U.S. patent application number 14/390356 was filed with the patent office on 2015-02-26 for methods and compositions for preparing a silk microsphere.
The applicant listed for this patent is TRUSTEES OF TUFTS COLLEGE. Invention is credited to David L. Kaplan, Michael Lovett, Xiaoqin Wang, Tuna Yucel.
Application Number | 20150056294 14/390356 |
Document ID | / |
Family ID | 49328198 |
Filed Date | 2015-02-26 |
United States Patent
Application |
20150056294 |
Kind Code |
A1 |
Kaplan; David L. ; et
al. |
February 26, 2015 |
METHODS AND COMPOSITIONS FOR PREPARING A SILK MICROSPHERE
Abstract
Provided herein relates to methods and compositions for
preparing a silk microsphere and the resulting silk microsphere. In
some embodiments, the methods and compositions described herein are
all aqueous, which can be used for encapsulating an active agent in
a silk microsphere, while maintaining activity of the active agent
during processing. In some embodiments, the resulting silk
microsphere can be used for sustained delivery of an active agent
encapsulated therein.
Inventors: |
Kaplan; David L.; (Concord,
MA) ; Yucel; Tuna; (Medford, MA) ; Wang;
Xiaoqin; (Winchester, MA) ; Lovett; Michael;
(Peabody, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TRUSTEES OF TUFTS COLLEGE |
Medford |
MA |
US |
|
|
Family ID: |
49328198 |
Appl. No.: |
14/390356 |
Filed: |
April 12, 2013 |
PCT Filed: |
April 12, 2013 |
PCT NO: |
PCT/US13/36356 |
371 Date: |
October 2, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61623970 |
Apr 13, 2012 |
|
|
|
Current U.S.
Class: |
424/499 ; 264/13;
264/5; 424/133.1; 514/662; 514/773 |
Current CPC
Class: |
A61K 9/0019 20130101;
A61K 31/13 20130101; A61P 35/00 20180101; A61P 43/00 20180101; A61P
25/28 20180101; A61K 9/1617 20130101; C07K 16/22 20130101; A61K
9/1682 20130101; A61K 47/38 20130101; A61P 37/04 20180101; A61K
9/1658 20130101 |
Class at
Publication: |
424/499 ;
514/773; 424/133.1; 514/662; 264/5; 264/13 |
International
Class: |
A61K 9/16 20060101
A61K009/16; A61K 31/13 20060101 A61K031/13; C07K 16/22 20060101
C07K016/22 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
No. P41 EB002520 awarded by the National Institutes of Health
(NIH). The U.S. government has certain rights in the invention.
Claims
1. A method of preparing a silk microsphere, the method comprising:
inducing formation of beta-sheet structure of fibroin in a silk
solution; and inducing formation of a microsphere from the silk
solution.
2. The method of claim 1, wherein said formation of the beta-sheet
structure of fibroin and the microsphere are induced
simultaneously.
3. The method of claim 1 or 2, wherein said formation of the
beta-sheet structure of fibroin in the silk solution is induced by
sonication.
4. The method of any of claims 1-3, wherein said formation of the
microsphere from the silk solution is induced by atomization of the
silk solution.
5. The method of claim 2, wherein said formation of the beta-sheet
structure of fibroin and the microsphere are induced simultaneously
by flowing the silk solution through a flow-through chamber that is
ultrasonically activated or an ultrasonic atomizer.
6. The method of claim 5, wherein the silk solution is flowed
through the flow-through chamber or the ultrasonic atomizer at a
flow rate of about 0.001 mL/min to about 5 mL/min.
7. The method of claim 6, wherein the silk solution is flowed
through the flow-through chamber or the ultrasonic atomizer at the
flow rate of about 0.05 mL/min to about 0.3 mL/min.
8. The method of any of claims 3-7, wherein the sonication is
performed at a frequency of at least about 10 kHz, or about 20 kHz
to about 40 kHz.
9. The method of any of claims 3-8, wherein the sonication power
output ranges from about 1 watt to about 50 watts, or from about 2
watts to about 20 watts.
10. The method of any of claims 1-9, further comprising freezing
the silk microsphere.
11. The method of claim 10, wherein the silk microsphere can be
frozen by exposing the silk microsphere to a sub-zero
temperature.
12. The method of claim 10 or 11, wherein the silk microsphere is
exposed to the sub-zero temperature by collecting the silk
microsphere in a container cooled by a cooling agent.
13. The method of any of claims 1-12, further comprising subjecting
the silk microsphere to lyophilization.
14. The method of any of claims 1-13, wherein the silk microsphere
has a porosity of at least about 30%.
15. The method of any of claims 1-14, wherein the silk microsphere
has a pore size of about 1 nm to about 500 .mu.m, or 10 nm to about
50 .mu.m.
16. The method of any of claims 1-15, wherein the silk solution
comprises silk fibroin at a concentration of about 1% (w/v) to
about 30% (w/v).
17. The method of claim 16, wherein the silk solution comprises
silk fibroin at a concentration of about 5% (w/v).
18. The method of any of claims 1-17, wherein the silk microsphere
comprises an active agent.
19. The method of claim 18, wherein the active agent includes a
temperature-sensitive active agent.
20. The method of claim 18 or 19, wherein the active agent is a
therapeutic agent.
21. The method of claim 20, wherein the therapeutic agent is
selected from the group consisting of small organic or inorganic
molecules; saccharides; oligosaccharides; polysaccharides;
biological macromolecules, e.g., peptides, proteins, and peptide
analogs and derivatives; peptidomimetics; nucleic acids; nucleic
acid analogs and derivatives; antibodies and antigen binding
fragments thereof; an extract made from biological materials such
as bacteria, plants, fungi, or animal cells; animal tissues;
naturally occurring or synthetic compositions; and any combinations
thereof.
22. The method of claim 20 or 21, wherein the therapeutic agent
includes bevacizumab, memantine, or a combination thereof.
23. The method of any of claims 18-22, wherein the active agent is
present in the silk microsphere in an amount of about 0.1% (w/w) to
about 50%(w/w).
24. The method of claim 23, wherein the active agent is present in
the silk microsphere in an amount of about 1%(w/w) to about
30%(w/w).
25. The method of any of claims 18-24, wherein the active agent is
present in the silk solution.
26. The method of any of claims 1-25, wherein the silk microsphere
comprises silk in an amount of about 30%(w/w) to about 100%(w/w),
of the total weight of the microsphere.
27. The method of any of claims 1-26, wherein the silk solution
further comprises an additive.
28. The method of claim 27, wherein a weight ratio of the additive
to silk in the silk solution is about 1:100 to about 100:1.
29. The method of claim 27 or 28, wherein the weight ratio of the
additive to silk in the silk solution is about 1:10 to about
10:1.
30. The method of any of claims 27-29, wherein the additive is
selected from the group consisting of a biopolymer, a porogen, a
magnetic particle, a plasticizer, a detection label, and any
combinations thereof.
31. The method of any of claims 27-30, wherein the additive is a
plasticizer.
32. The method of claim 30 or 31, wherein the plasticizer induces
formation of beta-sheet crystalline structure of fibroin in the
silk.
33. The method of any of claims 30-32, wherein the plasticizer is
selected from the group consisting of glycerol, polyvinyl alcohol,
collagen, gelatin, alginate, chitosan, hyaluronic acid,
polyethylene glycol, polyethylene oxide, and any combinations
thereof.
34. The method of any of claims 1-33, further comprising subjecting
the silk microsphere to a post-treatment.
35. The method of claim 34, wherein the post-treatment further
induces formation of beta-sheet crystalline structure of fibroin in
the silk microsphere.
36. The method of any of claims 34-35, wherein the post-treatment
is selected from the group consisting of alcohol immersion, water
vapor annealing, heat annealing, and any combinations thereof.
37. The method of any of claims 34-36, wherein the silk microsphere
prior to the post-treatment has a water solubility of less than
50%.
38. The method of any of claims 34-37, wherein the silk microsphere
prior to the post-treatment has a water solubility of less than
30%.
39. The method of any of claims 1-38, wherein the silk microsphere
has a size of about 10 .mu.m to about 1000 .mu.m.
40. The method of any of claims 1-39, wherein the silk microsphere
has a size of about 50 .mu.m to about 100 .mu.m.
41. The method of any of claims 4-40, wherein the atomization
comprises using a spray nozzle system of a droplet generator.
42. The method of any of claims 4-41, wherein the atomization
comprises syringe extrusion, coaxial air flow method, mechanical
disturbance method, electrostatic force method, or electrostatic
bead generator method.
43. The method of any of claims 4-42, wherein the atomization
comprises spraying the silk solution through a nozzle of an air
driven droplet generating encapsulation unit.
44. The method of any of claims 1-43, wherein a shape or a size of
the silk microsphere is varied by varying one or more parameters
selected from the group consisting of nozzle diameter; flow rate of
the spray; pressure of the spray; distance of the container
collecting the silk microsphere from the nozzle; concentration of
the silk solution; power of sonication waves; sonication treatment
time; and any combinations thereof.
45. A silk microsphere prepared using the method of any of claims
1-44.
46. The silk microsphere of claim 45, wherein the silk microsphere
releases at least about 5% of the active agent loaded therein over
a period of at least about 10 days.
47. A pharmaceutical composition comprising the silk microsphere of
any of claims 45-46 and a pharmaceutically acceptable
excipient.
48. The composition of claim 47, wherein the composition is
formulated to be injectable.
49. A method of sustained delivery in vivo of a therapeutic agent
comprising administering the pharmaceutical composition of any of
claims 47-48 to a subject in need thereof.
50. A composition comprising a silk microsphere having a size of
about 10 .mu.m to about 2000 .mu.m.
51. The composition of claim 50, wherein the size of the silk
microsphere is about 30 .mu.m to about 1000 .mu.m.
52. The composition of claim 50 or 51, wherein the silk microsphere
is water-insoluble.
53. The composition of any of claims 50-52, wherein the
water-insoluble silk microsphere has a beta sheet crystalline
content of at least about 50% or higher.
54. The composition of any of claims 50-53, wherein the silk
microsphere further comprises an active agent.
55. The composition of claim 54, wherein the active agent is
solvent-sensitive and/or temperature-sensitive active agent.
56. The composition of any of claims 50-55, wherein the active
agent is selected from the group consisting of small organic or
inorganic molecules; saccharides; oligosaccharides;
polysaccharides; biological macromolecules, e.g., peptides,
proteins, and peptide analogs and derivatives; peptidomimetics;
nucleic acids; nucleic acid analogs and derivatives; antibodies and
antigen binding fragments thereof; therapeutic agents; an extract
made from biological materials such as bacteria, plants, fungi, or
animal cells; animal tissues; naturally occurring or synthetic
compositions; and any combinations thereof.
57. The composition of claim 56, wherein the therapeutic agent
comprises bevacizumab, memantine, or a combination thereof.
58. The composition of any of claims 54-57, wherein the silk
microsphere comprising the active agent has a release profile of
about 1% release to about 50% release of the total loading of the
active agent over a period of 5 days.
59. The composition of claim 58, wherein the release profile
comprises a sustained release.
60. The composition of claim 59, wherein the release profile
further comprises an immediate release.
61. The composition of any of claims 50-60, wherein the active
agent is present in the silk microsphere in an amount of about 0.1%
(w/w) to about 50%(w/w).
62. The composition of any of claims 50-61, wherein the silk
microsphere comprises silk fibroin in an amount of about 10%(w/w)
to about 100%(w/w), of the total weight of the microsphere.
63. The composition of any of claims 50-62, wherein the silk
microsphere further comprises an additive.
64. The composition of claim 63, wherein a weight ratio of the
additive to silk fibroin in the silk microsphere is about 1:100 to
about 100:1.
65. The composition of claim 63 or 64, wherein the additive is
selected from the group consisting of a biopolymer, a porogen, a
magnetic particle, a plasticizer, a detection label, and any
combinations thereof.
66. The composition of claim 65, wherein the additive comprises a
plasticizer.
67. The composition of claim 66, wherein the plasticizer induces
formation of beta-sheet crystalline structure of fibroin in the
silk.
68. The composition of claim 66 or 67, wherein the plasticizer is
selected from the group consisting of glycerol, polyvinyl alcohol,
collagen, gelatin, alginate, chitosan, hyaluronic acid,
polyethylene glycol, polyethylene oxide, and any combinations
thereof.
69. The composition of claim 68, wherein the additive comprises
glycerol.
70. The composition of claim 69, wherein the ratio of glycerol to
silk fibroin the silk microsphere ranges from about 1:10 to about
10:1.
71. The composition of any of claims 50-70, wherein the composition
is injectable.
72. The composition of any of claims 50-71, wherein the composition
is a pharmaceutical composition.
73. The composition of claim 72, further comprises a
pharmaceutically acceptable excipient.
74. The composition of claim 72 or 73, wherein the pharmaceutical
composition is in a form of a tablet, a capsule, a lozenge, powder,
paste, granules, a liquid, a solution, gel, or any combinations
thereof.
75. The composition of any of claims 50-74, wherein the silk
microsphere is porous.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Application No. 61/623,970 filed
Apr. 13, 2012, the content of which is incorporated herein by
reference in its entirety.
TECHNICAL FIELD
[0003] Provided herein relates to methods and compositions for
preparing a silk microsphere, and uses of the silk microsphere. In
some embodiments, the silk microsphere can be used as a drug
delivery vehicle or reservoir for an active agent such as a
therapeutic agent.
BACKGROUND
[0004] Microspheres with a particle size from 1 to 1000 .mu.m have
been being used as drug delivery vehicles. Compared to nanospheres
(e.g., <1 .mu.m) that can more easily penetrate tissues and
enter cells, microspheres possess the advantage of higher drug
loading capacity due to their larger volumes. Furthermore,
microspheres can also present a more homogeneous distribution of
entrapped drug molecules throughout their matrices, rendering them
more suitable for use as sustained drug release reservoirs. The
entrapment of drug molecules is generally achieved during
microsphere preparation, and the subsequent drug release commonly
occurs once the dry microspheres are hydrated. See, e.g., Chiellini
F. et al. "Micro/nanostructured polymeric systems for biomedical
and pharmaceutical applications." Nanomed (2008) 3:367-93; Ranade V
V et al. "Drug delivery systems." 2nd ed. Boca Raton; CRC Press
(2004). If the microspheres are made from non-degradable materials,
drug release is generally driven by diffusion alone, e.g., due to a
drug concentration gradient from the microspheres to the release
medium. Id. For microspheres prepared from biodegradable materials,
the drug release pathways can include both material degradation and
diffusion (Id.; and Ye M. et al. "Issues in long-term protein
delivery using biodegradable microparticles." J Control Release
(2010)146:241-260).
[0005] However, existing methods for generating microspheres
generally require one or more organic solvents, and/or high
temperatures. Such conditions can cause degradation or inactivation
of an active agent (e.g., a therapeutic agent) during the
encapsulation process, resulting in a decrease in the effective
amount of the active agent available for administration to a
subject. Thus, there is a need for improved methods and
compositions for making a microsphere, such that an active agent
can maintain its bioactivity during the encapsulation process.
SUMMARY
[0006] A low yield of microspheres and drug deactivation due to
high temperatures and/or organic solvent treatments are generally
the main concerns associated with the existing methods for
microsphere production. Thus, these microspheres and/or fabrication
methods may not be suitable for delivery of temperature-sensitive
drugs, and there is a need to develop a novel method for producing
microspheres that is more suitable for drug encapsulation. Provided
herein generally relates to methods of preparing a silk-based
material, the silk-based material resulting therefrom, and uses of
the silk-based material, e.g., for drug delivery. In one
embodiment, the silk-based material is produced in a form of a
microsphere. Thus, methods of preparing a silk microsphere, the
silk microsphere resulting therefrom, and uses of the silk
microsphere, e.g., for drug delivery, are also provided herein. In
some embodiments, a silk-based material (e.g., a silk microsphere)
can be prepared in completely aqueous based solvents, and can thus
avoid or minimize the use of organic solvents or any harsh
chemicals that can degrade and/or deactivate therapeutic agent(s)
loaded therein. In some embodiments, an insoluble silk-based
material can be produced by the method described herein without
further post-treatment with an organic solvent, e.g., methanol. In
some embodiments, the silk-based material need not be exposed to a
high temperature during preparation, thus maintaining bioactivity
of a therapeutic agent encapsulated therein.
[0007] Inventors have developed, in some embodiments, a novel,
inexpensive, quick, simple, all-aqueous method to produce a
beta-sheet crystalline (water-insoluble) and porous silk-based
material. For example, to prepare a silk microsphere, a silk
fibroin solution can be sonicated (e.g., at a frequency of about 10
kHz or higher) to induce formation of beta-sheet structures of
fibroin, and simultaneously form a spray of silk microspheres rich
in beta-sheet crystalline structure. While it may not be necessary,
the silk microsphere can be further freeze-dried to induce a higher
degree of micro/nanoporosity. Further, the inventors have
demonstrated the feasibility of such preparation methods to
encapsulate a therapeutic agent (e.g., bevacizumab or memantine
hydrochloride) in a silk microsphere, its injectability, and its
applications for sustained delivery applications.
[0008] Accordingly, in one aspect, methods of preparing a silk
microsphere are provided herein. The method comprises inducing
formation of beta-sheet structure of fibroin in a silk solution;
and inducing formation of a microsphere from the silk solution.
[0009] The beta-sheet structure of fibroin can be generally formed
in a silk solution by any known methods in the art, e.g., but not
limited to, ultrasonic energy (e.g., by sonication), shear stress,
water immersion, heat treatment, solvent immersion, e.g., methanol
treatment, lyophilization, gas-drying, water annealing, water vapor
annealing, heat annealing, pH reduction (e.g., pH titration and/or
exposing a silk solution to an electric field), or any combinations
thereof. In some embodiments, e.g., where an active agent is
present in the silk solution, it can be less desirable to employ
heat treatment or alcohol treatment, e.g., methanol, to induce
formation of beta sheet structures of fibroin. In some embodiments,
formation of the beta-sheet structure of fibroin in the silk
solution is induced by sonication (or a high frequency of
ultrasound energy), which can be used to simultaneously form or
facilitate the formation of droplets or microspheres from the silk
solution.
[0010] Sonication can be generally performed at a frequency of
about 10 kHz or higher, e.g., at least about 20 kHz, at least about
30 kHz, at least about 40 kHz, at least about 50 kHz, at least
about 60 kHz, at least about 70 kHz, at least about 80 kHz or
higher. In some embodiments, sonication can be performed at a
frequency of about 20 kHz to about 40 kHz. Depending on desired
morphology and/or solubility of the silk microsphere, formation of
beta-sheet structure of fibroin can be induced at any sonication
power output. In one embodiment, the sonication power output can
range from about 1 watt to about 50 watts, or from about 2 watts to
about 20 watts.
[0011] A silk microsphere can be formed from the silk solution,
e.g., by atomization of the silk solution. Exemplary atomization
methods can include, but are not limited to, syringe extrusion,
coaxial air flow method, mechanical disturbance method,
electrostatic force method, electrostatic bead generator method,
spraying, sonication (ultrasonic energy), or any combinations
thereof.
[0012] In one embodiment, atomization of the silk solution to form
a silk microsphere can include spraying, e.g., by a spray nozzle
system of a droplet generator, or through a nozzle of an air driven
droplet generating encapsulation unit. In such embodiments, the
shape and/or size of the silk microsphere can be adjusted by
varying one or more parameters, including, without limitations,
nozzle diameter, flow rate of the spray, pressure of the spray,
distance of the container collecting the silk microsphere from the
nozzle, concentration of the silk solution, power of sonication
waves, sonication treatment time, and any combinations thereof. In
some embodiments, atomization of the silk solution to form a silk
microsphere can comprise ultrasonic spraying.
[0013] In some embodiments, formation of the beta-sheet structure
and the microsphere can be induced simultaneously and/or
concomitantly, e.g., in one single step. By way of example only,
formation of the beta-sheet structure of fibroin and the
microsphere in a silk solution can be induced simultaneously and/or
concomitantly by flowing the silk solution through a flow-through
chamber that can be ultrasonically activated. In such embodiment,
the flow-through chamber can contain a nozzle for droplet
generation.
[0014] A silk microsphere can be prepared in a batch process, a
continuous-flow process, or a combination thereof. In some
embodiments, a silk microsphere can be prepared in a
continuous-flow process. For example, the silk solution can be
flowed (e.g., through a flow-through chamber such as an ultrasonic
atomizer) at rate of about 0.0001 mL/min to about 5 mL/min, or
about 0.001 mL/min to about 5 mL/min, or about 0.05 mL/min to about
0.3 mL/min.
[0015] In some embodiments, the method can further comprise
freezing the silk microsphere. For example, in one embodiment, the
silk microsphere can be collected in a container maintained at a
sub-zero temperature, e.g., a temperature that is sufficient to
immediately freeze the silk microsphere. The container can be
pre-cooled to and/or maintained at the sub-zero temperature by a
cooling agent, e.g., but not limited to, dry ice, liquid
nitrogen.
[0016] To induce a micro- or nano-porous structure in a silk
microsphere, the method can further comprise subjecting the silk
microsphere, e.g., after atomization and optional freezing, to
lyophilization. The lyophilization condition (e.g., pressure and/or
temperature) can affect the porosity and/or pore size of the silk
microsphere. In some embodiments, the silk microsphere can be
subjected to lyophilization at a condition (e.g., pressure and/or
temperature) that yields a porosity of at least about 10% or more
(e.g., at least about 20%, at least about 30% or more). In some
embodiments, the silk microsphere can be subjected to
lyophilization at a condition (e.g., pressure and/or temperature)
that yields a pore size of about 1 nm to about 500 .mu.m, or 10 nm
to about 50 .mu.m.
[0017] A silk solution for use in the method described herein can
comprise fibroin at any concentration, depending on desired
characteristics of the silk microsphere, e.g., drug release profile
and/or its solubility, e.g., in water. In some embodiments, the
silk solution can comprise silk fibroin at a concentration of about
1%(w/v) to about 30%(w/v), or about 1% (w/v) to about 15% (w/v). In
one embodiment, the silk solution can comprise silk fibroin at a
concentration of about 5% (w/v). In some embodiments, the silk
solution can be sericin-depleted.
[0018] In some embodiments, the silk solution can further comprise
one or more additives, e.g., for various desired properties.
Exemplary additives can include, but are not limited to, a
biopolymer, a porogen, a magnetic particle, a plasmonic particle, a
metamaterial, an excipient, a plasticizer, a detection label, and
any combinations thereof. The additive can be present in the silk
solution at any ratio. For example, the total weight ratio of one
or more additives to silk present in the silk solution can range
from about 1:1000 to about 1000:1, or from about 1:100 to about
100:1, or from about 1:10 to about 10:1.
[0019] In some embodiments, the additive added into the silk
solution can include one or more plasticizers, e.g., an agent that
induces formation of beta-sheet crystalline structure in the silk.
In such embodiments, the total weight ratio of one or more
plasticizers to silk present in the silk solution can range from
about 1:20 to about 20:1 or about 1:10 to about 10:1. In some
embodiments, the total weight ratio of one or more plasticizers to
silk present in the silk solution can be about 1:3. Non-limiting
examples of a plasticizer can include glycerol, polyvinyl alcohol,
collagen, gelatin, alginate, chitosan, hyaluronic acid,
polyethylene glycol, polyethylene oxide, and any combinations
thereof. In one embodiment, glycerol is added into the silk
solution, e.g., to induce formation of beta-sheet crystalline
structure in the silk.
