U.S. patent application number 12/098354 was filed with the patent office on 2009-03-19 for poly(amino acid) targeting moieties.
This patent application is currently assigned to MASSACHUSETTS INSTITUTE OF TECHNOLOGY. Invention is credited to Frank Alexis, Pamela Basto, Juliana Chan, Omid C. Farokhzad, Frank X. Gu, Robert S. Langer, Etgar Levy-Nissenbaum, Aleksandar F. Radovic-Moreno, Liangfang Zhang.
Application Number | 20090074828 12/098354 |
Document ID | / |
Family ID | 39831375 |
Filed Date | 2009-03-19 |
United States Patent
Application |
20090074828 |
Kind Code |
A1 |
Alexis; Frank ; et
al. |
March 19, 2009 |
POLY(AMINO ACID) TARGETING MOIETIES
Abstract
The present invention generally relates to polymers and
macromolecules, in particular, to polymers useful in particles such
as nanoparticles. One aspect of the invention is directed to a
method of developing nanoparticles with desired properties. In one
set of embodiments, the method includes producing libraries of
nanoparticles having highly controlled properties, which can be
formed by mixing together two or more macromolecules in different
ratios. One or more of the macromolecules may be a polymeric
conjugate of a moiety to a biocompatible polymer. In some cases,
the nanoparticle may contain a drug. Other aspects of the invention
are directed to methods using nanoparticle libraries.
Inventors: |
Alexis; Frank; (Brighton,
MA) ; Zhang; Liangfang; (San Diego, CA) ;
Radovic-Moreno; Aleksandar F.; (Cambridge, MA) ; Gu;
Frank X.; (Waterloo, CA) ; Basto; Pamela;
(Somerville, MA) ; Levy-Nissenbaum; Etgar;
(Tel-Aviv, IL) ; Chan; Juliana; (Cambridge,
MA) ; Langer; Robert S.; (Newton, MA) ;
Farokhzad; Omid C.; (Chestnut Hill, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP;FLOOR 30, SUITE 3000
ONE POST OFFICE SQUARE
BOSTON
MA
02109
US
|
Assignee: |
MASSACHUSETTS INSTITUTE OF
TECHNOLOGY
Cambridge
MA
|
Family ID: |
39831375 |
Appl. No.: |
12/098354 |
Filed: |
April 4, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60910097 |
Apr 4, 2007 |
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60938590 |
May 17, 2007 |
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60985104 |
Nov 2, 2007 |
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60986202 |
Nov 7, 2007 |
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60990250 |
Nov 26, 2007 |
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Current U.S.
Class: |
424/422 ;
424/486; 424/489; 514/1.1; 514/178; 514/291; 514/449; 514/656;
514/773; 530/330 |
Current CPC
Class: |
A61K 9/5192 20130101;
A61P 9/10 20180101; A61P 9/00 20180101; A61P 9/08 20180101; A61P
9/14 20180101; A61P 37/04 20180101; A61K 47/62 20170801; B82Y 5/00
20130101; A61K 9/5153 20130101; A61K 47/6935 20170801; A61K 9/5123
20130101; A61K 47/6937 20170801; A61P 35/00 20180101 |
Class at
Publication: |
424/422 ;
424/489; 514/773; 424/486; 514/656; 514/449; 514/12; 514/291;
514/178; 530/330 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 47/42 20060101 A61K047/42; A61K 31/135 20060101
A61K031/135; A61K 31/337 20060101 A61K031/337; A61K 38/16 20060101
A61K038/16; A61K 31/4353 20060101 A61K031/4353; A61K 31/56 20060101
A61K031/56; A61K 9/00 20060101 A61K009/00; C07K 5/10 20060101
C07K005/10; A61P 9/00 20060101 A61P009/00 |
Claims
1. A controlled-release system, comprising a plurality of
target-specific stealth nanoparticles; wherein said nanoparticles
contain targeting moieties attached thereto, wherein the targeting
moiety is a poly(amino acid) that targets the basement membrane of
a blood vessel.
2. The controlled-release system of claim 1, wherein the
nanoparticle has an amount of targeting moiety effective for the
treatment of vulnerable plaque in a subject in need thereof.
3. The controlled-release system of claim 1, wherein the
nanoparticle has an amount of targeting moiety effective for the
treatment of restenosis.
4. The controlled-release system of claim 1, wherein the
nanoparticle has an amount of targeting moiety effective for the
treatment of cancer in a subject in need thereof.
5-6. (canceled)
7. The controlled-release system of claim 1, wherein the poly(amino
acid) comprises natural amino acids, unnatural amino acids,
modified amino acids, or protected amino acids.
8. The controlled-release system of claim 1, wherein the poly(amino
acid) is selected from the group consisting of a protein,
peptidomimetic, affibody or peptide.
9. The controlled-release system of claim 1, wherein the poly(amino
acid) binds to the basement membrane of a blood vessel.
10. The controlled-release system of claim 1, wherein the
poly(amino acid) binds to collagen.
11. The controlled-release system of claim 1, wherein the
poly(amino acid) binds to collagen IV.
12. (canceled)
13. The controlled-release system of claim 8, wherein the peptide
comprises a sequence selected from the group consisting of AKERC,
CREKA, ARYLQKLN and AXYLZZLN, wherein X and Z are variable amino
acids.
14. The controlled-release system of claim 1, wherein the
nanoparticle comprises a polymeric matrix.
15. The controlled-release system of claim 14, wherein the
polymeric matrix comprises two or more polymers.
16. The controlled-release system of claim 13, wherein the
polymeric matrix comprises polyethylenes, polycarbonates,
polyanhydrides, polyhydroxyacids, polypropylfumerates,
polycaprolactones, polyamides, polyacetals, polyethers, polyesters,
poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,
polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,
polycyanoacrylates, polyureas, polystyrenes, dextran or polyamines,
or combinations thereof.
17-19. (canceled)
20. The controlled-release system of claim 14, wherein at least one
polymer is a polyester.
21. The controlled-release system of claim 20, wherein the
polyester is selected from the group consisting of PLGA, PLA, PGA,
and polycaprolactones.
22. The controlled-release system of claim 20, wherein the
polyester is PLGA or PLA.
23. The controlled-release system of claim 14, wherein the
polymeric matrix comprises a copolymer of two or more polymers.
24. The controlled-release system of claim 23, wherein the
copolymer is a copolymer of a polyalkylene glycol and a
polyester.
25. The controlled-release system of claim 24, wherein the
copolymer is a copolymer of PLGA and PEG.
26. The controlled-release system of claim 24, wherein the
polymeric matrix comprises PLGA and a copolymer of PLGA and
PEG.
27. (canceled)
28. The controlled-release system of claim 14, wherein the
polymeric matrix comprises lipid-terminated PEG and PLGA.
29-34. (canceled)
35. The controlled-release system of claim 14, wherein the
nanoparticle has a ratio of ligand-bound polymer to
non-functionalized polymer effective for the treatment of
cancer.
36. The controlled-release system of claim 14, wherein the
nanoparticle has a ratio of ligand-bound polymer to
non-functionalized polymer effective for the treatment of a
vulnerable plaque.
37. The controlled-release system of claim 14, wherein the polymers
of the polymer matrix have a molecular weight effective for the
treatment of cancer.
38. The controlled-release system of claim 14, wherein the polymers
of the polymer matrix have a molecular weight effective for the
treatment of vulnerable plaque.
39-42. (canceled)
43. The controlled-release system of claim 1, wherein the
nanoparticle further comprises a therapeutic agent.
44-45. (canceled)
46. The controlled-release system of claim 43, wherein the
therapeutic agent is selected from the group consisting of
mitoxantrone and docetaxel.
47. The controlled-release system of claim 43, wherein the
therapeutic agent is selected from the group consisting of VEGF,
fibroblast growth factors, monocyte chemoatractant protein 1
(MCP-1), transforming growth factor alpha (TGF-alpha), transforming
growth factor beta (TGF-beta), DEL-1, insulin like growth factors
(IGF), placental growth factor (PLGF), hepatocyte growth factor
(HGF), prostaglandin E1 (PG-E1), prostaglandin E2 (PG-E2), tumor
necrosis factor alpha (THF-alpha), granulocyte stimulating growth
factor (G-CSF), granulocyte macrophage colony-stimulating growth
factor (GM-CSF), angiogenin, follistatin, and proliferin, PR39,
PR11, nicotine, hydroxy-methylglutaryl coenzyme A (HMG CoA)
reductase inhibitors, statins, niacin, bile acid resins, fibrates,
antioxidants, extracellular matrix synthesis promoters, inhibitors
of plaque inflammation and extracellular degradation, and
estradiol.
48. The controlled-release system of claim 43, wherein the
therapeutic agent is selected from the group consisting of
everolimus, paclitaxel, zotarolimus, pioglitazone, BO-653,
rosiglitazone, sirolimus, dexamethasone, rapamycin, tacrolimus,
biophosphonates, estrogen, angiopeptin, statin, PDGF inhibitors,
ROCK inhibitors, MMP inhibitors, 2-CdA, zotarolimus and
dexamethasone.
49. A method of treating breast cancer in a subject in need
thereof, comprising administering to the subject an effective
amount of the controlled-release system of claim 1.
50-54. (canceled)
55. A method of treating vulnerable plaque in a subject in need
thereof, comprising administering to the subject an effective
amount of the controlled-release system of claim 1.
56. A method of treating restenosis in a subject in need thereof,
comprising administering to the subject an effective amount of the
controlled-release system of claim 1.
57. The method of claim 55, wherein the controlled-release system
is locally administered to a designated region of the blood vessel
where the vulnerable plaque occurs.
58. The method of claim 55, wherein the controlled-release system
is administered via a medical device.
59. The method of claim 58, wherein the medical device is a drug
eluding stent, needle catheter, or stent graft.
60-62. (canceled)
63. A method of preparing a stealth nanoparticle, wherein the
nanoparticle has a ratio of ligand-bound polymer to
non-functionalized polymer effective for the treatment of a
disease, comprising: providing a therapeutic agent; providing a
first polymer; providing a poly(amino acid) ligand; reacting the
first polymer with the poly(amino acid) ligand to prepare a
ligand-bound polymer; and mixing the ligand-bound polymer with a
second, non-functionalized polymer, and the therapeutic agent; such
that the stealth nanoparticle is formed.
64-71. (canceled)
72. A stealth nanoparticle, comprising a copolymer of PLGA and PEG;
and a therapeutic agent; wherein said nanoparticle contains
targeting moieties attached thereto, wherein the targeting moiety
comprises AKERC or CREKA.
73. A stealth nanoparticle, comprising a polymeric matrix
comprising a complex of a phospholipid bound-PEG and PLGA; and a
therapeutic agent; wherein said nanoparticle contains targeting
moieties attached thereto, wherein the targeting moiety is a
poly(amino acid).
74-78. (canceled)
79. A controlled-release system, comprising a plurality of
target-specific stealth nanoparticles; wherein said nanoparticles
contain targeting moieties attached thereto, wherein the targeting
moiety is a poly(amino acid).
80. (canceled)
81. The compounds: ##STR00005## wherein n is 20 to 1720; and
##STR00006## wherein R.sub.7 is an alkyl groups, R.sub.8 is an
ester or amide linkage, X=0 to 1 mole fraction, Y=0 to 0.5 mole
fraction, X+Y=20 to 1720, and Z=25 to 455.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 60/910,097, Attorney Docket No. BBZ-011-1, filed
Apr. 4, 2007, titled "Amphiphilic compound assisted polymeric
particles for targeted delivery;" U.S. Provisional Application No.
60/985,104, Attorney Docket No. BBZ-011-2, filed Nov. 2, 2007,
titled "Lipid-Stabilized Polymeric Nanoparticles for Targeted Drug
Delivery;" U.S. Provisional Application No. 60/938,590, Attorney
Docket No. BBZ-012-1, filed May 17, 2007, titled "Poly(Amino
Acid)-Targeted Drug Delivery;" U.S. Provisional Application No.
60/986,202, Attorney Docket No. BBZ-012-2, filed Nov. 7, 2007,
titled "Poly(Amino Acid)-Targeted Drug Delivery;" and U.S.
Provisional Application No. 60/990,250, Attorney Docket No.
BBZ-012-3, filed Nov. 26, 2007, titled "Poly(Amino Acid)-Targeted
Drug Delivery;" all of which are incorporated herein by reference
in their entirety. Additionally, the contents of any patents,
patent applications, and references cited throughout this
specification are hereby incorporated by reference in their
entireties.
FIELD OF INVENTION
[0002] The present invention generally relates to targeted
nanoparticles that target tissue basement membrane.
BACKGROUND
[0003] The delivery of a drug to a patient with controlled-release
of the active ingredient has been an active area of research for
decades and has been fueled by the many recent developments in
polymer science. In addition, controlled release polymer systems
can be designed to provide a drug level in the optimum range over a
longer period of time than other drug delivery methods, thus
increasing the efficacy of the drug and minimizing problems with
patient compliance.
