U.S. patent application number 12/442595 was filed with the patent office on 2010-02-04 for silk microspheres for encapsulation and controlled release.
This patent application is currently assigned to TRUSTEES OF TUFTS COLLEGE. Invention is credited to David L. Kaplan, Xiaoqin Wang.
Application Number | 20100028451 12/442595 |
Document ID | / |
Family ID | 39789141 |
Filed Date | 2010-02-04 |
United States Patent
Application |
20100028451 |
Kind Code |
A1 |
Kaplan; David L. ; et
al. |
February 4, 2010 |
SILK MICROSPHERES FOR ENCAPSULATION AND CONTROLLED RELEASE
Abstract
A method was developed to prepare silk fibroin microspheres
using lipid vesicles as templates to efficiently load therapeutic
agents in active form for controlled release. The lipids are
subsequently removed through the use of a dehydration agent, such
as methanol or sodium chloride, resulting in .beta.-sheet structure
dominant silk microsphere structures having about 2 .mu.m in
diameter. The therapeutic agent can be entrapped in the silk
microspheres and used in pharmaceutical formulations for
controlled-release treatments.
Inventors: |
Kaplan; David L.; (Concord,
MA) ; Wang; Xiaoqin; (Winchester, MA) |
Correspondence
Address: |
DAVID S. RESNICK
NIXON PEABODY LLP, 100 SUMMER STREET
BOSTON
MA
02110-2131
US
|
Assignee: |
TRUSTEES OF TUFTS COLLEGE
Medford
MA
|
Family ID: |
39789141 |
Appl. No.: |
12/442595 |
Filed: |
September 26, 2007 |
PCT Filed: |
September 26, 2007 |
PCT NO: |
PCT/US07/20789 |
371 Date: |
July 7, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60847100 |
Sep 26, 2006 |
|
|
|
Current U.S.
Class: |
424/491 ;
424/130.1; 424/85.2; 424/85.5; 424/85.7; 424/94.1; 514/1.1;
514/44R |
Current CPC
Class: |
A61K 9/16 20130101; A61K
47/34 20130101; A61K 47/42 20130101; A61K 9/5052 20130101; A61K
9/19 20130101; A61K 31/7076 20130101; A61K 9/1658 20130101; A61K
38/44 20130101; A61K 9/127 20130101 |
Class at
Publication: |
424/491 ; 514/2;
514/44.R; 424/130.1; 424/94.1; 514/12; 424/85.7; 424/85.5;
424/85.2; 514/3 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 38/02 20060101 A61K038/02; A61K 31/7088 20060101
A61K031/7088; A61K 39/395 20060101 A61K039/395; A61K 38/43 20060101
A61K038/43; A61K 38/16 20060101 A61K038/16; A61K 38/21 20060101
A61K038/21; A61K 38/20 20060101 A61K038/20; A61K 38/28 20060101
A61K038/28 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was supported by the NIH under an
Osteochondral Plug Grant No. NIDCR. The Government has certain
rights to the invention.
Claims
1. A method of preparing silk fibroin microspheres, comprising: a.
mixing a silk fibroin solution with a lipid composition; b.
lyophilizing the mixture; c. combining the lyophilized material
with a dehydration medium for a sufficient period of time to at
least partially dehydrate the silk fibroin solution and induce
.beta.-sheet structures in the silk fibroin; and d. removing at
least a portion of the lipids to form silk fibroin
microspheres.
2. The method of claim 1, wherein the dehydration medium is
selected from the group consisting of methanol, ethanol, propanol,
acetone, chloroform, polyethylene glycol solutions, and salt
solutions.
3. The method of claim 2, wherein the dehydration medium is
methanol or a solution of sodium chloride.
4. The method of claim 1, further comprising the step of
freeze-thawing the silk fibroin-lipid composition mixture before
the lyophilization step.
5. The method of claim 1, further comprising the step of suspending
the microspheres in water or a buffer.
6. The method of claim 1, wherein all or substantially all of the
removable lipids have been removed.
7. The method of claim 1, wherein about 15 to about 20% of the
total lipids remain in the silk fibroin microspheres.
8. The method of claim 1; wherein less than about 5% of the total
lipids remain in the silk fibroin microspheres.
9. The method of claim 1, wherein the lipid composition is selected
from the group consisting of
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC);
1,2-dioleoyl-sn-glycero-3-phophoethanolamine (DOPE);
1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); and
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC).
10. The method of claim 1, wherein the silk fibroin solution
contains at least one therapeutic agent.
11. The method of claim 10, wherein the therapeutic agent is
encapsulated in the silk fibroin microspheres.
12. The method of claim 11, wherein the therapeutic agent is in an
active form and capable of administering a therapeutic effect upon
release from the microsphere.
13. The method of claim 10, wherein the therapeutic agent is
selected from the group consisting of proteins, peptides, nucleic
acids, peptide nucleic acids, aptamers, antibodies, growth factors,
cytokines, enzymes, small molecules, and combinations thereof.
14. The method of claim 10, wherein the therapeutic agent is
selected from the group consisting of morphogenetic protein 2
(BMP-2), insulin-like growth factor I and II (IGF-I and II),
epidermal growth factor (EGF), platelet-derived growth factor
(PDGF), fibroblast growth factors (FGFs), transforming growth
factors-.beta. (TGFs-.beta.), transforming growth factors-.alpha.,
erythropoietin (EPO), interferon .alpha. and .gamma., interleukins,
tumor necrosis factor .alpha. and .beta., insulin, antibiotics,
adenosine, and combinations thereof.
15. A pharmaceutical formulation comprising the silk fibroin
microspheres produced by the method of claim 11.
16. The formulation of claim 15, wherein the average size of the
microspheres is less than about 2.0 .mu.m.
17. A method of administering a therapeutic agent to a patient in
need thereof, comprising the step of administering the
pharmaceutical formulation of claim 15 to the patient.
18. The method of claim 17, wherein less than about 5% of the
therapeutic agent is released into the patient from the
microspheres over a period of one month.
19. The method of claim 17, wherein at least about 5% of the
therapeutic agent remains in the microspheres 10 days after
administration.
20. The method of claim 17, wherein the activity of the therapeutic
agent remains at least at 50% one month after administration.
21. A drug delivery composition comprising a therapeutic agent
encapsulated in crosslinked silk fibroin microspheres, wherein the
microspheres contain lipid components.
22. The composition of claim 21, wherein the lipid components
assist in controlling the release of the therapeutic agent from the
microspheres.
23. The composition of claim 21, wherein the microspheres contain
less than about 5% lipids by weight.
24. The composition of claim 21, wherein the weight percentage of
microspheres in the total silk is at least 50%.
25. The composition of claim 21, wherein the average size of the
microspheres is less than about 2.0 .mu.m.
26. The composition of claim 21, wherein the crosslinking of the
silk fibroin microspheres was induced by exposing the silk fibroin
to methanol or sodium chloride.
27. A method of encapsulating a biomaterial in silk fibroin
microcapsules, comprising: a. mixing a solution comprising silk
fibroin and a biomaterial with a lipid composition; b. lyophilizing
the mixture; c. dehydrating the lyophilized material in a
dehydration medium for a sufficient period of time to at least
partially dehydrate the silk fibroin solution and induce
.beta.-sheet structures in the silk fibroin; and d. removing at
least a portion of the lipids to produce a biomaterial that has
been encapsulated in silk fibroin microspheres.
28. The method of claim 27, wherein the dehydration medium is
selected from the group consisting of methanol, ethanol, propanol,
acetone, chloroform, polyethylene glycol solutions, and salt
solutions.
29. The method of claim 28, wherein the dehydration medium is
methanol or a solution of sodium chloride.
30. The method of claim 27, further comprising the step of
freeze-thawing the silk fibroin-lipid composition mixture before
the lyophilization step.
31. The method of claim 27, wherein the biomaterial is a
therapeutic agent.
32. The method of claim 27, wherein the biomaterial is an enzyme or
enzyme-based electrode.
33. The method of claim 32, wherein the enzyme or enzyme-based
electrode is used in a field selected from the group consisting of
tissue engineering, biosensors, the food industry, environmental
control, and biomedical applications.