[0020] In some embodiments, the silk microsphere described herein
can be used as a drug delivery vehicle and/or reservoir for an
active agent. The silk microsphere can comprise an active agent,
e.g., a temperature-sensitive active agent. The active agent can be
generally present in the silk microsphere in an amount of about
0.01% (w/w) to about 70%(w/w), or about 0.1% (w/w) to about
50%(w/w), or about 1%(w/w) to about 20%(w/w). The active agent can
be present on a surface of the silk microsphere and/or dispersed or
encapsulated in the silk microsphere homogeneously or
heterogeneously or in a gradient. In some embodiments, the active
agent can be added into the silk solution as an additive, prior to
forming the silk microsphere. In some embodiments, the active agent
can be coated on a surface of the silk microsphere after its
formation. In some embodiments, a silk microsphere can be incubated
in a solution of an active agent for a period of time, during which
an amount of the active agent diffuses into the silk
microsphere.
[0021] Depending on various applications of the silk microsphere,
different types of active agents can be included in the silk
microsphere. Without wishing to be bound, for example, the silk
microsphere can comprise one or more therapeutic agents, including
chemotherapeutic agents for treatment of a disease or disorder.
Examples of the therapeutic agent can include, but are not limited
to, small organic or inorganic molecules; saccharides;
oligosaccharides; polysaccharides; biological macromolecules, e.g.,
peptides, proteins, and peptide analogs and derivatives;
peptidomimetics; nucleic acids; nucleic acid analogs and
derivatives; antibodies and antigen binding fragments thereof; an
extract made from biological materials such as bacteria, plants,
fungi, or animal cells; animal tissues; naturally occurring or
synthetic compositions; and any combinations thereof. In one
embodiment, the therapeutic agent included in a silk microsphere
described herein can include bevacizumab, memantine, or a
combination thereof.
[0022] In some embodiments, the method can further comprise
subjecting the silk microsphere to a post-treatment. For example,
while the silk microsphere produced by the methods described herein
are generally water-insoluble or have a low water solubility and
thus does not require additional processing to induce beta-sheet
formation of fibroin, in some embodiments, the silk microsphere can
be subjected to a post-treatment that is generally used to induce
formation of beta-sheet crystalline structure, after the silk
microsphere is formed. Such post-treatment can include, without
limitations, solvent immersion, water annealing, water vapor
annealing, heat annealing, or any combination thereof. In some
embodiments, the method does not comprise solvent immersion, water
annealing, or water vapor annealing after the silk microsphere is
formed, and yet the silk microsphere is water-insoluble (e.g.,
maintaining original shape and volume after hydration, e.g., at
about 37.degree. C. for a period of time, e.g., for at least about
2 hours or longer) or has a lower water solubility (e.g., a water
solubility of less than 50%, less than 30% or lower).
[0023] The beta-sheet crystallinity--and the resulting water
insolubility, and/or the porous structure of the silk microsphere
can be controlled by changing various processing condition
parameters, such as sonication or flow parameters, silk
concentration, the composition and/or condition of the spray
solution, addition of an additive (e.g., a beta-sheet crystallinity
inducing agent such as glycerol), or any combinations thereof.
[0024] In some embodiments, as noted earlier, the silk microsphere
produced by the method described herein does not require a
post-treatment to induce additional formation of beta-sheet
crystalline structure, e.g., solvent immersion, water or water
vapor annealing and/or heat annealing. In some embodiments,
sonication of the silk solution can induce formation of beta-sheet
crystalline structure in an amount sufficient to prepare a silk
microsphere that is completely or partially insoluble in water. For
example, the silk microsphere prior to the beta-sheet
content-inducing post-treatment (e.g., solvent immersion, water or
water vapor annealing and/or heat annealing) can have a water
solubility of less than 50% or less than 30% or lower. In some
embodiments, the silk microsphere prior to the beta-sheet
content-inducing post-treatment (e.g., solvent immersion, water or
water vapor annealing and/or heat annealing) can be water
insoluble.
[0025] In some embodiments, the silk microsphere can have a beta
sheet crystalline content of at least about 10%, at least about
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70% or higher. In some embodiments,
the silk microsphere can have a beta sheet crystalline content of
at least about 20%, at least about 30%, at least about 40%, at
least about 50%, at least about 60%, at least about 70% or higher
without any post-treatment with solvent immersion or water-vapor
annealing. In some embodiments, the silk microsphere can have a
beta sheet crystalline content of at least about 50% or higher
without any post-treatment with solvent immersion or water-vapor
annealing.
[0026] Silk microspheres described herein can be used in any
applications where appropriate. For example, in some embodiments,
the silk microspheres can be used as drug-delivery vehicles. In
some embodiments, the silk microspheres can be used as a filling
material. In some embodiments, the silk microspheres can be used in
a composite material, e.g., silk microspheres encapsulated in a
matrix material, e.g., a silk-based material. Accordingly, another
aspect described herein relates to compositions comprising a silk
microsphere prepared by various embodiments of the methods
described herein. In some embodiments, the composition can be used
for administration of a therapeutic agent. For in vivo
administration, pharmaceutical compositions comprising a silk
microsphere described herein and a pharmaceutically acceptable
excipient are provided. Depending on various administration routes,
in some embodiments, the composition or pharmaceutical composition
can be formulated for injections.
[0027] In some embodiments of any aspects described herein, the
silk microsphere can have a size of about 10 .mu.m to about 1000
.mu.m, or about 50 .mu.m to about 100 .mu.m.
[0028] In some embodiments of any aspects described herein, the
silk microsphere can comprise silk in any amount. For example, the
silk microsphere can comprise silk in an amount of about 10% (w/w)
to about 100% (w/w), about 30% (w/w) to about 100% (w/w), or about
50% (w/w) to about 100% (w/w).
[0029] In some embodiments of any aspects described herein, the
silk microsphere comprising an active agent (e.g., a therapeutic
agent) can provide a sustained release of the active agent. For
example, the silk microsphere comprising an active agent (e.g., a
therapeutic agent) can release at least about 5% of the active
agent loaded therein over a period of at least about 10 days.
[0030] In another aspect, a silk microsphere and a composition
comprising one or more silk microspheres are also provided herein.
For example, provide herein relates to a composition comprising a
silk microsphere having a size of about 10 .mu.m to about 2000
.mu.m. In some embodiments, the silk microsphere is
water-insoluble, e.g., having a beta sheet crystalline sheet
content of at least about 50% or higher. In some embodiments, the
silk microsphere further comprises a solvent-sensitive or
temperature-sensitive active agent. In some embodiments, the silk
microsphere can further comprise an additive as described herein,
e.g., but not limited to glycerol. In some embodiments, the
composition is injectable. In some embodiments, the composition is
a pharmaceutical composition in a form of, e.g., but not limited
to, a tablet, a capsule, lozenge, powder, paste, granules, a
liquid, a solution, a gel, or any combinations thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 is an exemplary schematic of a setup for a
spray-crystallize-freeze-drying (SCFD) process for preparation of a
silk microsphere.
[0032] FIGS. 2A-2D are optical microscope images of silk SCFD
spheres in accordance with one or more embodiments described
herein. FIG. 2A is an optical microscope image of silk SCFD
microspheres before resuspension in water, where the silk SCFD
microspheres were prepared from a 5% (w/v) silk solution at 25%
sonication amplitude with a flow rate of about 0.1 mL/min. FIG. 2B
is an optical microscope image of silk SCFD microspheres before
resuspension in water, where the silk SCFD microspheres were
prepared from a 5% (w/v) silk solution at 25% sonication amplitude
with a flow rate of about 1 mL/min. FIG. 2C is an optical
microscope image of silk SCFD microspheres after resuspension in
water, where the silk SCFD microspheres were prepared from a 5%
(w/v) silk solution at 25% sonication amplitude with a flow rate of
about 0.1 mL/min. FIG. 2D is an optical microscope image of silk
SCFD microspheres after resuspension in water, where the silk SCFD
microspheres were prepared from a 5% (w/v) silk solution at 25%
sonication amplitude with a flow rate of about 1 mL/min. Bar=100
.mu.m.
[0033] FIGS. 3A-3B are optical microscope images of silk/glycerol
SCFD microspheres in accordance with one or more embodiments
described herein. FIG. 3A is an optical microscope image of
silk/glycerol (in a ratio of about 3/1) SCFD spheres before
suspension in water, where the silk/glycerol SCFD spheres were
prepared at 25% sonication amplitude and a flow rate of 0.17
mL/min. FIG. 3B is an optical microscope image of silk/glycerol (in
a ratio of about 3/1) SCFD spheres after suspension in water, where
the silk/glycerol SCFD spheres were prepared at 25% sonication
amplitude and a flow rate of 0.17 mL/min. Bar=100 .mu.m.
[0034] FIGS. 4A-4D are scanning electron microscopy (SEM) images of
silk/glycerol SCFD microspheres with or without memantine in
accordance with one or more embodiments described herein. FIGS. 4A
and 4C are SEM images of silk/glycerol SCFD microspheres without
memantine. FIGS. 4B and 4D are SEM images of silk/glycerol SCFD
microsphere loaded with memantine. FIGS. 4A and 4B were collected
from lyophilized powder. FIGS. 4C and 4D were collected from
resuspended and dried powder. Bar=100 .mu.m.
[0035] FIG. 5 is a line graph showing memantine release from silk
SCFD microspheres having different silk/glycerol ratios. The SG25M
samples contained 25% (w/w) glycerol (silk/glycerol=.about.3/1);
the SG15M samples contained 15% (w/w) glycerol; and the SM samples
contained no glycerol.
[0036] FIG. 6 is a line graph showing bevacizumab release from silk
SCFD microspheres having different silk/glycerol ratios. The SG25A
samples contained 25% (w/w) glycerol (silk/glycerol=3/1); the SG15A
samples contained 15% (w/w) glycerol; and the SA samples contained
no glycerol.
DETAILED DESCRIPTION
[0037] There is a need to develop novel methods for producing
higher yields of drug delivery vehicles or reservoirs, and/or
methods for encapsulating a drug in those vehicles or reservoirs
such that the drug can maintain its bioactivity during the
encapsulating process. Provided herein generally relates to methods
for preparing a silk matrix and uses thereof. In some embodiments,
the silk matrix can be produced in a form of a microsphere. Thus,
methods of preparing a silk microsphere, and uses of the silk
microsphere, e.g., for drug delivery such as sustained release, are
also provided herein. In some embodiments, an insoluble silk matrix
can be produced by the method described herein without further
post-treatment with an organic solvent, e.g., methanol.
Additionally, a silk matrix can be prepared in completely aqueous
based solvents, thus avoiding or minimizing the use of organic
solvents or any harsh chemicals that can degrade or deactivate any
therapeutic agent loaded therein. In other embodiments, the
preparation of the silk matrix does not require a high temperature,
thus allowing bioactivity of a therapeutic agent encapsulated
therein to be maintained. Accordingly, the methods for increasing
an effective amount of a therapeutic agent encapsulated in a silk
composition are also provided herein.
[0038] The inventors have demonstrated, in some embodiments, a
novel, inexpensive, simple, all-aqueous method to produce a
beta-sheet crystalline (water-insoluble) and porous, silk matrix.
For example, to prepare a silk microsphere, a silk fibroin solution
can be sonicated for inducing formation beta-sheet structure of
fibroin therein, which can be simultaneously and/or concomitantly
turned into a spray of silk microsphere rich in beta-sheet
crystalline structure. While it may not be necessary, the silk
microsphere can be further freeze-dried to induce a higher degree
of micro/nanoporosity. Further, the inventors have demonstrated the
feasibility of such preparation methods to encapsulate a
therapeutic agent (e.g., bevacizumab or memantine hydrochloride) in
a silk microsphere, its injectability, and its applications for
sustained delivery applications.
[0039] Accordingly, some embodiments of various aspects described
herein relates to a silk microsphere and a composition comprising
one or more silk microspheres and methods of making the same. For
example, provide herein relates to a composition comprising a silk
microsphere having a size of about 10 .mu.m to about 2000 .mu.m. In
some embodiments, the silk microsphere is water-insoluble, e.g.,
having a beta sheet crystalline sheet content of at least about 50%
or higher. In some embodiments, the silk microsphere further
comprises a solvent-sensitive or temperature-sensitive active
agent. In some embodiments, the silk microsphere can further
comprise an additive as described herein, e.g., but not limited to
glycerol. In some embodiments, the composition is injectable. In
some embodiments, the composition is a pharmaceutical composition
in a form of, e.g., but not limited to, a tablet, a capsule,
lozenge, powder, paste, granules, a liquid, a solution, a gel, or
any combinations thereof. In some embodiments, the silk microsphere
is porous.
Methods for Preparing a Silk-Based Material or Silk Matrix (e.g., a
Silk Microsphere) and Compositions Comprising a Silk
Microsphere
[0040] Accordingly, one aspect described herein relates to methods
of preparing a silk-based material (or silk matrix, which is used
interchangeably herein). The method comprises inducing formation of
beta-sheet structure in a silk solution; and inducing formation of
a silk matrix from the silk solution. In some embodiments,
formation of the beta-sheet structure in a silk solution can be
induced concurrently with formation of the silk matrix from the
silk solution. The silk matrix can include, e.g., but are not
limited to, a particle (including a microsphere and a nanosphere),
a fiber, a rod, a hydrogel, a film, a gel-like or gel particle, and
any combinations thereof.
[0041] Microspheres have been used widely as drug delivery vehicles
in a broad range of biomedical applications. In some embodiments,
the methods described herein can be used to produce a silk
microsphere. Accordingly, provided herein also relates to a method
of preparing a silk microsphere, the method comprising inducing
formation of beta-sheet structure in a silk solution; and inducing
formation of a microsphere from the silk solution.
[0042] As used interchangeably herein, the phrase "silk matrix" or
"silk-based material" generally refers to a matrix including a
microsphere comprising silk. A silk matrix can be present in any
form, including, but not limited to, a particle or a lyophilized
particle (e.g., a nanoparticle or a microparticle), a sphere or a
lyophilized sphere (e.g., a nanosphere or a microsphere), a fiber,
a gel or a gel-like particle, a hydrogel, a film, powder, and any
combinations thereof. In some embodiments, a silk matrix can be
present in a form of a microsphere or a lyophilized microsphere. In
some embodiments, silk can exclude sericin. In some embodiments,
silk can comprise silk fibroin, silk sericin or a combination
thereof. The phrase "silk matrix" or "silk microsphere" can refer
to a matrix or a microsphere in which silk (or silk fibroin)
constitutes at least about 10% (w/w) or more of the total matrix,
including at least at least about 20% (w/w), at least about 30%
(w/w), at least about 40% (w/w), at least about 50%(w/w), at least
about 60%(w/w), at least about 70% (w/w), at least about 80% (w/w),
at least about 90% (w/w), at least about 95% (w/w), up to and
including 100% (w/w) or any percentages between about 30% (w/w) and
about 100% (w/w), of the total matrix. In certain embodiments, the
silk matrix (e.g., a silk microsphere) can be substantially formed
from silk or silk fibroin. In various embodiments, the silk matrix
(e.g., a silk microsphere) can be substantially formed from silk or
silk fibroin comprising at least one active agent.
[0043] Formation of Beta-Sheet Structure:
[0044] As used herein, the phrase "inducing formation of beta-sheet
structure" refers to increasing an amount of beta-sheet structure
(e.g., silk II beta-sheet crystallinity structure) in a silk
solution by at least about 10%, at least about 20%, at least about
30%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90% or higher,
as compared to an original amount of beta-sheet structure present
in the silk solution. In some embodiments, the phrase "inducing
formation of beta-sheet structure" can refer to increasing an
amount of beta-sheet structure in a silk solution by at least about
1-fold, at least about 2-fold, at least about 3-fold, at least
about 4-fold, at least about 5-fold, or higher, as compared to an
original amount of beta sheet structure present in the silk
solution. Methods for determining the structure of silk protein
(e.g., random coil vs. beta-sheet) are well known in the art, e.g.,
but not limited to, circular dichroism.
[0045] In some embodiments, formation of beta-sheet structure in a
silk solution can be induced such that the silk composition (e.g.,
a silk microsphere) formed from the silk solution can become
insoluble, e.g., without any further post-treatment described
herein. By the term "insoluble" is generally meant a silk
composition (e.g., a silk microsphere) completely or partially
insoluble under a specified condition. Generally, solubility of a
substance depends on properties and/or compositions of solvents
(e.g., aqueous vs. non-aqueous solvents, and/or intermolecular
interaction of the substance with a solvent), temperatures,
pressures, or any combinations thereof. For example, a silk
composition (e.g., a silk microsphere) can have a higher solubility
in one solvent than another, and/or it can have a higher solubility
in a solvent at a higher temperature than at a lower temperature in
the same solvent. In some embodiments, a silk composition (e.g., a
silk microsphere) can be completely or partially insoluble in an
aqueous solution at a certain temperature, e.g., ranging from above
0.degree. C. to about room temperature or from about room
temperature to about body temperature of a subject (e.g., about
37.degree. C. for a normal healthy human being, or higher or lower
for other animals). An aqueous solution to which a silk composition
(e.g., a silk microsphere) is exposed can include any fluid that
comprises water, including, but not limited to, water, blood,
interstitial fluid and any other body fluid. In some embodiments, a
silk microsphere is water insoluble, e.g., being able to maintain
original shape and volume after hydration, e.g., at about
37.degree. C., for a period of time, e.g., for at least about 2
hours or longer).
[0046] The term "partially insoluble" as used herein refers to a
silk composition (e.g., a silk microsphere) having a solubility
with respect to a specified condition (e.g., an aqueous solution
such as water or a buffered solution at room temperature) of less
than 60%, less than 50%, less than 40%, less than 30%, less than
20%, less than 10%, less than 5% or lower. In some embodiments, the
silk composition (e.g., a silk microsphere) can have a solubility
of less than 30% in an aqueous solution such as water or a buffered
solution at room temperature. In some embodiments, when the silk
composition (e.g., a silk microsphere) is administered in vivo, the
silk composition (e.g., a silk microsphere) dispersed or
distributed in a body fluid and/or tissue can have a solubility of
less than 60%, less than 50%, less than 40%, less than 30%, less
than 20%, less than 10%, less than 5% or lower. As used herein,
solubility expressed in percentages refers to the maximum amount of
a substance that can be dissolved in .about.100 g solvent to form a
homogenous solution. For example, a silk microsphere having a water
solubility of 30% means that a maximum amount of 30 g of silk
microspheres can be dissolved in 100 g of water to form a
homogenous solution.
[0047] Beta-sheet structure can be formed in a silk solution by any
known methods in the art, e.g., but not limited to, sonication,
shear stress, water immersion, heat treatment, alcohol treatment,
e.g., methanol treatment, pH modulation, or any combinations
thereof. In some embodiments, formation of the beta-sheet structure
in the silk solution is not induced by heat treatment or alcohol
treatment, e.g., methanol.
[0048] In some embodiments, formation of beta-sheet structure in a
silk solution can be induced by sonication, e.g., sonicating a silk
solution comprising silk or silk fibroin at a concentration of
about 0.25%(w/v) to about 50%(w/v), about 0.25%(w/v) to about 30%
(w/v), about 0.5%(w/v) to about 20%(w/v) or about 1% (w/v) to about
15% (w/v). In some embodiments, the silk solution can contain silk
or silk fibroin at a concentration that allows injection, e.g., a
silk concentration of about 0.5% (w/v) to about 10% (w/v). In one
embodiment, the silk solution can comprise silk fibroin at a
concentration of about 3% (w/v) to about 10% (w/v). In one
embodiment, the silk hydrogel can comprise silk fibroin at a
concentration of about 5% (w/v) to about 8% (w/v) to about silk
fibroin. See, e.g., U.S. Pat. App. No. U.S. 2010/0178304 and
International App. No.: WO 2008/150861, the contents of which are
incorporated herein by reference, for methods of inducing
beta-structure formation using sonication.
[0049] Sonication is generally an act of subjecting a substance to
sound (acoustic) wave, e.g., ultrasound. Ultrasound generally spans
the frequency of about 15 kHz to 10 MHz. In accordance with some
embodiments of the methods described herein, the sonication can be
performed at a frequency of about 10 kHz or higher (e.g., 20 kHz or
higher) to induce formation of beta-sheet structure in the silk
solution. In some embodiments, sonication can be performed at a
frequency of about 20 kHz to about 40 kHz to induce formation of
beta-sheet structure in the silk solution. The sonication can be
applied to the silk solution in any fashion including, but not
limited to, continuous mode, pulse mode, and any combination
thereof.
[0050] Depending on desired morphology, solubility of the silk
microsphere, sonication frequency, and/or sonication duration, a
sonication power output of any level can be employed in inducing
formation of beta-sheet structure. In some embodiments, the
sonication power output can range from about 1 watt to about 50
watts, or from about 2 watts to about 20 watts. In one embodiment,
the sonication power output for inducing formation of beta-sheet
structure can vary from about 2 watts to about 20 watts.
[0051] The sonication or ultrasonication treatment of the silk
solution can generally last for a period of time sufficient to
induce formation of a desired amount of beta-sheet structure in the
silk solution, but not so long as to compromise the mechanical
properties of the silk matrix. Typically, depending on the
sonication power output and/or frequency, sonication or
ultrasonication treatment of the silk solution can last from about
5 seconds to about 60 seconds, depending on the silk concentration,
amounts of fibroin in the silk solution, presence of additives, if
any, and other factors appreciated by those of ordinary skill in
the art. For example, the sonication or ultrasonication treatment
can last from about 15 seconds to about 45 seconds. Formation of
beta-sheet structure in the silk solution can generally begin at
the onset of the sonication and/or ultrasonication treatment and
continues for a period of time after the treatment ends.
[0052] In some embodiments, the combination of the sonication
frequency, sonication duration and sonication power output used in
the method of preparing a silk matrix (e.g., a silk microsphere) as
described herein does not generate heat sufficient to degrade or
deactivate any active agent (e.g., therapeutic agent), if any,
encapsulated therein. In such embodiments, the bioactivity of an
active agent (e.g., a therapeutic agent) present in the silk matrix
(e.g., a microsphere) can maintain at least about 30% of its
original bioactivity, including at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 95% or higher, of its original
bioactivity. The phrase "original bioactivity" can refer to the
activity of an active agent when it is initially constituted in a
silk solution prior to processing by the method described
herein.
[0053] While not necessary, the sonication or ultrasonication
treatment can include additional treatments to facilitate formation
of beta-sheet structure in the silk solution. For example, the
additional treatment can include a salt solution. Salt solutions
are known in the art to assist in inducing gelation. In such
embodiments, addition of a salt into a silk solution can reduce the
sonication duration, frequency, and/or power output used to achieve
formation of a desired amount of beta-sheet structure in the silk
solution. Typical salt solutions containing ions of potassium,
calcium, sodium, magnesium, copper, and/or zinc can be used. In
some embodiments, potassium salt solution can be added in the silk
solution for sonication treatment.
[0054] In alternative embodiments, a shear stress can also be
applied to a silk solution during sonication to facilitate
formation of beta-sheet structure in the silk solution. See, e.g.,
International App. No.: WO 2011/005381, the content of which is
incorporated herein by reference for methods of producing
vortex-induced silk fibroin gelation for encapsulation and
delivery. In such embodiments, subjecting the silk solution to both
sonication and shear stress can reduce the sonication duration,
frequency, and/or power output used to achieve formation of a
desired amount of beta-sheet structure in the silk solution.