[0004] Nanoparticles have been developed as vehicles used in the
administration for the delivery of small molecule drugs as well as
proteins, peptide drugs and nucleic acids. The drugs are typically
encapsulated or conjugated in a polymer matrix which is
biodegradable and biocompatible. As the polymer is degraded and/or
as the drug diffuses out of the polymer, the drug is released into
the body. Typically, polymers used in preparing these particles are
polyesters such as poly(lactide-co-glycolide) (PLGA), polyglycolic
acid, poly-beta-hydroxybutyrate, polyacrylic acid ester,
polymetacrylate, polyglutamate, etc. These particles can also
protect the drug from degradation by the body. Furthermore, these
particles can be administered using a wide variety of
administration routes.
[0005] Targeting controlled release polymer systems (e.g., targeted
to a particular tissue or cell type or targeted to a specific
diseased tissue but not normal tissue) is desirable because it
reduces the amount of a drug present in tissues of the body that
are not targeted. This is particularly important when treating a
condition such as cancer where it is desirable that a cytotoxic
dose of the drug is delivered to cancer cells without killing the
surrounding non-cancerous tissue. Effective drug targeting should
reduce the undesirable and sometimes life threatening side effects
common in anticancer therapy.
[0006] Targeted delivery for diagnosis and therapeutic applications
has until recently largely been limited to receptor ligands such as
antibodies, modified-antibodies and nucleic acids. Antibodies are
the most widely used type of targeting agent today. The large size
of antibody molecules can be advantageous for bimodal binding
mechanisms but it may also lead to poor solid penetration and slow
elimination from the blood circulation. Unfortunately, slow
elimination kinetics can cause myelotoxicity. In addition, its in
vivo application has been proven more challenging because of cost
and potential immunogenicity after repeat injections of such
formulations. To avoid these problems, Fab's and scFv have
successfully been made but are still too large. The molecular
weight of fragments has been shown to be a major factor of
capillary permeability, so fragments can reach the interstitial
spaces more easily than whole antibody. However, the effects of the
increased permeability are offset by the more rapid excretion of
the antibody fragments which decreases the ability of the antibody
to cross membranes resulting in lower absolute tumor levels as well
as lower blood and tissue levels.
[0007] Accordingly, there is a need for developing new and
alternative targeting controlled release polymer systems,
especially those useful in the treatment of diseases, e.g., cancer,
restenosis, and vulnerable plaques.
SUMMARY OF THE INVENTION
[0008] There remains a need for compositions useful in the
treatment or prevention or amelioration of one or more symptoms of
cancer and cardiovascular disease. There also remains a need for
compositions useful in the treatment or prevention or amelioration
of one or more symptoms of vulnerable plaques. In one aspect, the
invention provides a controlled-release system, comprising a
plurality of target-specific stealth nanoparticles; wherein said
nanoparticles contain targeting moieties attached thereto, wherein
the targeting moiety targets the tissue basement membrane, such as
the vascular basement membrane.
[0009] In one aspect, the invention provides a controlled-release
system, comprising a plurality of target-specific stealth
nanoparticles; wherein said nanoparticles contain targeting
moieties attached thereto, wherein the targeting moiety is a
poly(amino acid) that targets the tissue basement membrane, such as
the vascular basement membrane. In one embodiment, the nanoparticle
has an amount of targeting moiety effective for the treatment of
vulnerable plaque in a subject in need thereof. In another
embodiment, the nanoparticle has an amount of targeting moiety
effective for the treatment of cancer in a subject in need thereof.
As discussed below, the nanoparticle has an amount of targeting
moiety effective for the treatment of restinosis in a subject in
need thereof. In one embodiment, the cancer to be treated is
selected from the group consisting of breast cancer, ovarian
cancer, brain cancer, colon cancer, renal cancer, lung cancer,
bladder cancer, prostate cancer and melanoma. In still another
embodiment, the cancer is breast cancer.
[0010] In one embodiment, the nanoparticles of the invention can be
used to treat ovarian cancer, breast cancer, and human pancreatic
cancer in a subject in need thereof.
[0011] In one embodiment of the controlled-release system of the
invention, the poly(amino acid) comprises natural amino acids,
unnatural amino acids, modified amino acids, protected amino acids
or mimetic of amino acids. In still another embodiment, the
poly(amino acid) is selected from the group consisting of a
glycoprotein, protein, peptidomimetic, affibody or peptide. In yet
another embodiment, the poly(amino acid) binds to the basement
membrane of tissues, such as the vascular basement of a blood
vessel. In another embodiment, the poly(amino acid) binds to
collagen. In still another embodiment, the poly(amino acid) binds
to collagen IV.
[0012] In one embodiment, the poly(amino acid) is an affibody,
wherein the affibody is an anti-HER2 affibody. In another
embodiment, the poly(amino acid) is a peptide, wherein the peptide
comprises a sequence selected from the group consisting of AKERC,
CREKA, ARYLQKLN and AXYLZZLN, wherein X and Z are variable amino
acids.
[0013] In one embodiment of the controlled-release system of the
invention, the nanoparticle comprises a polymeric matrix. In one
embodiment, the polymeric matrix comprises two or more synthetic or
natural polymers. In a particular embodiment, the polymeric matrix
comprises polyethylenes, polycarbonates, polyanhydrides,
polyhydroxyacids, polypropylfumerates, polycaprolactones,
polyamides, polyacetals, polyethers, polyesters, poly(orthoesters),
polycyanoacrylates, polyvinyl alcohols, polyurethanes,
polyphosphazenes, polyacrylates, polymethacrylates,
polycyanoacrylates, polyureas, polystyrenes, or polyamines,
polyglutamate, dextran, or combinations thereof.
[0014] In another embodiment, the polymeric matrix comprises one or
more polyesters, polyanhydrides, polyethers, polyurethanes,
polymethacrylates, polyacrylates or polycyanoacrylates. In another
embodiment, at least one polymer is a polyalkylene glycol. In still
another embodiment, the polyalkylene glycol is polyethylene glycol.
In yet another embodiment, at least one polymer is a polyester. In
one embodiment, the polyester is selected from the group consisting
of poly-lactic-co-glycolic acid (PLGA), polylactic acid (PLA),
polyglycolic acid (PGA), and polycaprolactones. In still another
embodiment, the polyester is PLGA or PLA.
[0015] In one embodiment of the controlled-release system of the
invention, the nanoparticle comprises a polymeric matrix, wherein
the polymeric matrix comprises a copolymer of two or more polymers.
In another embodiment, the copolymer is a copolymer of a
polyalkylene glycol and a polyester. In still another embodiment,
the copolymer is a copolymer of PLGA and PEG. In yet another
embodiment, the polymeric matrix comprises PLGA and a copolymer of
PLGA and PEG.
[0016] In another embodiment of the controlled-release system of
the invention, the nanoparticle comprises a polymeric matrix,
wherein the polymeric matrix comprises a lipid-terminated
polyalkylene glycol and a polyester. In one embodiment, the
polymeric matrix comprises lipid-terminated PEG and PLGA. In one
embodiment, the lipid is of the Formula V, and salts thereof. In
one one embodiment, the lipid is 1,2
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts
thereof.
[0017] In one embodiment of the controlled-release system, a
portion of the polymer matrix is covalently bound to the poly(amino
acid). In another embodiment, the polymer matrix is covalently
bound to the poly(amino acid) via the free terminus of PEG. In
still another embodiment, the polymer matrix is covalently bound to
the poly(amino acid) via a carboxyl group at the free terminus of
PEG. In yet another embodiment, the polymer matrix is covalently
bound to the poly(amino acid) via a maleimide functional group at
the free terminus of PEG.
[0018] In one embodiment of the controlled-release system, the
nanoparticle has a ratio of ligand-bound polymer to
non-functionalized polymer effective for the treatment of cancer.
In another embodiment, the nanoparticle has a ratio of ligand-bound
polymer to non-functionalized polymer effective for the treatment
of a vulnerable plaque. In still another embodiment, the polymers
of the polymer matrix have a molecular weight effective for the
treatment of cancer. In another embodiment, the polymers of the
polymer matrix have a molecular weight effective for the treatment
of vulnerable plaque.
[0019] In another embodiment of the controlled-release system, the
nanoparticle has a surface charge effective for the treatment of
cancer. In still another embodiment, the nanoparticle has a surface
charge effective for the treatment of vulnerable plaque. In yet
another embodiment, said system is suitable for target-specific
treatment of a disease or disorder and delivery of a therapeutic
agent.
[0020] In another embodiment of the controlled-release system, the
nanoparticle further comprises a therapeutic agent. In one
embodiment, the therapeutic agent is associated with the surface
of, encapsulated within, surrounded by, or dispersed throughout the
nanoparticle. In another embodiment, the therapeutic agent is
encapsulated within the hydrophobic core of the nanoparticle. In
still another embodiment, the therapeutic agent is selected from
the group consisting of mitoxantrone, platin and docetaxel. In yet
another embodiment, the therapeutic agent is selected from the
group consisting of VEGF, fibroblast growth factors, monocyte
chemoatractant protein 1 (MCP-1), transforming growth factor alpha
(TGF-alpha), transforming growth factor beta (TGF-beta), DEL-1,
insulin like growth factors (IGF), placental growth factor (PLGF),
hepatocyte growth factor (HGF), prostaglandin E1 (PG-E1),
prostaglandin E2 (PG-E2), tumor necrosis factor alpha (THF-alpha),
granulocyte stimulating growth factor (G-CSF), granulocyte
macrophage colony-stimulating growth factor (GM-CSF), angiogenin,
follistatin, and proliferin, PR39, PR11, nicotine,
hydroxy-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors,
statins, niacin, bile acid resins, fibrates, antioxidants,
extracellular matrix synthesis promoters, inhibitors of plaque
inflammation and extracellular degradation, and estradiol.
[0021] In another aspect, the invention provides a method of
treating breast cancer in a subject in need thereof, comprising
administering to the subject an effective amount of the
controlled-release system of the invention. In one embodiment for
the treatment of breast cancer, the controlled-release system is
administered systemically. In still another embodiment, the
controlled-release system is administered directly to breast cancer
cells. In another embodiment, the controlled-release system is
administered directly to breast cancer cells by injection into
tissue comprising the breast cancer cells. In another embodiment,
the controlled-release system is administered to the subject by
implantation of nanoparticles at or near breast cancer cells during
surgical removal of a tumor. In still another embodiment, the
controlled-release system is administered via intravenous
administration.
[0022] In another aspect, the invention provides a method of
treating vulnerable plaque in a subject in need thereof, comprising
administering to the subject an effective amount of the
controlled-release system of the invention. In one embodiment, the
controlled-release system is locally administered to a designated
region of the blood vessel where the vulnerable plaque occurs. In
still another embodiment, the controlled-release system is
administered via a medical device. In yet another embodiment, the
medical device is a drug eluding stent, needle catheter, or stent
graft.
[0023] In another aspect, the invention provides a method of
treating restenosis in a subject in need thereof, comprising
administering to the subject an effective amount of the
controlled-release system of the invention. In one embodiment, the
controlled-release system is locally administered to a designated
region of the blood vessel where the restenosis occurs. In still
another embodiment, the controlled-release system is administered
via a medical device. In yet another embodiment, the medical device
is a drug eluding stent, needle catheter, or stent graft. In
another embodiment, the invention provides a method of treating
restenosis in a subject in need thereof, comprising administering
to the subject an effective amount of the controlled-release system
of the invention wherein the controlled release system contains a
drug suitable for the treatment of restenosis. In another
embodiment, the invention provides a method of treating restenosis
in a subject in need thereof, comprising administering to the
subject an effective amount of the controlled-release system of the
invention wherein the controlled release system contains at least
two drugs suitable for the treatment of restenosis. In another
embodiment, the invention provides a method of treating restenosis
in a subject in need thereof, comprising administering to the
subject an effective amount of the controlled-release system of the
invention wherein the controlled release system contains
zotarolimus and dexamethasone.
[0024] In one embodiment, the nanoparticles of this invention are
delivered locally to the coronary arteries, central arteries,
peripheral arteries, veins, and bile ducts. In another embodiment,
the nanoparticles of this invention are delivered locally to the
coronary arteries, central arteries, peripheral arteries, veins,
and bile ducts after the implantation of a stent in such tissue in
a patient for the treatment of restenosis. In another embodiment,
the nanoparticles of this invention are administered to a patient
undergoing a coronary angioplasty, a peripheral angioplasty, a
renal artery angioplasty, or a carotid angioplasty in order to
prevent resenosis. In another embodiment, the nanoparticles of this
invention are administered within 12 hours of a patient undergoing
a coronary angioplasty, a peripheral angioplasty, a renal artery
angioplasty, or a carotid angioplasty in order to prevent
resenosis. In another embodiment, the nanoparticles of this
invention are administered locally to a patient undergoing a
coronary angioplasty, a peripheral angioplasty, a renal artery
angioplasty, or a carotid angioplasty in order to prevent
resenosis.
[0025] In one embodiment, the nanoparticles of this invention pass
through the endothelial layer of a blood vessel due to plaque
damage of the endothelial tissue and bind to collage 4 of the
basement membrane.
