the silk ear tube is less than 2 mm in length and
has an outer diameter of less than 1.5 mm, and wherein the silk ear
tube is resorbable. In some embodiments, the present invention also
provides methods for treating otitis media including the step of
introducing a silk ear tube into the ear canal of a subject,
wherein the silk ear tube is less than 2 mm in length and has an
outer diameter of less than 1.5 mm, and wherein the silk ear tube
is resorbed by the subject.
Inventors: |
Kaplan; David; (Concord,
MA) ; Whalen; Michael; (Needham, MA) ;
Hartnick; Christopher; (Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tufts University
Massachusetts Eye and Ear Infirmary
The General Hospital Corporation - dba Mass General
Hospital |
Medford
Boston
Boston |
MA
MA
MA |
US
US
US |
|
|
Family ID: |
57835176 |
Appl. No.: |
15/746141 |
Filed: |
July 20, 2016 |
PCT Filed: |
July 20, 2016 |
PCT NO: |
PCT/US2016/043166 |
371 Date: |
January 19, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62194423 |
Jul 20, 2015 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 31/047 20130101;
A61L 2300/43 20130101; A61L 2400/08 20130101; A61L 31/148 20130101;
A61L 31/16 20130101; A61F 2210/0076 20130101; A61F 2250/0067
20130101; A61L 29/16 20130101; A61F 11/002 20130101; A61L 2300/406
20130101; A61M 31/00 20130101; A61P 27/16 20180101; A61F 2210/0004
20130101; A61L 2300/402 20130101; A61L 31/146 20130101; A61L 29/048
20130101; A61L 29/148 20130101; A61F 2240/001 20130101; A61L 31/047
20130101; A61L 2430/14 20130101; A61L 2420/02 20130101; C08L 89/00
20130101; A61L 29/146 20130101 |
International
Class: |
A61L 29/14 20060101
A61L029/14; A61F 11/00 20060101 A61F011/00; A61L 29/04 20060101
A61L029/04; A61L 29/16 20060101 A61L029/16 |
Claims
1. A method for making resorbable ear tubes comprising providing a
silk fibroin solution; and forming a silk ear tube from the silk
fibroin solution, wherein the silk ear tube is less than 2 mm in
length and has an outer diameter of less than 1.5 mm, and wherein
the silk ear tube is resorbable.
2. The method of claim 1, wherein the silk ear tube is formed via
gel spinning or gel deposition.
3. The method of claim 1, wherein the silk ear tube is formed via
dip coating or solution deposition.
4. The method of claim 1, wherein the silk ear tube is formed via
micromolding.
5. The method of claim 1, wherein the method further comprises
introducing a plurality of pores into the silk ear tube.
6. The method of claim 5, wherein the pores are introduced by
associating a porogen with the silk fibroin solution prior to
forming the silk ear tube.
7. The method of claim 6, wherein the porogen is selected from
polyethylene oxide, NaCl, alkali metals, alkali earth metal
halides, phosphates, sulfates, sugar crystals, water-soluble
microspheres, polysaccharides, protein microspheres, wax particles,
and synthetic polymer particles.
8. The method of any one of claims 1-7, wherein the silk ear tube
comprises at least two layers.
9. The method of claim 1, further comprising associating at least
one therapeutic agent with the silk fibroin solution prior to or
during formation of the silk ear tube.
10. The method of claim 9, wherein the at least one therapeutic
agent is selected from the group consisting of antibiotics, pain
relievers, and steroids.
11. The method of any one of claims 1-10, wherein the silk fibroin
solution contains between 1%-30% wt silk fibroin.
12. The method of any one of the above claims, wherein the silk ear
tube has a resorption rate of between one day and one week,
inclusive.
13. The method of any one of the above claims, wherein the silk ear
tube has a resorption rate of between eight days and 2 years,
inclusive.
14. The method of any one of claims 1-13, wherein the silk ear tube
comprises beta-sheet content between 1-60%.
15. The method of claim 14, wherein the substantial beta sheet
content is introduced by at least one of autoclaving, water vapor
annealing and treatment with methanol.
16. A method for treating otitis media comprising introducing a
silk ear tube into the ear canal of a subject, wherein the silk ear
tube is less than 2 mm in length and has an outer diameter of less
than 1.5 mm, and wherein the silk ear tube is resorbed by the
subject.
17. The method of claim 16, further comprising administering at
least one therapeutic agent to the subject prior to introduction of
the silk ear tube.
18. The method of claim 16, further comprising administering at
least one therapeutic agent to the subject subsequent to
introduction of the silk ear tube.
19. The method of claim 16, further comprising administering at
least one therapeutic agent to the subject prior substantially
concurrently with introduction of the silk ear tube.
20. The method of any one of claims 16-19, wherein the at least one
therapeutic agent is an antibiotic, a pain reliever, or a
steroid.
21. The method of any one of claims 16-20, wherein the otitis media
is acute otitis media, otitis media with effusion, or chronic
suppurative otitis media.
22. The method of any one of claims 16-21, wherein the silk ear
tube is resorbed over a period of time of between one day and one
week, inclusive.
23. The method of any one of claims 16-21, wherein the silk ear
tube is resorbed over a period of time of between eight days and 2
years, inclusive.
24. The method of any one of claims 16-22, wherein the silk ear
tube comprises beta-sheet content between 1-60%.
25. The method of claim 24, wherein the substantial beta sheet
content is introduced by at least one of autoclaving, water vapor
annealing and treatment with methanol.
26. The method of any one of claims 16-25, wherein the silk ear
tube comprises at least two layers.
27. The method of claim 26, wherein the silk ear tube comprises at
least one porous layer.
28. The method of any one of the above claims, wherein the silk
fibroin is selected from the group consisting of spider silk,
silkworm silk, and recombinant silk.
Description
BACKGROUND
[0001] Tympanostomy tubes are small tubes inserted into the eardrum
in order to keep the middle ear aerated and to prevent the buildup
of fluid in the middle ear. Tympanostomy tube placement is the
single most common ambulatory surgical procedure performed under
general anesthesia in children, with 667,000 procedures done
annually in the United States, often as a treatment for otitis
media or barotrauma. At approximately $2,700 per procedure, the
total financial burden is approximated at nearly $1.8 billion
dollars, excluding perioperative visits and additional testing. By
age 3, nearly 7% of children will have tubes in place.
Unfortunately, previous tympanostomy tubes have proven
unsatisfactory for a variety of reasons including triggering of
chronic inflammation and scarring, and the need to conduct repeated
surgical interventions.
SUMMARY
[0002] Unlike previous attempts at producing tympanostomy tubes
(also referred to as "ear tubes"), the present invention provides
for bioresorbable tubes that do not result in a substantial
inflammatory reaction in a subject, and can be produced with any of
a variety of physical and mechanical characteristics. For example,
according to various embodiments, provided silk ear tubes may be
produced to last for weeks, months, or years in a subject before
being resorbed into the patient's body. Also, in some embodiments,
provided silk ear tubes include multiple layers, each of which may
include one or more of: pores, therapeutic agents, and varying
mechanical and/or physical properties. In some embodiments,
provided silk ear tubes may also have varying compositions and/or
properties along their length. In some embodiments, provided silk
ear tubes may comprise flanges, bevels, and/or other features.