[0055] Depending on stability of an active agent present in the
silk solution at various pHs, in some embodiments, the pH of the
silk solution prepared for sonication can be modulated. For
example, the pH of the silk solution can be altered by subjecting
the silk solution to an electric field and/or reducing the pH of
the silk solution with an acid. See, e.g., U.S. App. No.: US
2011/0171239, the content of which is incorporated herein by
reference, for details on methods of producing pH-induced silk
gels. In such embodiments, subjecting the silk solution to
sonication in combination with pH control can reduce the sonication
duration, frequency, and/or power output used to achieve formation
of a desired amount of beta-sheet structure in the silk
solution.
[0056] Formation of a Microsphere from the Silk Solution:
[0057] Formation of a microsphere from the silk solution can be
induced by any methods known in the art, e.g., but not limited to,
emulsification, atomization, sedimentation, dispersion and
precipitation methods. In emulsification, for example, the silk
aqueous solution can be mixed in a non-aqueous phase containing an
emulsifier to form emulsion droplets. The solution can then be
gelled with a gelling agent, e.g., a pH-reducing agent or any agent
that can induce silk gelation. In the dispersion method, direct
dispersion of a silk solution in a cross-linking solution e.g., PEG
solution, can lead to formation of microspheres. In the
sedimentation/precipitation method, mixing of a silk-based
ionomeric pair can lead to formation of microspheres (see, e.g.,
International Application No. WO 2011/109691, the content of which
is incorporated herein by reference).
[0058] In some embodiments, a microsphere can be formed from the
silk solution by atomization of the silk solution. Exemplary
atomization methods can include, but are not limited to, syringe
extrusion, coaxial air flow method, mechanical disturbance method,
electrostatic force method, electrostatic bead generator method,
spraying, atomization using a rotary or centrifugal atomizer, air
atomization (e.g., using a spray gun and air pressure), pressure
atomization, vacuum atomization (e.g., by spraying from high
pressure into low pressure zone), ultrasonic atomization,
sonication (ultrasonic energy), and any combinations thereof.
[0059] In air driven atomization, silk solution droplets can be
broken into fine droplets with the aid of air flow pressure. The
air flow pattern can be altered to form coaxial pattern for
formation of uniform microspheres or particles. Coaxial air flow
technique generally uses concentric streams of air which shear the
liquid droplets released from one or more needles.
[0060] Alternatives to the air driven mechanism include
electrostatic field, mechanical disturbance and electrostatic
force. Electrostatic mechanism generally utilizes a potential
difference between a capillary tip such as a nozzle and a flat
counter electrode to reduce the diameter of the droplets by
applying an additional force (i.e., electric force) in the
direction of gravitational force in order to overcome the upward
capillary force of liquid. Without wishing to be bound by theory,
these methods can be used to produce droplets smaller than 100
.mu.m from viscous liquids depending on their conductivity. In
mechanical disturbance method, liquid droplets can be broken into
fine droplets using a mechanical disturbance. Typically, vibrations
including ultrasonic atomization can be as a mechanical disturbance
to produce microspheres. In electrostatic force method,
electrostatic forces can destabilize a viscous jet, where the
electrostatic force can be used to disrupt the liquid surface
instead of a mechanical disturbance.
[0061] Depending on various atomization method, each atomization
conditions can be independently controlled to provide a desired
atomized droplet size, and, in turn, a desired size of a silk
microsphere. These atomization processes are known in the art and
any skilled artisan can readily perform and optimize these
atomization conditions for a silk solution to produce a microsphere
of a desirable size.
[0062] For example, the atomization of a silk solution can produce
a silk microsphere of different size and/or shape by changing
instrumental/process, and/or material parameters. Exemplary
instrumental/process parameters that can be varied include, but are
not limited to, air pressure of a spray, nozzle size (e.g., nozzle
diameter), sonication frequency, atomization power output (e.g.,
sonication power output), flow rate of a spray, height of a nozzle
head (e.g., distance of the nozzle head from a collection bath or
container), atomization duration (e.g., sonication treatment time),
and material parameters that can be varied include, but are not
limited to, concentration and/or viscosity of silk solution, and/or
concentration of a plasticizer, if any.
[0063] In some embodiments, the atomization of the silk solution
can comprise using a spray nozzle system of a droplet generator.
For example, the silk solution can be sprayed using an
encapsulation unit with a desired flow rate and/or air pressure. In
some embodiment, the silk solution can be sprayed through a nozzle
of an air-driven droplet-generating encapsulation unit. In such
embodiments, the silk solution can be sprayed with a flow rate of
about 0.05 ml/hour to about 1000 ml/hour, or about 10 ml/hr to
about 750 ml/hr, or about 25 ml/hr to about 500 ml/hr, or about 50
ml/hr to about 250 ml/hr. In other embodiments, the silk solution
can be sprayed with a flow rate of about 1 ml/hr to about 20 ml/hr
or about 5 ml/hr to about 10 ml/hr.
[0064] In some embodiments, the silk solution is sprayed using a
spray nozzle system of an air-driven droplet generator with an air
pressure ranging from about 0 bar-1 bar, from about 0 bar-500 mbar,
from about 0 mbar-250 mbar, or from about 0 mbar-100 mbar. In some
embodiments, the silk solution can be sprayed with an air pressure
of about 1 bar-500 bars; or about 1 bar-250 bars; or about 5
bars-100 bars, or about 10 bars to about 50 bars.
[0065] In some embodiments, the atomization of the silk solution
can comprise a spray nozzle system of an ultrasonic atomizer.
Ultrasonic atomization generally relies on an electromechanical
device that vibrates at a very high frequency, e.g., at about 20
kHz or higher. A silk solution passing over the vibrating surface
can be turned into droplets by the high-frequency vibration, e.g.,
ultrasonication. In such embodiments, the sonication can be
performed at a frequency of about 20 kHz or higher to form a
microsphere from the silk solution. In some embodiments, the
sonication can be performed at a frequency of about 20 kHz to about
10 MHz to form a microsphere from the silk solution. In some
embodiments, the sonication can be performed at a frequency of
about 20 kHz to about 40 kHz. The sonication can be applied to the
silk solution in any fashion including, but not limited to,
continuous mode, pulse mode, and any combination thereof.
[0066] Depending on desired morphology and/or solubility of the
silk microsphere, sonication frequency and/or duration, a
sonication power output of any level can be generally employed in
atomizing a silk solution. In some embodiments, the sonication
power output can range from about 1 watt to about 50 watts, or from
about 2 watts to about 20 watts. In some embodiments, the
sonication power output can be at least about 1 watt, at least
about 2 watts, at least about 3 watts, at least about 4 watts, at
least about 5 watts, at least about 10 watts, at least about 20
watts, at least about 30 watts, at least about 40 watts, at least
about 50 watts, at least about 60 watts, or more. In one
embodiment, the sonication power output for formation of
microspheres from the silk solution can vary from about 2 watts to
about 20 watts.
[0067] While formation of the beta-sheet structure in the silk
solution and formation of the microsphere from the silk solution
can be performed separately using different methods described
herein, it can be desirable to employ the same method and/or the
same instrument to achieve both purposes concomitantly. For
example, in some embodiments, formation of the beta-sheet structure
can be induced in the silk solution while one or more microspheres
can be concomitantly or concurrently formed from the silk solution.
In one embodiment, atomization and beta-sheet crystalline structure
can be achieved concomitantly one single step using a single
instrument. By way of example only, formation of the beta-sheet
structure and atomization of the silk solution can be performed
concomitantly or concurrently by flowing a silk solution through a
flow-through chamber that can be ultrasonically activated. In such
embodiments, the ultrasonically-activated flow-through chamber can
contain a nozzle for droplet generation, e.g., an ultrasonic
atomizer. Any commercial ultrasonic atomizers known in the art can
be used in some embodiments of the methods described herein,
including SONIFIER.RTM. cell disruptors adapted for atomization,
e.g., equipped with a flow-through horn for atomization and/or
spraying.
[0068] A silk microsphere can be prepared in a batch process, a
continuous-flow process, or a combination thereof. In some
embodiments, a silk microsphere can be prepared in a
continuous-flow process. For example, when sonication is used to
induce beta-sheet structure in the silk solution while
concomitantly or concurrently forming microspheres from the silk
solution, the flow rate of the silk solution can be adjusted to
provide a sufficient residence time of the silk solution under
sonication for inducing a desired amount of beta-sheet structure
(that can at least partly determines solubility) and/or
microspheres of desired size. For example, the silk solution can be
flowed (e.g., through an ultrasonic atomizer or an equivalent
thereof, e.g., a sonicator equipped with a flow-through chamber or
horn) at rate of about 0.0001 mL/min to about 5 mL/min, or about
0.001 mL/min to about 5 mL/min, or about 0.05 mL/min to about 0.3
mL/min. In some embodiments, the silk solution can be flowed (e.g.,
through a flow-through chamber or horn) at a rate of about 0.1
mL/min to about 0.2 mL/min. One skilled in the art can readily
recognize that the flow of silk solution can be accomplished by
various means, including, for example, a diaphragm pump, a
centrifugal pump, a gas-generation pump, a syringe pump, or by any
other suitable means known to those in the art, depending on the
scale of the process.
[0069] After atomization of the silk solution to form silk
microspheres (e.g., by ultrasonic atomization), in some
embodiments, the method can further comprise freezing the silk
microspheres. Means suitable for freezing silk microspheres are
known to a skilled artisan. For example, the silk microsphere can
be frozen by contacting the silk microspheres, directly or
indirectly, with a cooling agent. In one embodiment, the silk
microspheres can be frozen by contacting them directly with a
cooling agent, e.g., but not limited to, a cryogenic fluid such as
liquid nitrogen, and/or dry ice; or alternatively, the silk
microspheres can be collected in a pre-cooled container at a
sub-zero temperature, e.g., a cryogenic temperature, which is cold
enough to immediately freeze the silk microspheres. In one
embodiment, the silk microspheres can be collected in a container,
at least part of the outside surface of which is in contact with a
cryogenic fluid such as liquid nitrogen and/or dry ice. In these
embodiments, the distance between the tip of the spray nozzle and
the bottom of the container can be adjusted to ensure both
immediate freezing of the spray and the spray homogeneity. For
example, the tip of the spray nozzle can be at least about 10 cm,
at least about 20 cm, at least about 30 cm, at least about 40 cm or
more, apart from the bottom of the container.
[0070] In some embodiments, the method of producing a silk
microsphere can further comprise forming a porous structure in the
silk microsphere. Methods for forming pores in a silk matrix are
known in the art, e.g., porogen-leaching method, freeze-drying
method (e.g., lyophilization), and/or gas-forming method. Such
methods are described, e.g., in U.S. Pat. App. Nos.: US
2010/0279112, US 2010/0279112, and U.S. Pat. No. 7,842,780, the
contents of which are incorporated herein by reference in their
entirety.
[0071] In some embodiments, the silk microsphere can be subjected
to lyophilization to induce a high degree of micro- or
nano-porosity within the silk microsphere. In some embodiments, the
silk microsphere can be frozen prior to lyophilization. The
lyophilization condition (e.g., pressure and temperature) can
affect the porosity and/or pore size of the silk microsphere. In
some embodiments, the silk microsphere can be subjected to
lyophilization at a condition (e.g., pressure and/or temperature)
that yields a porosity of at least about 30%, at least about 40%,
at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, or higher. Too high porosity
can yield a silk composition (e.g., a silk microsphere) with lower
mechanical properties, but with faster release of any active agent
encapsulated therein. However, too low porosity can decrease the
release of an active agent encapsulated therein. One of skill in
the art can adjust the porosity accordingly, based on a number of
factors such as, but not limited to, desired release rates,
molecular size and/or diffusion coefficient of the active agent,
and/or concentrations and/or amounts of silk fibroin in a silk
matrix. The term "porosity" as used herein is a measure of void
spaces in a material, e.g., a matrix such as silk fibroin, and is a
fraction of volume of voids over the total volume, as a percentage
between 0 and 100% (or between 0 and 1). Determination of matrix
porosity is well known to a skilled artisan, e.g., using
standardized techniques, such as mercury porosimetry and gas
adsorption, e.g., nitrogen adsorption.
[0072] The porous silk matrix (e.g., a silk microsphere) can have
any pore size, e.g., ranging from about 1 nm to about 1000 .mu.m,
from about 1 nm to about 500 .mu.m, or from about 10 nm to about 50
.mu.m. In some embodiments, the pores of a silk matrix (e.g., a
silk microsphere) can have a size distribution or a size ranging
from about 1 nm to about 1000 nm, from about 10 nm to about 750 nm,
from about 25 nm to about 500 nm, from about 50 nm to about 250 nm.
In other embodiments, the pores of a silk matrix (e.g., a silk
microsphere) can have a size distribution or a size ranging from
about 1 .mu.m to about 1000 .mu.m, from about 5 .mu.m to about 750
.mu.m, from about 10 .mu.m to about 500 .mu.m, from about 25 .mu.m
to about 250 .mu.m, or from about 50 .mu.m to about 100 .mu.m. As
used herein, the term "pore size" refers to a diameter or an
effective diameter of the cross-section of a pore. The term "pore
size" can also refer to an average diameter or an average effective
diameter of the cross-sections of the pores, based on the
measurements of a plurality of pores. The effective diameter of a
cross-section that is not circular equals the diameter of a
circular cross-section that has the same cross-sectional area as
that of the non-circular cross-section. In some embodiments, the
silk fibroin can be swellable when the silk fibroin matrix is
hydrated. The sizes of the pores can then change depending on the
water content in the silk fibroin. The pores can be filled with a
fluid such as water or air.
[0073] The porous silk matrix (e.g., a porous silk microsphere) can
be used as a drug delivery vehicle or reservoir. Accordingly, in
some embodiments, the silk matrix (e.g., a silk microsphere) can
comprise one or more (e.g., one, two, three, four, five or more)
active agents. Exemplary active agents include, but are not limited
to, therapeutic agents, diagnostic agents (e.g., contrast agents),
and any combinations thereof. In some embodiments, the active agent
present in the silk matrix (e.g., a silk microsphere) can include a
labile active agent, e.g., an agent that can undergo chemical,
physical, or biological change, degradation and/or deactivation
after exposure to a specified condition, e.g., high temperatures,
high humidity, light exposure, and any combinations thereof. In
some embodiments, the active agent present in the silk matrix
(e.g., a silk microsphere) can include a temperature-sensitive
active agent, e.g., an active agent that will lose at least about
30% or more, of its original activity or bioactivity, upon exposure
to a temperature of at least about 10.degree. C. or above,
including at least about 15.degree. C. or above, at least about
room temperature or above, or at least about body temperature
(e.g., about 37.degree. C.) or above.
[0074] The active agent can be generally present in the silk matrix
(e.g., a silk microsphere) in an amount of about 0.01% (w/w) to
about 70%(w/w), or about 0.1% (w/w) to about 50%(w/w), or about 1%
(w/w) to about 30%(w/w). The active agent can be present on a
surface of the silk matrix (e.g., a silk microsphere) and/or
encapsulated and dispersed in the silk matrix (e.g., a silk
microsphere) homogeneously or heterogeneously or in a gradient. In
some embodiments, the active agent can be added into the silk
solution, which is then subjected to the methods described herein
for preparing a silk matrix (e.g., a silk microsphere). In some
embodiments, the active agent can be coated on a surface of the
silk matrix (e.g., a silk microsphere). In some embodiments, the
active agent can be loaded in a silk matrix (e.g., a silk
microsphere) by incubating the silk microsphere in a solution of
the active agent for a period of time, during which an amount of
the active agent can diffuse into the silk matrix (e.g., a silk
microsphere), and thus distribute within the silk matrix (e.g., a
silk microsphere).
[0075] In some embodiments, the method of preparing a silk matrix
(e.g., a silk microsphere) can further comprise subjecting the silk
matrix (e.g., a silk microsphere) to a post-treatment, e.g., to
further modify the surface and/or bulk properties of the silk
matrix (e.g., a silk microsphere). In some embodiments, the
post-treatment can include loading the silk matrix (e.g., a silk
microsphere) with an active agent, e.g., by coating a surface of
the silk matrix (e.g., a silk microsphere) with an active agent, or
diffusing an active agent into the silk matrix (e.g., a silk
microsphere).
[0076] In some embodiments, the post-treatment can include
modifying a surface of a silk matrix (e.g., a silk microsphere).
For example, the silk matrix (e.g., a silk microsphere) can be
coated with an active agent as described earlier. Additionally or
alternatively, the silk matrix (e.g., a silk microsphere) can be
coated with a ligand, e.g., a targeting ligand, or a cell-targeting
ligand. As used herein, the term "targeting ligand" refers to any
material or substance which can promote targeting of the silk
matrix to tissues and/or receptors in vivo and/or in vitro. The
targeting ligand can be synthetic, semi-synthetic, or
naturally-occurring. Materials or substances which can serve as
targeting ligands include, for example, proteins, including
antibodies, antibody fragments, hormones, hormone analogues,
glycoproteins and lectins, peptides, polypeptides, amino acids,
sugars, saccharides, including monosaccharides and polysaccharides,
carbohydrates, vitamins, steroids, steroid analogs, hormones,
cofactors, and genetic material, including nucleosides,
nucleotides, nucleotide acid constructs, peptide nucleic acids
(PNA), aptamers, and polynucleotides. Other targeting ligands that
can be used herein include cell adhesion molecules (CAM), among
which are, for example, cytokines, integrins, cadherins,
immunoglobulins and selectin. The silk matrix (e.g., silk
microspheres) can also encompass precursor targeting ligands. A
precursor to a targeting ligand refers to any material or substance
which can be converted to a targeting ligand. Such conversion can
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, and azide groups. The targeting ligand can be covalently
(e.g., cross-linked) or non-covalently linked to the silk matrix
(e.g., silk microsphere). For example, a targeting ligand can be
covalently linked to silk fibroin used for making the silk
matrix.
[0077] In some embodiments, the surface of the silk matrix (e.g., a
silk microsphere) can be modified, e.g., to facilitate the coating
of an active agent or a ligand. Exemplary surface modification of a
silk matrix (e.g., a silk microsphere) can include, but are not
limited to, carbodiimide coupling reaction (see, e.g. U.S. Patent
Application. No. US 2007/0212730), diazonium coupling reaction
(see, e.g., U.S. Patent Application No. US 2009/0232963),
avidin-biotin interaction (see, e.g., International Application
No.: WO 2011/011347). In other embodiments, the silk matrix (e.g.,
a silk microsphere) can be coated with a biocompatible polymer as
described herein, e.g., pegylation with a chemically active or
activated derivatives of the PEG polymer (see, e.g., International
Application No. WO 2010/057142). In some embodiments, the external
surface of a silk matrix (e.g., a silk microsphere) can be
deposited with one or more (e.g., one, two, three, four, five or
more) silk matrix layers. Each silk matrix layer can have a
different composition (e.g., but not limited to, different silk
concentration, different drug and/or concentration). An exemplary
method of stepwise deposition of one or more silk fibroin coatings
around the silk matrix (e.g., a silk microsphere) can be found in
U.S. App. No. US 2009/0202614, the content of which is incorporated
herein by reference.
[0078] Generally, the silk matrix (e.g., a silk microsphere)
produced by the method described herein need not a post-treatment
to further induce formation of beta-sheet crystalline structure of
fibroin in the silk matrix (e.g., a silk microsphere). For example,
sonication of the silk solution can induce formation of beta-sheet
crystalline fibroin sufficient to maintain the silk microsphere
completely or partially insoluble in water. In some embodiments,
the silk matrix (e.g., a silk microsphere) prior to the
beta-sheet-inducing post-treatment (e.g., solvent immersion, water
or water vapor annealing and/or heat annealing) can have a water
solubility of less than 50%, less than 40%, less than 30%, less
than 20%, less than 10%, less than 5%, or lower. In some
embodiments, the silk microsphere prior to the beta-sheet-inducing
post-treatment (e.g., solvent immersion, water or water vapor
annealing and/or heat annealing) can be water-insoluble.
[0079] In some embodiments, the silk microsphere can have a beta
sheet crystalline content of at least about 10%, at least about
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70% or higher. In some embodiments,
the silk microsphere can have a beta sheet crystalline content of
at least about 20%, at least about 30%, at least about 40%, at
least about 50%, at least about 60%, at least about 70% or higher
without any post-treatment with solvent immersion or water-vapor
annealing. In some embodiments, the silk microsphere can have a
beta sheet crystalline content of at least about 50% or higher
without any post-treatment with solvent immersion or water-vapor
annealing.
[0080] In some embodiments, the porous silk microsphere (e.g.,
lyophilized silk microsphere) can have a beta sheet crystalline
content of at least about 20%, at least about 30%, at least about
40%, at least about 50%, at least about 60%, at least about 70% or
higher without any post-treatment with solvent immersion or
water-vapor annealing. In some embodiments, the porous silk
microsphere (e.g., lyophilized silk microsphere) can have a beta
sheet crystalline content of at least about 50% or higher without
any post-treatment with solvent immersion or water-vapor
annealing.
[0081] While not necessary, in some embodiments, the silk matrix
(e.g., a silk microsphere) can be subjected to a post-treatment
that is generally used to induce formation of beta-sheet
crystalline structure of fibroin in the silk matrix (e.g., a silk
microsphere). For example, in some embodiments, the silk matrix
(e.g., a silk microsphere) can be subjected to a post-treatment for
inducing additional formation of beta-sheet crystalline structure
in the silk matrix (e.g., a silk microsphere) to further decrease
the solubility of the silk matrix (e.g., a silk microsphere).
Exemplary post-treatments for inducing formation of beta-sheet
crystalline structure in the silk matrix (e.g., a silk microsphere)
can include, but are not limited to, alcohol immersion, water vapor
annealing, heat annealing, and any combinations thereof. However,
in some embodiments where an active agent is present in the silk
matrix (e.g., a silk microsphere), it can be undesirable to expose
the silk matrix (e.g., a silk microsphere) to an organic solvent
and/or high temperature, due to possibilities of degradation and/or
deactivation of the active agent.
[0082] The beta-sheet crystallinity--and the resulting water
insolubility, and/or the porous structure of the silk microsphere
can be controlled by changing various processing condition
parameters, such as sonication or flow parameters, silk
concentration, the composition and/or condition of the spray
solution, addition of an additive (e.g., a beta-sheet crystallinity
inducing agent such as glycerol), or any combinations thereof.
[0083] Depending on the format and/or material state of the silk
matrix (e.g., a silk microsphere vs. a silk fiber), the silk matrix
can be of any size, ranging from nanometers in width to meters in
length. In some embodiments where the silk matrix is too big in
size for injection, the method can further comprise reducing the
silk matrix into smaller particles, e.g., by grinding, cutting,
and/or crushing. In some embodiments, the silk particles can be of
any size suitable for injection.