[0026] In another aspect, the invention provides a method of
preparing a stealth nanoparticle, wherein the nanoparticle has a
ratio of ligand-bound polymer to non-functionalized polymer
effective for the treatment of a disease, comprising: providing a
therapeutic agent; providing a polymer; providing a poly(amino
acid) ligand; mixing the polymer with the therapeutic agent to
prepare particles; and associating the particles with the
poly(amino acid) ligand. In one embodiment of the method, the
polymer comprises a copolymer of two or more polymers. In another
embodiment, the copolymer is a copolymer of PLGA and PEG.
[0027] In another aspect, the invention provides a method of
preparing a stealth nanoparticle, wherein the nanoparticle has a
ratio of ligand-bound polymer to non-functionalized polymer
effective for the treatment of a disease, comprising: providing a
therapeutic agent; providing a first polymer; providing a
poly(amino acid) ligand; reacting the first polymer with the
poly(amino acid) ligand to prepare a ligand-bound polymer; and
mixing the ligand-bound polymer with a second, non-functionalized
polymer, and the therapeutic agent; such that the stealth
nanoparticle is formed. In one embodiment of this method, the first
polymer comprises a copolymer of two or more polymers. In another
embodiment, the second, non-functionalized polymer comprises a
copolymer of two or more polymers. In another embodiment of this
method, the first polymer is first reacted with a lipid, to form a
polymer/lipid conjugate, which is then reacted with the poly(amino
acid). In still another embodiment, the lipid is 1,2
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts
thereof. In yet another embodiment, the copolymer is a copolymer of
PLGA and PEG. In still another embodiment, the first polymer is a
copolymer of PLGA and PEG, wherein the PEG has a carboxyl group at
the free terminus.
[0028] In one embodiment of the aforementioned methods, the disease
to be treated is cancer, vulnerable plaque, or restenosis.
[0029] In another aspect, the invention provides a stealth
nanoparticle, comprising a copolymer of PLGA and PEG; and a
therapeutic agent comprising mitoxantrone, platin or docetaxel;
wherein said nanoparticle contains targeting moieties attached
thereto, wherein the targeting moiety is an anti-HER2 affibody.
[0030] In yet another aspect, the invention provides a stealth
nanoparticle, comprising a copolymer of PLGA and PEG; and a
therapeutic agent; wherein said nanoparticle contains targeting
moieties attached thereto, wherein the targeting moiety comprises
AKERC or CREKA.
[0031] In another aspect, the invention provides a stealth
nanoparticle, comprising a polymeric matrix comprising a complex of
a phospholipid bound-PEG and PLGA; and a therapeutic agent; wherein
said nanoparticle contains targeting moieties attached thereto,
wherein the targeting moiety is a poly(amino acid).
[0032] In still another aspect, the invention provides a
controlled-release system, comprising a plurality of
target-specific stealth nanoparticles; wherein said nanoparticles
contain targeting moieties attached thereto, wherein the targeting
moiety is a basement membrane-targeting moiety. In one embodiment,
the basement membrane-targeting moiety is AKERC, CREKA, ARYLQKLN or
AXYLZZLN, wherein X and Z are variable amino acids.
[0033] In another embodiment of the controlled-release system of
the invention, the polymeric matrix is surrounded by a lipid
monolayer shell. In one embodiment, the lipid monolayer shell
comprises an amphiphilic compound. In another embodiment, the
amphiphilic compound is lecithin. In another embodiment, the lipid
monolayer is stabilized.
[0034] In another aspect, the invention provides a
controlled-release system, comprising a plurality of
target-specific stealth nanoparticles; wherein said nanoparticles
contain targeting moieties attached thereto, wherein the targeting
moiety is a poly(amino acid). In one embodiment, the nanoparticles
target antigen presenting cells and elicit an immunomodulatory
response.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIGS. 1A, 1B, and 2 show representative synthesis schematics
for the target-specific stealth nanoparticle of the invention.
[0036] FIG. 3 is a representative schematic of a nanoparticle of
the invention.
[0037] FIG. 4 shows a schematic illustration of amphiphilic
compound assisted polymeric nanoparticles for targeted drug
delivery.
[0038] FIGS. 5A and 5B show size and zeta-potential stabilities,
respectively, for nanoparticles prepared according to a process of
the invention.
[0039] FIGS. 6 and 7 demonstrate drug encapsulation efficiency of a
lipid assisted polymeric nanoparticle as compared with a non-lipid
assisted polymeric nanoparticle.
[0040] FIG. 8 shows a drug release profile for a nanoparticle
prepared according to a process of the invention.
[0041] FIGS. 9A and 9B demonstrate a lecithin concentration effect
on PLGA-Lipid-PEG nanoparticle size and zeta potential,
respectively.
[0042] FIG. 10 demonstrates a schematic illustration of a
CREKA-targeted PLGA-Lipid-PEG nanoparticle.
[0043] FIGS. 11A and 11B demonstrate that (A) CREKA-targeted
PLGA-Lipid-PEG nanoparticles effectively bind to collagen IV coated
surface and (B) bare (nontargeted) PLGA-Lipid-PEG nanoparticles
rarely bind to collagen IV coated surface.
[0044] FIGS. 12A and 12B demonstrate (A) H&E staining of normal
rat aorta; (B) H&E staining of balloon injured aorta
(endothelium layer was removed).
[0045] FIGS. 13A and 13B demonstrates that CREKA-targeted
PLGA-Lipid-PEG nanoparticles effectively bind to a balloon-injured
rat aorta.
[0046] FIGS. 14A and 14B demonstrate that D-CREKA-targeted
PLGA-Lipid-PEG nanoparticles (D-form of amino acids) do not bind to
balloon-injured rat aorta.
[0047] FIGS. 15A and 15B demonstrates that scrambled peptide
CEAKR-targeted PLGA-Lipid-PEG nanoparticles do not bind to
balloon-injured rat aorta.
[0048] FIGS. 16A and 16B demonstrate that CREKA-targeted
PLGA-Lipid-PEG nanoparticles do not bind to a normal rat aorta.
[0049] FIG. 17 is a schematic illustration of CREKA-targeted
PLGA-Lipid-PEG nanoparticle
[0050] FIG. 18 shows fluorescence images of ARYLQKLN-targeted
PLGA-Lipid-PEG nanoparticles incubating with basement membrane
proteins for 10 minutes: (A) PBS; (B) Collagen I; (C) Collagen II;
(D) Collagen IV; (E) Fibronectin; and (F) vitronectin.
[0051] FIG. 19 demonstrates that ARYLQKLN-targeted PLGA-Lipid-PEG
nanoparticles bind to a balloon-injured rat aorta.
[0052] FIG. 20 demonstrates that ARYLQKLN-targeted PLGA-Lipid-PEG
nanoparticles do not bind to a normal rat aorta.
[0053] FIGS. 21A, 21B and 21C demonstrate the size diameter
(<100 nm) and distribution as visualized by electron microscopy
of a nanoparticle of the invention; direct visualization of an
affibody on the surface of a nanoparticle of the invention carried
out using fluorescent imaging; and .sup.1H-NMR (proton nuclear
magnetic resonance) spectrum of a PLA-PEG-affibody nanoparticle of
the invention.
[0054] FIG. 22 shows fluorescent microscopy of
nanoparticle-affibody bioconjugates incubated with HER-2 positive
cell lines.
[0055] FIG. 23 shows combined fluorescent images (60.times.
magnification) of a single SK-BR-3 cell to reconstruct a
three-dimensional image of a cell, demonstrating the
internalization of targeted NP-affibody bioconjugates to the
cell.
[0056] FIG. 24 shows the results of a cell viability assay (MTS
assay) to evaluate the differential toxicity of targeted (Np-Affb)
and untargeted nanoparticles (Np) with and without encapsulated
paclitaxel (Ptxl).
DETAILED DESCRIPTION OF THE INVENTION
[0057] The present invention generally relates to particles, and,
in particular, nanoparticles, wherein the nanoparticles comprise a
drug delivery system for the targeted delivery of a therapeutic
agent.
[0058] In one embodiment, the nanoparticle of the controlled
release system has an amount of targeting moiety (i.e., a
poly(amino acid)) effective for the targeting of tissue basement
membrane. In one embodiment, the nanoparticle of the controlled
release system has an amount of targeting moiety (i.e., a
poly(amino acid)) effective for the targeting of vascular basement
membrane. In certain embodiments, the poly(amino acid) is
conjugated to a polymer, and the nanoparticle comprises a certain
ratio of ligand-conjugated polymer to non-functionalized polymer.
The nanoparticle can have an optimized ratio of these two polymers,
such that an effective amount of ligand is associated with the
nanoparticle for treatment of a disease, e.g., cancer (e.g., breast
cancer), vulnerable plaque, restenosis. For example, increased
ligand density (e.g., on a PLGA-PEG copolymer) will increase target
binding (cell binding/target uptake), making the nanoparticle
"target specific." Alternatively, a certain concentration of
non-functionalized polymer (e.g., non-functionalized PLGA-PEG
copolymer) in the nanoparticle can control inflammation and/or
immunogenicity (i.e., the ability to provoke an immune response),
and allow the nanoparticle to have a circulation half-life that is
therapeutically effective for the treatment of, e.g., cancer,
vulnerable plaque, or restenosis. Furthermore, the
non-functionalized polymer can lower the rate of clearance from the
circulatory system via the reticuloendothelial system. Thus, the
non-functionalized polymer gives the nanoparticle "stealth"
characteristics. Additionally, the non-functionalized polymer
balances an otherwise high concentration of ligands, which can
otherwise accelerate clearance by the subject, resulting in less
delivery to the target cells.
Target-Specific Stealth Nanoparticles Comprising Polymers
[0059] In preferred embodiments, the nanoparticles of the invention
comprise a matrix of polymers. In general, a "nanoparticle" refers
to any particle having a diameter of less than 1000 nm. In some
embodiments, a therapeutic agent and/or targeting moiety (i.e., a
poly(amino acid)) can be associated with the polymeric matrix. In
some embodiments, the targeting moiety can be covalently associated
with the surface of a polymeric matrix. In some embodiments,
covalent association is mediated by a linker. In some embodiments,
the therapeutic agent can be associated with the surface of,
encapsulated within, surrounded by, and/or dispersed throughout the
polymeric matrix.
[0060] A wide variety of polymers and methods for forming particles
therefrom are known in the art of drug delivery. In some
embodiments of the invention, the matrix of a particle comprises
one or more polymers. Any polymer may be used in accordance with
the present invention. Polymers may be natural or unnatural
(synthetic) polymers. Polymers may be homopolymers or copolymers
comprising two or more monomers. In terms of sequence, copolymers
may be random, block, or comprise a combination of random and block
sequences. Typically, polymers in accordance with the present
invention are organic polymers.
[0061] A "polymer," as used herein, is given its ordinary meaning
as used in the art, i.e., a molecular structure comprising one or
more repeat units (monomers), connected by covalent bonds. The
repeat units may all be identical, or in some cases, there may be
more than one type of repeat unit present within the polymer. In
some cases, the polymer is biologically derived, i.e., a
biopolymer. Non-limiting examples include peptides or proteins
(i.e., polymers of various amino acids), or nucleic acids such as
DNA or RNA. In some cases, additional moieties may also be present
in the polymer, for example biological moieties such as those
described below.
[0062] If more than one type of repeat unit is present within the
polymer, then the polymer is said to be a "copolymer." It is to be
understood that in any embodiment employing a polymer, the polymer
being employed may be a copolymer in some cases. The repeat units
forming the copolymer may be arranged in any fashion. For example,
the repeat units may be arranged in a random order, in an
alternating order, or as a "block" copolymer, i.e., comprising one
or more regions each comprising a first repeat unit (e.g., a first
block), and one or more regions each comprising a second repeat
unit (e.g., a second block), etc. Block copolymers may have two (a
diblock copolymer), three (a triblock copolymer), or more numbers
of distinct blocks.
[0063] It should be understood that, although the terms "first,"
"second," etc. may be used herein to describe various elements,
including polymeric components, these terms should not be construed
as being limiting (e.g., describing a particular order or number of
elements), but rather, as being merely descriptive, i.e., labels
that distinguish one element from another, as is commonly used
within the field of patent law. Thus, for example, although one
embodiment of the invention may be described as having a "first"
element present and a "second" element present, other embodiments
of the invention may have a "first" element present but no "second"
element present, a "second" element present but no "first" element
present, two (or more) "first" elements present, and/or two (or
more) "second" elements present, etc., and/or additional elements
such as a "first" element, a "second" element, and a "third"
element, without departing from the scope of the present
invention.
[0064] Various embodiments of the present invention are directed to
copolymers, which, in particular embodiments, describes two or more
polymers (such as those described herein) that have been associated
with each other, usually by covalent bonding of the two or more
polymers together. Thus, a copolymer may comprise a first polymer
and a second polymer, which have been conjugated together to form a
block copolymer where the first polymer is a first block of the
block copolymer and the second polymer is a second block of the
block copolymer. Of course, those of ordinary skill in the art will
understand that a block copolymer may, in some cases, contain
multiple blocks of polymer, and that a "block copolymer," as used
herein, is not limited to only block copolymers having only a
single first block and a single second block. For instance, a block
copolymer may comprise a first block comprising a first polymer, a
second block comprising a second polymer, and a third block
comprising a third polymer or the first polymer, etc. In some
cases, block copolymers can contain any number of first blocks of a
first polymer and second blocks of a second polymer (and in certain
cases, third blocks, fourth blocks, etc.). In addition, it should
be noted that block copolymers can also be formed, in some
instances, from other block copolymers.