[0003] In some embodiments, the present invention provides methods
for making resorbable ear tubes including the steps of providing a
silk fibroin solution, and forming a silk ear tube from the silk
fibroin solution, wherein the silk ear tube is less than 2 mm in
length and has an outer diameter of less than 1.5 mm, and wherein
the silk ear tube is resorbable.
[0004] In some embodiments, the present invention also provides
methods for treating otitis media including the step of introducing
a silk ear tube into the ear canal of a subject, wherein the silk
ear tube is less than 2 mm in length and has an outer diameter of
less than 1.5 mm, and wherein the silk ear tube is resorbed by the
subject. In some embodiments, the otitis media is acute otitis
media, otitis media with effusion, or chronic suppurative otitis
media.
[0005] According to various embodiments, provided silk ear tubes
may be formed via any known method. In some embodiments, provided
silk ear tubes are formed via gel spinning or gel deposition. In
some embodiments, provided silk ear tubes are formed via dip
coating or solution deposition. In some embodiments, provided silk
ear tubes are formed via injection molding, micromolding, or
machining.
[0006] In some embodiments, provided methods may allow for silk ear
tubes comprising multiple layers. In some embodiments, the silk ear
tube comprises at least two layers (e.g., at least 3, 4, 5, 6, 7,
8, 9, 10 or more layers). In some embodiments, provided silk ear
tubes comprise a single layer.
[0007] In some embodiments, provided methods comprise one or more
additional steps, for example, to conform particular silk ear
tube(s) to a particular application and/or subject. In some
embodiments, provided methods further comprise introducing a
plurality of pores into at least one layer of provided silk ear
tubes. In some embodiments, pores are introduced by associating a
porogen with the silk fibroin solution prior to forming the silk
ear tube. In some embodiments, the porogen is selected from
polyethylene oxide, NaCl, alkali metals, alkali earth metal
halides, phosphates, sulfates, sugar crystals, water-soluble
microspheres, polysaccharides, protein microspheres, wax particles,
and synthetic polymer particles In some embodiments, pores are
introduced using other known methods, for example, lyophilization
or gas evolution methods.
[0008] According to various embodiments, one or more properties of
the silk fibroin solution may be varied including, but not limited
to the amount of silk fibroin in the silk fibroin solution, the
presence or absence of additional materials in the silk fibroin
solution, and the composition of the solvent in the silk fibroin
solution, to name just a few. For example, in some embodiments, the
silk fibroin solution contains between 1%-30% wt silk fibroin in
water. In some embodiments, the silk fibroin solution may also
contain one or more other additives including, for example,
glycerol and/or glycol. In some embodiments, the addition of one or
more additives may be useful in controlling the formation and/or
morphology of provided silk ear tubes.
[0009] In some embodiments, silk ear tubes used in provided methods
comprise at least one therapeutic agent. In some embodiments,
provided methods may further comprise associating at least one
therapeutic agent with the silk fibroin solution prior to of during
formation of the silk ear tube. In some embodiments, provided
methods further comprise administering at least one therapeutic
agent to the subject prior to introduction of the silk ear tube. In
some embodiments, provided methods further comprise administering
at least one therapeutic agent to the subject subsequent to
introduction of the silk ear tube. In some embodiments, provided
methods further comprise administering at least one therapeutic
agent to the subject substantially concurrently with introduction
of the silk ear tube. In some embodiments, the at least one
therapeutic agent is selected from the group consisting of
antibiotics, pain relievers, and steroids.
[0010] According to various embodiments, provided silk ear tubes
are resorbable in a subject (i.e., are broken down and assimilated
into a subject's body). In some embodiments, the silk ear tube has
a resorption rate of between one day and one week, inclusive. In
some embodiments, the silk ear tube has a resorption rate of
between eight days and 2 years, inclusive.
[0011] It is contemplated that one or more physical properties of
provided silk ear tubes may be varied in order to suit a specific
application and/or subject's needs. For example, in some
embodiments, provided silk ear tubes comprise substantial
beta-sheet content. In some embodiments, the substantial beta sheet
content is introduced by at least one of autoclaving, water vapor
annealing and treatment with methanol. In some embodiments,
beta-sheet content may be between 1-75% (e.g., 1-10%, 1-20%, 1-30%,
1-40%, 1-50%, 1-60%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%,
20-60%, 20-50%, 20-40%, 20-30%, or 0-60%). Without wishing to be
held to a particular theory, it is contemplated that a higher
beta-sheet content will lead to a slower degradation of provided
silk ear tubes in vivo. In some embodiments, the beta-sheet content
of provided silk ear tubes may vary in any application-appropriate
manner. For example, in some embodiments, provided silk ear tubes
may comprise a gradient of beta-sheet content. In some embodiments,
a gradient may comprise a multi-layered silk ear tube with at least
one layer having a beta-sheet content that is different (e.g.,
higher or lower) than at least one other layer.
[0012] According to various embodiments, any silk fibroin may be
used in provided methods and silk ear tubes. In some embodiments,
the silk fibroin is selected from the group consisting of spider
silk (e.g., from Nephila ciavipes), silkworm silk (e.g., from
Bombyx mori), and recombinant silks from silkworm or spider
silks.
[0013] Any citations to publications, patents, or patent
applications herein are incorporated by reference in their
entirety. Any numerals used in this application with or without
about/approximately are meant to cover any normal fluctuations
appreciated by one of ordinary skill in the relevant art.
[0014] Other features, objects, and advantages of the present
invention are apparent in the detailed description that follows. It
should be understood, however, that the detailed description, while
indicating embodiments of the present invention, is given by way of
illustration only, not limitation. Various changes and
modifications within the scope of the invention will become
apparent to those skilled in the art from the detailed
description.
DEFINITIONS
[0015] In this application, unless otherwise clear from context,
(i) the term "a" may be understood to mean "at least one"; (ii) the
term "or" may be understood to mean "and/or"; (iii) the terms
"comprising" and "including" may be understood to encompass
itemized components or steps whether presented by themselves or
together with one or more additional components or steps; and (iv)
the terms "about" and "approximately" are used as equivalents and
may be understood to permit standard variation as would be
understood by those of ordinary skill in the art; and (v) where
ranges are provided, endpoints are included.
[0016] Agent: The term "agent" as used herein may refer to a
compound or entity of any chemical class including, for example,
polypeptides, nucleic acids, saccharides, lipids, small molecules,
metals, or combinations thereof. As will be clear from context, in
some embodiments, an agent can be or comprise a cell or organism,
or a fraction, extract, or component thereof. In some embodiments,
an agent is or comprises a natural product in that it is found in
and/or is obtained from nature. In some embodiments, an agent is or
comprises one or more entities that is man-made in that it is
designed, engineered, and/or produced through action of the hand of
man and/or is not found in nature. In some embodiments, an agent
may be utilized in isolated or pure form; in some embodiments, an
agent may be utilized in crude form. In some embodiments, potential
agents are provided as collections or libraries, for example that
may be screened to identify or characterize active agents within
them. Some particular embodiments of agents that may be utilized in
accordance with the present invention include small molecules,
antibodies, antibody fragments, aptamers, nucleic acids (e.g.,
siRNAs, shRNAs, DNA/RNA hybrids, antisense oligonucleotides,
ribozymes), peptides, peptide mimetics, etc. In some embodiments,
an agent is or comprises a polymer. In some embodiments, an agent
is not a polymer and/or is substantially free of any polymer. In
some embodiments, an agent contains at least one polymeric moiety.