[0084] In some embodiments where the silk matrix is a silk particle
or microsphere, the silk particle or microsphere can have a
dimension (e.g., a diameter) of about 0.5 .mu.m to about 2000
.mu.m, about 1 .mu.m to about 2000 .mu.m, about 10 .mu.m to about
1000 .mu.m, about 20 .mu.m to about 800 .mu.m, about 30 .mu.m to
about 500 .mu.m, about 40 .mu.m to about 250 .mu.m, or about 50
.mu.m to about 100 .mu.m. In some embodiments, the silk microsphere
can have a diameter of about 50 .mu.m to about 100 .mu.m. The term
"microsphere" as used herein is not meant to be construed as
limiting the shape of a silk particle to a sphere, but also
encompasses a particle with any shape, e.g., spherical, rod,
elliptical, cylindrical, capsule, or disc. It will be understood by
one of ordinary skill in the art that microspheres usually exhibit
a distribution of particle sizes around the indicated "size." In
some embodiments, the term "size" as used herein refers to the mode
of a size distribution of microspheres, i.e., the value that occurs
most frequently in the size distribution. Methods for measuring the
microsphere size are known to a skilled artisan, e.g., by dynamic
light scattering (such as photo-correlation spectroscopy, laser
diffraction, low-angle laser light scattering (LALLS), and
medium-angle laser light scattering (MALLS)), light obscuration
methods (such as Coulter analysis method), or other techniques
(such as rheology, and light or electron microscopy).
[0085] Accordingly, in another aspect, a silk microsphere and a
composition comprising one or more silk microspheres are also
provided herein. For example, provide herein relates to a
composition comprising a silk microsphere having a size of about 10
.mu.m to about 2000 .mu.m. In some embodiments, the silk
microsphere is water-insoluble, e.g., having a beta sheet
crystalline sheet content of at least about 50% or higher. In some
embodiments, the silk microsphere further can comprise a
solvent-sensitive or temperature-sensitive active agent. In some
embodiments, the silk microsphere can further comprise an additive
as described herein, e.g., but not limited to glycerol. In some
embodiments, the composition is injectable. In some embodiments,
the composition can be a pharmaceutical composition in a form of,
e.g., but not limited to, a tablet, a capsule, lozenge, powder,
paste, granules, a liquid, a solution, a gel, or any combinations
thereof, which is further described below.
[0086] Previous reports have indicated that silk microparticles,
generally having irregular shapes, can be fabricated directly by
milling raw or degummed silk fibers [Ref 6]. These microparticles
were used as an anti-oxidizing agent in cosmetic formulas, or as a
reinforcement additive for 3D porous silk scaffolds in tissue
engineering [Refs. 7,8]. For drug delivery purposes, degummed silk
fibers can be solubilized in an aqueous solution into which drug is
added and mixed with silk. The solution is then processed further
to obtain regenerated silk materials in a variety of formats, such
as films, gels, nanofibers or microspheres [Ref. 4]. However, it
would be desirable to have an even distribution of the drug
molecules in the silk material matrices to enable constant drug
release rates.
[0087] Spray-drying, a widely used method to prepare
microparticles, have been previously reported for preparing silk
microspheres [Refs. 9,10]. The preparation steps for spray-dried
microparticles included nozzle atomization of a silk solution, and
spray drying, both steps requiring high temperatures followed by
cyclonic separation[9,10]. Even these high temperatures could
induce some random coil to beta-sheet transition in the
microspheres and allow them to maintain their spherical shape for
short periods of time (i.e., a few hours) after hydration, they are
not suitable for the delivery of temperature sensitive drugs. A low
yield, especially for hydrophobic polymers, and possible drug
deactivation due to high temperatures and methanol treatment were
the main additional concerns associated with the spray drying
method. A modified spray-drying method to prepare silk microspheres
was previously reported [11], in which instead of using hot air to
dry the silk spray, a vibrating nozzle was used to obtain a spray,
which was directly collected and frozen in a liquid nitrogen
container. The vibrating nozzle was employed at a frequency far
below a typical range of sonication frequency (e.g., 20 kHz-40
kHz). After lyophilization, a subsequent methanol or water vapor
treatment was still necessary to keep the microspheres water
insoluble. Therefore, this reported technique required exposing the
silk microsphere to an organic solvent and thus lacked the
potential benefits of an all-aqueous microsphere preparation method
for drug delivery. In contrast to these existing and conventional
spray-drying methods as previously reported, some embodiments of
the method described herein require neither subjecting a silk
solution to a high temperature nor post-treating a silk microsphere
with methanol or water vapor for maintaining the microsphere
insoluble in water. Yet beta-sheet structures can be formed in the
silk microparticles (e.g., silk microspheres) produced by the
method described herein and allow them to maintain their shapes for
a period of time (e.g., for at least 24 hours or longer) after
hydration.
[0088] Other methods to prepare silk microspheres with about 2
.mu.m average size under mild conditions using phospholipids as
microsphere-forming templates have been previously described in
Refs [12,13]. A method that is based on phase separation between
silk and polyvinyl alcohol (PVA) has been previously described in
Ref. [14], in which PVA was used as the continuous phase to
separate silk droplets in the nano- to micro-scale in the blend
solution, and water-insoluble silk nanospheres (300-400 nm) and
microspheres (10-20 .mu.m average size) could be obtained directly
by rehydration of dried blend films. However, in contrast to some
embodiments of the methods described herein, none of these
previously-reported methods can achieve atomization and beta-sheet
crystalline formation concomitantly in one step, e.g., using a
single instrument.
[0089] For example, in one particular embodiment, the method can
utilize a flow-through sonication horn, through which a silk
solution is passed through. A relatively high silk, beta-sheet
content can be directly induced since the solution is sonicated as
it passes through the horn, and a fine spray of atomized silk
microparticles is obtained at the tip of the horn. The spray can be
collected directly upon exiting the horn in a liquid
nitrogen-cooled flask and optionally lyophilized for at least about
12 hours or longer. Subsequent freeze-drying of the spray can
induce a porous structure in the microspheres with pore sizes in
the nano- to microscale. Since the atomization and beta-sheet
crytalline formation can be achieved concomitantly in one step
using a single instrument, a minimal processing time of less than
24 hours including the lyophilization, and a low consumption of
energy and solvent can be achieved, indicating that the method can
be used for large-scale production of silk microspheres. Further,
the silk microspheres prepared using this particular embodiment of
the method described herein can have average sizes ranging from 50
to 100 .mu.m, which can be larger than the microspheres produced by
the existing methods and thus broaden the available size range and
provide a highly porous structural alternative for silk
microspheres.
Silk Fibroin and Silk Solution for Use in the Method Described
Herein:
[0090] Silk fibroin protein have unique chemical and physical
properties, e.g., tunable degradation rates, controllable
crystallinity due to hydrophobic beta-sheet segments--ideal
diffusion barriers for entrapped drug molecules, an amino acidic
nature that provides an inert microenvironment for drug
encapsulation, as well as an aqueous-based material processing that
is favorable for sensitive drug molecules. Silk-based biomaterials
have been previously reported for their biocompatibility and
biosafety for various in vivo applications, which is comparable
with or superior to other biodegradable materials, such as
collagen, hyaluronic acids, poly-lactic-co-glycolic acid (PLGA)
[Refs. 4,5].
[0091] As used herein, the term "silk fibroin" includes silkworm
fibroin and insect or spider silk protein. See e.g., Lucas et al.,
13 Adv. Protein Chem. 107 (1958). Any type of silk fibroin can be
used according to aspects provided herein. Silk fibroin produced by
silkworms, such as Bombyx mori, is the most common and represents
an earth-friendly, renewable resource. For instance, silk fibroin
used in a silk fibroin fiber can be attained by extracting sericin
from the cocoons of B. mori. Organic silkworm cocoons are also
commercially available. There are many different silks, however,
including spider silk (e.g., obtained from Nephila clavipes),
transgenic silks, genetically engineered silks, such as silks from
bacteria, yeast, mammalian cells, transgenic animals, or transgenic
plants (see, e.g., WO 97/08315; U.S. Pat. No. 5,245,012), and
variants thereof, that can be used. In some embodiments, silk
fibroin can be derived from other sources such as spiders, other
silkworms, bees, and bioengineered variants thereof. In some
embodiments, silk fibroin can be extracted from a gland of silkworm
or transgenic silkworms (see, e.g., WO 2007/098951).
[0092] 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. In one embodiment, 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. In some embodiments, the
extracted silk is dissolved in about 8M-12 M LiBr solution. The
salt is consequently removed using, for example, dialysis.
[0093] 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. In some embodiments,
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) can be used. However, any dialysis system may
be used. The dialysis can be performed for a time period sufficient
to result in a final stock concentration of aqueous silk solution
between about 6% (w/v)-about 30% (w/v). In one embodiment, the
dialysis can be performed for a time period sufficient to result in
a final stock concentration of aqueous silk solution of about 8%
(w/v). In most cases dialysis for 2-12 hours is sufficient. See,
for example, International Application No. WO 2005/012606, the
content of which is incorporated herein by reference.
[0094] 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'l Gakkaishi 1997, 54, 85-92; Nazarov, R. et
al., Biomacromolecules 2004 May-June; 5(3):718-26. For example, an
exemplary organic solvent that can be used to produce a silk
solution includes, but is not limited to,
hexafluoroisopropanol.
[0095] A silk solution subjected to the method of preparing a silk
matrix (e.g., a silk microsphere) described herein can comprise
fibroin at any concentration, depending on desired characteristics
of the silk microsphere, e.g., drug release profile and/or its
solubility, e.g., in water, and/or atomization method. In some
embodiments, the silk solution can comprise silk fibroin at a
concentration of about 0.1% (w/v) to about 30% (w/v), about 0.5%
(w/v) to about 20% (w/v), about 1% (w/v) to about 15% (w/v), or
about 2% (w/v) to about 10% (w/v). In some embodiments, the silk
solution can comprise silk fibroin at a concentration of about 5%
(w/v) to about 8% (w/v). In some embodiments, the silk solution can
comprise silk fibroin at a concentration of about 5% (w/v).
Generally, higher silk concentration can result in faster gelation.
Depending on processing methods such as atomization, a high silk
concentration can potentially clog a spray nozzle. A skilled
artisan can optimize the silk concentration for use in various
atomization methods and/or nozzle sizes.
[0096] In various embodiments, the silk fibroin can be modified for
different applications and/or desired mechanical or chemical
properties (e.g., to facilitate formation of a gradient of a
therapeutic agent in silk fibroin matrices). One of skill in the
art can select appropriate methods to modify silk fibroins, e.g.,
depending on the side groups of the silk fibroins, desired
reactivity of the silk fibroin and/or desired charge density on the
silk fibroin. In one embodiment, modification of silk fibroin can
use the amino acid side chain chemistry, such as chemical
modifications through covalent bonding, or modifications through
charge-charge interaction. Exemplary chemical modification methods
include, but are not limited to, carbodiimide coupling reaction
(see, e.g. U.S. Patent Application. No. US 2007/0212730), diazonium
coupling reaction (see, e.g., U.S. Patent Application No. US
2009/0232963), avidin-biotin interaction (see, e.g., International
Application No.: WO 2011/011347) and pegylation with a chemically
active or activated derivatives of the PEG polymer (see, e.g.,
International Application No. WO 2010/057142). Silk fibroin can
also be modified through gene modification to alter functionalities
of the silk protein (see, e.g., International Application No. WO
2011/006133). For instance, the silk fibroin can be genetically
modified, which can provide for further modification of the silk
such as the inclusion of a fusion polypeptide comprising a fibrous
protein domain and a mineralization domain, which can be used to
form an organic-inorganic composite. See WO 2006/076711. In some
embodiments, the silk fibroin can be genetically modified to be
fused with a protein, e.g., a therapeutic protein. Additionally,
the silk fibroin matrix can be combined with a chemical, such as
glycerol, that, e.g., affects flexibility and/or solubility of the
matrix. See, e.g., WO 2010/042798, Modified Silk films Containing
Glycerol.
[0097] In some embodiments, the silk solution for preparing a silk
matrix (e.g., a silk microsphere) can further comprise one or more
(e.g., one, two, three, four, five or more) additives, e.g., for
various desired properties and/or applications. Exemplary additives
can include, but are not limited to, a biopolymer, a porogen (e.g.,
a salt or polymeric particle), a magnetic particle, a plasmonic
particle, a metamaterial, an excipient, a plasticizer, a detection
label, and any combinations thereof. The additive(s) can be present
in the silk solution at any ratio. For example, the weight ratio of
the additive to silk in the silk solution can range from about
1:1000 to about 1000:1, or from about 1:100 to about 100:1, or from
about 1:10 to about 10:1. In some embodiments, total amount of
additives in the solution can be from about 0.1 wt % to about 70 wt
%, from about 5 wt % to about 60 wt %, from about 10 wt % to about
50 wt %, from about 15 wt % to about 45 wt %, or from about 20 wt %
to about 40 wt %, of the total silk fibroin in the solution.
[0098] In some embodiments, at least one additive added into the
silk solution can include one or more (e.g., one, two, three, four,
five or more) plasticizers, e.g., agent(s) that induce formation of
beta-sheet crystalline structure in the silk. In such embodiments,
the total weight ratio of the plasticizer(s) to silk in the silk
solution can range from about 1:20 to about 20:1 or about 1:10 to
about 10:1. In some embodiments, the total weight ratio of the
plasticizer(s) to silk in the silk solution can be about 1:3. In
some embodiments, the total amount of the plasticizer(s) can be
from about 10 wt % to about 50 wt %, from about 20 wt % to about 40
wt %, or from about 25 wt % to about 35 wt %, of the total silk
fibroin in the solution. Non-limiting examples of a plasticizer can
include glycerol, polyvinyl alcohol, collagen, gelatin, alginate,
chitosan, hyaluronic acid, polyethylene glycol, polyethylene oxide,
and any combinations thereof. In one embodiment, glycerol is added
into the silk solution, e.g., to induce formation of beta-sheet
crystalline structure in the silk. In such embodiments, the weight
ratio of glycerol to silk in the silk solution can range from about
1:10 to about 10:1. In one embodiment, the weight ratio of glycerol
to silk in the silk solution can be about 1:3. State another way,
the amount of glycerol in the solution can be about 20 wt % to
about 40 wt %, or from about 25 wt % to about 35 wt %, of the total
silk fibroin in the solution.
[0099] In some embodiments, the amount of a plasticizer (e.g.,
glycerol) added into the silk solution can be sufficient to induce,
during sonication, formation of a silk II beta-sheet crystallinity
content of at least about 5%, for example, a silk II beta-sheet
crystallinity content of at least about 10%, at least about 20%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, or at least about 95% but not 100% (i.e., all the silk is
present in a silk II beta-sheet conformation), in the silk
solution. In some embodiments, the silk in the silk matrix can be
completely in a silk II beta-sheet conformation after the silk
solution is atomized into a silk microsphere.
[0100] In some embodiments, at least one additive added into the
silk solution for preparing a silk matrix, e.g., a silk
microsphere, can include one or more (e.g., one, two, three, four,
five or more) biopolymers and/or biocompatible polymers. Exemplary
biopolymers and/or biocompatible polymers include, but are not
limited to, a poly-lactic acid (PLA), poly-glycolic acid (PGA),
poly-lactide-co-glycolide (PLGA), polyesters, poly(ortho ester),
poly(phosphazine), polyphosphate ester), polycaprolactone, gelatin,
collagen, fibronectin, keratin, polyaspartic acid, alginate,
chitosan, chitin, hyaluronic acid, pectin, polyhydroxyalkanoates,
dextrans, and polyanhydrides, polyethylene oxide (PEO),
poly(ethylene glycol) (PEG), triblock copolymers, polylysine,
alginate, polyaspartic acid, any derivatives thereof and any
combinations thereof. Other exemplary biocompatible polymers
amenable to use according to the present disclosure include those
described for example in U.S. Pat. No. 6,302,848; No. 6,395,734;
No. 6,127,143; No. 5,263,992; No. 6,379,690; No. 5,015,476; No.
4,806,355; No. 6,372,244; No. 6,310,188; No. 5,093,489; U.S. Pat.
No. 387,413; No. 6,325,810; No. 6,337,198; No. U.S. Pat. No.
6,267,776; No. 5,576,881; No. 6,245,537; No. 5,902,800; and No.
5,270,419, content of all of which is incorporated herein by
reference.
Exemplary Therapeutic Agents and Amounts Thereof in Silk Matrices,
e.g., Microspheres
[0101] Depending on various applications of the silk matrix (e.g.,
a silk microsphere), different types of the active agent can be
present in the silk matrix (e.g., a silk microsphere), e.g., by
encapsulation and/or coating. Without wishing to be bound, for
example, the silk matrix (e.g., a silk microsphere) can comprise
one or more active agents, including, but not limited to,
therapeutic agents, imaging agents or any combinations thereof.
[0102] In some embodiments, one or more imaging agents can be
included in a silk matrix (e.g., a silk microsphere). Examples of
imaging agents can include, but are not limited to, dyes,
fluorescent agents, radiological imaging agents, any art-recognized
contrast agents for imaging tissues and/or organs, and any
combinations thereof. Fluorescent agents are well known in the art.
Examples of fluorescent agents can include, but are not limited to,
fluoresceinisothiocyanato-dextran (FITC-dextran), ruthenium based
dye, or platinum porphyrin, or a mixture thereof.
[0103] As used herein, the term "therapeutic agent" means a
molecule, group of molecules, complex or substance administered to
an organism for diagnostic, therapeutic, preventative medical, or
veterinary purposes. As used herein, the term "therapeutic agent"
includes a "drug" or a "vaccine." This term include externally and
internally administered topical, localized and systemic human and
animal pharmaceuticals, treatments, remedies, nutraceuticals,
cosmeceuticals, biologicals, devices, diagnostics and
contraceptives, including preparations useful in clinical and
veterinary screening, prevention, prophylaxis, healing, wellness,
detection, imaging, diagnosis, therapy, surgery, monitoring,
cosmetics, prosthetics, forensics and the like. This term can also
be used in reference to agriceutical, workplace, military,
industrial and environmental therapeutics or remedies comprising
selected molecules or selected nucleic acid sequences capable of
recognizing cellular receptors, membrane receptors, hormone
receptors, therapeutic receptors, microbes, viruses or selected
targets comprising or capable of contacting plants, animals and/or
humans. This term can also specifically include nucleic acids and
compounds comprising nucleic acids that produce a therapeutic
effect, for example deoxyribonucleic acid (DNA), ribonucleic acid
(RNA), or mixtures or combinations thereof.
[0104] The term "therapeutic agent" also includes an agent that is
capable of providing a local or systemic biological, physiological,
or therapeutic effect in the biological system to which it is
applied. For example, the therapeutic agent can act to control
infection or inflammation, enhance cell growth and tissue
regeneration, control tumor growth, act as an analgesic, promote
anti-cell attachment, and enhance bone growth, among other
functions. Other suitable therapeutic agents can include anti-viral
agents, hormones, antibodies, or therapeutic proteins. Other
therapeutic agents include prodrugs, which are agents that are not
biologically active when administered but, upon administration to a
subject are converted to biologically active agents through
metabolism or some other mechanism. Additionally, a silk matrix
(e.g., a silk microsphere) can contain combinations of two or more
therapeutic agents.
[0105] A therapeutic agent can include a wide variety of different
compounds, including chemical compounds and mixtures of chemical
compounds, e.g., small organic or inorganic molecules; saccharines;
oligosaccharides; polysaccharides; biological macromolecules, e.g.,
peptides, proteins, and peptide analogs and derivatives;
peptidomimetics; antibodies and antigen binding fragments thereof;
nucleic acids; nucleic acid analogs and derivatives; an extract
made from biological materials such as bacteria, plants, fungi, or
animal cells; animal tissues; naturally occurring or synthetic
compositions; and any combinations thereof. In some embodiments,
the therapeutic agent is a small molecule.
[0106] As used herein, the term "small molecule" can refer to
compounds that are "natural product-like," however, the term "small
molecule" is not limited to "natural product-like" compounds.
Rather, a small molecule is typically characterized in that it
contains several carbon-carbon bonds, and has a molecular weight of
less than 5000 Daltons (5 kDa), preferably less than 3 kDa, still
more preferably less than 2 kDa, and most preferably less than 1
kDa. In some cases it is preferred that a small molecule have a
molecular weight equal to or less than 700 Daltons.
[0107] Exemplary therapeutic agents include, but are not limited
to, those found in Harrison's Principles of Internal Medicine,
13.sup.th Edition, Eds. T. R. Harrison et al. McGraw-Hill N.Y., NY;
Physicians Desk Reference, 50.sup.th Edition, 1997, Oradell N.J.,
Medical Economics Co.; Pharmacological Basis of Therapeutics,
8.sup.th Edition, Goodman and Gilman, 1990; United States
Pharmacopeia, The National Formulary, USP XII NF XVII, 1990, the
complete contents of all of which are incorporated herein by
reference.
[0108] Therapeutic agents include the herein disclosed categories
and specific examples. It is not intended that the category be
limited by the specific examples. Those of ordinary skill in the
art will recognize also numerous other compounds that fall within
the categories and that are useful according to the present
disclosure. Examples include a radiosensitizer, a steroid, a
xanthine, a beta-2-agonist bronchodilator, an anti-inflammatory
agent, an analgesic agent, a calcium antagonist, an
angiotensin-converting enzyme inhibitors, a beta-blocker, a
centrally active alpha-agonist, an alpha-1-antagonist, an
anticholinergic/antispasmodic agent, a vasopressin analogue, an
antiarrhythmic agent, an anti-parkinsonian agent, an
antiangina/antihypertensive agent, an anticoagulant agent, an
antiplatelet agent, a sedative, an ansiolytic agent, a peptidic
agent, a biopolymeric agent, an antineoplastic agent, a laxative,
an antidiarrheal agent, an antimicrobial agent, an antifingal
agent, a vaccine, a protein, or a nucleic acid. In a further
aspect, the pharmaceutically active agent can be coumarin, albumin,
steroids such as betamethasone, dexamethasone, methylprednisolone,
prednisolone, prednisone, triamcinolone, budesonide,
hydrocortisone, and pharmaceutically acceptable hydrocortisone
derivatives; xanthines such as theophylline and doxophylline;
beta-2-agonist bronchodilators such as salbutamol, fenterol,
clenbuterol, bambuterol, salmeterol, fenoterol; antiinflammatory
agents, including antiasthmatic anti-inflammatory agents,
antiarthritis antiinflammatory agents, and non-steroidal
antiinflammatory agents, examples of which include but are not
limited to sulfides, mesalamine, budesonide, salazopyrin,
diclofenac, pharmaceutically acceptable diclofenac salts,
nimesulide, naproxene, acetaminophen, ibuprofen, ketoprofen and
piroxicam; analgesic agents such as salicylates; calcium channel
blockers such as nifedipine, amlodipine, and nicardipine;
angiotensin-converting enzyme inhibitors such as captopril,
benazepril hydrochloride, fosinopril sodium, trandolapril,
ramipril, lisinopril, enalapril, quinapril hydrochloride, and
moexipril hydrochloride; beta-blockers (i.e., beta adrenergic
blocking agents) such as sotalol hydrochloride, timolol maleate,
esmolol hydrochloride, carteolol, propanolol hydrochloride,
betaxolol hydrochloride, penbutolol sulfate, metoprolol tartrate,
metoprolol succinate, acebutolol hydrochloride, atenolol, pindolol,
and bisoprolol fumarate; centrally active alpha-2-agonists such as
clonidine; alpha-1-antagonists such as doxazosin and prazosin;
anticholinergic/antispasmodic agents such as dicyclomine
hydrochloride, scopolamine hydrobromide, glycopyrrolate, clidinium
bromide, flavoxate, and oxybutynin; vasopressin analogues such as
vasopressin and desmopressin; antiarrhythmic agents such as
quinidine, lidocaine, tocainide hydrochloride, mexiletine
hydrochloride, digoxin, verapamil hydrochloride, propafenone
hydrochloride, flecainide acetate, procainamide hydrochloride,
moricizine hydrochloride, and disopyramide phosphate;
antiparkinsonian agents, such as dopamine, L-Dopa/Carbidopa,
selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine,
and bromocryptine; antiangina agents and antihypertensive agents
such as isosorbide mononitrate, isosorbide dinitrate, propranolol,
atenolol and verapamil; anticoagulant and antiplatelet agents such
as Coumadin, warfarin, acetylsalicylic acid, and ticlopidine;
sedatives such as benzodiazapines and barbiturates; ansiolytic
agents such as lorazepam, bromazepam, and diazepam; peptidic and
biopolymeric agents such as calcitonin, leuprolide and other LHRH
agonists, hirudin, cyclosporin, insulin, somatostatin, protirelin,
interferon, desmopressin, somatotropin, thymopentin, pidotimod,
erythropoietin, interleukins, melatonin,
granulocyte/macrophage-CSF, and heparin; antineoplastic agents such
as etoposide, etoposide phosphate, cyclophosphamide, methotrexate,
5-fluorouracil, vincristine, doxorubicin, cisplatin, hydroxyurea,
leucovorin calcium, tamoxifen, flutamide, asparaginase,
altretamine, mitotane, and procarbazine hydrochloride; laxatives
such as senna concentrate, casanthranol, bisacodyl, and sodium
picosulphate; antidiarrheal agents such as difenoxine
hydrochloride, loperamide hydrochloride, furazolidone,
diphenoxylate hdyrochloride, and microorganisms; vaccines such as
bacterial and viral vaccines; antimicrobial agents such as
penicillins, cephalosporins, and macrolides, antifungal agents such
as imidazolic and triazolic derivatives; and nucleic acids such as
DNA sequences encoding for biological proteins, and antisense
oligonucleotides.