[0065] For example, a first block copolymer may be conjugated to
another polymer (which may be a homopolymer, a biopolymer, another
block copolymer, etc.), to form a new block copolymer containing
multiple types of blocks, and/or to other moieties (e.g., to
non-polymeric moieties).
[0066] In some embodiments, the polymer (e.g., copolymer, e.g.,
block copolymer) is amphiphilic, i.e., having a hydrophilic portion
and a hydrophobic portion, or a relatively hydrophilic portion and
a relatively hydrophobic portion. A hydrophilic polymer is one
generally that attracts water and a hydrophobic polymer is one that
generally repels water. A hydrophilic or a hydrophobic polymer can
be identified, for example, by preparing a sample of the polymer
and measuring its contact angle with water (typically, the polymer
will have a contact angle of less than 60.degree., while a
hydrophobic polymer will have a contact angle of greater than about
60.degree.). In some cases, the hydrophilicity of two or more
polymers may be measured relative to each other, i.e., a first
polymer may be more hydrophilic than a second polymer. For
instance, the first polymer may have a smaller contact angle than
the second polymer.
[0067] In one set of embodiments, a polymer (e.g., copolymer, e.g.,
block copolymer) of the present invention includes a biocompatible
polymer, i.e., the polymer that does not typically induce an
adverse response when inserted or injected into a living subject,
for example, without significant inflammation and/or acute
rejection of the polymer by the immune system, for instance, via a
T-cell response. Accordingly, the nanoparticles of the present
invention can be "non-immunogenic." The term "non-immunogenic" as
used herein refers to endogenous growth factor in its native state
which normally elicits no, or only minimal levels of, circulating
antibodies, T-cells, or reactive immune cells, and which normally
does not elicit in the individual an immune response against
itself.
[0068] It will be recognized, of course, that "biocompatibility" is
a relative term, and some degree of immune response is to be
expected even for polymers that are highly compatible with living
tissue. However, as used herein, "biocompatibility" refers to the
acute rejection of material by at least a portion of the immune
system, i.e., a non-biocompatible material implanted into a subject
provokes an immune response in the subject that is severe enough
such that the rejection of the material by the immune system cannot
be adequately controlled, and often is of a degree such that the
material must be removed from the subject. One simple test to
determine biocompatibility is to expose a polymer to cells in
vitro; biocompatible polymers are polymers that typically will not
result in significant cell death at moderate concentrations, e.g.,
at concentrations of 50 micrograms/10.sup.6 cells. For instance, a
biocompatible polymer may cause less than about 20% cell death when
exposed to cells such as fibroblasts or epithelial cells, even if
phagocytosed or otherwise uptaken by such cells. Non-limiting
examples of biocompatible polymers that may be useful in various
embodiments of the present invention include polydioxanone (PDO),
polyhydroxyalkanoate, polyhydroxybutyrate, poly(glycerol sebacate),
polyglycolide, polylactide, PLGA, polycaprolactone, or copolymers
or derivatives including these and/or other polymers.
[0069] In certain embodiments, the biocompatible polymer is
biodegradable, i.e., the polymer is able to degrade, chemically
and/or biologically, within a physiological environment, such as
within the body. As used herein, "biodegradable" polymers are those
that, when introduced into cells, are broken down by the cellular
machinery (biologically degradable) and/or by a chemical process,
such as hydrolysis, (chemically degradable) into components that
the cells can either reuse or dispose of without significant toxic
effect on the cells.
[0070] In a preferred embodiment, the biodegradable polymer and
their degradation byproducts are biocompatible.
[0071] For instance, the polymer may be one that hydrolyzes
spontaneously upon exposure to water (e.g., within a subject), the
polymer may degrade upon exposure to heat (e.g., at temperatures of
about 37.degree. C.). Degradation of a polymer may occur at varying
rates, depending on the polymer or copolymer used. For example, the
half-life of the polymer (the time at which 50% of the polymer is
degraded into monomers and/or other nonpolymeric moieties) may be
on the order of days, weeks, months, or years, depending on the
polymer. The polymers may be biologically degraded, e.g., by
enzymatic activity or cellular machinery, in some cases, for
example, through exposure to a lysozyme (e.g., having relatively
low pH). In some cases, the polymers may be broken down into
monomers and/or other nonpolymeric moieties that cells can either
reuse or dispose of without significant toxic effect on the cells
(for example, polylactide may be hydrolyzed to form lactic acid,
polyglycolide may be hydrolyzed to form glycolic acid, etc.).
[0072] In some embodiments, polymers may be polyesters, including
copolymers comprising lactic acid and glycolic acid units, such as
poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide),
collectively referred to herein as "PLGA"; and homopolymers
comprising glycolic acid units, referred to herein as "PGA," and
lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid,
poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and
poly-D,L-lactide, collectively referred to herein as "PLA." In some
embodiments, exemplary polyesters include, for example,
polyhydroxyacids; PEGylated polymers and copolymers of lactide and
glycolide (e.g., PEGylated PLA, PEGylated PGA, PEGylated PLGA, and
derivatives thereof. In some embodiments, polyesters include, for
example, polyanhydrides, poly(ortho ester) PEGylated poly(ortho
ester), poly(caprolactone), PEGylated poly(caprolactone),
polylysine, PEGylated polylysine, poly(ethylene inline), PEGylated
poly(ethylene imine), poly(L-lactide-co-L-lysine), poly(serine
ester), poly(4-hydroxy-L-proline ester),
poly[a-(4-aminobutyl)-L-glycolic acid], and derivatives
thereof.
[0073] In some embodiments, a polymer may be PLGA. PLGA is a
biocompatible and biodegradable co-polymer of lactic acid and
glycolic acid, and various forms of PLGA are characterized by the
ratio of lactic acid:glycolic acid. Lactic acid can be L-lactic
acid, D-lactic acid, or D,L-lactic acid. The degradation rate of
PLGA can be adjusted by altering the lactic acid-.glycolic acid
ratio. In some embodiments, PLGA to be used in accordance with the
present invention is characterized by a lactic acid:glycolic acid
ratio of approximately 85:15, approximately 75:25, approximately
60:40, approximately 50:50, approximately 40:60, approximately
25:75, or approximately 15:85.
[0074] In particular embodiments, by optimizing the ratio of lactic
acid to glycolic acid monomers in the polymer of the nanoparticle
(e.g., the PLGA block copolymer or PLGA-PEG block copolymer),
nanoparticle parameters such as water uptake, therapeutic agent
release (e.g., "controlled release") and polymer degradation
kinetics can be optimized.
[0075] In some embodiments, polymers may be one or more acrylic
polymers. In certain embodiments, acrylic polymers include, for
example, acrylic acid and methacrylic acid copolymers, methyl
methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl
methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic
acid), poly(methacrylic acid), methacrylic acid alkylamide
copolymer, poly(methyl methacrylate), poly(methacrylic acid
polyacrylamide, aminoalkyl methacrylate copolymer, glycidyl
methacrylate copolymers, polycyanoacrylates, and combinations
comprising one or more of the foregoing polymers. The acrylic
polymer may comprise fully-polymerized copolymers of acrylic and
methacrylic acid esters with a low content of quaternary ammonium
groups.
[0076] In some embodiments, polymers can be cationic polymers. In
general, cationic polymers are able to condense and/or protect
negatively charged strands of nucleic acids (e.g. DNA, RNA, or
derivatives thereof). Amine-containing polymers such as
poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and
Kabanov et al., 1995, Bioconjugate Chem., 6:7), polyethylene imine)
(PEI; Boussif et al, 1995, Proc. Natl. Acad. Sci., USA, 1995,
92:7297), and poly(amidoamine) dendrimers (Kukowska-Latallo et al.,
1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al., 1996,
Bioconjugate Chem., 7:703; and Haensler et al., 1993, Bioconjugate
Chem., 4:372) are positively-charged at physiological pH, form ion
pairs with nucleic acids, and mediate transfection in a variety of
cell lines.
[0077] In some embodiments, polymers can be degradable polyesters
bearing cationic side chains (Putnam et al., 1999, Macromolecules,
32:3658; Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwonef
al, 1999, Macromolecules, 22325Q-, Urn et al., 1999, J. Am. Chem.
Soc., 121:5633; and Zhou et al, 1990, Macromolecules, 23:3399).
Examples of these polyesters include poly(L-lactide-co-L-lysine)
(Barrera et al, 1993, J. Am. Chem. Soc., 115:11010), poly(serine
ester) (Zhou et al, 1990, Macromolecules, 23:3399),
poly(4-hydroxy-L-proline ester) (Putnam et al, 1999,
Macromolecules, 32:3658; and Lim et al, 1999, J. Am. Chem. Soc.,
121:5633). Poly(4-hydroxy-L-proline ester) was demonstrated to
condense plasmid DNA through electrostatic interactions, and to
mediate gene transfer (Putnam et al, 1999, Macromolecules, 32:3658;
and Lim et al, 1999, J. Am. Chem. Soc., 121:5633). These new
polymers are less toxic than poly(lysine) and PEI, and they degrade
into non-toxic metabolites.
[0078] A polymer (e.g., copolymer, e.g., block copolymer)
containing poly(ethylene glycol) repeat units is also referred to
as a "PEGylated" polymer. Such polymers can control inflammation
and/or immunogenicity (i.e., the ability to provoke an immune
response) and/or lower the rate of clearance from the circulatory
system via the reticuloendothelial system (RES), due to the
presence of the poly(ethylene glycol) groups.
[0079] PEGylation may also be used, in some cases, to decrease
charge interaction between a polymer and a biological moiety, e.g.,
by creating a hydrophilic layer on the surface of the polymer,
which may shield the polymer from interacting with the biological
moiety. In some cases, the addition of poly(ethylene glycol) repeat
units may increase plasma half-life of the polymer (e.g.,
copolymer, e.g., block copolymer), for instance, by decreasing the
uptake of the polymer by the phagocytic system while decreasing
transfection/uptake efficiency by cells. Those of ordinary skill in
the art will know of methods and techniques for PEGylating a
polymer, for example, by using EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and
NHS (N-hydroxysuccinimide) to react a polymer to a PEG group
terminating in an amine, by ring opening polymerization techniques
(ROMP), or the like.
[0080] In another embodiment, the nanoparticle of the invention
does not contain PEG.
[0081] In addition, certain embodiments of the invention are
directed towards copolymers containing poly(ester-ether)s, e.g.,
polymers having repeat units joined by ester bonds (e.g.,
R--C(O)--O--R' bonds) and ether bonds (e.g., R--O--R' bonds). In
some embodiments of the invention, a biodegradable polymer, such as
a hydrolyzable polymer, containing carboxylic acid groups, may be
conjugated with poly(ethylene glycol) repeat units to form a
poly(ester-ether).
[0082] In a particular embodiment, the molecular weight of the
polymers of the nanoparticles of the invention are optimized for
effective treatment of cancer, e.g., breast cancer. For example,
the molecular weight of the polymer influences nanoparticle
degradation rate (particularly when the molecular weight of a
biodegradable polymer is adjusted), solubility, water uptake, and
drug release kinetics (e.g. "controlled release"). As a further
example, the molecular weight of the polymer can be adjusted such
that the nanoparticle biodegrades in the subject being treated
within a reasonable period of time (ranging from a few hours to 1-2
weeks, 3-4 weeks, 5-6 weeks, 7-8 weeks, etc.). In particular
embodiments of a nanoparticle comprising a copolymer of PEG and
PLGA, the PEG has a molecular weight of 1,000-20,000, e.g.,
5,000-20,000, e.g., 10,000-20,000, and the PLGA has a molecular
weight of 5,000-100,000, e.g., 20,000-70,000, e.g.,
20,000-50,000.
[0083] In certain embodiments, the polymers of the nanoparticles
may be conjugated to a lipid. The polymer may be, for example, a
lipid-terminated PEG. As described below, the lipid portion of the
polymer can be used for self assembly with another polymer,
facilitating the formation of a nanoparticle. For example, a
hydrophilic polymer could be conjugated to a lipid that will self
assemble with a hydrophobic polymer.
[0084] In some embodiments, lipids are oils. In general, any oil
known in the art can be conjugated to the polymers used in the
invention. In some embodiments, an oil may comprise one or more
fatty acid groups or salts thereof. In some embodiments, a fatty
acid group may comprise digestible, long chain (e.g.,
C.sub.8-C.sub.50), substituted or unsubstituted hydrocarbons. In
some embodiments, a fatty acid group may be a C.sub.10-C.sub.20
fatty acid or salt thereof. In some embodiments, a fatty acid group
may be a C.sub.15-C.sub.20 fatty acid or salt thereof. In some
embodiments, a fatty acid may be unsaturated. In some embodiments,
a fatty acid group may be monounsaturated. In some embodiments, a
fatty acid group may be polyunsaturated. In some embodiments, a
double bond of an unsaturated fatty acid group may be in the cis
conformation. In some embodiments, a double bond of an unsaturated
fatty acid may be in the trans conformation.