In some embodiments, an agent lacks or is substantially free of any
polymeric moiety.
[0017] Approximately: As used herein, the term "approximately" or
"about," as applied to one or more values of interest, refers to a
value that is similar to a stated reference value. In certain
embodiments, the term "approximately" or "about" refers to a range
of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,
13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in
either direction (greater than or less than) of the stated
reference value unless otherwise stated or otherwise evident from
the context (except where such number would exceed 100% of a
possible value).
[0018] Associated with: Two events or entities are "associated"
with one another, as that term is used herein, if the presence,
level and/or form of one is correlated with that of the other. For
example, a particular entity (e.g., polypeptide, genetic signature,
metabolite, etc) is considered to be associated with a particular
disease, disorder, or condition, if its presence, level and/or form
correlates with incidence of and/or susceptibility to the disease,
disorder, or condition (e.g., across a relevant population). In
some embodiments, two or more entities are physically "associated"
with one another if they interact, directly or indirectly, so that
they are and/or remain in physical proximity with one another. In
some embodiments, two or more entities that are physically
associated with one another are covalently linked to one another;
in some embodiments, two or more entities that are physically
associated with one another are not covalently linked to one
another but are non-covalently associated, for example by means of
hydrogen bonds, van der Waals interaction, hydrophobic
interactions, magnetism, and combinations thereof.
[0019] Biocompatible: The term "biocompatible", as used herein,
refers to materials that do not cause significant harm to living
tissue when placed in contact with such tissue, e.g., in vivo. In
certain embodiments, materials are "biocompatible" if they are not
toxic to cells. In certain embodiments, materials are
"biocompatible" if their addition to cells in vitro results in less
than or equal to 20% cell death, and/or their administration in
vivo does not induce significant inflammation or other such adverse
effects.
[0020] Biodegradable: As used herein, the term "biodegradable"
refers to materials that, when introduced into cells, are broken
down (e.g., by cellular machinery, such as by enzymatic
degradation, by hydrolysis, and/or by combinations thereof) into
components that cells can either reuse or dispose of without
significant toxic effects on the cells. In certain embodiments,
components generated by breakdown of a biodegradable material are
biocompatible and therefore do not induce significant inflammation
and/or other adverse effects in vivo. In some embodiments,
biodegradable polymer materials break down into their component
monomers. In some embodiments, breakdown of biodegradable materials
(including, for example, biodegradable polymer materials) involves
hydrolysis of ester bonds. Alternatively or additionally, in some
embodiments, breakdown of biodegradable materials (including, for
example, biodegradable polymer materials) involves cleavage of
urethane linkages. Exemplary biodegradable polymers include, for
example, polymers of hydroxy acids such as lactic acid and glycolic
acid, including but not limited to poly(hydroxyl acids),
poly(lactic acid)(PLA), poly(glycolic acid)(PGA),
poly(lactic-co-glycolic acid)(PLGA), and copolymers with PEG,
polyanhydrides, poly(ortho)esters, polyesters, polyurethanes,
poly(butyric acid), poly(valeric acid), poly(caprolactone),
poly(hydroxyalkanoates, poly(lactide-co-caprolactone), blends and
copolymers thereof. Many naturally occurring polymers are also
biodegradable, including, for example, proteins such as albumin,
collagen, gelatin and prolamines, for example, zein, and
polysaccharides such as alginate, cellulose derivatives and
polyhydroxyalkanoates, for example, polyhydroxybutyrate blends and
copolymers thereof. Those of ordinary skill in the art will
appreciate or be able to determine when such polymers are
biocompatible and/or biodegradable derivatives thereof (e.g.,
related to a parent polymer by substantially identical structure
that differs only in substitution or addition of particular
chemical groups as is known in the art).
[0021] Combination therapy: As used herein, the term "combination
therapy" refers to those situations in which a subject is
simultaneously exposed to two or more therapeutic regimens (e.g.,
two or more therapeutic agents). In some embodiments, two or more
agents or may be administered simultaneously; in some embodiments,
such agents may be administered sequentially; in some embodiments,
such agents are administered in overlapping dosing regimens.
[0022] Composition: A "composition" or a "pharmaceutical
composition" according to this invention refers to the combination
of two or more agents as described herein for co-administration or
administration as part of the same regimen. It is not required in
all embodiments that the combination of agents result in physical
admixture, that is, administration as separate co-agents each of
the components of the composition is possible; however many
patients or practitioners in the field may find it advantageous to
prepare a composition that is an admixture of two or more of the
ingredients in a pharmaceutically acceptable carrier, diluent, or
excipient, making it possible to administer the component
ingredients of the combination at the same time.
[0023] Improve, increase or reduce: as used herein or grammatical
equivalents thereof, indicate values that are relative to a
baseline measurement, such as a measurement in the same individual
prior to initiation of a treatment described herein, or a
measurement in a control individual (or multiple control
individuals) in the absence of the treatment described herein. In
some embodiments, a "control individual" is an individual afflicted
with the same form of disease or injury as an individual being
treated.
[0024] Subject: By "subject" is meant a mammal (e.g., a human). In
some embodiments, a subject is suffering from a relevant disease,
disorder or condition. In some embodiments, a subject is
susceptible to a disease, disorder, or condition. In some
embodiments, a subject displays one or more symptoms or
characteristics of a disease, disorder or condition. In some
embodiments, a subject does not display any symptom or
characteristic of a disease, disorder, or condition. In some
embodiments, a subject is someone with one or more features
characteristic of susceptibility to or risk of a disease, disorder,
or condition. In some embodiments, a subject is a patient. In some
embodiments, a subject is an individual to whom diagnosis and/or
therapy is and/or has been administered.
[0025] Substantially: As used herein, the term "substantially"
refers to the qualitative condition of exhibiting total or
near-total extent or degree of a characteristic or property of
interest. One of ordinary skill in the biological arts will
understand that biological and chemical phenomena rarely, if ever,
go to completion and/or proceed to completeness or achieve or avoid
an absolute result. The term "substantially" is therefore used
herein to capture the potential lack of completeness inherent in
many biological and chemical phenomena.
[0026] Therapeutic agent: As used herein, the phrase "therapeutic
agent" in general refers to any agent that elicits a desired
pharmacological effect when administered to an organism. In some
embodiments, an agent is considered to be a therapeutic agent if it
demonstrates a statistically significant effect across an
appropriate population. In some embodiments, the appropriate
population may be a population of model organisms. In some
embodiments, an appropriate population may be defined by various
criteria, such as a certain age group, gender, genetic background,
preexisting clinical conditions, etc. In some embodiments, a
therapeutic agent is a substance that can be used to alleviate,
ameliorate, relieve, inhibit, prevent, delay onset of, reduce
severity of, and/or reduce incidence of one or more symptoms or
features of a disease, disorder, and/or condition. In some
embodiments, a "therapeutic agent" is an agent that has been or is
required to be approved by a government agency before it can be
marketed for administration to humans. In some embodiments, a
"therapeutic agent" is an agent for which a medical prescription is
required for administration to humans.