34. A silk fibroin microsphere composition, comprising a
therapeutic agent encapsulated in crosslinked silk fibroin
microspheres, wherein at least 75% of the microspheres are
spherical or substantially spherical, and wherein at least 75% of
the microspheres have a diameter ranging from about 1.0 to about
3.0 microns.
35. The composition of claim 34, wherein at least 90% of the
microspheres are spherical or substantially spherical.
36. The composition of claim 34, wherein at least 90% of the
microspheres have a diameter ranging from about 1.0 to about 3.0
microns.
37. The composition of claim 36, wherein at least 95% of the
microspheres have a diameter ranging from about 1.0 to about 3.0
microns.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. provisional
application No. 60/847,100, filed Sep. 26, 2006, the content of
which is incorporated herein by reference in its entirety.
FIELD OF INVENTION
[0003] This invention relates to silk fibroin microspheres prepared
by mixing silk fibroin with lipids to form microspheres capable of
encapsulating therapeutic drugs and releasing the drugs in a
controlled manner.
BACKGROUND OF INVENTION
[0004] Controlled drug release involves a combination of a polymer
matrix with bioactive drugs such that the drugs can be delivered in
a predictable manner. Polymeric materials, including biodegradable
synthetic polymers such as poly(D,L-lactide-co-glycolide) (PLGA)
and natural polymer such as collagen and alginate have been used as
drug delivery matrices. These polymer matrices function in many
ways as an artificial extracellular matrix (ECM) to stabilize
encapsulated proteins, such as growth factors. See Jiang et al.,
"Biodegradable poly(lactic-o-glycolic acid) microparticles for
injectable delivery of vaccine antigens," Adv. Drug Deliv. Rev. 57
(2005) 391-410; see also Wee et al., "Protein release from alginate
matrices," Adv. Drug Deliv. Rev. 31 (1998) 267-285.
[0005] The release of encapsulated protein drugs are controlled by
both passive diffusion of protein drugs and degradation of polymer
matrices. Encapsulation and controlled release are of particular
importance for protein drugs with short half-lives when free in
solution, and for reduced systemic toxicity. However, preservation
of biological activity of incorporated protein drugs in a polymer
matrix and control of subsequent release remain major
challenges.
[0006] Silk fibroin has a long history in clinical applications
used as suture threads, and now it is finding new and important
applications in the tissue-engineering field as a scaffold support
for the growth of artificial tissues such as bone and cartilage.
Recently, the use of silk fibroin for controlled drug delivery has
been explored with electrospun silk fiber mats that encapsulated
bone morphogenetic protein 2 (BMP-2). See Li et al., "Electrospun
silk-BMP-2 scaffolds for bone tissue engineering," Biomaterials 27
(2006):3115-3124. Hoffman investigated the encapsulation and
release of different proteins such as horseradish peroxidase (HRP)
and lysozyme from silk films and the correlation between silk
crystallinity that were induced by methanol and protein release
behaviors. It was found that high silk crystallinity could
significantly retard the release of encapsulated proteins. See
Hofmann et al., "Silk fibroin as an organic polymer for controlled
drug delivery," J Control Release 111 (2006):219-227.
[0007] Thus, silk fibroin holds great promise for controlled drug
delivery due to its unique structure and crystallinity properties
as well as the other advantages discussed above. Silk microspheres
can be fabricated using physical methods such as spray-drying,
however, harsh conditions such as high temperature have prohibited
their uses as a protein drug delivery carrier. See Hino et al.,
"Change in secondary structure of silk fibroin during preparation
of its microspheres by spray-drying and exposure to humid
atmosphere," J Colloid Interface Sci. 266 (2003) 68-73. In
addition, conventional microspheres typically have a large size
(above 100 .mu.m), making them less useful as encapsulation
vehicles for many of the smaller drug molecules.
[0008] Accordingly, what is needed in the art is a way to prepare
silk fibroin microspheres under mild conditions so that protein
drugs and other therapeutic agents can be encapsulated in the
microspheres and released in their active forms. This invention
answers that need.
SUMMARY OF INVENTION
[0009] One embodiment of this invention relates to a method of
preparing silk fibroin microspheres. The method involves (a) mixing
a silk fibroin solution with a lipid composition; (b) lyophilizing
the mixture; (c) combining the lyophilized material with a
dehydration medium for a sufficient period of time to at least
partially dehydrate the silk fibroin solution and induce
.beta.-sheet structures in the silk fibroin; and (d) removing at
least a portion of the lipids to form silk fibroin
microspheres.
[0010] Another embodiment of this invention relates to a drug
delivery composition comprising a therapeutic agent encapsulated in
crosslinked silk fibroin microspheres, wherein the microspheres
contain lipid components.
[0011] Another embodiment of this invention relates to a method of
encapsulating a biomaterial in silk fibroin microcapsules. The
method comprises (a) mixing a solution comprising silk fibroin and
a biomaterial with a lipid composition; (b) lyophilizing the
mixture; (c) combining the lyophilized material with a dehydration
medium for a sufficient period of time to at least partially
dehydrate the silk fibroin solution and induce .beta.-sheet
structures in the silk fibroin; and (d) removing at least a portion
of the lipids to produce a biomaterial that has been encapsulated
in silk fibroin microspheres.
[0012] Another embodiment of this invention relates to a silk
fibroin microsphere composition, comprising a therapeutic agent
encapsulated in crosslinked silk fibroin microspheres, wherein at
least 75% of the microspheres are spherical or substantially
spherical, and wherein at least 75% of the microspheres have a
diameter ranging from 1.0 to 3.0 .mu.m.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 depicts photomicrographs of particle suspensions
obtained during silk microsphere preparation of: (A) DOPC film
hydrated by water; (B) DOPC film hydrated by silk solution; (C)
DOPC-silk mixture after freeze-thaw 3 times; and freeze-thawed and
lyophilized DOPC-silk suspended in saturated NaCl solution at 10
min (D), 20 min (E), and 40 min (F). The arrows indicate the fused
lipid vesicles. Bar indicates 50 .mu.m.
[0014] FIG. 2 represents charts depicting the yield of microspheres
when (A) silk and DOPC ratio were varied in MeOH-based silk
microspheres, and (B) NaCl treatment time was varied in NaCl-based
silk microspheres. Error bars represent standard deviations from
samples n=3.
[0015] FIG. 3 depicts SEM images of freeze-thawed and lyophilized
DOPC-silk silk microspheres when untreated (A-D), treated with
methanol (E-H), and treated with NaCl for 15 h (1-L). Bar indicates
20 .mu.m in A, E, I; 5 .mu.m in B, F, J; and 2 .mu.m in C, D, G, H,
K, and L.
[0016] FIG. 4 depicts confocal laser scanning microscopy images of
the silk microspheres containing fluorescein-labeled DOPE. Labeled
phospholipids remained in MeOH-based silk microspheres (A) and
NaCl-based silk microspheres (B), forming either multilamellar
structures (C) or non-lamellar structure (D). Bar indicates 75
.mu.m in A and B; 7.36 .mu.m in C; and 10.77 .mu.m in D.
[0017] FIG. 5 represents FTIR spectra (amide I band) of silk
microspheres prepared (a) as lyophilized DOPC-silk suspended in
water; (b), (c), and (e) as silk microspheres prepared with 1, 4,
and 15 h NaCl treatment and suspended in water, respectively; and
(d) as silk microspheres prepared with MeOH treatment.
[0018] FIG. 6 depicts confocal laser scanning microscopy images
showing the distribution of silk and drug in silk microspheres.
Fluorescein-labeled silk (left panels) and rhodamin B-labeled
dextran 40,000 (middle panels) are located in separate layers (A)
or domains (B) in MeOH-based microspheres prepared from lyophilized
DOPC, silk, and drug mixture. The same mixture when freeze-thawed
prior to lyophilization shows that the silk and drug are mixed in
the same layers (C) and domains (D) in both MeOH-based and
NaCl-based microspheres. Images in the left and middle panels are
merged into the right panels. Bar indicates 5.29, 1.49, 2.24, and
3.67 .mu.m in A, B, C and D, respectively.
[0019] FIG. 7 represents (A) the HRP release from MeOH-based silk
microspheres ( ) and DOPC-silk mixture prior to MeOH treatment
(.box-solid.) and (B) the HRP release from NaCl-based silk
microspheres after NaCl treatment for 1 h (.box-solid.), 4 h ( ),
and 15 h (.tangle-solidup.). Error bars represent standard
deviations from samples n=3.