[0109] As noted above, any therapeutic agent can be included in a
silk matrix (e.g., a silk microsphere), e.g., by encapsulation
and/or coating. In some embodiments, it is desirable to include in
a silk matrix (e.g., a silk microsphere) materials to promote the
growth of the agent (for biological agents), promote the
functionality of the agent after it is released from the
encapsulation, or increase the agent's ability to survive or retain
its efficacy during the encapsulation period. Materials known to
promote cell growth include cell growth media, such as Dulbecco's
Modified Eagle Medium (DMEM), fetal bovine serum (FBS),
non-essential amino acids and antibiotics, and growth and morphogen
factors such as basic fibroblast growth factor (bFGF), transforming
growth factors (TGFs), Vascular endothelial growth factor (VEGF),
insulin-like growth factor (IGF-I), bone morphogenetic growth
factors (BMPs), nerve growth factors and related proteins.
[0110] Additional options for delivery via the silk matrix (e.g., a
silk microsphere) described herein can include DNA, siRNA,
antisense, plasmids, liposomes and related systems for delivery of
genetic materials; antibodies and antigen binding fragment thereof;
peptides and proteins to active cellular signaling cascades;
peptides and proteins to promote mineralization or related events
from cells; adhesion peptides and proteins to improve gel-tissue
interfaces; antimicrobial peptides; and proteins and related
compounds.
[0111] In some embodiments, the therapeutic agent(s) for use in the
present disclosure include, but are not limited to, those requiring
relatively frequent dosing. For example, those used in the
treatment of chronic disorders or conditions.
[0112] In some embodiments, the therapeutic agent includes
2-[4-[3-[2-(trifluoromethyl)-10H-phenothiazin-10-yl]propyl]piperazin-1-yl-
]ethanol (fluphenazine), 3,5-dimethyltricyclo[3.3.1.1]decan-lamine
(3,5-dimethyladamantan-1-amine, memantine) or memantine chloride.
Fluphenazine is presently available in oral and injectable dosage
forms. Disadvantageously, fluphenazine has an incomplete oral
bioavailability of 40% to 50% (due to extensive first pass
metabolization in the liver) such that its half-life is 15 to 30
hours. Memantine is presently available in oral dosage form as
tablets, capsules or solution, under the brand Namenda by Forest
Labs. In some embodiment, memantine can be administered or included
in the silk matrix (e.g., a silk microsphere) in combination with
one or more cholinesterase inhibitors (e.g., donepezil, razadyne
and rivastigmin).
[0113] In some embodiments, the therapeutic agent includes
bevacizumab (AVASTIN.RTM.), ranibizumab (LUCENTIS.RTM.), or a
combination thereof. In some embodiments, bevacizumab and/or
ranibizumab can be administered or included in the silk matrix
(e.g., a silk microsphere) in combination with one or more
antiangiogenic agents known in the art, e.g., anti-VEGF agents.
[0114] In some embodiments, the therapeutic agent is a cell, e.g. a
biological cell. In such embodiments, the cells can be distributed
within a silk matrix (e.g., a silk microsphere) by incubating the
silk matrix (e.g., a silk microsphere) in a cell suspension, where
the cells can migrate from the suspension into the pores of the
silk matrix (e.g., a silk microsphere). Cells amenable to be
incorporated into the silk matrix (e.g., a silk microsphere)
include, but are not limited to, stem cells (embryonic stem cells,
mesenchymal stem cells, bone-marrow derived stem cells and
hematopoietic stem cells), chrondrocytes progenitor cells,
pancreatic progenitor cells, myoblasts, fibroblasts, keratinocytes,
neuronal cells, glial cells, astrocytes, pre-adipocytes,
adipocytes, vascular endothelial cells, hair follicular stem cells,
endothelial progenitor cells, mesenchymal cells, neural stem cells
and smooth muscle progenitor cells.
[0115] In some embodiments, the cell is a genetically modified
cell. A cell can be genetically modified to express and secrete a
desired compound, e.g. a bioactive agent, a growth factor,
differentiation factor, cytokines, and the like. Methods of
genetically modifying cells for expressing and secreting compounds
of interest are known in the art and easily adaptable by one of
skill in the art.
[0116] Differentiated cells that have been reprogrammed into stem
cells can also be used. For example, human skin cells reprogrammed
into embryonic stem cells by the transduction of Oct3/4, Sox2,
c-Myc and Klf4 (Junying Yu, et. al., Science, 2007, 318, 1917-1920
and Takahashi K. et. al., Cell, 2007, 131, 1-12).
[0117] Cells useful for incorporation into the silk matrix (e.g., a
silk microsphere) can come from any source, for example human, rat
or mouse. Human cells include, but are not limited to, human
cardiac myocytes-adult (HCMa), human dermal fibroblasts-fetal
(HDF-f), human epidermal keratinocytes (HEK), human mesenchymal
stem cells-bone marrow, human umbilical mesenchymal stem cells,
human hair follicular inner root sheath cells, human umbilical vein
endothelial cells (HUVEC), and human umbilical vein smooth muscle
cells (HUVSMC), human endothelial progenitor cells, human
myoblasts, human capillary endothelial cells, and human neural stem
cells.
[0118] Exemplary rat and mouse cells include, but not limited to,
RN-h (rat neurons-hippocampal), RN-c (rat neurons-cortical), RA
(rat astrocytes), rat dorsal root ganglion cells, rat
neuroprogenitor cells, mouse embryonic stem cells (mESC) mouse
neural precursor cells, mouse pancreatic progenitor cells mouse
mesenchymal cells and mouse endodermal cells.
[0119] In some embodiments, tissue culture cell lines can be used
in the silk matrix (e.g., a silk microsphere) described herein.
Examples of cell lines include, but are not limited to, C166 cells
(embryonic day 12 mouse yolk), C6 glioma Cell line, HL1 (cardiac
muscle cell line), AML12 (nontransforming hepatocytes), HeLa cells
(cervical cancer cell line) and Chinese Hamster Ovary cells (CHO
cells).
[0120] An ordinary skill artisan in the art can locate, isolate and
expand such cells. In addition, the basic principles of cell
culture and methods of locating, isolation and expansion and
preparing cells for tissue engineering are described in "Culture of
Cells for Tissue Engineering" Editor(s): Gordana Vunjak-Novakovic,
R. Ian Freshney, 2006 John Wiley & Sons, Inc., and Heath C. A.,
Trends in Biotechnology, 2000, 18, 17-19, content of both of which
is herein incorporated by reference in its entirety.
[0121] Generally, any amount of the therapeutic agent can be
dispersed or encapsulated in the silk matrix, depending on a number
of factors, including, but not limited to, desirable release
profile (e.g., release rates and/or duration), properties (e.g.,
half-life and/or molecular size) and/or potency of the therapeutic
agent, severity of a subject's disease or disorder to be treated,
desirable administration schedule, loading capacity of the silk
matrix, and any combinations thereof. For example, in some
embodiments, a therapeutic agent can be present in a silk matrix
(e.g., about 10 mg of silk microspheres) in an amount of about 1 ng
to about 100 mg, about 500 ng to about 90 mg, about 1 .mu.g to
about 75 mg, about 0.01 mg to about 50 mg, about 0.1 mg to about 50
mg, about 1 mg to about 40 mg, about 5 mg to about 25 mg. In some
embodiments, a therapeutic agent can be present in a silk matrix
(e.g., about 10 mg of silk microspheres) in an amount of about
0.01% (w/w) to about 90% (w/w) of the total weight (i.e., the
combined weight of the silk matrix and the therapeutic agent), for
example, including, about 0.01% (w/w) to about 70% (w/w), about
0.1% (w/w) to about 50% (w/w), about 1% (w/w) to about 30% (w/w),
about 5% (w/w) to about 25% (w/w), or about 7.5% (w/w) to about 20
(w/w) of the total weight. In some embodiments, the therapeutic
agent can be present in a silk matrix in an amount of about 0.5%
(w/w) to about 20% (w/w) of the total weight. In some embodiments,
the therapeutic agent can be present in a silk matrix in an amount
of about 2% (w/w) to about 20% (w/w) of the total weight. In one
embodiment, the therapeutic agent (e.g., bevacizumab, ranibizumab,
or a mixture thereof) can be present in a silk matrix in an amount
of about 1% (w/w) to about 20% (w/w) of the total weight. In one
embodiment, the therapeutic agent (e.g., memantine) can be present
in a silk matrix in an amount of about 0.1% (w/w) to 5% (w/w) of
the total weight.
[0122] Without wishing to be bound by theory, the duration of a
therapeutic effect on a target site to be treated is generally
correlated with how long an amount of the therapeutic agent
delivered to the target site can be maintained at a therapeutically
effective amount. Thus, in some embodiments, a pharmaceutical
composition described herein can comprise a therapeutic agent
dispersed or encapsulated in a silk matrix (e.g., a dosage of silk
microspheres), wherein the therapeutic agent is present in an
amount sufficient to maintain a therapeutically effective amount
thereof delivered to treat a target site, upon administration, over
a specified period of time, e.g., over more than 1 week, or more
than 1 month.
[0123] The term "therapeutically effective amount" as used herein
refers to an amount of a therapeutic agent which is effective for
producing a beneficial or desired clinical result in at least a
sub-population of cells in a subject at a reasonable benefit/risk
ratio applicable to any medical treatment. For example, a
therapeutically effective amount delivered to a target site is
sufficient to, directly or indirectly, produce a statistically
significant, measurable therapeutic effect as defined herein. By
way of example only, the therapeutically effective amount delivered
to a target site for treatment is sufficient to reduce at least one
symptom or marker associated with the disease or disorder to be
treated (e.g., but not limited to, cancer, ocular diseases such as
age-related macular degeneration, or neurodegenerative diseases
such as Alzheimer's disease) by at least about 10%, at least about
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60% or higher, as compared to absence of the
therapeutic agent. In some embodiments, the therapeutically
effective amount delivered to a target site for treatment is
sufficient to reduce at least one symptom or marker associated with
the disease or disorder to be treated (e.g., but not limited to,
cancer, ocular diseases such as age-related macular degeneration,
or neurodegenerative diseases such as Alzheimer's disease) by at
least about 60%, at least about 70%, at least about 80% or higher,
as compared to absence of the therapeutic agent. In some
embodiments, the therapeutically effective amount delivered to a
target site is sufficient to reduce at least one symptom or marker
associated with the disease or disorder to be treated (e.g., but
not limited to, cancer, ocular diseases such as age-related macular
degeneration, or neurodegenerative diseases such as Alzheimer's
disease) by at least about 80%, at least about 90%, at least about
95%, at least about 98%, at least about 99%, up to and including
100%, as compared to absence of the therapeutic agent.
[0124] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art. Generally, a
therapeutically effective amount can vary with the subject's
history, age, condition, sex, as well as the severity and type of
the medical condition in the subject, and administration of other
pharmaceutically active agents. Furthermore, therapeutically
effective amounts will vary, as recognized by those skilled in the
art, depending on the specific disease treated, the route of
administration, the excipient selected, and the possibility of
combination therapy. In some embodiments, the therapeutically
effective amount can be in a range between the ED50 and LD50 (a
dose of a therapeutic agent at which about 50% of subjects taking
it are killed). In some embodiments, the therapeutically effective
amount can be in a range between the ED50 (a dose of a therapeutic
agent at which a therapeutic effect is detected in at least about
50% of subjects taking it) and the TD50 (a dose at which toxicity
occurs at about 50% of the cases). In alternative embodiments, the
therapeutically effective amount can be an amount determined based
on the current dosage regimen of the same therapeutic agent
administered in a non-silk matrix. For example, an upper limit of
the therapeutically effective amount can be based on a
concentration or an amount of the therapeutic agent delivered to a
target site, on the day of administration with the current dosage
of the therapeutic agent in a non-silk matrix; while the lower
limit of the therapeutically effective amount can be based on a
concentration or an amount of the therapeutic agent delivered to a
target site, on the day at which a fresh dosage of the therapeutic
agent in a non-silk matrix is required.
[0125] As used herein, the term "maintain" is used in reference to
sustaining a concentration or an amount of a therapeutic agent
delivered to a target site at least about or above the
therapeutically effective amount over a specified period of time.
In some embodiments, the term "maintain" as used herein can refer
to keeping the concentration or amount of a therapeutic agent at an
essentially constant value over a specified period of time. In some
embodiments, the term "maintain" as used herein can refer to
keeping the concentration or amount of a therapeutic agent within a
range over a specified period of time. For example, the
concentration or amount of a therapeutic agent delivered to a
target site can be maintained within a range between about the ED50
and about the LD50 or between about the ED50 and about the TD50
over a specified period of time. In such embodiments, the
concentration or amount of a therapeutic agent delivered to a
target site can vary with time, but is kept within the
therapeutically effective amount range for at least 90% of the
specified period of time (e.g., at least about 95%, about 98%,
about 99%, up to and including 100%, of the specified period of
time).
[0126] In some embodiments, the therapeutic agent can be present in
an amount sufficient to maintain a therapeutically effective amount
thereof delivered to a target site, upon administration, over a
period of more than 1 week, including, e.g., at least about 2
weeks, at least about 3 weeks, at least about 1 month, at least
about 2 months, at least about 3 months, at least about 6 months,
at least about 12 months or longer. Such amounts of the therapeutic
agent present in a dosage of a silk matrix (e.g., a dosage of silk
microspheres) can be generally smaller, e.g., at least about 10%
smaller, than the amount of the therapeutic agent present in the
current dosage of the treatment regimen (i.e., without silk matrix)
required for producing essentially the same therapeutic effect.
Accordingly, a dosage of silk matrix (e.g., a dosage of silk
microspheres) can comprise the therapeutic agent in an amount which
is less than the amount recommended for one dosage of the
therapeutic agent. For example, if the recommended dosage of the
therapeutic agent is X amount then the silk matrix can comprise a
therapeutic agent in an amount of about 0.9.times., about
0.8.times., about 0.7.times., about 0.6.times., about 0.5.times.,
about 0.4.times., about 0.3.times., about 0.2.times., about
0.1.times. or less. Without wishing to be bound by a theory, this
can allow administering a lower dosage of the therapeutic agent in
a silk matrix to obtain a therapeutic effect which is similar to
when a higher dosage is administered without the silk matrix.
[0127] In some embodiments, an amount of the therapeutic agent
dispersed or encapsulated in a dosage of a silk matrix (e.g., a
dosage of silk microspheres) can be more than the amount generally
recommended for one dosage of the same therapeutic agent
administered for a particular indication. Administration of a
therapeutic agent (e.g., bevacizumab) in solution does not
generally allow controlled and sustained release. Thus, release
rate of a therapeutic agent in solution can generally create a
higher initial burst and/or overall faster release kinetics than
that of the same amount of the therapeutic agent loaded in silk
matrix. However, the silk matrix can act as a depot such that an
amount of the therapeutic agent loaded in a silk matrix can be
higher than the amount generally recommended for one dosage of the
same therapeutic agent and release the therapeutic agent over a
period of time, thus providing a longer therapeutic effect with
lower frequency of administration. Accordingly, if the recommended
dosage of the therapeutic agent is X amount then the silk matrix
can encapsulate a therapeutic agent in an amount of about
1.25.times., about 1.5.times., about 1.75.times., about 2.times.,
about 2.5.times., about 3.times., about 4.times., about 5.times.,
about 6.times., about 7.times., about 8.times., about 9.times.,
about 10.times. or more. Without wishing to be bound by a theory,
this can allow administering the therapeutic agent in a silk matrix
to obtain a therapeutic effect which is similar to one obtained
with multiple administration of the therapeutic agent administered
without the silk matrix described herein.
[0128] In some embodiments, an amount of the therapeutic agent
encapsulated or dispersed in a dosage of the silk matrix (e.g., a
dosage of silk microspheres) can be essentially the same amount
recommended for one dosage of the therapeutic agent. For example,
if the recommended dosage of the therapeutic agent is X amount,
then the silk-based composition can comprise about X amount of the
therapeutic agent. Without wishing to be bound by a theory, this
can allow less frequent administration of the therapeutic agent to
obtain a therapeutic effect over a longer period of time.
[0129] As used herein, the term "sustained delivery" refers to
continual delivery of a therapeutic agent in vivo or in vitro over
a period of time following administration. For example, sustained
release can occur over a period of at least about 3 days, at least
about a week, at least about two weeks, at least about three weeks,
at least about four weeks, at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, at least about 12 months or
longer. In some embodiments, the sustained release can occur over a
period of more than one month or longer. In some embodiments, the
sustained release can occur over a period of at least about three
months or longer. In some embodiments, the sustained release can
occur over a period of at least about six months or longer. In some
embodiments, the sustained release can occur over a period of at
least about nine months or longer. In some embodiments, the
sustained release can occur over a period of at least about twelve
months or longer.
[0130] Sustained delivery of the therapeutic agent in vivo can be
demonstrated by, for example, the continued therapeutic effect of
the agent over time. Alternatively, sustained delivery of the
therapeutic agent can be demonstrated by detecting the presence or
level of the therapeutic agent in vivo over time. The release rate
of a therapeutic agent can be adjusted by a number of factors such
as silk matrix composition and/or concentration, porous property of
the silk matrix, molecular size of the therapeutic agent, and/or
interaction of the therapeutic agent with the silk matrix. For
example, if the therapeutic agent has a higher affinity with the
silk matrix, the release rate is usually slower than the one with a
lower affinity with the silk matrix. Additionally, when a silk
matrix has larger pores, the encapsulated therapeutic agent is
generally released from the silk matrix faster than from a silk
matrix with smaller pores.
[0131] In some embodiments, the therapeutic agent can be present in
an amount to provide a release profile of the therapeutic agent
from the silk matrix such that the amount of the therapeutic agent
delivered to a target site is maintained within a therapeutically
effective amount range over a period of time. In some embodiments,
the therapeutic agent can be present in an amount to provide a
release profile of the therapeutic agent with release rates ranging
from about 0.01 ng/day to about 1000 mg/day, from about 0.1 ng/day
to about 500 mg/day, or from about 1 ng/day to about 250 mg/day
over a period of time. Without wishing to be bound by theory, upon
administration of a therapeutic agent encapsulated or dispersed in
a silk matrix or a composition described herein, there is generally
an initial spike in the amount of the therapeutic agent delivered
to a target site, and then the release rate of the therapeutic
agent from the silk matrix is decreasing over a period of time.
Thus, the therapeutic agent can be released initially at a rate as
high as mg/day, and later released in a slower rate, e.g., in
.mu.g/day or ng/day. Accordingly, in some embodiments, the
therapeutic agent can be present in an amount to provide a release
profile such that daily release of the therapeutic agent can range
from about 1 ng/day to about 1000 mg/day. For example, amount
released can be in a range with a lower limit of from 1 to 1000
(e.g., every integer from 1 to 1000) and upper limit of from 1 to
1000 (e.g. every integer from 1 to 1000), wherein the lower and
upper limit units can be selected independently from ng/day,
.mu.g/day, mg/day, or any combinations thereof.
[0132] In some embodiments, daily release can vary from about 1
.mu.g/day to about 10 mg/day, from about 0.25 .mu.g/day to about
2.5 mg/day, or from about 0.5 .mu.g/day to about 5 mg/day. In some
embodiments, daily release of the therapeutic agent can range from
about 100 ng/day to 1 mg/day, for example, or about 500 ng/day to 5
mg/day, or about 100 .mu.g/day.
[0133] Stated another way, the therapeutic agent can be released
from the silk matrix at a rate such that at least about 5%,
including, e.g., at least about 10%, at least about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90% or
more, of the therapeutic agent initially present in the silk matrix
can be released over a period of about 3 days, about 1 week, about
10 days, about 20 days, about 1 month, about 2 months, about 3
months, about 4 months, about 5 months, about 6 months, about 7
months, about 8 months, about 9 months, about 10 months, about 11
months, about 12 months or longer. In some embodiments, the
therapeutic agent (e.g., bevacizumab) can be released from the silk
matrix at a rate such that about 5-30% of the therapeutic agent
initially present in the silk matrix can be released over a period
of about 3-20 days. In some embodiments, the therapeutic agent
(e.g., memantine) can be released from the silk matrix at a rate
such that about 40-90% of the therapeutic agent initially present
in the silk matrix can be released over a period of about 3-30
days.
[0134] The release profiles of the therapeutic agent from a dosage
of silk matrix (e.g., a dosage of silk microspheres) or a
pharmaceutical composition can be modulated by a number of factors
such as amounts and/or molecular size of the therapeutic agents
loaded in a silk matrix, porosity of the silk matrix, amounts of
silk fibroin in a silk matrix and/or contents of beta-sheet
conformation structures in a silk matrix, binding affinity of the
therapeutic agent to a silk matrix, and any combinations
thereof.
[0135] In addition, silk matrix can stabilize the bioactivity of a
therapeutic agent under a certain condition, e.g., under an in vivo
physiological condition. See, e.g., U.S. Provisional Application
No. 61/477,737, the content of which is incorporated herein by
reference, for additional details on compositions and methods of
stabilization of active agents. Accordingly, in some embodiments,
encapsulating a therapeutic agent in a silk matrix can increase the
in vivo half-life of the therapeutic agent. For example, in vivo
half-life of a therapeutic agent dispersed or encapsulated in a
silk matrix can be increased by at least about 5%, at least about
10%, at least about 15%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 90%, at least about 1-fold, at least about 1.5-folds relative
to the therapeutic agent without the silk matrix. Without wishing
to be bound by theory, an increase in in vivo half-life of a
therapeutic agent dispersed or encapsulated in a silk matrix can
provide a longer therapeutic effect. Stated another way, an
increase in in vivo half-life of a therapeutic agent dispersed or
encapsulated in a silk matrix can allow loading of a smaller amount
of the therapeutic agent for the same duration of therapeutic
effect.