BRIEF DESCRIPTION OF THE DRAWING
[0027] FIG. 1 shows images of silk tubes generated using
dip-coating. (panel a) Dip-coated for 3 times, (panels b-d)
dip-coated for 5 times, (panels a,b) fractured cross-section,
(panel c) magnified fractured cross-section, (panel d) side
view.
[0028] FIG. 2 shows portions of an exemplary fabrication process.
(panel A) Digital pictures of the fabrication of gel spun silk
tubes. In the left image, a polypropylene tube (1090 .mu.m outer
diameter) was assembled to make one end of tube with a larger inner
lumen fitting. In the middle image, highly concentrated silk
solution was spun on the rotating metal wire. In the right image,
the gel spun silk tube was obtained after freeze-drying. (panel B)
The schematic of the silk catheter. The other end of tube was
capped with silk by dipping one end into concentrated silk solution
and drying. (panel C) The pictures demonstrate that the gel spun
tubes are flexible even when dried.
[0029] FIG. 3 shows an exemplary provided method for ear tube
fabrication. (panel A) shows a schematic for producing ear tubes
via a provided injection molding process. Following fabrication
steps of injection, freeze-drying, methanol treatment, drying
(panel B) and machining, tubes were fabricated (shown in side-view
(panel C), and top view (panel D)). (panel E) SEM images show a low
degree of porosity in this exemplary embodiment.
[0030] FIG. 4 panel (A) shows the results of Instron mechanical
testing was performed and compressive modulus was calculated for
exemplary ear tubes shown in FIG. 3 (shown in FIG. 3, panels C, D,
and E). Panel (B) exemplary compressive modulus is shown, panel (C)
as is the compressive modulus of the current PTFE tubes on the
market, which is much less than the silk bone screws that are
fabricated using a similar method (panel C).
[0031] FIG. 5 shows (panel A) results of bench top lyophilization
of exemplary ear tubes, (panel B) and (panel C) show exemplary
methanol-based dehydration of provided ear tubes for 6 hours at a
temperature between -20.degree. C. and -30.degree. C. with (panel
B) showing the results for ear tubes made with a 25% silk fibroin
solution and (panel C) showing the results for ear tubes made with
a 33% silk fibroin solution.
[0032] FIG. 6 shows exemplary photographs of a provided coating
process that includes steps of mixing a PEO/silk solution (panel
A), painting the coating on with a paintbrush (panel B), methanol
treating (panel C), and drying (panel D) to achieve an ear tube
with a porous coating.
[0033] FIG. 7 shows exemplary photographs of certain provided
compositions made in accordance with the methods shown in FIG. 6,
including a PEO coating on ear tube, which provides a porous layer
for, inter alia, tympanic membrane attachment and degradation.
[0034] FIG. 8 shows exemplary images and dimensions of certain
provided embodiments shown in FIG. 7 as measured in Image J
software post-sterilization (inner diameter was adjusted to 1.1
mm).
[0035] FIG. 9 shows exemplary results relating to the efficacy of
certain drug eluting tubes via dip coating method post-incubation
in 37.degree. C. for 24 hours with 5.21*10.sup.7 CFU/mL of gram
negative moraxella catarrhalis bacterial strain (top row). As is
shown in the top row of FIG. 9, exemplary provided silk ear tubes
coated with coated drug (Tube +D), show antimicrobial activity
compared to a similar tube without a drug coating (Tube -D) and
positive control of lysogeny broth with gram negative bacteria (LB,
G-). Subsequent culture of media for an additional 24 hours shows
the drug eluting tube was effective at both preventing growth and
killing the bacteria (bottom row, right) compared to the tube
without drug (bottom row, middle) and the positive control (bottom
row, left)
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0036] The present invention provides, inter alfa, resorbable silk
ear tubes with highly customizable physical and mechanical
properties, as well as methods of making and using provided silk
ear tubes.
[0037] According to various embodiments, the present invention
provides methods for making resorbable ear tubes including the
steps of providing a silk fibroin solution, and forming a silk ear
tube from the silk fibroin solution, wherein the silk ear tube is
less than 2 mm in length and has an outer diameter of less than 1.5
mm, and wherein the silk ear tube is resorbable. In some
embodiments, provided silk ear tubes are substantially completely
resorbable. In some embodiments, provided silk ear tubes are only
partially resorbable.
Silk Fibroin Solutions
[0038] The silk fibroin solutions used in methods and compositions
described herein may be obtained from a solution containing a
dissolved silkworm silk, such as, for example, from Bombyx mori.
Alternatively, the silk fibroin solution may be obtained from a
solution containing a dissolved spider silk, such as, for example,
from Nephila clavipes. The silk fibroin solution can also 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. In some embodiments, genetically engineered silk
can, for example, comprise a therapeutic agent, e.g., a fusion
protein with a cytokine, an enzyme, or any number of hormones or
peptide-based drugs, antimicrobials and related substrates.
[0039] According to various embodiments, the silk fibroin solution
can be prepared by any conventional method known to one skilled in
the art. In some embodiments, the solution is an aqueous solution.
For example, B. mori cocoons are boiled for about 30 minutes in an
aqueous solution. In some embodiments, the aqueous solution is
about 0.02M Na.sub.2CO.sub.3. The cocoons may be rinsed, for
example, with water to extract the sericin proteins and the
extracted silk is dissolved in an aqueous salt solution. Exemplary
salts useful for this purpose include lithium bromide, lithium
thiocyanate, calcium nitrate or other chemicals capable of
solubilizing silk. Preferably, in some embodiments, the extracted
silk is dissolved in about 9-12 M LiBr solution. The salt is
consequently removed using, for example, dialysis.
[0040] 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. 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 can 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.
[0041] In accordance with various embodiments, a silk solution may
comprise any of a variety of concentrations of silk fibroin. In
some embodiments, a silk solution may comprise 0.1 to 40% by weight
silk fibroin. In some embodiments, a silk solution may comprise
between about 0.5% and 40% (e.g., 0.5% to 25%, 0.5% to 20%, 0.5% to
15%, 0.5% to 10%, 0.5% to 5%, 0.5% to 1.0%) by weight silk fibroin,
inclusive. In some embodiments, a silk solution may comprise
between 15-30% (e.g., 20-25%) by weight silk fibroin, inclusive. In
some embodiments, a silk solution may comprise at least 0.1% (e.g.,
at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,
25%, 30%, 35%, 40%) by weight silk fibroin. In some embodiments, a
silk solution may comprise at most 30% (e.g., at most 40%, 35%,
30%, 25%, 20%, 15%, 14%, 13%, 12% 11%, 10%, 5%, 4%, 3%, 2%, 1%) by
weight silk fibroin
[0042] In some embodiments, the silk ear tube compositions
described herein, and the methods using them can be performed in
the absence of any organic solvent. Thus, these compositions and
methods are particularly amenable to the incorporation of labile
molecules, such as bioactive agents or therapeutics, and can, in
certain embodiments, be used to produce controlled release
biomaterials. Preferably, the methods are performed in water
only.