[0020] FIG. 8 represents a schematic showing the process of
preparing MeOH-based and NaCl-based microspheres.
DETAILED DESCRIPTION
[0021] This invention relates to a method of preparing silk fibroin
microspheres. The method involves (a) mixing a silk fibroin
solution with a lipid composition; (b) lyophilizing the mixture;
(c) combining the lyophilized material with a dehydration medium
for a sufficient period of time to at least partially dehydrate the
silk fibroin solution and induce .beta.-sheet structures in the
silk fibroin; and (d) removing at least a portion of the lipids to
form silk fibroin microspheres.
[0022] Silkworm fibroin is the structural protein of silk fibers.
Silk fibroin can be fabricated easily into desired shapes, such as
films, 3-dimensional porous scaffolds, electrospun fibers, and
hydrogels. These materials have the advantage of excellent
mechanical properties, biocompatibility and biodegradability. Silk
fibroin solutions may be prepared as aqueous stock solution in
accordance with the procedures used by Sofia et al.,
"Functionalized silk-based biomaterials for bone formation," J
Biomed Mater Res. 54 (2001) 139-148, herein incorporated by
reference in its entirety.
[0023] As used herein, the term "fibroin" includes silkworm fibroin
and insect or spider silk protein (Lucas et al., Adv. Protein Chem
13: 107-242 (1958)). Preferably, fibroin is obtained from a
solution containing a dissolved silkworm silk or spider silk. The
silkworm silk protein is obtained, for example, from Bombyx mori,
and the spider silk is obtained from Nephila clavipes. In the
alternative, suitable silk proteins can be obtained from a solution
containing a genetically engineered silk, such as from bacteria,
yeast, mammalian cells, transgenic animals or transgenic plants.
See, for example, WO 97/08315 and U.S. Pat. No. 5,245,012.
[0024] In addition to the silk fibroin, the silk fibroin solution
may also contain one or more therapeutic agents. The therapeutic
agent may be any agent known by those of skill in the art to have
therapeutic properties. Suitable therapeutic agents include
proteins, peptides (preferably therapeutic peptides), nucleic
acids, PNA, aptamers, antibodies, growth factors, cytokines,
enzymes, and small molecules (preferably small medicinal drug
compounds having a molecular weight of less than 1000 Da).
Preferred therapeutic agents include morphogenetic protein 2
(BMP-2), insulin-like growth factor I and II (IGF-I and II),
epidermal growth factor (EGF), platelet-derived growth factor
(PDGF), fibroblast growth factors (FGFs), transforming growth
factors-.beta. (TGFs-.beta.), transforming growth factors-.alpha.,
erythropoietin (EPO), interferon .alpha. and .gamma., interleukins,
tumor necrosis factor .alpha. and .beta., insulin, antibiotics, and
adenosine.
[0025] The therapeutic agent, when mixed with the silk fibroin
solution, can be encapsulated in the silk fibroin microspheres. The
encapsulated therapeutic agent can then be released from the
microspheres through typical release mechanisms known in the art.
Preferably, the therapeutic agent is in an active form when added
to the silk fibroin and in an active form when encapsulated in the
silk fibroin microspheres. Keeping the therapeutic agent in an
active form throughout the microsphere preparation process enables
it to be therapeutically effective upon release from the
microsphere.
[0026] Biocompatible polymers can also be added to the silk fibroin
solution to generate composite matrices. Useful biocompatible
polymers include, for example, polyethylene oxide (PEO) (U.S. Pat.
No. 6,302,848), polyethylene glycol (PEG) (U.S. Pat. No.
6,395,734), collagen (U.S. Pat. No. 6,127,143), fibronectin (U.S.
Pat. No. 5,263,992), keratin (U.S. Pat. No. 6,379,690),
polyaspartic acid (U.S. Pat. No. 5,015,476), polylysine (U.S. Pat.
No. 4,806,355), alginate (U.S. Pat. No. 6,372,244), chitosan (U.S.
Pat. No. 6,310,188), chitin (U.S. Pat. No. 5,093,489), hyaluronic
acid (U.S. Pat. No. 6,387,413), pectin (U.S. Pat. No. 6,325,810),
polycaprolactone (U.S. Pat. No. 6,337,198), polylactic acid (U.S.
Pat. No. 6,267,776), polyglycolic acid (U.S. Pat. No. 5,576,881),
polyhydroxyalkanoates (U.S. Pat. No. 6,245,537), dextrans (U.S.
Pat. No. 5,902,800), polyanhydrides (U.S. Pat. No. 5,270,419), and
combinations thereof (all parenthetical references are to U.S. Pat.
Nos., which illustrate an example of the referenced polymer).
[0027] Lipid vesicles are used in the process as templates to
assist in modeling the microspheres into preferred shapes and
sizes. The lipid composition may include any lipid or combination
of lipids that can form liposomes. Suitable lipids in the lipid
composition include 1,2-dioleoyl-sn-glycero-3-phosphocholine
(DOPC); 1,2-dioleoyl-sn-glycero-3-phophoethanolamine (DOPE);
1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); and
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Other lipid
compositions known in the art may also be used.
[0028] The silk fibroin solution and lipid composition should be
mixed in a manner that integrates the silk fibroin and lipids. When
a therapeutic agent is present in the silk fibroin solution, the
therapeutic agent, silk fibroin, and lipids are all mixed together.
Preferably, the mixing takes place for a sufficient period of time
and under conditions so that the various components are
significantly integrated.
[0029] Sufficient mixing is sometimes difficult to achieve. In such
cases, a freeze-thaw step may be used, which promotes mixing among
the lipids, silk fibroin, and therapeutic agents, when present. A
freeze-thaw step can break larger multilamellar lipid vesicles into
smaller, unilamellar structures that have more homogeneous size
distributions. It can also be used to facilitate silk self assembly
and enhance the encapsulation of the therapeutic agent in the
liposomes.
[0030] Any freeze-thaw treatment known in the art may by used. See,
e.g., Colletier et al., "Protein encapsulation in liposomes:
efficiency depends on interactions between protein and phospholipid
bilayer," BMC Biotechnology 2 (2002) 9-17, herein incorporated by
reference in its entirety, for suitable freeze-thaw techniques. The
freeze-thaw may be repeated one or more times to promote further
mixing and size homogeneity. Freeze-thawing is not deemed necessary
when using certain dehydrating mediums, such as methanol, where the
particle-size distribution and integration level achieved through
mixing alone is usually adequate.
[0031] The amount of silk fibroin solution and lipid composition
that is mixed is dependent on the dehydrating medium used and the
desired structural formation of the microspheres. Typically, 0.1 to
2 ml of 8 (w/v) % silk solution is used for every 100 mg of lipids.
However, these amounts may vary depending on the exact make up of
the silk solution and lipid composition. Additionally, depending on
the dehydrating medium used, each medium will have a threshold
ratio. For instance, when methanol is used as the dehydrating
medium, the threshold ratio is 0.2 ml of 8 (w/v) % silk solution
for every 100 mg of lipids, and when sodium chloride is used as the
dehydrating medium, the threshold ratio is 0.5 ml of 8 (w/v) % silk
solution for every 100 mg of lipids. When the amount of lipids are
above the threshold ratio, multilamellar structures predominately
form in the microspheres; when the amount of lipids are below the
threshold ratio, unilamellar structures predominately form in the
microspheres.
[0032] The lipid components that remain in the microspheres will
form as either uni- or multilamellar structures. Compared to
multilamellar lipid vesicles, unilamellar vesicles offer higher
encapsulation capacity for hydrophilic drugs, more reproducible
rates of release, and less lipid content in the microspheres. On
the other hand, multilamellar vesicles are suitable for
encapsulating both lipophilic and hydrophilic drugs and are more
resistant to enzyme digestion, resulting in a longer circulation
time in the body. Therefore, unilamellar-structured microspheres
are generally preferred when higher drug loading is needed or when
hydrophilic drugs are used; multilamellar-structured microspheres
are generally preferred when lipophilic drugs are used and in cases
when drug loading is not important or when a slower degradation of
microspheres is desired. In addition to vesicle structure (uni- or
multilamellar), the drug release rate is also governed by
lipophilicity of drug molecules, the composition of the
encapsulation device, and the lipid composition.