[0136] In some embodiments, at least one therapeutic agent can be
dispersed or encapsulated in the silk matrix. In some embodiments,
at least two or more therapeutic agents can be dispersed or
encapsulated in the silk matrix. The therapeutic agent can be in
any form suitable for a particular method to be used for
encapsulation and/or dispersion. For example, the therapeutic agent
can be in the form of a solid, liquid, or gel. In some embodiments,
the therapeutic agent can be in the form of a powder or a pellet.
In some embodiments, the therapeutic agent can be dispersed or
encapsulated in a silk solution or matrix before forming the silk
matrix. In some embodiments, the therapeutic agent can be dispersed
or encapsulated in a silk solution or matrix after forming the silk
matrix. For example, the therapeutic agent can be dispersed
homogeneously or heterogeneously within the silk matrix, or
dispersed in a gradient, e.g., using the carbodiimide-mediated
modification method described in the U.S. Patent Application No. US
2007/0212730. In some embodiments, the therapeutic agent can be
coated on a surface of the silk matrix, e.g., via diazonium
coupling reaction (see, e.g., U.S. Patent Application No. US
2009/0232963), and/or avidin-biotin interaction (see, e.g.,
International Application No.: WO 2011/011347). In some
embodiments, the therapeutic agent can be encapsulated in the silk
matrix, e.g., by blending the therapeutic agent into a silk
solution before processing into a desired material state, e.g., a
hydrogel, or a microsphere or a nanosphere. In some embodiments,
the therapeutic agent can be present in a form of a fusion protein
with silk protein, e.g., by genetically engineering silk to
generate a fusion protein comprising the therapeutic agent.
[0137] In some embodiments, the therapeutic agent can be dispersed
or encapsulated in a silk matrix after the silk matrix is formed,
e.g., by placing the formed silk matrix in a therapeutic agent
solution and allowing the therapeutic agent diffuse into the silk
matrix over a period of time. In some embodiments, the silk matrix
can be optionally hydrated before loading with the therapeutic
agent. For example, the silk matrix can be incubated in deionized
water until completely hydrated.
Pharmaceutical Compositions and Administration
[0138] In yet another aspect, provided herein is a pharmaceutical
composition comprising one or a plurality of (e.g., two or more)
microspheres described herein, and a pharmaceutically acceptable
excipient. In some embodiments, a pharmaceutical composition can
comprise a plurality (e.g., two or more) of silk microspheres
described herein embedded in a biocompatible polymer as listed
herein. In some embodiments, a pharmaceutical composition can
comprise a plurality (e.g., two or more) of silk microspheres
embedded in a silk hydrogel. The silk hydrogel can be produced by
any methods known in the art. Depending on various administration
routes, in some embodiments, the pharmaceutical composition can be
formulated to be injectable.
[0139] The pharmaceutical composition can be formulated for
administration in solid or liquid form, including those adapted for
the following: (1) oral administration, for example, drenches
(aqueous or non-aqueous solutions or suspensions), lozenges,
dragees, capsules, pills, tablets (e.g., those targeted for buccal,
sublingual, and systemic absorption), boluses, powders, granules,
pastes for application to the tongue; (2) parenteral
administration, for example, by subcutaneous, intramuscular,
intravenous or epidural injection as, for example, a sterile
solution or suspension, or sustained-release formulation; (3)
topical application, for example, as a cream, ointment, or a
controlled-release patch or spray applied to the skin; (4)
intravaginally or intrarectally, for example, as a pessary, cream
or foam; (5) sublingually; (6) ocularly or intraocularly (e.g.,
intravitreous administration); (7) transdermally; (8)
transmucosally; or (9) nasally. Additionally, one or more
therapeutic agents can be implanted into a patient or injected
using a pharmaceutical composition described herein.
[0140] As used here, the term "pharmaceutically acceptable" refers
to those compounds, materials, compositions, and/or dosage forms
which are, within the scope of sound medical judgment, suitable for
use in contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk
ratio.
[0141] As used herein, the term "pharmaceutically acceptable
carrier" refers to a pharmaceutically-acceptable material,
composition or vehicle for administration of a therapeutic agent
and/or imaging agent. Pharmaceutically acceptable carriers include
any and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like which are compatible with the activity of the active agent and
are physiologically acceptable to the subject. Each carrier must be
"acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, methylcellulose, ethyl cellulose,
microcrystalline cellulose and cellulose acetate; (4) powdered
tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as
magnesium stearate, sodium lauryl sulfate and talc; (8) excipients,
such as cocoa butter and suppository waxes; (9) oils, such as
peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,
corn oil and soybean oil; (10) glycols, such as propylene glycol;
(11) polyols, such as glycerin, sorbitol, mannitol and polyethylene
glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate;
(13) agar; (14) buffering agents, such as magnesium hydroxide and
aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water;
(17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol;
(20) pH buffered solutions; (21) polyesters, polycarbonates and/or
polyanhydrides; (22) bulking agents, such as polypeptides and amino
acids (23) serum component, such as serum albumin, HDL and LDL;
(22) C.sub.2-C.sub.12 alchols, such as ethanol; and (23) other
non-toxic compatible substances employed in pharmaceutical
formulations. Wetting agents, coloring agents, release agents,
coating agents, sweetening agents, flavoring agents, perfuming
agents, preservative and antioxidants can also be present in the
formulation. The terms such as "excipient", "carrier",
"pharmaceutically acceptable carrier" or the like are used
interchangeably herein.
[0142] Pharmaceutically-acceptable antioxidants include, but are
not limited to, (1) water soluble antioxidants, such as ascorbic
acid, cysteine hydrochloride, sodium bisulfate, sodium
metabisulfite, sodium sulfite and the like; (2) oil-soluble
antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole
(BHA), butylated hydroxytoluene (BHT), lectithin, propyl gallate,
alpha-tocopherol, and the like; and (3) metal chelating agents,
such as citric acid, ethylenediamine tetraacetic acid (EDTA),
sorbitol, tartaric acid, phosphoric acids, and the like.
[0143] As used herein, the term "administered" refers to the
placement of a pharmaceutical composition into a subject by a
method or route which results in at least partial localization of
the pharmaceutically active agent at a desired site. A
pharmaceutical composition described herein can be administered by
any appropriate route which results in effective treatment in the
subject, i.e. administration results in delivery to a desired
location in the subject where at least a portion of the
pharmaceutically active agent is delivered. Exemplary modes of
administration include, but are not limited to, implant, injection,
infusion, instillation, implantation, or ingestion. "Injection"
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intraventricular, intracapsular,
intraorbital, intracardiac, intradermal, intraperitoneal,
transtracheal, subcutaneous, subcuticular, intraarticular, sub
capsular, subarachnoid, intraspinal, intracerebro spinal,
intraocular (e.g., intravitreous) and intrasternal injection and
infusion.
[0144] In some embodiments, a pharmaceutical composition described
herein can be implanted in a subject. As used herein, the term
"implanted," and grammatically related terms, refers to the
positioning of the pharmaceutical composition in a particular locus
in the subject, either temporarily, semi-permanently, or
permanently. The term does not require a permanent fixation of the
pharmaceutical composition in a particular position or location.
Exemplary in vivo loci include, but are not limited to site of a
wound, trauma or disease.
Method of Use
[0145] In another aspect described herein, the silk matrix (e.g.,
silk microsphere) and/or pharmaceutical composition described
herein can be used in various applications, e.g., but not limited
to, as a filler to fill a void, e.g., a wound, for medical
treatment or for cosmetic applications, or as a carrier to deliver
an active agent, e.g., a therapeutic agent, a diagnostic agent, or
as a reinforcing material, e.g., in a composite.
[0146] In some embodiments, provided herein is a method for imaging
at least one cell (including part of a tissue or an organ) in a
human or an animal subject by administering a diagnostically
effective amount of a pharmaceutical composition comprising a silk
microsphere as described herein. For example, the silk microsphere
can comprise a contrast agent suitable for the imaging method,
e.g., a gadolinium-based contrast agent; a radiocontrast agent such
as iodine or barium compounds; iron oxide, iron platinum, manganese
or any combinations thereof. After administration of the silk
microsphere and/or the pharmaceutical composition described herein,
the body of the subject can be examined with a diagnostic device or
an imaging system, including, but not limited to, X-ray scanner,
magnetic resonance imaging (MRI), and/or computerized axial
tomography (CAT scan).
[0147] A "diagnostically effective amount" refers to the amount of
a silk microsphere or pharmaceutical composition to facilitate a
desired diagnostic result. Diagnostics includes testing that is
related to the in vitro, ex vivo, or in vivo diagnosis of disease
states or biological status (e.g. diabetic, glucose intolerance,
iron deficiency, tumor detection, blood flow, etc.) in mammals, for
example, but not limited to, humans. The diagnostically effective
amount will vary depending upon the specific silk microsphere or
composition used, the dosing regimen, timing of administration, the
subject and disease condition being diagnosed, the weight and age
of the subject, the severity of the disease condition, the manner
of administration and the like, all of which can be determined
readily by one of ordinary skill in the art.
[0148] These imaging methods, as described herein, can be used to
diagnose or monitor treatment for conditions such as, but are not
limited to, brain tumor; tumors of the chest, abdomen or pelvis;
heart problems such as vessel blockage or infarction; diseases of
the liver, such as cirrhosis; diagnosis of other abdominal organs,
including the bile ducts, gallbladder, and pancreatic ducts; cysts
and solid tumors in the kidneys and other parts of the urinary
tract; blockages or enlargements of blood vessels, including the
aorta, renal arteries, and arteries in the legs; tumors and other
abnormalities of the reproductive organs (e.g., uterus, ovaries,
testicles, prostate); causes of pelvic pain in women, such as
fibroids, endometriosis and adenomyosis; suspected uterine
congenital abnormality in women undergoing evaluation for
infertility; breast cancer; and breast implants.
[0149] In some embodiments, provided herein is also a method for
treating a subject with a disease or disorder in a subject by
administering to the subject a therapeutically effective amount of
a silk microsphere or pharmaceutical composition described herein.
In some embodiments, the disease or disorder to be treated include,
but not limited to, chronic diseases which can benefit from a
treatment involving sustained-release drug delivery, for example,
without limitations, cancer, ocular disease such as age-related
macular degeneration, neurodegenerative disease such as Alzheimer's
disease. Additional exemplary chronic diseases include, but are not
limited to, autoimmune disease including autoimmune vasculitis,
cartilage damage, chronic inflammatory polyneuropathy (CIDP),
cystic fibrosis, diabetes (e.g., insulin diabetes), graft vs. host
disease, hemophilia, infection or other disease processes,
inflammatory arthritis, inflammatory bowel disease, inflammatory
conditions resulting from strain, inflammatory joint disease,
lupus, multiple sclerosis, myasthenia gravis, myositis, orthopedic
surgery, osteoarthritis, Parkinson's disease, psioriatic arthritis,
rheumatoid arthritis, sickle cell anemia, sprain, transplant
rejection, trauma, and the like.
[0150] In some embodiments, a therapeutically effective amount of a
silk microsphere comprising an anti-angiogenic agent (e.g., but not
limited to, bevacizumab) or pharmaceutical composition comprising
such silk microsphere can be administered to a subject for
treatment of cancer. Examples of cancers amenable for the treatment
described herein include, but are not limited to, solid tumors
including malignancies (e.g., sarcomas and carcinomas (e.g.,
adenocarcinoma or squamous cell carcinoma)) of the various organ
systems, such as those of brain, lung, breast, lymphoid,
gastrointestinal (e.g., colon), and genitourinary (e.g., renal,
urothelial, or testicular tumors) tracts, pharynx, prostate, and
ovary. Exemplary adenocarcinomas include colorectal cancers,
renal-cell carcinoma, liver cancer, non-small cell carcinoma of the
lung, and cancer of the small intestine. The cancer can be a
carcinoma, a sarcoma, a myeloma, a leukemia, a lymphoma or a mixed
type.
[0151] In some embodiments, a therapeutically effective amount of a
silk microsphere comprising an anti-angiogenic agent (e.g., but not
limited to, bevacizumab, ranibizumab, or a mixture thereof) or
pharmaceutical composition comprising such silk microsphere can be
administered to a subject for treatment of age-related macular
degeneration.
[0152] In some embodiments, a therapeutically effective amount of a
silk microsphere comprising a NMDA receptor antagonist (e.g., but
not limited to, memantine) or pharmaceutical composition comprising
such silk microsphere can be administered to a subject for
treatment of neurodegenerative disease or disorder such as
Alzheimer's disease.
[0153] In some embodiments of the methods described herein can
further comprise selecting a subject diagnosed with or suspected of
having a chronic disease or disorder. A subject suffering from a
chronic disease or disorder can be selected based on manifestation
of at least one symptoms associated with the chronic disease or
disorder.
[0154] In some embodiments, provided herein is a method for
sustained delivery of one or more (e.g., one, two, three, four or
more) therapeutic agents to a target site in a subject in need
thereof by administering to the subject a pharmaceutical
composition comprising a silk matrix or silk microspheres of one or
more therapeutic agents. Without wishing to be bound by theory, the
therapeutic agent can be released daily from the silk matrix (e.g.
silk microsphere) in a therapeutically effective amount as
described earlier. Generally, a therapeutically effective amount
can vary with the subject's history, age, condition, sex, as well
as the severity and type of the medical condition in the subject,
and administration of other agents for treatment. Guidance
regarding the efficacy and dosage which will deliver a
therapeutically effective amount of a compound can be readily
obtained from animal models of a condition to be treated by one of
skill in the art.
[0155] The dosage for the methods of treatment or sustained
delivery can be determined by a physician and adjusted, as
necessary, to suit observed effects of the treatment. Generally,
the therapeutic agents are administered so that the therapeutic
agent is given at a dose from 1 .mu.g/kg to 100 mg/kg, 1 .mu.g/kg
to 50 mg/kg, 1 .mu.g/kg to 20 mg/kg, 1 .mu.g/kg to 10 mg/kg, 1
.mu.g/kg to 1 mg/kg, 100 .mu.g/kg to 100 mg/kg, 100 mg/kg to 50
mg/kg, 100 .mu.g/kg to 20 mg/kg, 100 .mu.g/kg to 10 mg/kg, 100
.mu.g/kg to 1 mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg, 1
mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10
mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg. For antibody compounds,
one preferred dosage is 0.1 mg/kg of body weight (generally 10
mg/kg to 20 mg/kg).
[0156] Without limitations, the method of treatment or sustained
delivery described herein can be used for administering, to a
subject, a therapeutic agent that requires relatively frequent
administration. For example, a therapeutic agent that requires
administration at least once a day, at least once every 2 days, at
least once every 3 days, at least once every 4 days, at least once
every 5 days, at least once every 6 days, at least once every 1
week, at least once every 2 weeks, at least once every 3 weeks, at
least once 1 month, at least once every 2 months, at least once
every three months, for a period of time, for example over a period
of at least one week, at least two weeks, at least three weeks, at
least four weeks, at least one month, at least two months, at least
three months, at least four months, at least five months, at least
six months, at least one years, at least two years or longer.
[0157] By "treatment" is meant delaying or preventing the onset of
such a disorder or reversing, alleviating, ameliorating,
inhibiting, slowing down or stopping the progression, aggravation
or deterioration the progression or severity of such a condition.
In some embodiments, at least one symptom is alleviated by at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, or at least 95% but not
100%, i.e. not a complete alleviation. In some embodiments, at
least one symptom is completely alleviated.
[0158] As used herein, a "subject" can mean a human or an animal.
Examples of subjects include primates (e.g., humans, and monkeys).
Usually the animal is a vertebrate such as a primate, rodent,
domestic animal or game animal. Primates include chimpanzees,
cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus.
Rodents include mice, rats, woodchucks, ferrets, rabbits and
hamsters. Domestic and game animals include cows, horses, pigs,
deer, bison, buffalo, feline species, e.g., domestic cat, and
canine species, e.g., dog, fox, wolf. A patient or a subject
includes any subset of the foregoing, e.g., all of the above, or
includes one or more groups or species such as humans, primates or
rodents. In certain embodiments of the aspects described herein,
the subject is a mammal, e.g., a primate, e.g., a human. The terms,
"patient" and "subject" are used interchangeably herein. A subject
can be male or female. In some embodiments, a subject can be of any
age, including infants.
[0159] In one embodiment, the subject is a mammal. The mammal can
be a human, non-human primate, mouse, rat, dog, rabbit, cat, horse,
or cow, but are not limited to these examples. Mammals other than
humans can be advantageously used as subjects that represent animal
models of treatment of a specific disease or disorder. In addition,
the methods and compositions described herein can be employed in
domesticated animals and/or pets.
Drug Delivery Devices and Kits
[0160] Drug delivery devices and kits, e.g., to facilitate
administering any embodiments of the compositions and/methods of
use are also provided herein. In some embodiments, a drug delivery
device can comprise any embodiment of the composition described
herein. An drug delivery device can exist in any form, e.g., in
some embodiments, the device can be a syringe with an injection
needle, e.g., having a gauge of about 25 to about 34 or of about 27
to about 30. Other examples of a drug delivery device that can be
used to apply the silk matrix (e.g., silk microsphere) and/or the
pharmaceutical composition can include, but are not limited to, a
contact lens, a dropper, a microneedle (e.g., a silk microneedle),
an implant, and any combinations thereof.
[0161] In any embodiment of the drug delivery device, the
therapeutic agent dispersed or encapsulate in a silk matrix can
vary with desirable administration schedule, and/or release
profiles of the therapeutic agent. For example, the therapeutic
agent can be present in a silk matrix in an amount sufficient to
maintain a therapeutically effective amount thereof delivered to a
target site, upon administration, over a period of more than 2
days, including, e.g., more than 3 days, more than 1 week, more
than 2 weeks, more than 3 weeks, more than 1 month, more than 2
months, more than 3 months, more than 4 months, more than 5 months,
more than 6 months, more than 9 months, more than 12 months or
longer. In general, the longer the sustained release of the
therapeutic agent to a target site, the less frequently the
administration needs to be performed. Amounts or dosages of the
therapeutic agent encapsulated or dispersed in a silk matrix as
described in any embodiment of the compositions described herein
can be applicable to any embodiment of the drug delivery device
described herein.
[0162] A kit provided herein can generally comprise at least one
container containing one or more embodiments of the composition
described herein, or at least one drug delivery device in
accordance with any embodiments described herein. In some
embodiments, e.g., where the composition is not provided or
pre-loaded in a delivery device, the kit can further comprise,
e.g., a syringe and an injection needle. In some embodiments, the
kit can further comprise an anesthetic. In some embodiments, the
kit can further an antiseptic agent, e.g., to sterilize an
administration site. In some embodiments, the kit can further
comprise one or more swabs to apply the antiseptic agent onto the
administration site.
[0163] Without limitations, methods of sustained delivery described
herein, drug delivery devices and/or kits can be applicable for
administering, to a subject, a therapeutic agent that requires
relatively frequent administration. For example, a therapeutic
agent that requires administration at least once every three
months, at least once every two months, at least once every week,
at least once daily for a period of time, for example over a period
of at least one week, at least two weeks, at least three weeks, at
least four weeks, at least one month, at least two months, at least
three months, at least four months, at least five months, at least
six months, at least one years, at least two years or longer.
[0164] Embodiments of various aspects described herein can be
defined in any of the following numbered paragraphs: [0165] 1. A
method of preparing a silk microsphere, the method comprising:
[0166] inducing formation of beta-sheet structure of fibroin in a
silk solution; and [0167] inducing formation of a microsphere from
the silk solution. [0168] 2. The method of paragraph 1, wherein
said formation of the beta-sheet structure of fibroin and the
microsphere are induced simultaneously. [0169] 3. The method of
paragraph 1 or 2, wherein said formation of the beta-sheet
structure of fibroin in the silk solution is induced by sonication.
[0170] 4. The method of any of paragraphs 1-3, wherein said
formation of the microsphere from the silk solution is induced by
atomization of the silk solution. [0171] 5. The method of paragraph
2, wherein said formation of the beta-sheet structure of fibroin
and the microsphere are induced simultaneously by flowing the silk
solution through a flow-through chamber that is ultrasonically
activated or an ultrasonic atomizer. [0172] 6. The method of
paragraph 5, wherein the silk solution is flowed through the
flow-through chamber or the ultrasonic atomizer at a flow rate of
about 0.001 mL/min to about 5 mL/min. [0173] 7. The method of
paragraph 6, wherein the silk solution is flowed through the
flow-through chamber or the ultrasonic atomizer at the flow rate of
about 0.05 mL/min to about 0.3 mL/min. [0174] 8. The method of any
of paragraphs 3-7, wherein the sonication is performed at a
frequency of at least about 10 kHz, or about 20 kHz to about 40
kHz. [0175] 9. The method of any of paragraphs 3-8, wherein the
sonication power output ranges from about 1 watt to about 50 watts,
or from about 2 watts to about 20 watts. [0176] 10. The method of
any of paragraphs 1-9, further comprising freezing the silk
microsphere. [0177] 11. The method of paragraph 10, wherein the
silk microsphere can be frozen by exposing the silk microsphere to
a sub-zero temperature. [0178] 12. The method of paragraph 10 or
11, wherein the silk microsphere is exposed to the sub-zero
temperature by collecting the silk microsphere in a container
cooled by a cooling agent. [0179] 13. The method of any of
paragraphs 1-12, further comprising subjecting the silk microsphere
to lyophilization. [0180] 14. The method of any of paragraphs 1-13,
wherein the silk microsphere has a porosity of at least about 30%.
[0181] 15. The method of any of paragraphs 1-14, wherein the silk
microsphere has a pore size of about 1 nm to about 500 .mu.m, or 10
nm to about 50 .mu.m. [0182] 16. The method of any of paragraphs
1-15, wherein the silk solution comprises silk fibroin at a
concentration of about 1% (w/v) to about 30% (w/v). [0183] 17. The
method of paragraph 16, wherein the silk solution comprises silk
fibroin at a concentration of about 5% (w/v). [0184] 18. The method
of any of paragraphs 1-17, wherein the silk microsphere comprises
an active agent. [0185] 19. The method of paragraph 18, wherein the
active agent includes a temperature-sensitive active agent. [0186]
20. The method of paragraph 18 or 19, wherein the active agent is a
therapeutic agent. [0187] 21. The method of paragraph 20, wherein
the therapeutic agent is selected from the group consisting of
small organic or inorganic molecules; saccharides;
oligosaccharides; polysaccharides; biological macromolecules, e.g.,
peptides, proteins, and peptide analogs and derivatives;
peptidomimetics; nucleic acids; nucleic acid analogs and
derivatives; antibodies and antigen binding fragments thereof; an
extract made from biological materials such as bacteria, plants,
fungi, or animal cells; animal tissues; naturally occurring or
synthetic compositions; and any combinations thereof. [0188] 22.