[0043] Alternatively, in some embodiments, the silk fibroin
solution can be produced using organic solvents, for example
hexafluoroisopropanol (HFIP). Such methods have been described, for
example, in Li, M. , et al., J. Appi. Poly Sci. 2001, 79,
2192-2199; Mm, s., et al. Sen I Gakkaishi 1997, 54, 85-92; Nazarov,
R. et al., Biomacromolecules 2004 May-Jun.; 5 (3):71 8-26.
[0044] In some embodiments, non-silk biocompatible polymers can
also be added to the silk solution to generate composite matrices
in the silk ear tubes described herein. Biocompatible polymers
useful in the compositions described herein 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 (US 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). In some embodiments, two or more biocompatible
polymers can be used.
Dimensions
[0045] The outer diameter of provided silk ear tubes can vary
according to the needs of a specific application and/or subject,
for example, from about 0.1 mm to about 4 mm or more. As described
herein, in some embodiments, silk ear tubes or constructs of
specific inner lumen diameters may be prepared by using a rod of
the desired diameter in the process of making silk ear tubes.
Specific sizes include, for example, 0.1 mm, 0.2 mm, 0.3 mm, 0.4
mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.2 mm, 1.4 mm,
1.6 mm, 1.8 mm, 2.0 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm, 3.0 mm, 3.2
mm, 3.4 mm, 3.6 mm, and 3.8 mm. The preferred sizes can also be
expressed as a range, e.g., 0.1 to 2.9 mm, 0.1 to 2.5 mm, 0.1 to 2
mm, 0.1 to 1.5 mm, 0.1 to 1 mm, 1.0 to 3 mm, 1.0 to 2 mm, 1.0 to
1.5 mm, etc. In some embodiments, provided embodiments may vary
from one or more of these values by at most 20% (e.g., at most 15%,
10%, 5%).
[0046] The length of provided silk ear tubes may also vary in an
application and/or subject-specific manner. In some embodiments,
provided silk ear tubes may have a length between 0.5 and 3 mm
(e.g., between 0.5 mm and 2.5 mm, 0.5 and 2 mm, 0.5 and 1 mm, 1 and
2.5 mm, 1 and 2 mm, 1 and 1.5 mm). Specific lengths include, for
example, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm,
0.8 mm, 0.9 mm, 1.0 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2.0 mm, 2.2
mm, 2.4 mm, 2.6 mm, 2.8 mm, and 3.0 mm. In some embodiments,
provided embodiments may vary from one or more of these values by
at most 20% (e.g., at most 15%, 10%, 5%).
[0047] As used herein, concentrations, amounts, sizes, porosities,
and other numerical data may be presented herein in a range format.
It is to be understood that such range format is used merely for
convenience and brevity and should be interpreted flexibly to
include not only the numerical values explicitly recited as the
limits of the range, but also to include all the individual
numerical values or sub-ranges encompassed within that range as if
each numerical value and sub-range is explicitly recited.
[0048] For example, a range of about 1.0 to about 3.0 mm should be
interpreted to include not only the explicitly recited size limits
of 1.0 to about 3.0 mm, but also to include individual dimensions
such as 1.4 mm, 1.8 mm, 2.0 mm, and 2.7 mm, as well as sub-ranges
such as 1.0-1.4 mm, 1.0-1.8 mm, 1.8-2.4 mm, 1.4-3.0 mm, etc.
Further, such an interpretation should apply regardless of the
breadth of the range or the characteristic being described, such as
protein concentration, tubular porosity, lumen diameter, porogen
concentration and amounts and concentrations of other ingredients
or agents.
[0049] According to various embodiments, provided silk ear tubes
may vary in shape in any of a variety of ways. For example, in some
embodiments, provided silk ear tubes may comprise beveled edges on
one or both ends, comprise one end that has a larger outer diameter
than the other end, comprise one or more flanges, and/or comprises
a smooth inner surface and a porous outer surface.
Layers
[0050] According to various embodiments, provided silk ear tubes
may comprise two or more layers. In some embodiments, the thickness
of each deposited layer can also be controlled, inter alfa, by
adjusting the concentration of fibroin in the silk fibroin solution
used to form the layer. For example, in embodiments where provided
silk ear tubes are formed through dip coating, the more
concentrated the fibroin in the aqueous silk fibroin solution is,
the more fibroin that is deposited on the rod or on the previous
layer of silk fibroin and a more compact structure is formed.
[0051] In some embodiments, each layer of a silk ear tube has a
substantially similar thickness and/or functionalization. In some
embodiments, at least one layer of a silk ear tube has a thickness
and/or functionalization that is different form at least one other
layer. According to various embodiments, functionalization may
include the addition of one or more therapeutic agent.
[0052] In some embodiments, a silk ear tube may have between 1 and
10 layers (e.g., between 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1
to 4, 1 to 3, or 1 to 2 layers). In some embodiments, provided silk
ear tubes comprise a single layer. In some embodiments, each layer
of a silk ear tube may be between 1 nanometer (nm) and 1 millimeter
(mm). For example, in some embodiments, each layer of a silk ear
tube may be between 10 nm and 1 mm (e.g., between 25 nm and 1 mm,
50 nm and 1 mm, 100 nm and 1 mm, 200 nm and 1 mm, 30 nm and 1 mm,
400 nm and 1 mm, 500 nm and 1 mm, or 600 nm and 1 mm). In some
embodiments, each layer of a silk ear tube may be between 10 nm and
1 mm (e.g., between 10 nm and 900 nm, 10 nm and 800 nm, 10 nm and
700 nm, 10 nm and 500 nm, 10 nm and 400 nm, 10 nm and 300 nm, or 10
nm and 200 nm).
Pores
[0053] In some embodiments, provided silk ear tubes may be made
porous through the use of one or more porogens. It is contemplated
that any known porogen may be suitable for use according to various
embodiments. In some embodiments, a porogen may be or comprise
crystals (e.g., sodium chloride crystals, sugar crystals), micro-
and/or nano-spheres, polymers (such as polyethylene oxide, or PEO),
ice crystals, sulfates, phosphates, alkali metals, alkali earth
metal halides, polysaccharides, wax particles, synthetic polymer
particles, and/or a laser. In some embodiments a porogen may
comprise mechanical introduction of pores (e.g., using a needle or
other article or device to pierce a silk ear tube one or more
times, or using stress to introduce one or more tears in the silk
ear tube).
[0054] As used herein, the term "porous" refers to the property of
at least one layer of a silk ear tube described herein to permit
the passage of materials through the wall of the tube (in contrast
to their passage through or along the lumen of the tube). Silk ear
tubes described herein may encompass a range of porosities, from
those that do not substantially permit the passage of cells or
proteins, to those that substantially permit the passage of
proteins, but not cells, to those that permit the passage of both.