[0033] After the silk fibroin solution and the lipid composition
have been mixed and optionally freeze-thawed, the mixture is
lyophilized. Lyophilization techniques known in the art may be
used. Typically, the mixture is lyophilized for three days and
stored at temperatures around 4.degree. C.
[0034] The lyophilized material is then combined with a dehydration
medium. The dehydration medium may be any medium that can both
dehydrate the silk fibroin solution and induce .beta.-sheet
structures in the silk fibroin. Dehydrating the silk fibroin
solution extracts water from the silk fibroin and causes the silk
to self assemble and form crystalline .beta.-sheet structures. The
.beta.-sheet structures are physical crosslinks in the silk fibroin
that provide the silk with stability and unique mechanical features
in the fibers. The physical crosslinks also promote the entrapment
of therapeutic agents, when present, in the silk fibroin.
Beta-sheet structures in the silk fibroin may also be induced by
changes in salt concentration and shear forces.
[0035] Microspheres will form upon crosslinking of the silk
fibroin. Preferably, the weight percentage of microspheres in the
total silk is at least about 50%. The amount of microspheres in the
silk is dependent on various factors, such as the dehydration agent
used to induce .beta.-sheet structure, the type of silk fibroin
used, the amount of time the silk is exposed to the dehydration
agent, etc. If a therapeutic agent was introduced in the process,
then the silk fibroin microspheres can encapsulate the therapeutic
agent during microsphere formation.
[0036] The dehydration medium should at least partially dehydrate
the silk fibroin. Preferably, the silk fibroin is sufficiently
dehydrated so that significant amounts (e.g. 50% or more) of
.beta.-sheet structures form in the silk. The amount of dehydration
time necessary to induce .beta.-sheet formation is readily
determinable by one skilled in the art and will depend, in part, on
the dehydration medium used. Because high crystallinity can
significantly retard the release of encapsulated therapeutic
agents, such as proteins, inducing large amounts of .beta.-sheet
formation is preferable when forming microspheres designed for
control release.
[0037] Any known dehydration medium that does not destroy or
otherwise damage the silk fibroin may be used as the dehydration
medium. Polar alcohols, such as methanol and ethanol, are
particularly effective at inducing dehydration of the silk. Other
polar solvents, such as acetone, are also effective. Solvents and
alcohols with lower polarity, such as chloroform and propanol, may
also be used, but are not as effective at stabilizing the silk
structure. Additionally, many salts, such as sodium chloride and
potassium chloride, can dehydrate the silk fibroin as well change
the salt concentration, both of which induce .beta.-sheet
formation. Other suitable dehydration mediums include polyethylene
glycol solutions, desiccants, and dry gas. Preferably, the
dehydration medium is a polar solvent, such as methanol, ethanol,
and acetone, or a salt, such as sodium chloride or potassium
chloride. Methanol and solutions of sodium chloride are
particularly preferred.
[0038] The lyophilized material and dehydration medium may be
combined through any method known in the art. Preferably, the
dehydration medium is in a solution and the lyophilized material is
combined with it by adding the lyophilized material to the solution
containing the dehydration medium. Combining the two components in
this manner will typically form a suspension of the lyophilized
material in the dehydration medium solution. When the lyophilized
material is suspended in the solution, it allows for easier removal
of the lipids.
[0039] At least some of the lipids should be removed after the
lyophilized material has been combined with the dehydration medium.
The lipids may be removed through any technique known in the art.
Centrifugation may be used when the lyophilized material is
suspended in a solution containing the dehydration medium, however,
other removal or extraction techniques may be better suited to
remove the lipids depending on the dehydration medium utilized.
[0040] Certain dehydration mediums can function to remove the
lipids. For instance, a high concentration of methanol or sodium
chloride enables each medium to function as both a dehydration
medium and lipid remover. Additional removal steps, such as
centrifugation, are nonetheless still preferred even when using
methanol or sodium chloride. Other dehydration mediums, such as
desiccants or dry gas, function little if at all as a lipid
remover. These type of dehydration mediums, therefore, may have to
be combined with a more rigorous lipid extraction or removal step,
or multiple extraction/removal steps.
[0041] It is preferable to remove all or substantially all of the
removable lipids. Depending on the removal techniques and
dehydration medium used, complete lipid removal may not be
possible. For instance, when using methanol as the dehydration
medium, about 99% of the lipids are able to be removed; when using
sodium chloride as the dehydration medium, about 83% of the lipids
are able to be removed. In these cases, all or substantially all of
the removable lipids are considered to have been removed because
further removal techniques would not lead to any substantial amount
of additional lipids being removed.
[0042] While it is preferable to remove most of the lipids, it is
also believed that the lipid components, when present in a
relatively small amount, can be beneficial. In particular, it is
believed that the lipid component can assist in controlling the
release of the therapeutic agent from the microspheres. Therefore,
according to an embodiment of the invention, it is preferable to
have a microsphere composition where about 15 to about 20% of the
total lipids remain in the silk fibroin microspheres. It is also
preferable to have a microsphere composition where less than about
5% of the total lipids remain in the silk fibroin microspheres.
More preferably, less than about 2% of the total lipids remain in
the microspheres.
[0043] After the desired amount of lipids have been removed, the
composition is typically in a dehydrated pellet form. The
composition may be hydrated by suspending or resuspending the
microsphere composition in water or a buffer solution. Suspending
the microspheres in water or a buffer is often done before the
microsphere composition is used in a commercially viable manner.
For instance, if the silk fibroin microspheres are used in a
formulation suitable for administration, the formulation will
typically contain hydrated microspheres.
[0044] A pharmaceutical formulation may be prepared that contains
the silk fibroin microspheres having encapsulated therapeutic
agents. The formulation can be administered to a patient in need of
the particular therapeutic agent that has been encapsulated in the
microspheres.
[0045] The pharmaceutical formulation may be administered by a
variety of routes known in the art including topical, oral,
parenteral (including intravenous, intraperitoneal, intramuscular
and subcutaneous injection as well as intranasal or inhalation
administration) and implantation. The delivery may be systemic,
regional, or local. Additionally, the delivery may be intrathecal,
e.g., for CNS delivery.
[0046] In addition to the silk microspheres, the pharmaceutical
formulation may also contain a targeting ligand. Targeting ligand
refers to any material or substance which may promote targeting of
the pharmaceutical formulation to tissues and/or receptors in vivo
and/or in vitro with the formulations of the present invention. The
targeting ligand may be synthetic, semi-synthetic, or
naturally-occurring. Materials or substances which may serve as
targeting ligands include, for example, proteins, including
antibodies, antibody fragments, hormones, hormone analogues,
glycoproteins and lectins, peptides, polypeptides, amino acids,
sugars, saccharides, including monosaccharides and polysaccharides,
carbohydrates, vitamins, steroids, steroid analogs, hormones,
cofactors, and genetic material, including nucleosides,
nucleotides, nucleotide acid constructs, peptide nucleic acids
(PNA), aptamers, and polynucleotides. Other targeting ligands in
the present invention include cell adhesion molecules (CAM), among
which are, for example, cytokines, integrins, cadherins,
immunoglobulins and selectin.
[0047] The pharmaceutical formulations may also encompass precursor
targeting ligands. A precursor to a targeting ligand refers to any
material or substance which may be converted to a targeting ligand.
Such conversion may involve, for example, anchoring a precursor to
a targeting ligand. Exemplary targeting precursor moieties include
maleimide groups, disulfide groups, such as ortho-pyridyl
disulfide, vinylsulfone groups, azide groups, and iodo acetyl
groups.
[0048] The pharmaceutical formulations may contain common
components found in other pharmaceutical formulations, such as
known excipients. Exemplary excipients include diluents, solvents,
buffers, solubilizers, suspending agents, viscosity controlling
agents, binders, lubricants, surfactants, preservatives and
stabilizers. The formulations may also include bulking agents,
chelating agents, and antioxidants. Where parenteral formulations
are used, the formulation may additionally or alternately include
sugars, amino acids, or electrolytes.
[0049] Suitable excipients include polyols, for example, of a
molecular weight less than about 70,000 kD, such as trehalose,
mannitol, and polyethylene glycol. See for example, U.S. Pat. No.