[0085] In some embodiments, a fatty acid group may be one or more
of butyric, caproic, caprylic, capric, lauric, myristic, palmitic,
stearic, arachidic, behenic, or lignoceric acid. In some
embodiments, a fatty acid group may be one or more of palmitoleic,
oleic, vaccenic, linoleic, alpha-linolenic, gamma-linoleic,
arachidonic, gadoleic, arachidonic, eicosapentaenoic,
docosahexaenoic, or erucic acid.
[0086] In a particular embodiment, the lipid is of the Formula
V:
##STR00001##
and salts thereof, wherein each R is, independently, C.sub.1-30
alkyl. In one embodiment of Formula V, the lipid is 1,2
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts
thereof, e.g., the sodium salt.
[0087] The properties of these and other polymers and methods for
preparing them are well known in the art (see, for example, U.S.
Pat. Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404;
6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600;
5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and
4,946,929; Wang et al, 2001, J. Am. Chem. Soc., 123:9480; Lim et
al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Ace. Chem.
Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and Uhrich et
al., 1999, Chem. Rev., 99:3181). More generally, a variety of
methods for synthesizing suitable polymers are described in Concise
Encyclopedia of Polymer Science and Polymeric Amines and Ammonium
Salts, Ed. by Goethals, Pergamon Press, 1980; Principles of
Polymerization by Odian, John Wiley & Sons, Fourth Edition,
2004; Contemporary Polymer Chemistry by Allcock et al.,
Prentice-Hall, 1981; Deming et al, 1997, Nature, 390:386; and in
U.S. Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.
[0088] In still another set of embodiments, a particle (comprising,
e.g., a copolymer, e.g., a block copolymer) of the present
invention includes a therapeutic moiety, i.e., a moiety that has a
therapeutic or prophylactic effect when given to a subject.
Examples of therapeutic moieties to be used with the nanoparticles
of the present invention include antineoplastic or cytostatic
agents or other agents with anticancer properties, or a combination
thereof.
[0089] Thus, in certain embodiments, a library of such particles
may be created, as discussed herein.
[0090] In some cases, the particle is a nanoparticle, i.e., the
particle has a characteristic dimension of less than about 1
micrometer, where the characteristic dimension of a particle is the
diameter of a perfect sphere having the same volume as the
particle. For example, the particle may have a characteristic
dimension of the particle may be less than about 300 nm, less than
about 200 nm, less than about 150 nm, less than about 100 nm, less
than about 50 nm, less than about 30 nm, less than about 10 nm,
less than about 3 nm, or less than about 1 nm in some cases. In
particular embodiments, the nanoparticle of the present invention
has a diameter of 50 nm-200 nm.
[0091] In some cases, a population of particles may be present. For
example, a population of particles may include at least 20
particles, at least 50 particles, at least 100 particles, at least
300 particles, at least 1,000 particles, at least 3,000 particles,
or at least 10,000 particles. Various embodiments of the present
invention are directed to such populations of particles. For
instance, in some embodiments, the particles may each be
substantially the same shape and/or size ("monodisperse"). For
example, the particles may have a distribution of characteristic
dimensions such that no more than about 5% or about 10% of the
particles have a characteristic dimension greater than about 10%
greater than the average characteristic dimension of the particles,
and in some cases, such that no more than about 8%, about 5%, about
3%, about 1%, about 0.3%, about 0.1%, about 0.03%, or about 0.01%
have a characteristic dimension greater than about 10% greater man
the average characteristic dimension of the particles. In some
cases, no more than about 5% of the particles have a characteristic
dimension greater than about 5%, about 3%, about 1%, about 0.3%,
about 0.1%, about 0.03%, or about 0.01% greater than the average
characteristic dimension of the particles.
[0092] In one set of embodiments, the particles may have an
interior and a surface, where the surface has a composition
different from the interior, i.e., there may be at least one
compound present in the interior but not present on the surface (or
vice versa), and/or at least one compound is present in the
interior and on the surface at differing concentrations. For
example, in one embodiment, a compound, such as a targeting moiety
(i.e., a poly(amino acid)) of a polymeric conjugate of the present
invention, may be present in both the interior and the surface of
the particle, but at a higher concentration on the surface than in
the interior of the particle, although in some cases, the
concentration in the interior of the particle may be essentially
nonzero, i.e., there is a detectable amount of the compound present
in the interior of the particle.
[0093] In some cases, the interior of the particle is more
hydrophobic than the surface of the particle. For instance, the
interior of the particle may be relatively hydrophobic with respect
to the surface of the particle, and a drug or other pay load may be
hydrophobic, and readily associates with the relatively hydrophobic
center of the particle. The drug or other payload may thus be
contained within the interior of the particle, which may thus
shelter it from the external environment surrounding the particle
(or vice versa). For instance, a drug or other payload contained
within a particle administered to a subject will be protected from
a subject's body, and the body will also be isolated from the drug.
A targeting moiety present on the surface of the particle may allow
the particle to become localized at a particular targeting site,
for instance, a tumor, a disease site, a tissue, an organ, a type
of cell, etc. The drug or other payload may then, in some cases, be
released from the particle and allowed to interact locally with the
particular targeting site. Yet another aspect of the invention is
directed to polymer particles having more than one polymer or
macromolecule present, and libraries involving such polymers or
macromolecules. For example, in one set of embodiments, particles
may contain more than one distinguishable polymers (e.g.,
copolymers, e.g., block copolymers), and the ratios of the two (or
more) polymers may be independently controlled, which allows for
the control of properties of the particle. For instance, a first
polymer may be a polymeric conjugate comprising a targeting moiety
and a biocompatible portion, and a second polymer may comprise a
biocompatible portion but not contain the targeting moiety, or the
second polymer may contain a distinguishable biocompatible portion
from the first polymer. Control of the amounts of these polymers
within the polymeric particle may thus be used to control various
physical, biological, or chemical properties of the particle, for
instance, the size of the particle (e.g., by varying the molecular
weights of one or both polymers), the surface charge (e.g., by
controlling the ratios of the polymers if the polymers have
different charges or terminal groups), the surface hydrophilicity
(e.g., if the polymers have different molecular weights and/or
hydrophilicities), the surface density of the targeting moiety
(e.g., by controlling the ratios of the two or more polymers),
etc.
[0094] As a specific example, a particle may comprise a first
polymer comprising a poly(ethylene glycol) and a targeting moiety
conjugated to the poly(ethylene glycol), and a second polymer
comprising the poly(ethylene glycol) but not the targeting moiety,
or comprising both the poly(ethylene glycol) and the targeting
moiety, where the poly(ethylene glycol) of the second polymer has a
different length (or number of repeat units) than the poly(ethylene
glycol) of the first polymer. As another example, a particle may
comprise a first polymer comprising a first biocompatible portion
and a targeting moiety, and a second polymer comprising a second
biocompatible portion different from the first biocompatible
portion (e.g., having a different composition, a substantially
different number of repeat units, etc.) and the targeting moiety.
As yet another example, a first polymer may comprise a
biocompatible portion and a first targeting moiety, and a second
polymer may comprise a biocompatible portion and a second targeting
moiety different from the first targeting moiety.
[0095] Libraries of such particles may also be formed. For example,
by varying the ratios of the two (or more) polymers within the
particle, libraries of particles may be formed, which may be
useful, for example, for screening tests, high-throughput assays,
or the like. Entities within the library may vary by properties
such as those described above, and in some cases, more than one
property of the particles may be varied within the library.
Accordingly, one embodiment of the invention is directed to a
library of nanoparticles having different ratios of polymers with
differing properties. The library may include any suitable ratio(s)
of the polymers.
[0096] In another embodiment, the nanoparticle is associated with
(e.g., surrounded by) a small molecule amphiphilic compound, giving
the "amphiphilic-nanoparticle" three main components: 1) a
biodegradable polymeric material that forms the core of the
particle, which can carry bioactive drugs and release them at a
sustained rate after cutaneous, subcutaneous, mucosal,
intramuscular, ocular, systemic, oral or pulmonary administration;
2) a small molecule amphiphilic compound that surrounds the
polymeric material forming a shell for the particle; and 3) a
stealth material that can allow the particles to evade recognition
by immune system components and increase particle circulation half
life. This embodiment may also include a fourth component: 4) a
targeting molecule that can bind to a unique molecular signature on
cells, tissues, or organs of the body. In a preferred embodiment,
these particles would be useful in drug delivery for therapeutic
applications. In an alternative preferred embodiment, these
particles would be useful for molecular imaging, for diagnostic
applications, or for a combination thereof.
[0097] In another embodiment, the amphiphilic-nanoparticle of the
invention comprises a: 1) a biodegradable polymeric core which can
carry bioactive drugs and release them at a sustained rate; 2) a
lipid monolayer shell which can prevent the carried agents from
freely diffusing out of the nanoparticle and reduce water
penetration rate into the nanoparticle, thereby enhancing drug
encapsulation efficiency and slowing drug release; 3) a stealth
material that can allow the particles to evade recognition by
immune system components and increase particle circulation half
life; and 4) a targeting molecule that can bind to a unique
molecular signature on cells, tissues, or organs of the body.
[0098] In a preferred embodiment of the amphiphilic-nanoparticle, a
poly(amino acid) targeting molecule is first chemically conjugated
to the hydrophilic region of a small molecule amphiphilic compound.
This conjugate is then mixed with a certain ratio of unconjugated
small molecule amphiphilic compounds in an aqueous solution
containing one or more water-miscible solvents. In a preferred
embodiment, the poly(amino acid) targeting molecule is one or a
plurality of antibodies, aptamers, peptides, small molecules, or
combinations thereof. The amphiphilic compound can be, but is not
limited to, one or a plurality of the following: naturally derived
lipids, surfactants, or synthesized compounds with both hydrophilic
and hydrophobic moieties. The water miscible solvent can be, but is
not limited to: acetone, ethanol, methanol, and isopropyl alcohol.
Separately, a biodegradable polymeric material is mixed with the
agent or agents to be encapsulated in a water miscible or partially
water miscible organic solvent. In a preferred embodiment, the
biodegradable polymer can be any of the biodegradable polymers
disclosed herein, for example, poly(D,L-lactic acid),
poly(D,L-glycolic acid), poly(.epsilon.-caprolactone), or their
copolymers at various molar ratios. The carried agent can be, but
is not limited to, one or a plurality of the following therapeutic
agents discussed below, including, for example, therapeutic drugs,
imaging probes, or hydrophobic or lipophobic molecules for medical
use. The water miscible organic solvent can be but is not limited
to: acetone, ethanol, methanol, or isopropyl alcohol. The partially
water miscible organic solvent can be, but is not limited to:
acetonitrile, tetrahydrofuran, ethyl acetate, isopropyl alcohol,
isopropyl acetate, or dimethylformamide. The resulting polymer
solution is then added to the aqueous solution of conjugated and
unconjugated amphiphilic compound to yield nanoparticles by the
rapid diffusion of the organic solvent into the water and
evaporation of the organic solvent.
[0099] As used herein, the term "amphiphilic" refers to a property
where a molecule has both a polar portion and a non-polar portion.
Often, an amphiphilic compound has a polar head attached to a long
hydrophobic tail. In some embodiments, the polar portion is soluble
in water, while the non-polar portion is insoluble in water. In
addition, the polar portion may have either a formal positive
charge, or a formal negative charge. Alternatively, the polar
portion may have both a formal positive and a negative charge, and
be a zwitterion or inner salt. For purposes of the invention, the
amphiphilic compound can be, but is not limited to, one or a
plurality of the following: naturally derived lipids, surfactants,
or synthesized compounds with both hydrophilic and hydrophobic
moieties.
[0100] Specific examples of amphiphilic compounds include, but are
not limited to, phospholipids, such as 1,2
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),
dipalmitoylphosphatidylcholine (DPPC),
distearoylphosphatidylcholine (DSPC),
diarachidoylphosphatidylcholine (DAPC),
dibehenoylphosphatidylcholine (DBPC),
ditricosanoylphosphatidylcholine (DTPC), and
dilignoceroylphatidylcholine (DLPC), incorporated at a ratio of
between 0.01-60 (weight lipid/w polymer), most preferably between
0.1-30 (weight lipid/w polymer). Phospholipids which may be used
include, but are not limited to, phosphatidic acids, phosphatidyl
cholines with both saturated and unsaturated lipids, phosphatidyl
ethanolamines, phosphatidylglycerols, phosphatidylserines,
phosphatidylinositols, lysophosphatidyl derivatives, cardiolipin,
and .beta.-acyl-y-alkyl phospholipids. Examples of phospholipids
include, but are not limited to, phosphatidylcholines such as
dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine,
dipentadecanoylphosphatidylcholine dilauroylphosphatidylcholine,
dipalmitoylphosphatidylcholine (DPPC),
distearoylphosphatidylcholine (DSPC),
diarachidoylphosphatidylcholine (DAPC),
dibehenoylphosphatidylcho-line (DBPC),
ditricosanoylphosphatidylcholine (DTPC),
dilignoceroylphatidylcholine (DLPC); and phosphatidylethanolamines
such as dioleoylphosphatidylethanolamine or
1-hexadecyl-2-palmitoylglycerophos-phoethanolamine. Synthetic
phospholipids with asymmetric acyl chains (e.g., with one acyl
chain of 6 carbons and another acyl chain of 12 carbons) may also
be used.