As used herein, the term "not porous" means that a tube as
described herein does not substantially permit the passage of
Alexa-Fluor-488-labeled BSA through the wall of the tube over the
course of a 20 minute assay. By "not substantially permit" is meant
that under the detection conditions described herein, no labeled
BSA from inside the tube is detected outside the tube after a 20
minute assay. Alternatively, the porosity of a tubular composition
as described herein can be expressed in terms of a permeability
coefficient, measured/calculated as described herein or otherwise
known in the art. Tubular compositions as described herein are
considered "not porous" to the passage of proteins or cells if the
permeability coefficient for Alexa-Fluor-488-labeled BSA is
7.3.times.10.sup.4.+-.1.5.times.10.sup.-4 cm/s or lower. As used
herein, the term "permeable to proteins" means that a tubular
composition as described herein permits the passage of
Alexa-Fluor-488-labeled BSA through the tube wall with a
permeability coefficient, measured as described herein, of at least
8.9.times.10.sup.-4 cm/s. Other modes of assessment of porosity may
include Scanning Electron Microscopy assessment of cross sections
of provided silk ear tubes followed by image processing; or mercury
porisimetry measurements.
[0055] Various embodiments may comprise silk ear tubes comprising
pores of various sizes. In some embodiments, pores in a silk ear
tube may have a diameter between about 1-100 .mu.m, inclusive. In
some embodiments, pores in a silk ear tube may have a diameter
between about 1-50 .mu.m (e.g., 1-40, 1-35, 1-30, 1-25, 1-20, 1-15
.mu.m), inclusive. In some embodiments, pores in a silk ear tube
may have a diameter between about 5-25 .mu.m, inclusive. In some
embodiments, pores in a silk ear tube may have a diameter between
about 1-10 .mu.m, inclusive.
Methods of Forming Silk Ear Tubes
[0056] According to various embodiments, provided silk ear tubes
may be formed via any application-appropriate method. In some
embodiments, for example, methods of making provided silk ear tubes
include, but are not limited to, injection molding, dip coating,
gel spinning, 3D printing, and machining, layer by layer
techniques, and filling molds. Additionally, certain exemplary
methods for forming silk ear tubes are shown in the Examples
below.
[0057] In some embodiments, provided silk ear tubes as described
herein may be sterilized using conventional sterilization process
such as radiation based sterilization (i.e. gamma-ray), chemical
based sterilization or autoclaving. In some embodiments, the
sterilization process may be with ethylene oxide at a temperature
between 52-55.degree. C. for a time of 8 hours or less. In some
embodiments, provided silk ear tubes may be sterilized via
autoclaving using high temperature and pressure. After
sterilization the biomaterials may be packaged in an appropriate
sterilized moisture resistant package for shipment.
Therapeutic Agents
[0058] In some embodiments, provided silk ear tubes comprise one or
more therapeutic agents. In some embodiments, the silk fibroin
solution may be contacted with a therapeutic agent prior to forming
the silk ear tube, or can be loaded onto the silk ear tube after it
is formed (e.g., as a coating). In some embodiments, at least one
therapeutic agent is entrapped in the silk during formation of the
silk ear tube, for example, in some embodiments, drying of an
aqueous fibroin layer with a stream of gas, e.g., dehydrating a
silk fibroin layer with N.sub.2 gas induces a conformation change
of the fibroin to the beta sheet structure, which entraps the
agent. In some embodiments, additional layers may then be added
either with the same agent, a different agent, or no agent. In some
embodiments, this stepwise deposition approach also allows
entrapment of varied concentrations of therapeutics within each
layer. According to various embodiments, any pharmaceutical carrier
may optionally be used that does not dissolve the silk material. In
some embodiments, a therapeutic agents may be present as a liquid,
a finely divided solid, or any other appropriate physical form.
[0059] The variety of different therapeutic agents that can be used
in conjunction with the silk ear tubes of the present invention is
vast and includes small molecules, proteins, peptides and nucleic
acids. In general, therapeutic agents which can be associated with
tubular compositions described herein include, without limitation:
anti-infectives such as antibiotics (e.g., ciprofloxacin) and
antiviral agents; anti-rejection agents; analgesics and analgesic
combinations; anti-inflammatory agents (e.g., dexamethasone);
hormones such as steroids; growth factors (bone morphogenic
proteins (i.e. BMP's 17), 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)); nerve growth factors, anti-angiogenic proteins such
as endostatin, and other naturally derived or genetically
engineered proteins, polysaccharides, glycoproteins, or
lipoproteins.
[0060] In some embodiments, provided silk ear tubes may comprise
one or more proteases. In some embodiments a protease may be one or
more of a serine protease (e.g., proteinase K, proteinase XIV, or
a-chymotrypsin), collagenase, or a matrix metalloproteinase (MMP)
(e.g., MMP_1, MMP-2, etc). In some embodiments, one or more
proteases are embedded in the silk ear tube or are associated with
the silk ear tube after implantation in a subject. In some
embodiments, one or more proteases may be used that require one or
more activating events in order to cause protease activation. In
some embodiments an activating event may be, for example,
hydration, change in pH (i.e., raising or lowering), the addition
of a co-factor, and/or any other application-appropriate activating
event. Additional information may be found in Brown et al., Impact
of silk biomaterial structure on proteolysis, Acta Biomaterialia,
11:212-224, (2014), the disclosure of which is hereby incorporated
in its entirety.
Methods of Use
[0061] One of skill in the art will be able to envision several
uses for provided silk ear tubes. In some embodiments, the present
invention provides methods for treating otitis media including the
step of introducing a silk ear tube into the ear canal of a
subject, wherein the silk ear tube is less than 2 mm in length and
has an outer diameter of less than 1.5 mm, and wherein the silk ear
tube is resorbed by the subject. In some embodiments, the otitis
media is acute otitis media, otitis media with effusion and
conductive hearing loss, or chronic suppurative otitis media.
[0062] Provided silk ear tubes provide several advantages over
previous tympanostomy tubes including the prevention of many common
complications of previous tympanostomy tubes and methods of
insertion. Amongst the common complications of tympanostomy tubes
are delayed extrusion of the tubes with the need for a subsequent
second surgery to remove the tubes as well as tympanosclerosis and
granulation tissue that can develop around non-extruded tubes. A
more ominous complication is the development of an atrophic region
of the tympanic membrane adjacent to the tympanostomy tube, and can
in turn lead to cholesteatoma formation. Various embodiments of the
present invention are able to reduce or eliminate one or more of
these exemplary complications stemming, at least in part, from use
of previously known tympanostomy tubes. In some embodiments,
provided insertion or implantation of silk ear tubes does not
result in an inflammatory reaction in a subject.
[0063] According to various embodiments, provided silk ear tubes
may be designed to last in vivo or on a shelf for extended periods
of time. For example, in some embodiments, provided silk ear tubes
may be produced that maintain substantial integrity in vivo for a
period of time between 6 months and 5 years, in other words, these
exemplary silk ear tubes have a resorption rate of between 6 months
and 5 years. In some embodiments, provided silk ear tubes may have
a resorption rate between one day and one week, between eight days
and two years, between one year and four years, between one year
and three years, or between one year and two years. In some
embodiments, provided silk ear tubes may be produced that are shelf
stable for one year, two years, three years, four years, five
years, or more.