5,589,167, the disclosure of which is incorporated by reference
herein. Exemplary surfactants include nonionic surfactants, such as
Tweeng surfactants, polysorbates, such as polysorbate 20 or 80,
etc., and the poloxamers, such as poloxamer 184 or 188, Pluronic
polyols, and other ethylene/polypropylene block polymers, etc.
Suitable buffers include Tris, citrate, succinate, acetate, or
histidine buffers. Suitable preservatives include phenol, benzyl
alcohol, metacresol, methyl paraben, propyl paraben, benzalconium
chloride, and benzethonium chloride. Other additives include
carboxymethylcellulose, dextran, and gelatin. Suitable stabilizing
agents include heparin, pentosan polysulfate and other heparinoids,
and divalent cations such as magnesium and zinc.
[0050] The pharmaceutical formulations containing the microspheres
can be administered in a controlled-release manner so that portions
of the therapeutic agent are released in the patient over a period
of time. The therapeutic agent may release quickly or slowly. For
instance, the pharmaceutical formulation can be administered so
that less than about 5% of the therapeutic agent is released in the
patient from the microspheres over a period of one month.
Alternatively, a larger portion of the therapeutic agent may be
released initially, with a smaller portion retained in the
microspheres and released later. For example, the pharmaceutical
formulation can be administered so that at least 5% of the
therapeutic agent remains in the microspheres 10 days after
administration.
[0051] When administering the therapeutic agent in a
controlled-release manner, the therapeutic agent preferably remains
active in the microspheres so that it can perform its therapeutic
function upon release. Certain therapeutic agents become inactive
when exposed to encapsulation conditions for a significant period
time. Of course, the release of inactive therapeutic agents is of
little or no value to the patient, who is not able to receive the
benefits of an active therapeutic agent. A preferred pharmaceutical
formulation contains microspheres where the activity of the
therapeutic agent in the microspheres remains at least 50% one
month after administration to the patient.
[0052] Controlled release permits dosages to be administered over
time, with controlled release kinetics. In some instances, delivery
of the therapeutic agent is continuous to the site where treatment
is needed, for example, over several weeks. Controlled release over
time, for example, over several days or weeks, or longer, permits
continuous delivery of the therapeutic agent to obtain preferred
treatments. The controlled delivery vehicle is advantageous because
it protects the therapeutic agent from degradation in vivo in body
fluids and tissue, for example, by proteases.
[0053] Controlled release from the pharmaceutical formulation may
be designed to occur over time, for example, for greater than about
12 or 24 hours. The time of release may be selected, for example,
to occur over a time period of about 12 hours to 24 hours; about 12
hours to 42 hours; or, e.g., about 12 to 72 hours. In another
embodiment, release may occur for example on the order of about 2
to 90 days, for example, about 3 to 60 days. In one embodiment, the
therapeutic agent is delivered locally over a time period of about
7-21 days, or about 3 to 10 days. In other instances, the
therapeutic agent is administered over 1, 2, 3 or more weeks in a
controlled dosage. The controlled release time may be selected
based on the condition treated. For example, longer times may be
more effective for wound healing, whereas shorter delivery times
may be more useful for some cardiovascular applications.
[0054] Another embodiment of this invention relates to a drug
delivery composition comprising a therapeutic agent encapsulated in
crosslinked silk fibroin microspheres, wherein the microspheres
contain lipid components. The silk fibroin microspheres may be
crosslinked by exposing the silk fibroin to a dehydrating medium,
such as methanol or sodium chloride, which induces .beta.-sheet
formation, or the crosslinking of the silk fibroin.
[0055] When silk fibroin microspheres are prepared with a process
that utilizes lipid components, a portion of the lipid components
is typically present in the silk fibroin microspheres, even when
all of the removable lipid components have been removed. Depending
on the process used to incorporate and/or remover the lipids, lipid
components will typically be present in the microspheres from about
1 to about 25%, by weight. Preferably, the microspheres contain
less than about 20% lipids by weight, more preferably less than
about 5% lipids by weight. It is believed that the lipids, when
present in relatively small amounts, assist in controlling the
release of the therapeutic agent from the microspheres. When the
microspheres contain too high a percentage of lipids, the structure
and physical parameters of the silk fibroin microspheres can be
compromised, resulting in less effective microspheres or
microspheres with insufficient structural integrity.
[0056] Another embodiment of this invention relates to a method of
encapsulating a biomaterial in silk fibroin microcapsules. The
method comprises (a) mixing a solution comprising silk fibroin and
a biomaterial with a lipid composition; (b) lyophilizing the
mixture; (c) combining the lyophilized material with a dehydration
medium for a sufficient period of time to at least partially
dehydrate the silk fibroin solution and induce .beta.-sheet
structures in the silk fibroin; and (d) removing at least a portion
of the lipids to produce a biomaterial that has been encapsulated
in silk fibroin microspheres.
[0057] The biomaterial may be a therapeutic agent, such one or more
of the therapeutic agents discussed above. However, the
encapsulation process does not have to be used in the field of
pharmaceutical formulations and controlled-release methods. The
silk fibroin microcapsules may encapsulate various other
biomaterials useful in a variety of fields. For instance, the
biomaterial may be an enzyme or an enzyme-based electrode. The
enzyme or enzyme-based electrode may be used in the field of tissue
engineering, biosensors, the food industry, environmental control,
or biomedical applications. The system can also be used as a
reservoir for a variety of needs, such as in the food industry to
harbor vitamins, nutrients, antioxidants and other additives; in
the environmental field to harbor microorganisms for remediation or
water treatments; in materials as additives to serve as a source of
in situ detection and repair components, such as for nondestructive
evaluation of material failures and self-repairs of the materials;
and for biodetection schemes to help stabilize cells, molecules and
related systems.
[0058] The silk fibroin microspheres of the invention form in a
manner that provides them with advantageous physical properties
that are particularly useful for encapsulating therapeutic agents
for uses in controlled-release pharmaceutical formulations. The
microspheres exhibit a more homogeneous shape and size, especially
when compared to microspheres prepared via conventional techniques,
such as spray-dry methods. Exhibiting a homogeneous spherical
shape, the microspheres are less likely to experience aggregation,
which occurs more commonly when the microspheres are in a funicular
(fibrillar or elongated) state. The smaller and more narrow
diameter range of microspheres also provides a more consistent and
controlled release.
[0059] Accordingly, an embodiment of this invention relates to a
silk fibroin microsphere composition, comprising a therapeutic
agent encapsulated in crosslinked silk fibroin microspheres,
wherein at least 75% of the microspheres are spherical or
substantially spherical, and wherein at least 75% of the
microspheres have a diameter ranging from about 1.0 to about 3.0
.mu.m. Preferably, at least 90% of the microspheres are spherical
or substantially spherical, and at least 90% of the microspheres
have a diameter ranging from about 1.0 to about 3.0 .mu.m. More
preferably, at least 95% of the microspheres have a diameter
ranging from about 1.0 to about 3.0 .mu.m. The average size of the
microspheres is preferably less than about 2.0 .mu.m. The silk
microspheres with small sizes are of more interest for biomedical
applications
[0060] The size and shape of the microsphere will be dependent, to
some degree, on what techniques are used to crosslink the silk
fibroin. For instance, dehydrating the silk fibroin in methanol in
the above-described methods will typically produce microspheres
wherein about 90% of the microspheres are substantially spherical
and about 90% have a diameter ranging from 1.0 to 3.0 .mu.m. The
term "substantially spherical," as used herein, means spherical
microspheres that contain small blemishes in the surface or on the
edges of the microspheres, but that would otherwise be considered
spherical as opposed to funicular. See FIG. 3E, depicting
substantially spherical microspheres. Using these methods with
sodium chloride will typically produce microspheres wherein about
90% of the microspheres are spherical and about 98% have a diameter
ranging from 1.0 to 3.0 .mu.m. See FIG. 31, depicting spherical
microspheres.
[0061] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. Although methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of the invention, the preferred methods and materials
are described below. All publications, patent applications, patents
and other references mentioned herein are incorporated by
reference. In addition, the materials, methods and examples are
illustrative only and not intended to be limiting. In case of
conflict, the present specification, including definitions,
controls.
[0062] The invention will be further characterized by the following
examples which are intended to be exemplary of the invention.