[0101] In a particular embodiment, an amphiphilic component that
can be used to form an amphiphilic layer is lecithin, and, in
particular, phosphatidylcholine. Lecithin is an amphiphilic lipid
and, as such, forms a phospholipid bilayer having the hydrophilic
(polar) heads facing their surroundings, which are oftentimes
aqueous, and the hydrophobic tails facing each other. Lecithin has
an advantage of being a natural lipid that is available from, e.g.,
soybean, and already has FDA approval for use in other delivery
devices. In addition, a mixture of lipids such as lethicin is more
advantageous than one single pure lipid.
[0102] In certain embodiments of the invention, the amphiphilic
layer of the nanoparticle, e.g., the layer of lecithin, is a
monolayer, meaning the layer is not a phospholipid bilayer, but
exists as a single continuous or discontinuous layer around, or
within, the nanoparticle. A monolayer has the advantage of allowing
the nanoparticles to be smaller in size, which makes them easier to
prepare. The amphiphilic layer is "associated with" the
nanoparticle of the invention, meaning it is positioned in some
proximity to the polymeric matrix, such as surrounding the outside
of the polymeric matrix (e.g., PLGA), or dispersed within the
polymers that make up the nanoparticle.
[0103] By covering the polymeric nanoparticles with a thin film of
small molecule amphiphilic compounds and conjugating poly(amino
acid) targeting molecules to the amphiphilic compounds before
formulating nanoparticles, the disclosed invention has merits of
both polymer- and lipid-based nanoparticles, while excluding some
of their limitations. The amphiphilic compounds form a tightly
assembled monolayer around the polymeric core. This monolayer
effectively prevents the carried agents from freely diffusing out
of the nanoparticle, thereby enhancing the encapsulation yield and
slowing drug release. Moreover, the amphiphilic monolayer also
reduces water penetration rate into the nanoparticle, which slows
hydrolysis rate of the biodegradable polymers, thereby increasing
particle stability and lifetime. In addition, by conjugating
targeting ligands to the amphiphilic component prior to
incorporating them into the nanoparticle, the composition of the
nanoparticle and its surface properties can be more accurately
quantified.
[0104] In one embodiment, upon being administered to a subject, the
amphipilic layer of the nanoparticle of the invention can degrade,
such that the polymer core is eventually "unshielded." Such a
process, particularly when occurring after penetration into target
tissue, can lead to more efficient delivery of the therapeutic
agent, thereby affording an enhanced therapeutic effect. Without
being bound by theory, in the case of basement membrane targeting,
the nanoparticle may aggregate at the first collagen IV at the
surface of the basement membrane. By "shedding" the lipid shell,
the drug/polymer core can more deeply penetrate into the basement
membrane,
[0105] The surface of the nanoparticles of the invention can also
be modified to enhance their arterial uptake. Nanoparticle surface
modifying agents include, but are not limited to, heparin,
L-R-phosphatidylethanolamine, cyanoacrylate, epoxide, fibronectin,
fibrinogen, ferritin, lipofectin, didodecyldimethylammonium
bromide, and DEAE-Dextran, and any other surface modifying agent
disclosed in J Pharm Sci. 1998 October; 87(10):1229-34, which is
incorporated herein by reference in it entirety. The
nanoparticulate system of the invention can also be manipulated to
have better compatibility with a drug delivery device, e.g., stent.
For example, viscosity can be adjusted to adjust the drag force of
the nanoparticulate system.
[0106] In general, the nanoparticles of the present invention are
about 40 nm to about 500 nm in size. In one embodiment, the
nanoparticles of the invention are less than or equal to about 90
nm in size, e.g., about 40 nm to about 80 nm, e.g., about 40 nm to
about 60 nm. Because the nanoparticles of the invention can be less
than 90 nm in size, liver uptake by the subject is reduced, thereby
allowing longer circulation in the bloodstream.
[0107] In one embodiment, a nanoparticle of this invention is
between 40 nm and 80 nm in diameter and contains an amphiphilic
component to polymer ration of between 14:1 to 34:1. In one
embodiment, a nanoparticle will have approximately 10% to 40% lipid
(by weight). In another embodiment, the nanoparticle will have a
size of about 90 nm to about 40 nm. In one embodiment, a
nanoparticle that is approximately 10% to 40% lipid (by weight)
will have a corresponding size of about 90 nm to about 40 nm.
[0108] The nanoparticles of the invention also have a surface zeta
potential ranging from about -80 mV to 50 mV. Zeta potential is a
measurement of surface potential of a particle. In some
embodiments, the particles have a zeta potential ranging between 0
mV and -50 mV, e.g., between -1 mV and -50 mV. In some embodiments,
the particles have a zeta potential ranging between -1 mV and -25
mV. In some embodiments, the particles have a zeta potential
ranging between -1.1 mV and -10 mV.
[0109] In other embodiments, the nanoparticles of the invention are
liposomes, liposome polymer combinations, dendrimers, and albumin
particles that are functionalized with a poly(amino acid) ligand.
These nanoparticles can be used to deliver a therapeutic agent to a
subject, such as an anti-cancer agent like mitoxantrone or
docetaxel.
[0110] As used herein, the term "liposome" refers to a generally
spherical vesicle or capsid generally comprised of amphipathic
molecules (e.g., having both a hydrophobic (nonpolar) portion and a
hydrophilic (polar) portion). Typically, the liposome can be
produced as a single (unilamellar) closed bilayer or a
multicompartment (multilamellar) closed bilayer. The liposome can
be formed by natural lipids, synthetic lipids, or a combination
thereof. In a preferred embodiment, the liposome comprises one or
more phospholipids. Lipids known in the art for forming liposomes
include, but are not limited to, lecithin (soy or egg;
phosphatidylcholine), dipalmitoylphosphatidylcholine,
dimyristoylphosphatidylcholine, distearoylphosphatidylcholine,
dicetylphosphate, phosphatidylglycerol, hydrogenated
phosphatidylcholine, phosphatidic acid, cholesterol,
phosphatidylinositol, a glycolipid, phosphatidylethanolamine,
phosphatidylserine, a maleimidyl-derivatized phospholipid (e.g.,
N-[4(p-malei-midophenyl)butyryl] phosphatidylethanolamine),
dioleylphosphatidylcholine, dipalmitoylphosphatidylglycerol,
dimyristoylphosphatidic acid, and a combination thereof. Liposomes
have been used to deliver therapeutic agents to cells.
[0111] The nanoparticles of the invention can also be "stealth
liposomes," which comprise lipids wherein the head group is
modified with PEG. This results in extended circulating half life
in the subject.
[0112] Dendritic polymers (otherwise known as "dendrimers") are
uniform polymers, variously referred to in the literature as
hyperbranched dendrimers, arborols, fractal polymers and starburst
dendrimers, having a central core, an interior dendritic
(hyperbranched) structure and an exterior surface with end groups.
These polymers differ from the classical linear polymers both in
form and function. Dendrimer chemistry constructs macromolecules
with tight control of size, shape topology, flexibility and surface
groups (e.g., a poly(amino acid) ligand). In what is known as
divergent synthesis, these macromolecules start by reacting an
initiator core in high-yield iterative reaction sequences to build
symmetrical branches radiating from the core with well-defined
surface groups. Alternatively, in what is known as convergent
synthesis, dendritic wedges are constructed from the periphery
inwards towards a focal point and then several dendritic wedges are
coupled at the focal points with a polyfunctional core. Dendritic
syntheses form concentric layers, known as generations, with each
generation doubling the molecular mass and the number of reactive
groups at the branch ends so that the end generation dendrimer is a
highly pure, uniform monodisperse macromolecule that solubilizes
readily over a range of conditions. For the reasons discussed
below, dendrimer molecular weights range from 300 to 700,000
daltons and the number of surface groups (e.g., reactive sites for
coupling) range significantly.
[0113] "Albumin particles" (also referred to as "albumin
microspheres") have been reported as carriers of pharmacological or
diagnostic agents (see, e.g., U.S. Pat. Nos. 5,439,686; 5,498,421;
5,560,933; 5,665,382; 6,096,331; 6,506,405; 6,537,579; 6,749,868;
and 6,753,006; all of which are incorporated herein by reference).
Microspheres of albumin have been prepared by either heat
denaturation or chemical crosslinking. Heat denatured microspheres
are produced from an emulsified mixture (e.g., albumin, the agent
to be incorporated, and a suitable oil) at temperatures between
100.degree. C. and 150.degree. C. The microspheres are then washed
with a suitable solvent and stored. Leucuta et al. (International
Journal of Pharmaceutics 41:213-217 (1988)) describe the method of
preparation of heat denatured microspheres.
Poly(Amino Acid) Targeting Moieties
[0114] In yet another set of embodiments, the nanoparticles of the
present invention includes a poly(amino acid) targeting moiety,
i.e., a moiety able to bind to or otherwise associate with a
biological entity, for example, a membrane component, a cell
surface receptor, Her-2, the basement membrane of a blood vessel,
basement membrane proteins, collagen, collagen IV or the like. In
the case of the instant invention, the targeting moiety is a
poly(amino acid) ligand. The term "bind" or "binding," as used
herein, refers to the interaction between a corresponding pair of
molecules or portions thereof that exhibit mutual affinity or
binding capacity, typically due to specific or non-specific binding
or interaction, including, but not limited to, biochemical,
physiological, and/or chemical interactions. "Biological binding"
defines a type of interaction that occurs between pairs of
molecules including proteins, nucleic acids, glycoproteins,
carbohydrates, hormones, or the like. The term "binding partner"
refers to a molecule that can undergo binding with a particular
molecule. "Specific binding" refers to molecules, such as
polynucleotides, that are able to bind to or recognize a binding
partner (or a limited number of binding partners) to a
substantially higher degree than to other, similar biological
entities. In one set of embodiments, the targeting moiety has an
affinity (as measured via a disassociation constant) of less than
about 1 micromolar, at least about 10 micromolar, or at least about
100 micromolar.
[0115] The term "poly(amino acid)" as used herein, refers to a
protein, affibody, peptide, or peptidomimetic containing natural
and unnatural amino acids, modified amino acids or protected amino
acids. The agents to be incorporated in the polymeric nanocarrier
and delivered to a target cell or tissue by a conjugate of the
present invention may be therapeutic, diagnostic, prophylactic or
prognostic agents. Any chemical compound to be administered to an
individual may be delivered using the conjugates of the invention.
The agent may be a small molecule, organometallic compound,
radionuclides, nucleic acid, protein, peptide, polynucleotide,
metal, an isotopically labeled chemical compound, drug, vaccine,
immunological agent, etc.
[0116] The term "affibody" (see, e.g., U.S. Pat. No. 5,831,012,
incorporated herein by reference) refers to highly specific
affinity proteins that can be designed to bind to any desired
target molecule. These antibody mimics can be manufactured to have
the desired properties (specificity and affinity), while also being
highly robust to withstand a broad range of analytical conditions,
including pH and elevated temperature. The specific binding
properties that can be engineered into each protein allow it to
have very high specificity and the desired affinity for a
corresponding target protein. A specific target protein will thus
bind only to its corresponding capture protein.
[0117] The present invention further provides a nanoparticle
conjugated to a poly(amino acid) that selectively targets tumor
vasculature and selectively binds collagen, such as non-helical
collagen. In another embodiment, the invention provides a
nanoparticle conjugated to a poly(amino acid) that selectively
targets breast tumor vasculature and that selectively binds
collagen, e.g., collagen IV, e.g., denatured collagen IV or native
collagen IV. In a one embodiment, the invention provides a
nanoparticle conjugated to a poly(amino acid) that selectively
targets tumor vasculature and that selectively binds the alpha 2
chain of collagen IV.
[0118] In preferred embodiments, the poly(amino acid) targeting
moiety targets tissue basement membrane, such as the basement
membrane of a blood vessel. A "basement membrane" refers to a thin
membrane upon which is posed a single layer of cells. The basement
membrane is made up of proteins held together by type IV collagen.
The epithelial cells are anchored with hemidesmosome to the
basement membrane. The end result resembles a layer of tiles
attached to a thin sheet. As discussed below, in cases where the
endothelium is disrupted (by disease or trauma), the basement
membrane may be exposed and accessible to particles.