[0064] 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.
EXAMPLES
Example 1
Exemplary Dip Coating and Gel Spinning Methods
[0065] Preparation of silk solution--Silk solution may be generated
from Bombyx mori silkworm cocoons according to the procedures
described in previous studies. Cocoons of B. mori silkworm silk can
be supplied by Tajima Shoji Co (Yokohama, Japan). Briefly, the
cocoons are degummed in a boiled 0.02 M Na.sub.2CO.sub.3
(Sigma-Aldrich, St Louis, Mo.) solution for 20 min. The fibroin
extract is then rinsed three times in Milli-Q water, dissolved in a
9.3-M LiBr solution yielding a 20% (w/v) solution, and subsequently
dialyzed (MWCO 3,500) against distilled water for 2 days to obtain
silk fibroin aqueous solution (ca. 8 wt/vol %).
[0066] Preparation of silk tubes--In this Example, silk tubes are
formed using exemplary multiple dip-coating or gel spinning
methods. For the multiple dip-coating process, a mixture of silk
fibroin (10 ml of 8 wt/vol %) and PEO (MW=900,000; Sigma-Aldrich)
(4 ml of 5 wt/vol %) solution (Silk:PEO 4:1 w/w) is prepared (if
pore formation within the silk tubular matrix is desired, if no
pores are needed then the PEO need not be included in the process).
Silk tubes are generated by dip-coating nitinol, Teflon or other
wires or dowels (0.76 mm diameter) into the solution, treating the
coated mixture solution on the wires or dowels in MeOH for 30 sec
to stabilize the silk on the wire surface, and then drying the
coated layer in air for 1 hour. This dipping process is repeated to
generate around 1.36 mm outer diameter tubular matrices on the
wires. Post-dip coating, the tubes are treated in MeOH for 2 hours
and then placed into a water bath for 2 days to extract out the PEO
(if used). The tubes are removed from the wire and dried in air.
FIG. 1 shows exemplary images of silk ear tubes produced according
to this dip coating method. For the gel spinning process, the silk
solution is further concentrated (25.about.30 w/v %) by using a
CentriVap vacuum concentrator (Labconco, Kansas City, Mo.). Tubular
scaffolds are produced by spinning the concentrated silk solutions
[25-30% (w/v), 0.1 ml/5 cm of scaffold] onto a rotating (200 rpm)
and axially reciprocating wire with an axial slew rate (ASR) of 2
mm/sec using a custom gel spinning platform and program as
described previously [10]. The tubes are lyophilized and treated
with methanol for 2 hours and removed from the wires. FIG. 2 shows
exemplary images and a schematic of silk ear tubes produced
according to this gel-spinning method as well as some of the
mechanical characteristics of such silk ear tubes (see panel C of
FIG. 2).
[0067] Described in this Example are exemplary, non-limiting
methods for producing silk fibroin solutions and forming silk ear
tubes according to the present invention.
Example 2
Exemplary Production of Silk Ear Tubes via Injection Molding
[0068] In this Example, silk ear tubes were produced using an
injection molding process.
[0069] A mold was made of wax that was prepared from
MachinableWax.com (USA) according to previously described methods.
The molds were 3.30 cm in height and had a 0.76 cm diameter. The
mold had two pieces, a 2 mm thick top piece that was placed on top
of a 1 mm bottom wafer, providing supportive holes that were 0.76
mm in diameter corresponding the desirable placement of the PTFE in
the top portion. The molds also had a thin wafer attached to a
6-well top piece, having a height of 0.25 cm. Prior to silk
injection, the two pieces were placed on top of one another and
sealed together with parafilm and the PTFE was secured in the
middle of the well, via placement into the bottom-well socket.
Next, 30 minute boil silk that was concentrated to 25-33 w/v %
using the CentriVap bench top vacuum was loaded into each well of
the mold. Following silk addition to the well, the mold was placed
in -20.degree. C. for 4 hours and then placed in a lyophilizer on
vacuum at -30.degree. C. until completely dry.
[0070] The molds were then placed in a methanol bath for 12 hours
(FIG. 3, panel A), washed in deionized (DI) water, and allowed to
air dry prior to machining. It is important to note that in some
embodiments, injected samples may go directly into methanol without
the intervening lyophilization step. The machined tubes had an
inner diameter of 0.8 mm, outer diameter of 2 mm and length of 1.7
mm, which were measured using ImageJ software of scanning electron
microscopy images (SEM) (FIG. 3, panel E). In some embodiments, the
dimensions could be adjusted, such as to 0.79 mm inner diameter and
1.36 mm outer diameter. However, in some embodiments, sterilization
techniques such as autoclaving may cause the tube to shrink, so
this reduction in size should be accounted for prior to machining
to a different dimension. Drying in a 60.degree. C. oven may be
used, in some embodiments, to reduce shrinking, and/or the silk ear
tube may be designed to take the shrinking into account. In this
Example, a collar-button flange was used, though in other
embodiments, other designs may be useful, for example, a
pop-beveled grommet. Two of the silk ear tubes were subjected to
Instron mechanical testing to gauge the mechanics of tubes produced
with this method. As shown in FIG. 4, panels A and B, the
compressive moduli of the tubes were between 624 and 717 MPa, which
is in line with commercialized PTFE tube mechanics and much lower
than screws that are produced using a similar approach (FIG. 4,
panel C).
[0071] In addition, sample silk ear tubes produced as described
above were dried using a) a bench top lyophilizer which did not
simultaneously freeze the sample while on vacuum, b) methanol
treatment while timing, and c) large batch silk concentration
methods.
[0072] Without wishing to be held to a particular theory, it
appears that the molds used in this Example require a lyophilizer
that can dry the samples in vacuum, while maintaining a temperature
between -20 and -30.degree. C. to ensure the samples remains frozen
during the drying process. Placing the sample in the bench-top
lyophilizer resulted in partially defrosted tubes during the drying
process (FIG. 5, panel A). Further, methanol treatment was
performed for 6 hours, and this methanol treatment time resulted in
a tube that resembled more of a styro-foam material (see FIG. 5,
panels B and C), rather than the mechanically superior material
revealed from steps that achieve the tube shown in FIG. 3. The
mechanics were not tested on the tubes with shorter methanol time,
as they lacked the ability to be machined. In some embodiments,
therefore, it may be advantageous to administer methanol treatment
for at least 12 hours to encourage additional (.beta.-sheet
formation. Additionally, large-batch silk concentration methods
were experimented with, where silk with 5-7 w/v % was added to
dialysis tubing and placed in a hood for 1 day to pre-concentrate
the silk, prior to placement and concentration in 2 mL tubes in the
CentriVap machine. According to some embodiments, the transition to
larger batch concentration methods may allow for larger quantities
of silk to be recovered post-CentriVap spinning, helping to ensure
enough silk is extruded from the 2 mL tubes to reduce air bubble
accumulation in subsequent injection steps.