EXAMPLES
Materials
[0063] Cocoons of B. mori silkworm silk were supplied by M. Tsukada
(Institute of Sericulture, Tsukuba, Japan).
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein)
(fluorescein-DOPE) were purchased from Avanti Polar Lipids
(Alabaster, Ala.). 5-(Aminoacetamido)fluorescein (fluoresceinyl
glycine amide) was purchased from Molecular Probes (Carlsbad,
Calif.). Rhodamine .beta. isothiocyanate-Dextran (M.W. 40,000 Da),
horseradish peroxidase (HRP), .beta.-galactosidase, and other
chemicals were obtained from Sigma Aldrich (St. Louis, Mo.).
3,3'5,5' Tetramethylbenzidine (TMB) solution was purchased from
BioFX laboratories (Owing Mills, Md.).
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC),
N-hydroxysuccinimide (NHS), and hydroxylamine hydrochloride were
purchased from Pierce Biotechnology (Rockford, Ill.). All other
chemicals were purchased from Sigma-Aldrich (St. Louis, Mo.).
Purification and Fluorescent Labeling of Silk Fibroin:
[0064] Silk fibroin aqueous stock solutions were prepared as
described by Brandl, "Liposomes as drug carriers: a technological
approach," Biotechnol. Ann. Rev. 7 (2001) 59-85, herein
incorporated by reference in its entirety. Briefly, cocoons of B.
mori were boiled for 20 min in an aqueous solution of 0.02 M sodium
carbonate, and then rinsed thoroughly with pure water. After
drying, the extracted silk fibroin was dissolved in 9.3 M LiBr
solution at 60.degree. C. for 4 hours, yielding a 20% (w/v)
solution. This solution was dialyzed against distilled water using
Slide-a-Lyzer dialysis cassettes (MWCO 3,500, Pierce) for 3 days to
remove the salt. The solution was clear after dialysis and was
centrifuged to remove silk aggregates (small amount) that formed
during the dialysis and some dirt from cocoons. The final
concentration of silk fibroin aqueous solution was approximately 8%
(w/v). This concentration was determined by weighing the residual
solid of a known volume of solution after drying.
[0065] For fluorescent labeling, the silk fibroin stock solution
was diluted to 2% (w/v) with water, and 10 ml of the diluted
solution was dialyzed against 500 ml of 0.1 M
2-(morpholino)ethanesulfonic acid (MES) solution (pH 5.6) (Pierce,
Chemicals, IL) supplemented with 0.9% NaCl overnight. Eighty mg EDC
(2 mM) and 220 mg NHS (5 mM) were added to the buffered silk
solution with stirring and the reaction was continued for 15 min.
.beta.-mercaptoethanol was added to a final concentration of 20 mM
to quench the unreacted EDC. The carboxyl groups on silk fibroin
were activated for reacting with primary amines. After the
reaction, 10 mg of fluoresceinyl glycine amide was added to the
solution so that the molar ratio between fluorescent probe and silk
fibroin was about 40:1. The coupling reaction went for 2 hours
under slow stirring at room temperature and then 8 mg hydroxylamine
hydrochloride was added to quench the reaction. Finally the
solution was dialyzed exhaustively against water. The final
concentration of fluorescent silk fibroin was approximately 1.5%
(w/v) using the same weighing method.
Preparation of Silk Microspheres:
[0066] One hundred mg of DOPC was dissolved in 1 ml chloroform in a
glass tube and dried into a film under a flow of nitrogen gas. 8%
(w/v) silk fibroin solution with volume of 0.33 ml, 0.5 ml, and 1
ml was added to hydrate the lipid film, and the mixture was diluted
to 2 ml with water and moved to a plastic tube. The sample was
frozen in liquid nitrogen for 15 min and then thawed at 37.degree.
C. for 15 min. This freeze-thaw cycle was repeated 3 times and then
the thawed solution was slowly pipetted into a glass beaker
containing 50 ml water with fast stirring. For methanol-treated
microspheres, the freeze-thaw treatment was skipped and the 0.5 ml
of DOPC-silk mixture was diluted to 50 ml water directly. The
resulting solution was lyophilized for 3 days and stored at
4.degree. C.
[0067] To prepare MeOH-- based microspheres, 20 mg lyophilized
material was suspended in 2 ml MeOH in an Eppendorf tube and the
suspension was incubated for 30 min at room temperature followed by
centrifugation at 10,000 rpm for 5 min at 4.degree. C. (Eppendorf
5417R centrifuge). The pellet obtained was dried in air and stored
at 4.degree. C. To generate a suspension of silk microspheres, the
dried pellet was washed once with 2 ml of water by centrifugation,
and then resuspended in the desired water or buffer. The clustered
microspheres were dispersed by ultrasonication for 10 sec at 30%
amplitude (approximately 20 W) using a Branson 450 ultrasonicator
(Branson Ultrasonics Co., Danbury, Conn.).
[0068] To prepare NaCl-based microspheres, 20 mg lyophilized
material was suspended in 2 ml saturated NaCl solution in an
Eppendorf tube and the suspension was incubated at room temperature
for 1 h, 4 h, and 15 h followed by centrifugation at 10,000 rpm for
5 min at 4.degree. C. (Eppendorf 5417R centrifuge). The supernatant
and the white viscous material floating on the top were carefully
removed, and the pellet was washed once with 2 ml water by
centrifugation and then resuspended in water or buffer.
Phospholipids Quantification
[0069] Phospholipids remained in the silk microspheres and were
estimated by phosphorus determination through an acidic digestion.
See Rouser et al., "Two dimensional then layer chromatographic
separation of polar lipids and determination of phospholipids by
phosphorus analysis of spots," Lipids 5 (1970):494-496, and Zhou et
al., "Improved procedures for the determination of lipid phosphorus
by malachite green," J Lipid Res. 33 (1992):1233-1236, both of
which are herein incorporated by reference in their entirety. The
released phosphorus was reacted with ammonium molybdate to form a
strong blue color.
[0070] Dried MeOH-based and NaCl-based silk microspheres were
weighed and transferred into clean glass tubes. 0.65 ml perchloric
acid was added to each sample, and the tubes were heated at
180.degree. C. until the yellow color in all the tubes disappeared.
When cool, the tubes were supplemented with 3.3 ml water, 0.5 ml
2.5% (w/v) molybdate solution and 0.5 ml 10% (w/v) ascorbic acid
solution. The tubes were agitated on a vortex after each addition.
The samples were then boiled in a water bath for 5 min, and the
absorbance of cool samples (including the standards) was read at
800 nm. Potassium phosphate monobasic (KH.sub.2PO.sub.4) solution
was used as a standard. The stock solution of 439 mg per liter
water (i.e., 100 .mu.g phosphorus per milliliter water) was diluted
in 3.3 ml water and 0.65 ml perchloric acid. Digestion at
180.degree. C. was not necessary before adding reagents. The amount
of phospholipids was calculated directly on a weight basis after
multiplying the amount of phosphorus by 25.38 (DOPC contains 3.94%
w/w phosphorus).
Dynamic Light Scattering (DLS)
[0071] Microspheres were diluted in 10 ml water in a glass vial and
analyzed immediately at 25.degree. C. using a BIC BI-200 SM
research goniometer and laser light scattering system (Brookhaven
Instrument, Holtsville, N.Y.). Laser light at 532 nm was used to
measure the fluctuation in intensity of light scattered by
particles. Data were collected for 5 min for each sample, and the
mean diameter of particles was calculated using the BIC dynamic
light scattering software supplied by the manufacturer of the
above-referenced system.
Fourier Transform Infrared (FTIR) Spectroscopy
[0072] FTIR studies were performed using a Bruker Equinox 55 FTIR
spectrometer. A drop of microsphere suspension was added to the
zinc selenide (ZnSe) crystal cell and examined with the FTIR
microscope in the reflection mode. Background measurements were
taken with an empty cell and subtracted from the sample reading.
DOPC suspended in water did not show peaks at the amide I band
region, meaning that its influence was negligible. Deconvolution of
the fibroin amide I spectra was performed using
Gaussian.times.Lorentzian function in the spectroscopic software
from Briler (version 4.2). The curves that had absorption bands at
the frequency range of 1620-1630 cm.sup.-1 and 1695-1700 cm.sup.-1
represented enriched .beta.-sheet structure in silk II form (23).