[0119] A variety of poly(amino acids) that selectively target tumor
vasculature are useful targeting moieties for the nanoparticles of
the invention. Such poly(amino acids) include, without limitation,
targeting peptides and peptidomimetics. In one embodiment, the
targeting peptide or peptidomimetic portion of the nanoparticle has
a length of at most 200 residues. In another embodiment, the
targeting peptide or peptidomimetic portion of the nanoparticle has
a length of at most 50 residues. In a further embodiment, a
nanoparticle of the invention contains a targeting peptide or
peptidomimetic that includes the amino acid sequence AKERC, CREKA,
ARYLQKLN or AXYLZZLN, wherein X and Z are variable amino acids, or
conservative variants or peptidomimetics thereof. In particular
embodiments, the poly(amino acid) targeting moiety is a peptide
that includes the amino acid sequence AKERC, CREKA, ARYLQKLN or
AXYLZZLN, wherein X and Z are variable amino acids, and has a
length of less than 20, 50 or 100 residues. The CREKA peptide is
known in the art, and is described in U.S. Patent Application No.
2005/0048063, which is incorporated herein by reference in its
entirety. The octapeptide AXYLZZLN is described in Dinkla et al.,
The Journal of Biological Chemistry, Vol. 282, No. 26, pp.
18686-18693, which is incorporated herein by reference in its
entirety.
[0120] Moreover, the authors of The Journal of Biological
Chemistry, Vol. 282, No. 26, pp. 18686-18693 describe a binding
motif in streptococci that forms an autoantigenic complex with
human collagen IV. Accordingly, any peptide, or conservative
variants or peptidomimetics thereof, that binds or forms a complex
with collagen IV, or the basement membrane of a blood vessel, can
be used as a targeting moiety for the nanoparticles of the
invention.
[0121] In one embodiment, the targeting moiety is an isolated
peptide or peptidomimetic that has a length of less than 100
residues and includes the amino acid sequence CREKA (Cys Arg Glu
Lys Ala) or a peptidomimetic thereof. Such an isolated peptide- or
peptidomimetic can have, for example, a length of less than 50
residues or a length of less than 20 residues. In particular
embodiments, the invention provides a peptide that includes the
amino acid sequence CREKA and has a length of less than 20, 50 or
100 residues.
[0122] As used herein in reference to a specified amino acid
sequence, a "conservative variant" is a sequence in which a first
amino acid is replaced by another amino acid or amino acid analog
having at least one biochemical property similar to that of the
first amino acid; similar properties include, for example, similar
size, charge, hydrophobicity or hydrogen-bonding capacity.
[0123] The peptides and peptidomimetics of the invention to be used
as poly(amino acid) targeting moieties are provided in isolated
form. As used herein in reference to a peptide or peptidomimetic of
the invention, the term "isolated" means a peptide or
peptidomimetic that is in a form that is relatively free from
material such as contaminating polypeptides, lipids, nucleic acids
and other cellular material that normally is associated with the
peptide or peptidomimetic in a cell or that is associated with the
peptide or peptidomimetic in a library or in a crude
preparation.
[0124] The peptides and peptidomimetics of the invention to be used
as poly(amino acid) targeting moieties, including the bifunctional,
multivalent and targeting peptides and peptidomimetics discussed
below, can have a variety of lengths. A peptide or peptidomimetic
of the invention can have, for example, a relatively short length
of less than six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35
or 40 residues. A peptide or peptidomimetic of the invention also
can be useful in the context of a significantly longer sequence. In
another embodiment, a peptide or peptidomimetic of the invention
can have, for example, a length of up to 50, 100, 150, 200, 250,
300, 400, 500, 1000 or 2000 residues. In particular embodiments, a
peptide or peptidomimetic of the invention has a length of at least
10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 200 residues. In further
embodiments, a peptide or peptidomimetic of the invention has a
length of 5 to 200 residues, 5 to 100 residues, 5 to 90 residues, 5
to 80 residues, 5 to 70 residues, 5 to 60 residues, 5 to 50
residues, 5 to 40 residues, 5 to 30 residues, 5 to 20 residues, 5
to 15 residues, 5 to 10 residues, 10 to 200 residues, 10 to 100
residues, 10 to 90 residues, 10 to 80 residues, 10 to 70 residues,
10 to 60 residues, 10 to 50 residues, 10 to 40 residues, 10 to 30
residues, 10 to 20 residues, 20 to 200 residues, 20 to 100
residues, 20 to 90 residues, 20 to 80 residues, 20 to 70 residues,
20 to 60 residues, 20 to 50 residues, 20 to 40 residues or 20 to 30
residues. As used herein, the term "residue" refers to an amino
acid or amino acid analog.
[0125] As used herein, the term "peptide" is used broadly to mean
peptides, proteins, fragments of proteins and the like. The term
"peptidomimetic," as used herein, means a peptide-like molecule
that has the activity of the peptide upon which it is structurally
based. Such peptidomimetics include chemically modified peptides,
peptide-like molecules containing non-naturally occurring amino
acids, and peptoids and have an activity such as selective
targeting activity of the peptide upon which the peptidomimetic is
derived (see, for example, Goodman and Ro, Peptidomimetics for Drug
Design, in "Burger's Medicinal Chemistry and Drug Discovery" Vol. 1
(ed. M. E. Wolff; John Wiley & Sons 1995), pages 803-861).
[0126] In another embodiment, the poly(amino acid) targeting moiety
targets Her-2. In a particular embodiment, the poly(amino acid)
targeting moiety is an affibody that is an anti-HER2 affibody.
[0127] A polymeric conjugate to be used in the preparation of a
nanoparticle of the present invention may be formed using any
suitable conjugation technique. For instance, two components such
as a targeting moiety and a biocompatible polymer, a biocompatible
polymer and a poly(ethylene glycol), etc., may be conjugated
together using techniques such as EDC-NHS chemistry
(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and
N-hydroxysuccinimide) or a reaction involving a maleimide or a
carboxylic acid, which can be conjugated to one end of a thiol, an
amine, or a similarly functionalized polyether. The conjugation of
such polymers, for instance, the conjugation of a poly(ester) and a
poly(ether) to form a poly(ester-ether), can be performed in an
organic solvent, such as, but not limited to, dichloromethane,
acetonitrile, chloroform, dimethylformamide, tetrahydrofuran,
acetone, or the like. Specific reaction conditions can be
determined by those of ordinary skill in the art using no more than
routine experimentation.
[0128] In another set of embodiments, a conjugation reaction may be
performed by reacting a polymer that comprises a carboxylic acid
functional group (e.g., a poly(ester-ether) compound) with a
polymer or other moiety (such as a targeting moiety) comprising an
amine. For instance, a targeting moiety, such as a poly(amino-acid)
ligand, may be reacted with an amine to form an amine-containing
moiety, which can then be conjugated to the carboxylic acid of the
polymer. Such a reaction may occur as a single-step reaction, i.e.,
the conjugation is performed without using intermediates such as
N-hydroxysuccinimide or a maleimide. The conjugation reaction
between the amine-containing moiety and the carboxylic
acid-terminated polymer (such as a poly(ester-ether) compound) may
be achieved, in one set of embodiments, by adding the
amine-containing moiety, solubilized in an organic solvent such as
(but not limited to) dichloromethane, acetonitrile, chloroform,
tetrahydrofuran, acetone, formamide, dimethylformamide, pyridines,
dioxane, or dimethysulfoxide, to a solution containing the
carboxylic acid-terminated polymer. The carboxylic acid-terminated
polymer may be contained within an organic solvent such as, but not
limited to, dichloromethane, acetonitrile, chloroform,
dimethylformamide, tetrahydrofuran, or acetone. Reaction between
the amine-containing moiety and the carboxylic acid-terminated
polymer may occur spontaneously, in some cases. Unconjugated
reactants may be washed away after such reactions, and the polymer
may be precipitated in solvents such as, for instance, ethyl ether,
hexane, methanol, or ethanol.
[0129] As a specific example, a poly(amino acid) ligand may be
prepared as a targeting moiety in a particle as follows. Carboxylic
acid modified poly(lactide-co-glycolide) (PLGA-COOH) may be
conjugated to an amine-modified heterobifunctional poly(ethylene
glycol) (NH.sub.2--PEG-COOH) to form a copolymer of PLGA-PEG-COOH.
By using an amine-containing poly(amino acid) ligand
(NH.sub.2-Lig), a triblock polymer of PLGA-PEG-Lig may be formed by
conjugating the carboxylic acid end of the PEG to the amine
functional group on the ligand. The multiblock polymer can then be
used, for instance, as discussed below, e.g., for therapeutic
applications.
[0130] Another aspect of the invention is directed to particles
that include polymer conjugates such as the ones described above.
The particles may have a substantially spherical (i.e., the
particles generally appear to be spherical), or non-spherical
configuration. For instance, the particles, upon swelling or
shrinkage, may adopt a non-spherical configuration. In some cases,
the particles may include polymeric blends. For instance, a polymer
blend may be formed that includes a first polymer comprising a
targeting moiety (i.e., a poly(amino acid) ligand) and a
biocompatible polymer, and a second polymer comprising a
biocompatible polymer but not comprising the targeting moiety. By
controlling the ratio of the first and second polymers in the final
polymer, the concentration and location of targeting moiety in the
final polymer may be readily controlled to any suitable degree.
[0131] Accordingly, the present invention provides poly(amino acid)
targeting moieties bound to a polymer. For example, the invention
provides CREKA bound to PEG (CREKA-PEG), CREKA bound to PEG that is
bound to a lipid (e.g., CREKA-PEG-DSPE), and CREKA bound to
PEG-PLGA (CREKA-PEG-PLGA). The invention also provides the
following conjugates:
##STR00002##
wherein n is 20 to 1720; and
##STR00003##
wherein R.sub.7 is an alkyl group, R.sub.8 is an ester or amide
linkage, X=0 to 1 mole fraction, Y=0 to 0.5 mole fraction, X+Y=20
to 1720, and Z=25 to 455.
Preparation of Target-Specific Stealth Nanoparticles
[0132] Another aspect of the invention is directed to systems and
methods of producing such target-specific stealth nanoparticles. In
some embodiments, a solution containing a polymer is contacted with
a liquid, such as an immiscible liquid, to form nanoparticles
containing the polymeric conjugate. Other aspects of the invention
are directed to methods of using such libraries, methods of using
or administering such polymeric conjugates, methods of promoting
the use of such polymeric conjugates, kits involving such polymeric
conjugates, or the like.
[0133] As mentioned, one aspect of the invention is directed to a
method of developing nanoparticles with desired properties, such as
desired chemical, biological, or physical properties. In one set of
embodiments, the method includes producing libraries of
nanoparticles having highly controlled properties, which can be
formed by mixing together two or more polymers in different ratios.
By mixing together two or more different polymers (e.g.,
copolymers, e.g., block copolymers) in different ratios and
producing particles from the polymers (e.g., copolymers, e.g.,
block copolymers), particles having highly controlled properties
may be formed. For example, one polymer (e.g., copolymers, e.g.,
block copolymers) may include a poly(amino acid) ligand, while
another polymer (e.g., copolymers, e.g., block copolymers) may be
chosen for its biocompatibility and/or its ability to control
immunogenicity of the resultant particle.
[0134] Another aspect of the invention is directed to systems and
methods of making such particles. In one set of embodiments, the
particles are formed by providing a solution comprising one or more
polymers, and contacting the solution with a polymer nonsolvent to
produce the particle. The solution may be miscible or immiscible
with the polymer nonsolvent. For example, a water-miscible liquid
such as acetonitrile may contain the polymers, and particles are
formed as the acetonitrile is contacted with water, a polymer
nonsolvent, e.g., by pouring the acetonitrile into the water at a
controlled rate. The polymer contained within the solution, upon
contact with the polymer nonsolvent, may then precipitate to form
particles such as nanoparticles. Two liquids are said to be
"immiscible" or not miscible, with each other when one is not
soluble in the other to a level of at least 10% by weight at
ambient temperature and pressure. Typically, an organic solution
(e.g., dichloromethane, acetonitrile, chloroform, tetrahydrofuran,
acetone, formamide, dimethylformamide, pyridines, dioxane,
dimethysulfoxide, etc.) and an aqueous liquid (e.g., water, or
water containing dissolved salts or other species, cell or
biological media, ethanol, etc.) are immiscible with respect to
each other. For example, the first solution may be poured into the
second solution (at a suitable rate or speed). In some cases,
particles such as nanoparticles may be formed as the first solution
contacts the immiscible second liquid, e.g., precipitation of the
polymer upon contact causes the polymer to form nanoparticles while
the first solution poured into the second liquid, and in some
cases, for example, when the rate of introduction is carefully
controlled and kept at a relatively slow rate, nanoparticles may
form. The control of such particle formation can be readily
optimized by one of ordinary skill in the art using only routine
experimentation.
[0135] By creating a library of such particles, particles having
any desirable properties may be identified. For example, properties
such as surface functionality, surface charge, size, zeta (.zeta.)
potential, hydrophobicity, ability to control immunogenicity, and
the like, may be highly controlled. For instance, a library of
particles may be synthesized, and screened to identify the
particles having a particular ratio of polymers that allows the
particles to have a specific density of moieties (e.g., poly(amino
acid) ligands) present on the surface of the particle. This allows
particles having one or more specific properties to be prepared,
for example, a specific size and a specific surface density of
moieties, without an undue degree of effort. Accordingly, certain
embodiments of the invention are directed to screening techniques
using such libraries, as well as any particles identified using
such libraries. In addition, identification may occur by any
suitable method. For instance, the identification may be direct or
indirect, or proceed quantitatively or qualitatively.