[0073] Scanning electron microscope (SEM) imaging of the exemplary
ear tubes produced in this Example revealed a very low degree of
porosity. Without wishing to be held to a particular theory, a
higher degree of porosity may be desirable for some embodiments,
for example, for biodegradable drug-eluting silk ear tubes.
Although porosity was minimal, the exemplary technique of this
Example provides a reproducible, scalable, machinable, and more
reliable drug-loading method than previously observed.
[0074] This Example shows, inter alfa, that silk ear tubes having
mechanical characteristics similar to currently marketed PTFE ear
tubes may be produced in a rapid and economical manner. It also
shows that these tubes may be further customized to suit a
particular application or even a particular subject.
Example 3
Exemplary Production of Porous Silk Ear Tubes via Aqueous
Process
[0075] Preparation of silk solution--Silk solution may be generated
from Bombyx mori silkworm cocoons according to the procedures
described in previous studies. Cocoons of B. mori silkworm silk can
be supplied by Tajima Shoji Co (Yokohama, Japan). Briefly, the
cocoons are degummed in a boiled 0.02 M Na.sub.2CO.sub.3
(Sigma-Aldrich, St Louis, Mo.) solution for 30 min to provide a
5-7% w/v silk solution. Solutions were then allowed to concentrate
fo 1 day to a solution of approximately 8-9% w/v silk.
Subsequently, silk solutions were further concentrated using a
CentriVAP vacuum concentrator to achieve silk solutions with a
concentration of silk between 20-25% w/v.
[0076] Preparation of Silk tubes--the concentrated silk solutions
described above were then injected into 1 mL wax molds, and the
molds were then placed in a methanol bath for 48 hours. After 48
hours, silk cylinders were removed from the mold and placed in a
beaker of deionized water (DI) and stirred for 24 hours. Then, the
washed silk cylinders were removed from the molds and placed into 1
mL wax molds to dry in a hood. Without wishing to be held to a
particular theory, it is contemplated that drying the silk
cylinders in a mold may help to avoid warping of material during
the drying process. After drying, the silk cylinders were machined
to the desired ear tube dimensions.
Coating Tubes with Silk and Polyethylene Oxide (PEO)
[0077] PEO solution preparation--a 6% wt solution of PEO was
prepared by adding 60 mg/mL of Poly(ethylene) Oxide (PEO) to 50 mL
of deionized (DI) water (total of 300 mg of PEO). The DI water was
heated on a hot plate with a stir bar at about 80.degree. C. for 10
minutes before adding PEO. Once the PEO was added to the DI water,
the mixture was stirred for an hour and became a viscous,
homogenous solution. Concurrently, a 20% w/v silk solution was
prepared.
[0078] Preparation of coating solution and coating/induction of
pore formation of coating on silk tubes--Next, an 80/20 w/w mixture
of silk/PEO was made by calculating the volume of silk and PEO to
mix as follows: [0079] Using 1 mL of 20% silk:
[0080] a. (x total mg)*0.8=200 mg of silk
[0081] b. x total=250 mg, so mg of PEO=250 mg total-200 mg silk=50
mg PEO
[0082] c. 50 mg PEO/(60 mg/mL PEO)=0.83 mL of PEO
[0083] After adding the silk and PEO volumes to a 2 mL eppendorf
tube, the mixture was stirred with a needle and then vortexed until
the solution was completely mixed. Next, and prior to coating the
silk tubes: the tubes were placed on a polytetrafluoroethylene
(PTFE) coated rod with a diameter of 0.79 mm with the silk tubes
spaced evenly. To ensure minimal movement of tubes while PEO/silk
solution is painted on, tape was placed on either side of each of
the tubes, which also prevented coated tubes from sliding into each
other (see FIG. 6). Next, the rod was placed on a weigh boat and a
touchup paint brush was used to apply an even layer of the silk/PEO
solution onto each tube (see FIG. 1, panel B). Then, immediately
after coating, 100% methanol was poured into a dish and the tubes
were allowed to sit horizontally therein for 1 hour, where (3-sheet
formation occurred in the silk and the PEO was highly soluble,
creating a porous coating on the outside of the ear tube.
Approximately one hour after placement into the methanol bath, the
tubes were placed in DI water in a Falcon tube and shaken for 48
hours. After shaking, the tubes were removed from the water and
allowed to dry for 24 hours in a fume hood.
[0084] After drying, the silk tubes were autoclaved and examined
using scanning electron microscopy (SEM). As is shown in FIGS. 7-8,
provided methods are able to provide porous silk ear tubes.
Specifically, FIG. 7 shows a plurality of pores present in tubes
produced in this Example. Without wishing to be held to a
particular theory, it is contemplated that the presence and nature
of these pores may facilitate tympanic membrane attachment and/or
degradation of the tubes in vivo after a period of time. FIG. 8
shows exemplary SEM photographs of tubes provided in this Example,
including views of the significant degree of porosity achievable
via provided methods.
Example 4
Coating Tubes with Silk and Ciprofloxacin
[0085] Coating of porous tubes with Ciprodex (exemplary active
agent)--9 mg/mL ciprofloxacin HCL and 3 mg/mL dexamethasone were
added to a silk solution concentrated to between 20-25% silk w/v.
Next silk tubes prepared as described above in Example 3, without
the PEO coating, were placed on a Teflon rod. The silk tubes and
Teflon rod were then dipped into the
silk-ciprofloxacin-dexamethasone solution and then placed
horizontally in a weigh boat filled with 100% methanol for 1 hour.
Importantly, the methanol bath was saturated with both
ciprofloxacin and dexamethasone to prevent drug release from the
silk-drug dip coat into the methanol solution. After methanol
treatment, the silk tubes were dried in a fume hood. Subsequent to
drying, the silk tubes were sterilized using ethylene oxide
sterilization.
[0086] To test the effectiveness of provided silk ear tubes coated
with at least one active agent, here ciprofloxacin and
dexamethasone, these drug coated silk tubes were compared against
silk ear tubes with no such coating and also against a positive
control containing lysogeny broth in the ability to prevent and/or
slow the growth of bacteria. To test this, each of the drug coated
silk ear tubes, non-drug coated silk ear tubes, and positive
control ear tubes were incubated with approximately 5.21*107 CFU/mL
of the gram negative bacteria Moraxella catarrhalis for 24 hours in
a multi well plate (see FIG. 9 top row). After approximately 24
hours, the media was removed from the multi-well plate and
incubated at 37.degree. C. on a culture plate for an additional 24
hours. FIG. 9 shows that the drug coatings were effective in
reducing and/or preventing the growth of bacteria for at least 24
hours.
[0087] As shown in these Examples, provided methods may be sued to
create any of a variety of silk ear tubes, In some embodiments,
provided silk ear tubes may include physical and/or mechanical
characteristics similar to that of previously known, non-silk ear
tubes, while being biodegradable and amenable to including one or
more active agents, such as in a coating or in pores of a provided
embodiment.
EQUIVALENTS AND SCOPE
[0088] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. The scope of the present invention is not intended to be
limited to the above Description, but rather is as set forth in the
following claims:
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