The contribution of these curves .beta.-sheet structure content) to
the amide I band was assessed by integrating the area under the
curve and then normalizing to the total area under the amide I band
region (1600-1700 cm.sup.-1)
Scanning Electron Microscopy (SEM)
[0073] For lyophilized DOPC-silk and MeOH-based microspheres, dried
materials were directly mounted on samples mounts. For NaCl-based
microspheres, the solution containing microspheres were dried on
plastic slides which were further cut and mounted. Specimens were
then sputter-coated with Au using a Ploaron SC502 Sputter Coater
(Fison Instruments, UK), and examined using a JEOL JSM 840A
Scanning Electron Microscope (Peabody, Mass.) at 15 KV.
Phase Contrast and Confocal Laser Scanning Microscopy
[0074] Microspheres were suspended in pure water and approximately
20 .mu.l of suspension was put on a glass slide and covered with a
cover-slip. The samples were analyzed by a phase contrast light
microscope (Carl Zeiss, Jena, Germany) equipped with a Sony Exwave
HAD 3CCD color video camera, or a confocal laser scanning
microscope (TCS Leica SP2, Welzlar, Germany) with Leica Confocal
Software, version 2.5 (Leica Microsystems, Heidelberg,
Germany).
HRP Loading and Release
[0075] Silk microspheres were prepared as described above except
that 10 .mu.l of rhodamine B-labeled dextran, 40,000 Da or HRP
stock solution at 12.5 mg/ml in buffer, were mixed with 0.5 ml of
8% (w/v) silk solution prior to microsphere formation. Dulbecco's
phosphate buffer, pH 7.2 (Invitrogen, Carlsbad, Calif.) was used
for all HRP determinations. For loading and release, 40 mg of
lyophilized DOPC-silk fibroin was treated with MeOH or NaCl as
described. After washing with buffer, the microspheres were
suspended in 2 ml of phosphate buffer, pH 7.2. One ml aliquots of
the suspension were used for HRP loading, and the other 1 ml
aliquot for HRP release. TMB (HRP substrate, Mw=240 Da) was
oxidized during the enzymatic degradation of H.sub.2O.sub.2 by HRP.
The oxidized product of TMB exhibited a deep blue color which
turned to yellow upon addition of the acidic stop solution.
[0076] For loading determinations, 5 .mu.l of the suspension was
mixed with 100 .mu.l of TMB solution in 96-well standard microplate
wells for 1 min at room temperature. The reaction was stopped by
the addition of 100 .mu.l 0.1 M sulfuric acid. Absorbance was
detected at 450 nm by using a VersaMax microplate reader (Molecular
devices, Sunnyvale, Calif.). The HRP content was obtained using a
HRP standard curve generated under the same condition. The
remaining microspheres (995 .mu.l) were spun down, dried and
weighed. The loading was obtained as follows:
Loading ( g / mg ) = HRP content ( g ) .times. 199 Weight of
microspheres ( mg ) The loading efficiency was calculated as
follows : Loading efficiency ( % ) = HRP loading ( g / mg ) .times.
Total mircospheres ( mg ) Total HRP ( g ) ##EQU00001##
[0077] To determine HRP release, 1 ml suspensions of silk
microspheres were incubated at room temperature. At desired time
points, the suspensions were centrifuged at 10,000 rpm for 2 min.
The supernatant was carefully moved to another tube and the pellet
was resuspended in 1 ml fresh buffer. HRP content in the
supernatant was determined as described above and the percentage of
release was obtained by comparing this data with the loading data.
All experiments were performed in triplicate. Statistical analysis
of data was performed using the Student's t-test. Differences were
considered significant when p.ltoreq.0.05.
Liposome-Assisted Silk Microsphere Preparation
[0078] FIG. 1 shows the microscopic images of particle suspensions
that were generated in the different steps. As a control, hydration
of the DOPC film with water resulted in highly dispersed vesicles
with a heterogeneous size distribution (FIG. 1A). Hydration of DOPC
films with silk fibroin solution resulted in clustered vesicles
with similar heterogeneous size distributions (FIG. 1B). Once the
DOPC-silk mixture was freeze-thawed 3 times, the water suspension
was dominated by highly dispersed particles with a homogeneous size
distribution (FIG. 1C). Once the freeze-thawed and lyophilized
DOPC-silk was suspended in saturated NaCl solution, some particles
fused in time into larger lipid vesicles (FIGS. 1D-E). During
preparation, these larger lipid vesicles floated on top of the NaCl
solution and could be removed by subsequent centrifugation. The
reason that some vesicles tend to fuse in this case is probably due
to high lipid content within the vesicles. Those with low lipid but
high silk content could survive and be treated into solid
NaCl-based microspheres that were precipitated by centrifugation.
Similarly, MeOH dissolved those lipid-rich vesicles but treated
those silk-rich vesicles into MeOH-based microspheres.
Yield of Silk Microspheres
[0079] The lipid-to-silk ratio was adjusted to obtain a high yield
of microspheres (the weight percentage of microspheres in the total
silk). In this example, MeOH treatment was used, and the weights of
microspheres were compared with the total silk that was originally
added. As shown in FIG. 2A, a yield of about 55% was obtained when
40 mg of silk (0.5 ml 8% w/v silk solution) was mixed with 100 mg
DOPC. Silk was encapsulated to a saturated level in the lipid
vesicles at this ratio but it was diluted when below the ratio and,
therefore, was easier to be dispersed by MeOH. Thus, 0.5 ml 8% w/v
silk solution and 100 mg DOPC was used as a standard condition for
other preparations. Thirty-minute treatment time was used to
prepare MeOH-based microspheres, which was found to be sufficient
to induce characteristic silk II .beta.-sheet structures. For
NaCl-based preparations, the yields were reported in FIG. 2B. The
yield of microspheres was significantly increased with NaCl
treatment time, indicating that long NaCl treatment time (at least
15 h) is preferred for lipid removal and silk self-assembly, which
is consistent with the observation by microscopic study (FIG. 1)
and FTIR study (FIG. 3).
Particle Sizes
[0080] MeOH-based microspheres had an average size of 1.7 .mu.m, as
determined by dynamic light scattering (Table 1). The average size
of NaCl-based microspheres decreased with time of NaCl treatment,
from 2.7 .mu.m for 1 hour to 1.6 .mu.m when treated for 15 hours
(Table 1), indicating that the silk microspheres became more
condensed upon NaCl-treatment.
[0081] As shown in FIGS. 3 E-H, approximately 90% of the
methanol-based microspheres have a particle size ranging from 1.0
.mu.m to 3.0 .mu.m. As shown in FIGS. 3 I-L, approximately 98% of
the sodium chloride-based microspheres have a particle size ranging
from 1.0 .mu.m to 3.0 .mu.m.
Phospholipid Content
[0082] The phospholipids contents remained in the silk microspheres
were determined by phosphorus assay as described in the materials
and methods. The result showed that the MeOH-based and NaCl-based
microspheres contained about 1% w/w and 17% w/w DOPC, respectively
(Table 1).
TABLE-US-00001 TABLE 1 Characteristics of silk microspheres
DOPC-silk DOPC-silk MeOH-based 1 h NaCl 4 h NaCl 15 h NaCl
DOPC-silk MeOH Freeze-thaw MS MS MS MS Particle size (.mu.m).sup.1
-- -- -- 1.73 .+-. 0.11 2.70 .+-. 0.35 2.24 .+-. 0.17 1.60 .+-.
0.09 (mean .+-. SD) n = 3 Phospholipids content.sup.2 (%) -- -- --
0.965 .+-. 0.16 -- -- 17.13 .+-. 2.14 HRP loading.sup.3 0.082 .+-.
0.006 0.086 .+-. 0.013 0.165 .+-. 0.012 0.173 .+-. 0.02 0.062 .+-.
0.007 0.109 .+-. 0.013 0.148 .+-. 0.019 (ug/mg silk MS) HRP Loading
efficiciency.sup.4 (%) 9.8 .+-. 0.7 9.6 .+-. 1.4 19.8 .+-. 1.4 20.8
.+-. 2.4 7.4 .+-. 0.8 13.1 .+-. 1.6 17.8 .+-. 2.2 .sup.1Determined
by dynamic lighter scattering. Standard deviation (SD) obtained
based on three measurements. .sup.2Phospholipids content represents
the weight percentage of phospholipids in microspheres.