The method of paragraph 20 or 21, wherein the therapeutic agent
includes bevacizumab, memantine, or a combination thereof [0189]
23. The method of any of paragraphs 18-22, wherein the active agent
is present in the silk microsphere in an amount of about 0.1% (w/w)
to about 50%(w/w). [0190] 24. The method of paragraph 23, wherein
the active agent is present in the silk microsphere in an amount of
about 1%(w/w) to about 30%(w/w). [0191] 25. The method of any of
paragraphs 18-24, wherein the active agent is present in the silk
solution. [0192] 26. The method of any of paragraphs 1-25, wherein
the silk microsphere comprises silk in an amount of about 30%(w/w)
to about 100%(w/w), of the total weight of the microsphere. [0193]
27. The method of any of paragraphs 1-26, wherein the silk solution
further comprises an additive. [0194] 28. The method of paragraph
27, wherein a weight ratio of the additive to silk in the silk
solution is about 1:100 to about 100:1. [0195] 29. The method of
paragraph 27 or 28, wherein the weight ratio of the additive to
silk in the silk solution is about 1:10 to about 10:1. [0196] 30.
The method of any of paragraphs 27-29, wherein the additive is
selected from the group consisting of a biopolymer, a porogen, a
magnetic particle, a plasticizer, a detection label, and any
combinations thereof. [0197] 31. The method of any of paragraphs
27-30, wherein the additive is a plasticizer. [0198] 32. The method
of paragraph 30 or 31, wherein the plasticizer induces formation of
beta-sheet crystalline structure of fibroin in the silk. [0199] 33.
The method of any of paragraphs 30-32, wherein the plasticizer is
selected from the group consisting of glycerol, polyvinyl alcohol,
collagen, gelatin, alginate, chitosan, hyaluronic acid,
polyethylene glycol, polyethylene oxide, and any combinations
thereof. [0200] 34. The method of any of paragraphs 1-33, further
comprising subjecting the silk microsphere to a post-treatment.
[0201] 35. The method of paragraph 34, wherein the post-treatment
further induces formation of beta-sheet crystalline structure of
fibroin in the silk microsphere. [0202] 36. The method of any of
paragraphs 34-35, wherein the post-treatment is selected from the
group consisting of alcohol immersion, water vapor annealing, heat
annealing, and any combinations thereof [0203] 37. The method of
any of paragraphs 34-36, wherein the silk microsphere prior to the
post-treatment has a water solubility of less than 50%. [0204] 38.
The method of any of paragraphs 34-37, wherein the silk microsphere
prior to the post-treatment has a water solubility of less than
30%. [0205] 39. The method of any of paragraphs 1-38, wherein the
silk microsphere has a size of about 10 .mu.m to about 1000 .mu.m.
[0206] 40. The method of any of paragraphs 1-39, wherein the silk
microsphere has a size of about 50 .mu.m to about 100 .mu.m. [0207]
41. The method of any of paragraphs 4-40, wherein the atomization
comprises using a spray nozzle system of a droplet generator.
[0208] 42. The method of any of paragraphs 4-41, wherein the
atomization comprises syringe extrusion, coaxial air flow method,
mechanical disturbance method, electrostatic force method, or
electrostatic bead generator method. [0209] 43. The method of any
of paragraphs 4-42, wherein the atomization comprises spraying the
silk solution through a nozzle of an air driven droplet generating
encapsulation unit. [0210] 44. The method of any of paragraphs
1-43, wherein a shape or a size of the silk microsphere is varied
by varying one or more parameters selected from the group
consisting of nozzle diameter; flow rate of the spray; pressure of
the spray; distance of the container collecting the silk
microsphere from the nozzle; concentration of the silk solution;
power of sonication waves; sonication treatment time; and any
combinations thereof. [0211] 45. A silk microsphere prepared using
the method of any of paragraphs 1-44. [0212] 46. The silk
microsphere of paragraph 45, wherein the silk microsphere releases
at least about 5% of the active agent loaded therein over a period
of at least about 10 days. [0213] 47. A pharmaceutical composition
comprising the silk microsphere of any of paragraphs 45-46 and a
pharmaceutically acceptable excipient. [0214] 48. The composition
of paragraph 47, wherein the composition is formulated to be
injectable. [0215] 49. A method of sustained delivery in vivo of a
therapeutic agent comprising administering the pharmaceutical
composition of any of paragraphs 47-48 to a subject in need thereof
[0216] 50. A composition comprising a silk microsphere having a
size of about 10 .mu.m to about 2000 .mu.m. [0217] 51. The
composition of paragraph 50, wherein the size of the silk
microsphere is about 30 .mu.m to about 1000 .mu.m. [0218] 52. The
composition of paragraph 50 or 51, wherein the silk microsphere is
water-insoluble. [0219] 53. The composition of any of paragraphs
50-52, wherein the water-insoluble silk microsphere has a beta
sheet crystalline content of at least about 50% or higher. [0220]
54. The composition of any of paragraphs 50-53, wherein the silk
microsphere further comprises an active agent. [0221] 55. The
composition of paragraph 54, wherein the active agent is
solvent-sensitive and/or temperature-sensitive active agent. [0222]
56. The composition of any of paragraphs 50-55, wherein the active
agent is selected from the group consisting of small organic or
inorganic molecules; saccharides; oligosaccharides;
polysaccharides; biological macromolecules, e.g., peptides,
proteins, and peptide analogs and derivatives; peptidomimetics;
nucleic acids; nucleic acid analogs and derivatives; antibodies and
antigen binding fragments thereof; therapeutic agents; an extract
made from biological materials such as bacteria, plants, fungi, or
animal cells; animal tissues; naturally occurring or synthetic
compositions; and any combinations thereof [0223] 57. The
composition of paragraph 56, wherein the therapeutic agent
comprises bevacizumab, memantine, or a combination thereof [0224]
58. The composition of any of paragraphs 54-57, wherein the silk
microsphere comprising the active agent has a release profile of
about 1% release to about 50% release of the total loading of the
active agent over a period of 5 days. [0225] 59. The composition of
paragraph 58, wherein the release profile comprises a sustained
release. [0226] 60. The composition of paragraph 59, wherein the
release profile further comprises an immediate release. [0227] 61.
The composition of any of paragraphs 50-60, wherein the active
agent is present in the silk microsphere in an amount of about 0.1%
(w/w) to about 50%(w/w). [0228] 62. The composition of any of
paragraphs 50-61, wherein the silk microsphere comprises silk
fibroin in an amount of about 10%(w/w) to about 100%(w/w), of the
total weight of the microsphere. [0229] 63. The composition of any
of paragraphs 50-62, wherein the silk microsphere further comprises
an additive. [0230] 64. The composition of paragraph 63, wherein a
weight ratio of the additive to silk fibroin in the silk
microsphere is about 1:100 to about 100:1. [0231] 65. The
composition of paragraph 63 or 64, wherein the additive is selected
from the group consisting of a biopolymer, a porogen, a magnetic
particle, a plasticizer, a detection label, and any combinations
thereof. [0232] 66. The composition of paragraph 65, wherein the
additive comprises a plasticizer. [0233] 67. The composition of
paragraph 66, wherein the plasticizer induces formation of
beta-sheet crystalline structure of fibroin in the silk. [0234] 68.
The composition of paragraph 66 or 67, wherein the plasticizer is
selected from the group consisting of glycerol, polyvinyl alcohol,
collagen, gelatin, alginate, chitosan, hyaluronic acid,
polyethylene glycol, polyethylene oxide, and any combinations
thereof. [0235] 69. The composition of paragraph 68, wherein the
additive comprises glycerol. [0236] 70. The composition of
paragraph 69, wherein the ratio of glycerol to silk fibroin the
silk microsphere ranges from about 1:10 to about 10:1. [0237] 71.
The composition of any of paragraphs 50-70, wherein the composition
is injectable. [0238] 72. The composition of any of paragraphs
50-71, wherein the composition is a pharmaceutical composition.
[0239] 73. The composition of paragraph 72, further comprises a
pharmaceutically acceptable excipient. [0240] 74. The composition
of paragraph 72 or 73, wherein the pharmaceutical composition is in
a form of a tablet, a capsule, a lozenge, powder, paste, granules,
a liquid, a solution, gel, or any combinations thereof [0241] 75.
The composition of any of paragraphs 50-74, wherein the silk
microsphere is porous.
Some Selected Definitions
[0242] Unless stated otherwise, or implicit from context, the
following terms and phrases include the meanings provided below.
Unless explicitly stated otherwise, or apparent from context, the
terms and phrases below do not exclude the meaning that the term or
phrase has acquired in the art to which it pertains. The
definitions are provided to aid in describing particular
embodiments, and are not intended to limit the claimed invention,
because the scope of the invention is limited only by the claims.
Further, unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the
singular.
[0243] As used herein the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are essential to the invention, yet open to the
inclusion of unspecified elements, whether essential or not.
[0244] The singular terms "a," "an," and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word "or" is intended to include "and" unless the context
clearly indicates otherwise.
[0245] Other than in the operating examples, or where otherwise
indicated, all numbers expressing quantities of ingredients or
reaction conditions used herein should be understood as modified in
all instances by the term "about." The term "about" when used in
connection with percentages may mean.+-.5% of the value being
referred to. For example, about 100 means from 95 to 105.
[0246] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
this disclosure, suitable methods and materials are described
below. The term "comprises" means "includes." The abbreviation,
"e.g." is derived from the Latin exempli gratia, and is used herein
to indicate a non-limiting example. Thus, the abbreviation "e.g."
is synonymous with the term "for example."
[0247] As used herein, the terms "proteins" and "peptides" are used
interchangeably herein to designate a series of amino acid residues
connected to the other by peptide bonds between the alpha-amino and
carboxy groups of adjacent residues. The terms "protein", and
"peptide", which are used interchangeably herein, refer to a
polymer of protein amino acids, including modified amino acids
(e.g., phosphorylated, glycated, etc.) and amino acid analogs,
regardless of its size or function. Although "protein" is often
used in reference to relatively large polypeptides, and "peptide"
is often used in reference to small polypeptides, usage of these
terms in the art overlaps and varies. The term "peptide" as used
herein refers to peptides, polypeptides, proteins and fragments of
proteins, unless otherwise noted. The terms "protein" and "peptide"
are used interchangeably herein when referring to a gene product
and fragments thereof. Thus, exemplary peptides or proteins include
gene products, naturally occurring proteins, homologs, orthologs,
paralogs, fragments and other equivalents, variants, fragments, and
analogs of the foregoing.
[0248] The term "nucleic acids" used herein refers to
polynucleotides such as deoxyribonucleic acid (DNA), and, where
appropriate, ribonucleic acid (RNA), polymers thereof in either
single- or double-stranded form. Unless specifically limited, the
term encompasses nucleic acids containing known analogs of natural
nucleotides, which have similar binding properties as the reference
nucleic acid and are metabolized in a manner similar to naturally
occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence also implicitly encompasses conservatively
modified variants thereof (e.g., degenerate codon substitutions)
and complementary sequences, as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer, et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka, et al., J. Biol. Chem. 260:2605-2608
(1985), and Rossolini, et al., Mol. Cell. Probes 8:91-98 (1994)).
The term "nucleic acid" should also be understood to include, as
equivalents, derivatives, variants and analogs of either RNA or DNA
made from nucleotide analogs, and, single (sense or antisense) and
double-stranded polynucleotides.
[0249] The term "short interfering RNA" (siRNA), also referred to
herein as "small interfering RNA" is defined as an agent which
functions to inhibit expression of a target gene, e.g., by RNAi. An
siRNA can be chemically synthesized, it can be produced by in vitro
transcription, or it can be produced within a host cell. siRNA
molecules can also be generated by cleavage of double stranded RNA,
where one strand is identical to the message to be inactivated. The
term "siRNA" refers to small inhibitory RNA duplexes that induce
the RNA interference (RNAi) pathway. These molecules can vary in
length (generally 18-30 base pairs) and contain varying degrees of
complementarity to their target mRNA in the antisense strand. Some,
but not all, siRNA have unpaired overhanging bases on the 5' or 3'
end of the sense 60 strand and/or the antisense strand. The term
"siRNA" includes duplexes of two separate strands, as well as
single strands that can form hairpin structures comprising a duplex
region.
[0250] The term "shRNA" as used herein refers to short hairpin RNA
which functions as RNAi and/or siRNA species but differs in that
shRNA species are double stranded hairpin-like structure for
increased stability. The term "RNAi" as used herein refers to
interfering RNA, or RNA interference molecules are nucleic acid
molecules or analogues thereof for example RNA-based molecules that
inhibit gene expression. RNAi refers to a means of selective
post-transcriptional gene silencing. RNAi can result in the
destruction of specific mRNA, or prevents the processing or
translation of RNA, such as mRNA.
[0251] The term "enzymes" as used here refers to a protein molecule
that catalyzes chemical reactions of other substances without it
being destroyed or substantially altered upon completion of the
reactions. The term can include naturally occurring enzymes and
bioengineered enzymes or mixtures thereof. Examples of enzyme
families include kinases, dehydrogenases, oxidoreductases, GTPases,
carboxyl transferases, acyl transferases, decarboxylases,
transaminases, racemases, methyl transferases, formyl transferases,
and .alpha.-ketodecarboxylases.
[0252] The term "vaccines" as used herein refers to any preparation
of killed microorganisms, live attenuated organisms, subunit
antigens, toxoid antigens, conjugate antigens or other type of
antigenic molecule that when introduced into a subjects body
produces immunity to a specific disease by causing the activation
of the immune system, antibody formation, and/or creating of a
T-cell and/or B-cell response. Generally vaccines against
microorganisms are directed toward at least part of a virus,
bacteria, parasite, mycoplasma, or other infectious agent.
[0253] As used herein, the term "aptamers" means a single-stranded,
partially single-stranded, partially double-stranded or
double-stranded nucleotide sequence capable of specifically
recognizing a selected non-oligonucleotide molecule or group of
molecules. In some embodiments, the aptamer recognizes the
non-oligonucleotide molecule or group of molecules by a mechanism
other than Watson-Crick base pairing or triplex formation. Aptamers
can include, without limitation, defined sequence segments and
sequences comprising nucleotides, ribonucleotides,
deoxyribonucleotides, nucleotide analogs, modified nucleotides and
nucleotides comprising backbone modifications, branchpoints and
nonnucleotide residues, groups or bridges. Methods for selecting
aptamers for binding to a molecule are widely known in the art and
easily accessible to one of ordinary skill in the art.
[0254] As used herein, the term "antibody" or "antibodies" refers
to an intact immunoglobulin or to a monoclonal or polyclonal
antigen-binding fragment with the Fc (crystallizable fragment)
region or FcRn binding fragment of the Fc region. The term
"antibodies" also includes "antibody-like molecules", such as
portions of the antibodies, e.g., antigen-binding fragments.
Antigen-binding fragments can be produced by recombinant DNA
techniques or by enzymatic or chemical cleavage of intact
antibodies. "Antigen-binding fragments" include, inter alia, Fab,
Fab', F(ab')2, Fv, dAb, and complementarity determining region
(CDR) fragments, single-chain antibodies (scFv), single domain
antibodies, chimeric antibodies, diabodies, and polypeptides that
contain at least a portion of an immunoglobulin that is sufficient
to confer specific antigen binding to the polypeptide. Linear
antibodies are also included for the purposes described herein. The
terms Fab, Fc, pFc', F(ab') 2 and Fv are employed with standard
immunological meanings (Klein, Immunology (John Wiley, New York,
N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations of
Modern Immunology (Wiley & Sons, Inc., New York); and Roitt, I.
(1991) Essential Immunology, 7th Ed., (Blackwell Scientific
Publications, Oxford)). Antibodies or antigen-binding fragments
specific for various antigens are available commercially from
vendors such as R&D Systems, BD Biosciences, e-Biosciences and
Miltenyi, or can be raised against these cell-surface markers by
methods known to those skilled in the art.
[0255] As used herein, the term "Complementarity Determining
Regions" (CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino
acid residues of an antibody variable domain the presence of which
are necessary for antigen binding. Each variable domain typically
has three CDR regions identified as CDR1, CDR2 and CDR3. Each
complementarity determining region may comprise amino acid residues
from a "complementarity determining region" as defined by Kabat
(i.e. about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the
light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102
(H3) in the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (i.e. about residues 26-32
(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain
and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain
variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917
(1987)). In some instances, a complementarity determining region
can include amino acids from both a CDR region defined according to
Kabat and a hypervariable loop.
[0256] The expression "linear antibodies" refers to the antibodies
described in Zapata et al., Protein Eng., 8(10):1057-1062 (1995).
Briefly, these antibodies comprise a pair of tandem Fd segments
(VH--CH1-VH--CH1) which, together with complementary light chain
polypeptides, form a pair of antigen binding regions. Linear
antibodies can be bispecific or monospecific.
[0257] The expression "single-chain Fv" or "scFv" antibody
fragments, as used herein, is intended to mean antibody fragments
that comprise the VH and VL domains of antibody, wherein these
domains are present in a single polypeptide chain. Preferably, the
Fv polypeptide further comprises a polypeptide linker between the
VH and VL domains which enables the scFv to form the desired
structure for antigen binding. (The Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag,
New York, pp. 269-315 (1994)).
[0258] The term "diabodies," as used herein, refers to small
antibody fragments with two antigen-binding sites, which fragments
comprise a heavy-chain variable domain (VH) Connected to a
light-chain variable domain (VL) in the same polypeptide chain
(VH-VL). By using a linker that is too short to allow pairing
between the two domains on the same chain, the domains are forced
to pair with the complementary domains of another chain and create
two antigen-binding sites. (EP 404,097; WO 93/11161; Hollinger et
ah, Proc. Natl. Acad. Sd. USA, P0:6444-6448 (1993)).
[0259] The term "antibiotics" is used herein to describe a compound
or composition which decreases the viability of a microorganism, or
which inhibits the growth or reproduction of a microorganism. As
used in this disclosure, an antibiotic is further intended to
include an antimicrobial, bacteriostatic, or bactericidal agent.
Exemplary antibiotics include, but are not limited to, penicillins,
cephalosporins, penems, carbapenems, monobactams, aminoglycosides,
sulfonamides, macrolides, tetracyclines, lincosides, quinolones,
chloramphenicol, vancomycin, metronidazole, rifampin, isoniazid,
spectinomycin, trimethoprim, sulfamethoxazole, and the like.
[0260] As used herein, the term "antigens" refers to a molecule or
a portion of a molecule capable of being bound by a selective
binding agent, such as an antibody, and additionally capable of
being used in an animal to elicit the production of antibodies
capable of binding to an epitope of that antigen. An antigen may
have one or more epitopes. The term "antigen" can also refer to a
molecule capable of being bound by an antibody or a T cell receptor
(TCR) if presented by MHC molecules. The term "antigen", as used
herein, also encompasses T-cell epitopes. An antigen is
additionally capable of being recognized by the immune system
and/or being capable of inducing a humoral immune response and/or
cellular immune response leading to the activation of B- and/or
T-lymphocytes. This may, however, require that, at least in certain
cases, the antigen contains or is linked to a Th cell epitope and
is given in adjuvant. An antigen can have one or more epitopes (B-
and T-epitopes). The specific reaction referred to above is meant
to indicate that the antigen will preferably react, typically in a
highly selective manner, with its corresponding antibody or TCR and
not with the multitude of other antibodies or TCRs which may be
evoked by other antigens. Antigens as used herein may also be
mixtures of several individual antigens.
[0261] The term "immunogen" refers to any substance, e.g.,
vaccines, capable of eliciting an immune response in an organism.
An "immunogen" is capable of inducing an immunological response
against itself on administration to a subject. The term
"immunological" as used herein with respect to an immunological
response, refers to the development of a humoral (antibody
mediated) and/or a cellular (mediated by antigen-specific T cells
or their secretion products) response directed against an immunogen
in a recipient subject. Such a response can be an active response
induced by administration of an immunogen or immunogenic peptide to
a subject or a passive response induced by administration of
antibody or primed T-cells that are directed towards the immunogen.
A cellular immune response is elicited by the presentation of
polypeptide epitopes in association with Class I or Class II MHC
molecules to activate antigen-specific CD4+ T helper cells and/or
CD8+ cytotoxic T cells. Such a response can also involve activation
of monocytes, macrophages, NK cells, basophils, dendritic cells,
astrocytes, microglia cells, eosinophils or other components of
innate immunity.
[0262] The term "statistically significant" or "significantly"
refers to statistical significance and generally means at least two
standard deviation (2SD) away from a reference level. The term
refers to statistical evidence that there is a difference. It is
defined as the probability of making a decision to reject the null
hypothesis when the null hypothesis is actually true.
[0263] As used interchangeably herein, the terms "essentially" and
"substantially" means a proportion of at least about 60%, or
preferably at least about 70% or at least about 80%, or at least
about 90%, at least about 95%, at least about 97% or at least about
99% or more, or any integer between 70% and 100%. In some
embodiments, the term "essentially" means a proportion of at least
about 90%, at least about 95%, at least about 98%, at least about
99% or more, or any integer between 90% and 100%. In some
embodiments, the term "essentially" can include 100%.
[0264] Although preferred embodiments have been depicted and
described in detail herein, it will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be made without departing from the
spirit of the invention and these are therefore considered to be
within the scope of the invention as defined in the claims which
follow. Further, to the extent not already indicated, it will be
understood by those of ordinary skill in the art that any one of
the various embodiments herein described and illustrated may be
further modified to incorporate features shown in any of the other
embodiments disclosed herein.
[0265] The disclosure is further illustrated by the following
examples which should not be construed as limiting. The examples
are illustrative only, and are not intended to limit, in any
manner, any of the aspects described herein. The following examples
do not in any way limit the invention.
EXAMPLES
Example 1
Exemplary Materials and Methods
[0266] Materials:
[0267] Degummed silk fibers were purchased from Suho Biomaterials
Technology (Suzhou, China). Bevacizumab (AVASTIN.RTM., Genentech,
South San Francisco, Calif.) was purchased from CuraScript Inc.
(Orlando, Fl). Memantine hydrochloride and all other chemicals were
purchased from Sigma-Aldrich (St. Louis, Mo.).
[0268] Silk Fibroin Protein Purification.
[0269] To obtain silk fibroin solution from degummed silk fibers,
multiple purification steps including lithium bromide dissolution,
dialysis and centrifugation were performed. Briefly, 5 g of
degummed fibers were weighed and added to a container, e.g., a
glass beaker, containing 20 ml of freshly prepared 9.3 M lithium
bromide solution. The final concentration of silk was approximately
20% (w/v). The mixture was then heated until the silk fibers were
completely dissolved. For example, the container was covered with
an aluminum foil and placed in an oven at 60.degree. C. for 4 hours
until the silk fibers were completely dissolved. The solution was
dialyzed against ultrapure water (e.g., with an electrical
resistivity of about 18.2 MS2 cm), for example, using Slide-a-Lyzer
dialysis cassettes (MWCO 3,500, Pierce/Thermo Scientific, Rockford,
Ill.) for 48 hours, to remove the lithium bromide salt. The
dialyzed solution was centrifuged twice at 8,700 rpm and 4.degree.
C. for about 20 minutes using 50-ml conical tubes in an Eppendorf
5804R centrifuge. The final concentration of silk fibroin aqueous
solution was approximately 8% (w/v), as measured by drying a known
volume of solution at 60.degree. C. overnight and weighing the
residual solid. The 8% silk stock solution was stored at 4.degree.
C. and diluted with ultrapure water before use.
[0270] Spray-Crystallize-Freeze-Drying (SCFD) Set-Up.