.sup.3Determined by directly mixing substrate TMB with the
microspheres suspended in buffer. .sup.4Calculated by comparing the
amount of HRP determined in the silk microspheres with the total
amount of HRP added at the beginning.
Surface Morphology
[0083] The lyophilized DOPC-silk microspheres showed a smooth
surface by SEM (FIG. 3, A-D). A similar surface morphology was
observed for the NaCl-treated microspheres (FIG. 3, I-L). The
MeOH-based microspheres exhibited a rougher surface that displayed
minor defects at the sub-micron level (FIG. 3, E-H). It is believed
that the difference in surface morphology between MeOH-- and
NaCl-based microspheres might have reflected their difference in
phospholipids contents.
[0084] As shown in FIGS. 3 E-H, approximately 90% of the
methanol-based microspheres have a substantially spherical shape.
As shown in FIGS. 3 I-L, approximately 90% of the sodium
chloride-based microspheres have a spherical shape.
Lamellar Structures
[0085] Fluorescent probe (fluorescein)-labeled DOPE was used to
trace the phospholipids remaining in the microspheres using
confocal laser scanning microscopy. Phospholipids remained in the
MeOH-- and NaCl-based silk microspheres (FIGS. 4A and B), forming
either multilamellar (FIG. 4C) or unilamellar structures (FIG. 4D).
The formation of lamellar structure is believed to be influenced by
the ratio between lipid and silk in a microsphere: Once the ratio
is above a critical level, lipid will dominate the formation of
multilamellar structures, while below this level the silk fibroin
would dominate the formation of unilamellar structures.
Silk .beta.-Sheet Structures
[0086] The .beta.-sheet content in the MeOH-- and NaCl-based
microspheres was assessed by FTIR (FIG. 5). When the NaCl treatment
time was increased, the absorbance at the region of random coil,
.alpha.-helix, and turn and bend (1640-1690 cm.sup.-1)
significantly decreased (curve b, c, e in FIG. 5), indicating that
the .beta.-sheet structure (silk II band at characteristic region
(1620-1630 cm.sup.-1)) was increasing. Deconvolution of the curves
showed that the initial material, freeze-thawed and lyophilized
DOPC-silk, contained about 29% .beta.-sheet structure, which is
slightly higher than the 25% content that has been reported for
soluble silk fibroin in an aqueous solution. This indicates that
the protein structure was not significantly influenced by the
freeze-thaw and lyophilization process under the experimental
condition (mixed with lipids). The NaCl-based microspheres with 1
h, 4 h, and 15 h NaCl treatment showed .alpha.-sheet contents of
about 34%, 51%, and 67%, respectively. MeOH-based microspheres also
showed high .beta.-sheet content of about 58%. These trends
indicate that the .beta.-sheet content in silk microspheres
increases as the micropshere size decreases.
Controlled Drug Release
1. Silk and Drug Distribution in Silk Microspheres
[0087] The distribution of fluorescein-labeled silk (green) and
rhodamine B labeled dextran 40,000 (red) in microspheres was
studied by confocal laser scanning microscopy. When the freeze-thaw
step was not included in the preparation, silk and dextran were
found to locate in separate layers (FIG. 6A) or domains (FIG. 6B)
in the MeOH-based microspheres. Once the freeze-thaw treatment was
performed before lyophilization, in both MeOH-- and NaCl-based
microspheres, the silk and dextran were mixed in the layers (FIG. 6
C, D). Freeze-thaw was used to promote mixing between the silk
fibroin and the rhodamine B-labeled dextran 40,000.
2. HRP Loading in Silk Microspheres
[0088] Loading was determined in lyophilized DOPC-silk with and
without freeze-thaw. The freeze-thaw step increased the loading and
loading efficiency by approximately two-fold when compared to the
non-freeze-thawed samples (first and third columns in Table 1).
This might be because the freeze-thaw treatment helped mix silk and
drug in the microspheres so that more drug molecules could be
packed into the microspheres. MeOH treatment on both samples did
not deactivate the HRP and, therefore, the loading and loading
efficiency were not changed in the corresponding MeOH-based
microspheres (first and second, third and fourth, columns in Table
1). The loading and loading efficiency in the NaCl-based
microspheres with 1 h treatment were much lower than those in the
original material (third and fifth columns in Table 1), but
increased with time of NaCl treatment. The 15 h treatment led to
the loading of about 0.15 .mu.g of HRP per mg of silk microspheres,
close to the level in the original material (third and last columns
in Table 1). It is likely that some empty lipid vesicles that were
not yet fused after shorter NaCl treatments were co-precipitated
with silk microspheres, which contributed to the measured weights
and lowered the loading as a result.
3. HRP Release from Silk Microspheres
[0089] HRP that was encapsulated in lyophilized DOPC-silk displayed
a significant release once the material was suspended in PBS buffer
(FIG. 7A). In contrast, less than 5% HRP was released from the
MeOH-based microspheres (with or without freeze-thaw treatment)
into the surrounding buffer over a period of one month (FIG. 7A).
The activity in the microspheres, however, dropped slowly, with
about 50% remaining after one month (data not shown). NaCl-based
microspheres released encapsulated HRP at different release rates,
depending on the NaCl treatment time used. When the treatment
lasted for 15 h, a sustained release which reached maximal level
after 15 days was achieved (FIG. 7B). The 1 h and 4 h treated
samples released HRP more quickly. For all these three samples, the
HRP release reached about 200%. It is known that HRP activity can
be inhibited by many factors, including metal ions like Mn.sup.2+,
Co.sup.2+, Ni.sup.2+, and Cu.sup.2+, L-cystine and sulfide, and
surfactants and lipids. Therefore, it is likely that some of these
factors within the microspheres inhibited certain HRP activity,
resulting in an underestimation of HRP loading (Table 1). Once
released to the buffer, the inhibited HRP activity was
restored.
[0090] The HRP release as calculated by dividing the amount of
release by the loading, which produced values higher than 100%. It
is hard to determine the absolute HRP loading in this case since it
is difficult to extract HRP from silk microspheres while keeping
the enzyme active. NaCl treatment induces the formation of
.beta.-sheet structures, as demonstrated in FIG. 5, with beta sheet
content dependent on time of treatment, which produced different
drug release profiles.
[0091] The encapsulated HRP was released slower from MeOH-based
microspheres as compared to NaCl-based microspheres with 15 h
treatment, despite the fact that their .beta.-sheet contents were
both high (58% and 67%, respectively). The discrepancy might be due
to the different amount of phospholipids in MeOH-based microspheres
(1%) and NaCl-based microspheres (17%). It is believed that having
more phospholipids in microspheres provided more channels for HRP
to escape.
[0092] Because of its excellent entrapment capability, MeOH-based
silk microspheres are the preferred long-term drug delivery and
enzyme immobilizations. It is believed that other alcohol- or
solvent-based silk microspheres, such as ethanol, propanol,
acetone, chloroform, or polyethylene glycol solutions, would
provide similar entrapment capabilities for drug delivery. Because
of its mild preparation condition and controllable crystalline
.beta.-sheet structure formation, NaCl-based microspheres are the
preferred microspheres for those applications in which protein
drugs or other therapeutic drugs are susceptible to methanol or
alcohol treatment alternative drug release kinetics are needed. It
is believed that other salt-based silk microspheres, such as
potassium chloride, would also be suitable for these
applications.
[0093] The silk microspheres may also be used for tissue
engineering applications. For instance, by combining silk scaffolds
with the microspheres, the system can be used to deliver growth
factors in a time- and/or spatial-controllable manner so that the
artificial tissues like bone and cartilage can be generated with
more localized control from these scaffolds. Depending on the
processing, MeOH-based and NaCl-based silk microspheres released
encapsulated HRP with different kinetics, suggesting that the silk
microspheres can be useful and can carry sufficient growth factors
for tissue engineering applications.
[0094] Aside from controlled drug delivery, silk microspheres can
also be used to immobilize enzymes for biosensor purposes. For
instance, by combining silk microspheres and layer-by-layer coating
techniques using silk fibroin, enzyme-based electrodes can be
envisioned for use in a variety of applications, such as in the
food industry, environmental control, and biomedical
applications.
* * * * *