[0271] While an exemplary SCFD set-up was described in the
Examples, any other modifications within one of skill in the art
are still within the scope described herein. In one embodiment, a
SONIFIER.RTM. cell disruptor (Branson, Danbury, Conn.) equipped
with a flow-through horn and a syringe pump (KDS230, KD Scientific,
Holliston, Mass.) were used in the preparation to atomize the silk
solution and to induce formation of beta-sheet crystalline
structure in silk fibroin simultaneously. Silk solution was
injected into the flow-through horn via the syringe pump at desired
flow rates and sprayed through the horn directly into a fast-freeze
container (e.g., a 600-ml fast-freeze flask, which can be obtained
from Labconco Corp., Kansas City, Mo.). The flask was kept floating
in liquid nitrogen (FIG. 1), while the distance between the tip of
the horn and the bottom of the flask was adjusted to ensure both
immediate freezing of the spray and spray homogeneity. After
spraying, the flask was immediately loaded into a Virtis Genesis
lyophilizer (SP Scientific, Warminster, Pa.) and lyophilized
overnight.
[0272] Preparation of SCFD Microspheres.
[0273] The composition of the silk solution, the flow rate
(controlled via the syringe pump) and the sonication power output
were varied for preparing different SCFD microspheres. To
facilitate comparison among different SCFD microspheres, the
solution volume was kept constant at 5 mL for all batches, while
other variables such as amounts of silk, amounts of an additive
(e.g., glycerol to decrease solubility of silk microspheres), flow
rates, and sonication power were adjusted. Some exemplary values of
those variables for production of silk microspheres are listed in
Table 1.
TABLE-US-00001 TABLE 1 Exemplary parameters for production of silk
microspheres described herein (based on a ~5-mL solution volume)
Amount of silk ~50-~400 mg Amount of glycerol ~0-~170 mg Flow rate
~0.1-~1.0 mL/min Sonication power ~25-~55% amplitude
[0274] Microsphere Characterization (Size, Morphology and
Solubility).
[0275] The size and surface morphology of SCFD microspheres were
assessed in both freeze-dried (powder) form and after suspension of
dried powder in ultrapure water, e.g., using an inverted optical
microscope (Carl Zeiss, Jena, Germany) and a Scanning Electron
Microscope (SEM, JSM 840A, JEOL Peabody, Mass.). For SCFD
microsphere assessment using an optical microscope, the dried
powder or approximately 20 .mu.L of a water suspension of
microspheres was directly added on top of a glass slide. For SEM,
the dried powder was applied directly onto an SEM stub covered with
a conductive tape (JEOL Peabody, Mass.), while the water suspension
of microspheres was loaded onto an SEM stub with a conductive tape
and dried overnight at ambient temperature. The samples were
sputter-coated with approximately 20 nm of gold prior to SEM
analysis.
[0276] The solubility of SCFD microspheres can be estimated by any
art-recognized method. In one embodiment, the solubility of SCFD
microspheres was estimated, e.g., using the following protocol.
First, an aqueous microsphere suspension (1% w/v) was centrifuged
at 15000 rpm for about 10 minutes (e.g., using an Eppendorf 5424
microcentrifuge) after incubation at 37.degree. C. for about 2
hours with agitation (e.g., placing the suspension on a shaker).
After removal of the supernatant, the remaining microspheres were
dried at 60.degree. C. overnight and subsequently weighed to obtain
the weight of the dried pellet. The microsphere solubility was
estimated from the ratio of the difference between the initial
microsphere mass and the dried pellet mass to the initial
microsphere mass.
[0277] Preparation of Drug-Loaded SCFD Spheres.
[0278] For therapeutic drug-loaded SCFD spheres (e.g.,
bevacizumab-loaded SCFD spheres: A-sphere; or memantine
hydrochloride-loaded SCFD spheres: M-sphere), the drug solution was
mixed with silk and glycerol solutions prior to spraying. The total
solution volume was kept constant at 5 mL, while the mixing ratio
of different components (e.g., drug, silk and glycerol) and the
sonication power were varied, as shown in Table 2.
TABLE-US-00002 TABLE 2 Exemplary parameters for preparation of
drug-loaded silk-glycerol microspheres Amount of Sonication Amount
of Amount of glycerol power (% Flow rate Batch drug (mg) silk (mg)
(mg) amplitude) (ml/min) M-sphere 1 62.5 250.0 0.0 25 0.17 M-sphere
2 62.5 250.0 45.0 25 0.17 M-sphere 3 62.5 250.0 83.5 25 0.17
A-sphere 1 10.0 250.0 0.0 25 0.17 A-sphere 2 10.0 250.0 45.0 25
0.17 A-sphere 3 10.0 250.0 83.5 25 0.17 A-sphere 4 10.0 250.0 83.5
20 0.17 A-sphere 5 10.0 62.5 21.0 25 0.17 A-sphere 6 10.0 30.0 10.0
25 0.17
[0279] Drug Release from SCFD Microspheres.
[0280] Silk microspheres were stored in sealed glass vials at
4.degree. C. prior to release studies. Before use, approximately 10
mg of powder was weighed and added to a 15-ml plastic tube, to
which 4 ml of PBS buffer, pH 7.4 containing 0.02% (w/v) sodium
azide was added. The microsphere suspension was then incubated at
37.degree. C. At desired time points, the tubes containing silk
microspheres were centrifuged at 10,000 rpm for about 10 min (e.g.,
using Eppendorf 5804R centrifuge), and the supernatants were
collected and stored at 4.degree. C. for analysis. The microsphere
pellets were resuspended with 4 ml of PBS/sodium azide buffer, pH
7.4 and incubated until the next time point. Memantine
concentration in the release medium was determined using a
modification of the method previously described in Suckow R F et
al. "Sensitive and selective liquid chromatographic assay of
memantine in plasma with fluorescence detection after pre-column
derivatization." J Chromatogr B Biomed Sci Appl 1999; 729:217-224.
Some modifications include a fluorescence labeling reaction with
dansyl chloride and High Pressure Liquid Chromatography (HPLC)
using an Agilent 1200 series HPLC (Agilent, Santa Clara, Calif.)
instrument equipped with a reverse phase column (Agilent Eclipse
plus C-18 column, 4.6 mm I.D..times.75 mm L). Bevacizumab
concentration was analyzed using the same HPLC system equipped with
an Agilent Bio SEC-3 column (300 angstrom pore size, 4.6 mm I.
D..times.300 mm L).
Example 2
Role of Sonication in Silk SCFD Microsphere Preparation
[0281] Sonication has been used to induce silk fibroin gelation,
e.g., as reported in Wang X et al. "Sonication-induced gelation of
silk fibroin for cell encapsulation." Biomaterials 2008;
29:1054-64. The time for silk gelation was dependent on silk
solution concentration, sonication power output, and sonication
duration. Id. However, the Wang X et al. reference does not
describe the use of sonication to produce silk microspheres.
Presented herein is one embodiment of the methods for preparing a
silk microsphere, in which a sonicator (Branson SONIFIER.RTM. cell
disruptor) equipped with a flow-through horn was utilized to allow
silk solution to be continuously sonicated as it passed through the
inner channel of the horn, while concomitantly being atomized into
a fine spray at the nozzle (e.g., tip) of the horn (FIG. 1). The
atomized spray was collected as frozen particles in a flask that
was at least partially surrounded by liquid nitrogen, and the
frozen particles were subsequently lyophilized into dry
particles.
[0282] Without wishing to be bound by theory, dry silk particles
after lyophilization can gain certain amount of beta-sheet
crystalline structure due to sonication, which can result in
formation of water-insoluble particles. Accordingly, further
solvent treatment to induce crystallization can be unnecessary. It
has been previously reported that silk microspheres that were
fabricated by a spray-drying process gained a certain amount of
beta-sheet structure either due to the high temperature in the
spray-dryer (Hino T. et al. "Silk microspheres prepared by
spray-drying of an aqueous system." Pharm Pharmacol Commun 2000;
6:335-339; Yeo J H. et al. "Simple preparation and characteristics
of silk fibroin microsphere." Eur Polym J 2003; 39:1195-1199) or
due to a post-lyophilization treatment using methanol or water
vapor (Wenk E. et al. "Silk fibroin spheres as a platform for
controlled drug delivery." J Control Release 2008; 132:26-34).
However, unlike the methods described herein, these previous
reports show that heat and/or post-treatment with methanol or water
vapor are required to induce sufficient amounts of beta-sheet
structure of silk fibroin present in silk microspheres such that
the silk microspheres have a low solubility or become insoluble in
water.
[0283] Without wishing to be bound by theory, the increase in
beta-sheet content in silk fibroin resulted in the preservation of
shape and size of microspheres in water. The water solubility of
SCFD microparticles was estimated by comparing the particle size
and morphology in dry and wet states using both optical and
scanning electron microscopy. The weight loss of silk material due
to dissolution in water was further quantified. The results of
microscopy and dissolution tests were summarized in Table 3.
TABLE-US-00003 TABLE 3 Exemplary parameters for preparation of silk
microspheres Sonication Morphol- Morphol- Solu- Amount Flow power
ogy/ ogy bil- of silk rate (% ampli- Poly- after ity (mg) (ml/min)
tude) dispersity hydration* (%) 1 50 0.1 25 Fibers/ N.T. N.T.
Aggregates 2 250 0.1 25 Spherical/ Dissolved 91 .+-. 4 50-100 .mu.m
3 400 0.1 25 Spherical/ Dissolved 87 .+-. 5 50-100 .mu.m 4 250 0.5
25 Spherical/ Dissolved 93 .+-. 3 100-500 .mu.m 5 250 1.0 25
Spherical/ Dissolved 90 .+-. 3 100-800 .mu.m 6 250 0.5 35
Spherical/ Deformed** 21 .+-. 9 100-800 .mu.m 7 250 0.1 35
Spherical/ Deformed** 9 .+-. 2 100-500 .mu.m 8 250 0.5 45
Aggregates/ Deformed** 8 .+-. 2 100-800 .mu.m/ 9 250 0.5 55 Fibers/
N.T. N.T. Aggregates N.T. = not tested *Determined via optical
microscopy. **Microspheres lost their spherical shape and formed
aggregated clumps that floated in water.
[0284] As shown in Table 3, silk microspheres were generally
obtained at higher silk solution concentrations, e.g., .about.5-8%
(w/v), but not at lower concentrations (e.g. .about.1% (w/v)). With
too high silk concentration, however, the silk solution was more
prone to faster gelation, which could cause clogging in the
flow-through horn. Therefore, .about.5% (w/v) silk concentration
was used in the Examples described herein. In addition to silk
concentrations, the flow rates and/or sonication power output can
have significant influence on microsphere microstructure and/or
water solubility. In some embodiments, silk microspheres prepared
at a flow rate higher than 0.1 ml/min and a sonication power output
lower than 35% amplitude were highly soluble in water, likely due
to their low beta-sheet contents. These microspheres collapsed and
eventually deformed into aggregated fibers within a few minutes
upon hydration, with a high solubility in water of above 80% by
mass (FIGS. 2C and 2D, Table 3 above). In some embodiments, as
shown in Table 3, silk microspheres prepared at a sonication power
output higher than 35% amplitude had a lower yield, likely due to
gelation during sonication, but had significantly lower solubility
(.about.8-20% by mass), indicating that a significant amount of
beta-sheet crystalline structure could have formed under these
conditions. However, upon hydration, the silk microspheres formed
aggregated, low density clumps that floated in water. In order to
obtain a non-aggregated suspension of silk microspheres with low
water solubility, in one embodiment, at least one additive capable
of enhancing silk beta-sheet crystallinity can be added into the
silk solution prior to flow sonication (Lu S. et al. "Insoluble and
flexible silk films containing glycerol." Biomacromolecules 2010;
11:143-150).
Example 3
Role of Beta-Sheet Structure-Inducing Additives in Silk SCFD
Microsphere Preparation
[0285] It was next sought to determine the effects of various
beta-sheet structure-inducing additives on solubility of silk SCFD
microspheres. Poly(vinyl alcohol) (PVA) has been previously used to
obtain water-insoluble silk nano-/microspheres via phase separation
(See, e.g., Wang X. et al. "Silk nanospheres and microspheres from
silk/PVA blend films for drug delivery." Biomaterials 2010;
31:1025-1035). However, poly(vinyl alcohol) (PVA) did not
significantly affect the solubility of the microspheres produced by
the methods described herein. Glycerol is an additive previously
used to produce insoluble and flexible silk films (Lu S. et al.
"Insoluble and flexible silk films containing glycerol."
Biomacromolecules 2010; 11:143-150). The inventors have
demonstrated that, unlike PVA, glycerol can decrease the solubility
of silk microspheres produced by the methods described herein,
while preserving the non-aggregated spherical morphology in water.
Table 4 shows some exemplary parameters (e.g., but not limited to,
flow rate, sonication power, silk to glycerol ratio, and silk and
glycerol concentrations) varied to optimize processing conditions
for microsphere production.
TABLE-US-00004 TABLE 4 Exemplary parameters for preparation of
silk-glycerol microspheres Soni- A- cation A- mount Flow power
Morphol- Morphol- mount of rate (% ogy/ ogy Solu- of silk glycerol
(ml/ ampli- Poly- after bility (mg) (mg) min) tude) dispersity
hydration* (%) 1 250 83.50 0.10 25 Spherical/ Aggregated/ 23 .+-. 6
30-100 Partially .mu.m dissolved 2 250 83.50 0.17 25 Spherical/
Maintained 24 .+-. 6 50-100 original .mu.m shape 3 250 83.50 0.25
25 Spherical/ Aggregated/ 25 .+-. 3* 50-100 Partially .mu.m
dissolved 4 250 83.50 0.17 15 Fibers & N.T. N.T. aggregates 5
250 83.50 0.17 40 Spherical/ Maintained 28 .+-. 1 500-800 original
.mu.m shape 6 250 41.75 0.17 25 Spherical/ Partially 53 .+-. 5
100-800 dissolved .mu.m 7 250 167.00 0.17 25 Non- Partially 52 .+-.
2 spherical/ dissolved <100 .mu.m 9 125 41.75 0.17 25 Non-
Maintained 24 .+-. 4 spherical/ original 50-100 shape .mu.m 10 400
134.00 0.17 25 Spherical/ Maintained 22 .+-. 2 100-500 original
.mu.m shape** N.T. = not analyzed *Determined by optical
microscope. **Silk gelled in the flow-through horn during
sonication.
[0286] Compared to silk alone (Table 3), the silk/glycerol mixed
solution (Table 4) was more sensitive to sonication power output
and/or flow rate changes. In some embodiments, a flow rate of about
0.17 ml/min and a sonication amplitude of about 25% amplitude were
determined to be the optimum processing conditions, in which a
predominantly microspherical morphology with less than 30% water
solubility was produced, indicating a relatively high beta-sheet
content (Table 4, FIGS. 3A-3B). From previous reports on
silk-glycerol blend films (Id.), almost all glycerol was dissolved
in water in about one hour after film hydration, during which the
overall silk beta-sheet content increased from about 10% to above
50%, as determined by Fourier Transform Infrared (FTIR)
spectroscopy. As a result, these films not only preserved their
original dimensions but also had an improved mechanical strength.
Id. In the Example described herein, without wishing to be bound by
theory, the 30% mass loss upon hydration of microspheres could be
attributed mainly to dissolution of glycerol in water. Accordingly,
in some embodiments, the silk microspheres can have an effective
solubility of higher than 90%. In other embodiments, there can be
an overall beta-sheet crystalline content of over 50% in the
silk/glycerol microspheres. Without wishing to be by theory, higher
flow rates (>.about.0.17 ml/min) and/or lower sonication
amplitudes (<25%) resulted either in the lack of a spray or high
water solubility of microspheres, while lower flow rates and/or
higher sonication amplitudes generally resulted in premature silk
gelation, e.g., in a sonifier. In the previous reports on
silk/glycerol blend films, the weight ratio of glycerol to silk was
reported to be over 1/3 in order to prepare water-insoluble films.
Id. However, those previous reports do not indicate the weight
ratio of glycerol to silk in microspheres. It was determined herein
that a ratio of glycerol to silk at about 1/3 can produce a
microspherical morphology and low water solubility. On the other
hand, ratios of glycerol to silk much higher than about 1/3 can
cause premature silk/glycerol gelation in the sonifier and/or
formation of non-spherical particles. However, in particular
embodiments, water insoluble microsphere preparation was not
possible when ratios of glycerol to silk was below about 1/3 at a
flow rate of about 0.17 ml/min and a sonication amplitude of about
25%. Therefore, in one embodiment, .about.5% silk/.about.1.67%
glycerol (w/v) is used to encapsulate a drug for drug delivery
applications. In such embodiments, the microspheres can have sizes
ranging from about 50 .mu.m to about 100 .mu.m with high
nano-/micro-porosity, as visualized via optical and scanning
electron microscopy (FIGS. 4A-4D).
Example 4
Exemplary Silk SCFD Microspheres for Drug Delivery
[0287] Memantine-Silk SCFD Microspheres.
[0288] FIG. 5 shows the release kinetics of an FDA approved drug
for treatment of Alzheimer's disease (Memantine, e.g.,
NAMENDA.RTM.; MW (Memantine hydrochloride)=215.76 g/mole, water
solubility.apprxeq.50 mg/mL) from SCFD microspheres prepared from
.about.5% silk solutions having different glycerol contents (0%,
.about.15% and .about.25% glycerol) at .about.25% sonication
amplitude and .about.0.17 mL/min flow rate (Table 2). For the
assessed formulations, as indicated in FIG. 5, the release of
memantine was sustained for at least over 17 days. The initial
burst, e.g., the percentage of the encapsulated drug initially
released from the SCFD microsphere (e.g., measured at the first
time point (3 days) as shown in FIG. 5), and the cumulative
percentage of drug released after 17 days were the lowest in the
case of silk-alone SCFD spheres (e.g., silk SCFD spheres without
glycerol), while both the initial burst (.about.50.8%, .about.57.4%
and .about.67.3% for 0%, .about.15% and .about.25% glycerol,
respectively) and the cumulative release after 17 days
(.about.66.4%, .about.76% and .about.81.9%, for 0%, .about.15% and
.about.25% glycerol, respectively) increased with increasing
glycerol content. Unexpectedly, silk/memantine SCFD microspheres
were less soluble, e.g., in the release medium, than silk SCFD
microspheres prepared without memantine. This indicates that
memantine encapsulated in the silk SCFD microspheres can increase
the overall beta-sheet crystalline content and/or decrease the
water solubility of the microspheres. Furthermore, the data shown
in FIG. 5 indicates that the release kinetics of a drug, e.g., an
FDA approved small drug, can be controlled effectively, at least
partly, via the glycerol content in the formulation. Other factors
that can affect small drug release kinetics include, but are not
limited to, drug loading, silk concentration, or a combination
thereof.
[0289] Avastin-Silk SCFD Microspheres.
[0290] FIG. 6 shows the release kinetics of an FDA approved drug
for treatment of age-related (wet) macular degeneration
(Bevacizumab, e.g., AVASTIN.RTM.; MW=149 KDa, .about.25 mg/ml stock
solution) from SCFD microspheres prepared from .about.5% silk
solutions having different glycerol contents (0%, .about.15% and
.about.25% glycerol) at .about.25% sonication amplitude and
.about.0.17 mL/min flow rate (Table 2). Compared to preparation of
memantine-encapsulated SCFD silk microspheres, the silk/bevacizumab
solution was more prone to gelation or aggregation during
sonication when making bevacizumab-encapsulated SCFD silk
microspheres, indicating a stronger intermolecular interaction
between the silk and bevacizumab molecules. Furthermore, both the
initial burst (13.8%, 18.3% and 6.5% for 0%, .about.15% and
.about.25% glycerol, respectively) and the cumulative release from
bevacizumab-silk microspheres after 13 days (15.6%, 20% and 6.5%,
for 0%, .about.15% and .about.25% glycerol, respectively) were
significantly lower than those from memantine-silk microspheres
(FIG. 6). In addition, increasing the glycerol content in
bevacizumab-silk microspheres to about 25% did not show a trend of
increasing the drug release rate, as compared to the release from
memantine-silk microspheres. The bevacizumab-silk microspheres with
the highest glycerol content (.about.25%) showed the lowest level
of initial burst and the lowest sustained release after 13 days, as
compared to the bevacizumab-silk microspheres with 0% or about 15%
glycerol content (FIG. 6). Without wishing to be bound by theory,
it is likely that a high concentration of glycerol (rich hydroxyl
groups) could reinforce the interaction between bevacizumab and
silk through hydrogen bonding. The strong binding of protein
molecules to silk materials has been previously reported but the
underlying mechanism is not yet clear (Wang X. et al. "Silk
microspheres for encapsulation and controlled release." J Control
Release (2007)117:360-70 Wang X et al. "Silk nanospheres and
microspheres from silk/PVA blend films for drug delivery."
Biomaterials (2010) 31:1025-1035; Lu Q. et al. "Stabilization and
release of enzymes from silk films." Macromol Biosci (2010)
10:359-368). Due to the overall hydrophobic nature of silk
materials, hydrophobic interactions were contemplated to be the
predominant force for the binding, even though electrostatic
interactions (the pI value of silk fibroin is about 3) and hydrogen
bonding can also play important roles (Lu Q. et al., Id). In some
embodiments, adjusting other factors that can influence
intermolecular interaction between silk and protein drugs (e.g.,
bevacizumab), including, but not limited to, silk concentration
and/or the presence of affinity-interfering additives, can control
release of protein drugs from silk material carriers.
Example 5
Syringe Injectability of Silk SCFD Microspheres
[0291] Lyophilized silk microspheres, or silk-drug microspheres
(e.g., silk-memantine and silk-bevacizumab microspheres) were able
to be suspended in about 1% to about 3% sodium
carboxymethylcellulose solutions (CMC, viscosity=50-200 cP for 4%
solution in water, 25.degree. C.), forming homogeneous suspensions.
The CMC suspension of the silk microspheres, including silk-drug
microspheres, can be injected through a needle depending on size of
the silk microspheres, e.g., a 21 gauge needle, indicating the
feasibility of applying silk SCFD microsphere formulation through
non-invasive administration routes, e.g., subcutaneous,
intramuscular injections, for clinical applications.
[0292] Silk microspheres prepared through a novel
spray-crystallize-freeze-drying method described herein can
preserve their size (.about.50-.about.100 .mu.m) and microspherical
morphology upon hydration. In some embodiments, a beta-sheet
structure-inducing additive, e.g. glycerol, can be blended with
silk prior to preparation of silk microspheres, further preserving
their size and morphology upon hydration. The methods of producing
silk microspheres described herein are time, energy and cost
efficient, thus suitable for large-scale production of silk
microspheres. In some embodiments, the methods described herein can
all-aqueous processes. In some embodiments, high temperature and/or
organic solvents are not needed during the methods described herein
(e.g., all-aqueous processes), thus allowing encapsulation of
sensitive or labile drugs (e.g., heat-labile drugs) at a high yield
(e.g., up to 100%). The porous nature of the microspheres described
herein can increase the surface area available for drug release.
The SCFD microsphere preparation methods can be modified readily,
within one of skill in the art, to optimize the drug loading and
release profile of a specific therapeutic drug.
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[0311] All patents and other publications identified in the
specification and examples are expressly incorporated herein by
reference for all purposes. These publications are provided solely
for their disclosure prior to the filing date of the present
application. Nothing in this regard should be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior invention or for any other reason.
All statements as to the date or representation as to the contents
of these documents is based on the information available to the
applicants and does not constitute any admission as to the
correctness of the dates or contents of these documents.
* * * * *