[0136] In some embodiments, already-formed nanoparticles are
functionalized with a targeting moiety using procedures analogous
to those described for producing ligand-functionalized polymeric
conjugates. As a specific, non-limiting example, this embodiment is
exemplified schematically in FIG. 1A. In this figure, a first
copolymer (PLGA-PEG, poly(lactide-co-glycolide) and poly(ethylene
glycol)) is mixed with a therapeutic agent to form particles. The
particles are then associated with a poly(amino acid) ligand to
form nanoparticles that can be used for the treatment of cancer.
The particles can be associated with varying amounts of poly(amino
acid) ligands in order to control the poly(amino acid) ligand
surface density of the nanoparticle, thereby altering the
therapeutic characteristics of the nanoparticle. Furthermore, for
example, by controlling parameters such as PLGA molecular weight,
the molecular weight of PEG, and the nanoparticle surface charge,
very precisely controlled particles may be obtained using this
method of preparation.
[0137] As a specific, non-limiting example, another embodiment is
shown schematically in FIG. 1B. In this figure, a first copolymer
(PLGA-PEG, poly(lactide-co-glycolide) and poly(ethylene glycol)) is
conjugated to a poly(amino acid) ligand (PAALig) to form a
PLGA-PEG-PAALig polymer. This ligand-bound polymer is mixed with a
second, non-functionalized polymer (PLGA-PEG in this example) at
varying ratios to form a series of particles having different
properties, for example, different surface densities of PSMA ligand
as shown in this example. For example, by controlling parameters
such as PLGA molecular weight, the molecular weight of PEG, the
PSMA ligand surface density, and the nanoparticle surface charge,
very precisely controlled particles may be obtained using this
method of preparation. As shown in FIG. 1B, the resulting
nanoparticle can also include a therapeutic agent.
[0138] In another embodiment, the invention provides a method of
preparing a stealth nanoparticle wherein the nanoparticle has a
ratio of ligand-bound polymer to non-functionalized polymer
effective for the treatment of breast cancer, wherein the
hydrophilic, ligand-bound polymer is conjugated to a lipid that
will self assemble with the hydrophobic polymer, such that the
hydrophobic and hydrophilic polymers that constitute the
nanoparticle are not covalently bound. "Self-assembly" refers to a
process of spontaneous assembly of a higher order structure that
relies on the natural attraction of the components of the higher
order structure (e.g., molecules) for each other. It typically
occurs through random movements of the molecules and formation of
bonds based on size, shape, composition, or chemical properties.
For example, such a method comprises providing a first polymer that
is reacted with a lipid, to form a polymer/lipid conjugate. The
polymer/lipid conjugate is then reacted with the poly(amino acid)
ligand to prepare a ligand-bound polymer/lipid conjugate; and
mixing the ligand-bound polymer/lipid conjugate with a second,
non-functionalized polymer, and the therapeutic agent; such that
the stealth nanoparticle is formed. In certain embodiments, the
first polymer is PEG, such that a lipid-terminated PEG is formed.
In one embodiment, the lipid is of the Formula V, e.g., 2
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts
thereof, e.g., the sodium salt. The lipid-terminated PEG can then,
for example, be mixed with PLGA to form a nanoparticle.
[0139] More generally, the polymers chosen to be used to create the
library of particles may be any of a wide variety of polymers, such
as described in detail below. Generally, two, three, four, or more
polymers are mixed, in a wide range of ratios (e.g., each ranging
from 0% to 100%), to form particles such as nanoparticles having
different ratios of each of the polymers. The two or more polymers
may be distinguishable in some fashion, e.g., having different
polymeric groups, having the same polymeric groups but with
different molecular weights, having some polymeric groups in common
but having others that are different (e.g., one may have a
polymeric group that the other does not have), having the same
polymeric groups but in different orders, etc. The library of
particles may have any number of members, for example, the library
may have 2, 3, 5, 10, 30, 100, 300, 1000, 3000, 10,000, 30,000,
100,000, etc. members, which can be identified in some fashion. In
some cases, the library may exist contemporaneously; for example,
the library may be contained in one or more microtiter plates,
vials, etc., or in some embodiments, the library may have include
members created at different times.
[0140] The library of particles can then be screened in some
fashion to identify those particles having one or more desired
properties, for example, surface functionality, surface charge,
size, zeta (.zeta.) potential, hydrophobicity, ability to control
immunogenicity, and the like. One or more of the macromolecules
within the particles may include one or more polymers chosen to be
biocompatible or biodegradable, one or more polymers chosen to
reduce immunogenicity, and/or one or more poly(amino acid) ligands.
These are discussed in detail below. The macromolecules within the
library may comprise some or all of these polymers, in any suitable
combination (including, but not limited to, combinations in which a
first polymer comprises a poly(amino acid) ligand and a second
polymer does not contain any of these species).
[0141] As a specific example, in one embodiment, the particles may
include a first macromolecule comprising a biocompatible polymer,
and a poly(amino acid) ligand, and a second macromolecule
comprising a biocompatible polymer, which may or may not be the
same as that of the first macromolecule. As another example, a
first macromolecule may be a block copolymer comprising a
biocompatible hydrophobic polymer, a biocompatible hydrophilic
polymer, and a poly(amino acid) ligand; and a second macromolecule
distinguishable from the first macromolecule in some fashion. For
instance, the second macromolecule may comprise the same (or a
different) biocompatible hydrophobic polymer and the same (or a
different) biocompatible hydrophilic polymer, but a different
poly(amino acid) ligand (or no ligand at all) than the first
macromolecule.
[0142] The nanoparticle of the invention may also be comprised of,
as another example, a first macromolecule comprising a
biocompatible hydrophobic polymer, a biocompatible hydrophilic
polymer, and a poly(amino acid) ligand, and a second macromolecule
that is distinguishable from the first macromolecule. For instance,
the second macromolecule may contain none of the polymers of the
first macromolecule, the second macromolecule may contain one or
more polymers of the first macromolecule and one or more polymers
not present in the first macromolecule, the second macromolecule
may lack one or more of the polymers of the first macromolecule,
the second macromolecule may contain all of the polymers of the
first macromolecule, but in a different order and/or with one or
more of the polymers having different molecular weights, etc.
[0143] As yet another example, the first macromolecule may comprise
a biocompatible hydrophobic polymer, a biocompatible hydrophilic
polymer, and a poly(amino acid) ligand, and the second
macromolecule may comprise the biocompatible hydrophobic polymer
and the biocompatible hydrophilic polymer, and be distinguishable
from the first macromolecule in some fashion. As still another
example, the first macromolecule may comprise a biocompatible
hydrophobic polymer and a biocompatible hydrophilic polymer, and
the second macromolecule may comprise the biocompatible hydrophobic
polymer and a poly(amino acid) ligand, where the second
macromolecule is distinguishable from the first macromolecule in
some fashion.
[0144] The nanoparticles described above may also contain
therapeutic agents. Examples of therapeutic agents include, but are
not limited to, a chemotherapeutic agent, a radioactive agent, a
nucleic acid-based agent, a lipid-based agent, a carbohydrate based
agent, a natural small molecule, or a synthetic small molecule.
[0145] The polymers or macromolecules may then be formed into a
particle, using techniques such as those discussed in detail below.
The geometry formed by the particle from the polymer or
macromolecule may depend on factors such as the polymers that form
the particle.
[0146] FIG. 2 illustrates that libraries can be produced using
polymers such as those described above. For example, in FIG. 2,
polymeric particles comprising a first macromolecule comprising a
biocompatible hydrophobic polymer, a biocompatible hydrophilic
polymer, and a poly(amino acid) ligand, and a second macromolecule
that comprises a biocompatible hydrophobic polymer and a
biocompatible hydrophilic polymer may be used to create a library
of particles having different ratios of the first and second
macromolecules.
[0147] Such a library may be useful in achieving particles having
any number of desirable properties, for instance properties such as
surface functionality, surface charge, size, zeta (.zeta.)
potential, hydrophobicity, ability to control immunogenicity, or
the like. In FIG. 2, different ratios of the first and second
macromolecules (including ratios where one of the macromolecules is
absent) are combined to produce particles that form the basis of
the library.
[0148] For instance, as shown in FIG. 2, as the amount of the first
macromolecule is increased, relative to the second macromolecule,
the amount of moiety (e.g., poly(amino acid) ligand) present on the
surface of the particle may be increased. Thus, any suitable
concentration of moiety on the surface may be achieved simply by
controlling the ratio of the first and second macromolecules in the
particles. Accordingly, such a library of particles may be useful
in selecting or identifying particles having a particular
functionality.
[0149] As specific examples, in some embodiments of the present
invention, the library includes particles comprising polymeric
conjugates of a biocompatible polymer and a poly(amino acid)
ligand, as discussed herein. Referring now to FIG. 3, one such
particle is shown as a non-limiting example. In this figure, a
polymeric conjugate of the invention is used to form a particle 10.
The polymer forming particle 10 includes a poly(amino acid) 15,
present on the surface of the particle, and a biocompatible portion
17. In some cases, as shown here, targeting moiety 15 may be
conjugated to biocompatible portion 17. However, not all of
biocompatible portion 17 is shown conjugated to targeting moiety
15. For instance, in some cases, particles such as particle 10 may
be formed using a first polymer comprising biocompatible portion 17
and poly(amino acid) ligand 15, and a second polymer comprising
biocompatible portion 17 but not targeting moiety 15. By
controlling the ratio of the first and second polymers, particles
having different properties may be formed, and in some cases,
libraries of such particles may be formed. In addition, contained
within the center of particle 10 is drug 12. In some cases, drug 12
may be contained within the particle due to hydrophobic effects.
For instance, the interior of the particle may be relatively
hydrophobic with respect to the surface of the particle, and the
drug may be a hydrophobic drug that associates with the relatively
hydrophobic center of the particle. In one embodiment, the
therapeutic agent is associated with the surface of, encapsulated
within, surrounded by, or dispersed throughout the nanoparticle. In
another embodiment, the therapeutic agent is encapsulated within
the hydrophobic core of the nanoparticle.
[0150] As a specific example, particle 10 may contain polymers
including a relatively hydrophobic biocompatible polymer and a
relatively hydrophilic targeting moiety 15, such that, during
particle formation, a greater concentration of the hydrophilic
targeting moiety is exposed on the surface and a greater
concentration of the hydrophobic biocompatible polymer is present
within the interior of the particle.
[0151] In some embodiments, the biocompatible polymer is a
hydrophobic polymer. Non-limiting examples of biocompatible
polymers include polylactide, polyglycolide, and/or
poly(lactide-co-glycolide).
[0152] In some cases, the polymeric conjugate is part of a
controlled release system. A "controlled release system," as used
herein, is a polymer combined with an active agent or a drug or
other payload, such as a therapeutic agent, a diagnostic agent, a
prognostic, a prophylactic agent, etc., and the active agent is
released from the controlled release system in a predesigned or
controlled manner. For example, the active agent may be released in
a constant manner over a predetermined period of time, the active
agent may be released in a cyclic manner over a predetermined
period of time, or an environmental condition or external event may
trigger the release of the active agent. The controlled release
polymer system may include a polymer that is biocompatible, and in
some cases, the polymer is biodegradable.
Therapeutic Agents
[0153] Another aspect of the present invention is directed to a
therapeutic "payload," or a species (or more than one species)
contained within a particle, such as those described above. For
instance, the targeting moiety may target or cause the particle to
become localized at specific portions within a subject, and the
payload may be delivered to those portions. In a particular
embodiment, the drug or other payload is released in a controlled
release manner from the particle and allowed to interact locally
with the particular targeting site (e.g., a tumor). The term
"controlled release" (and variants of that term) as used herein
(e.g., in the context of "controlled-release system") is generally
meant to encompass release of a substance (e.g., a drug) at a
selected site or otherwise controllable in rate, interval, and/or
amount. Controlled release encompasses, but is not necessarily
limited to, substantially continuous delivery, patterned delivery
(e.g., intermittent delivery over a period of time that is
interrupted by regular or irregular time intervals), and delivery
of a bolus of a selected substance (e.g., as a predetermined,
discrete amount if a substance over a relatively short period of
time (e.g., a few seconds or minutes)).
[0154] For example, a targeting portion may cause the particles to
become localized to a tumor, a disease site, a tissue, an organ, a
type of cell, etc. within the body of a subject, depending on the
targeting moiety used. For example, a poly(amino acid) ligand may
become localized to Her-2, the basement membrane of a blood vessel,
collagen, collagen IV or the like. The subject may be a human or
non-human animal. Examples of subjects include, but are not limited
to, a mammal such as a dog, a cat, a horse, a donkey, a rabbit, a
cow, a pig, a sheep, a goat, a rat, a mouse, a guinea pig, a
hamster, a primate, a human or the like.
[0155] In one set of embodiments, the payload is a drug or a
combination of more than one drug. Such particles may be useful,
for example, in embodiments where a targeting moiety may be used to
direct a particle containing a drug to a particular localized
location within a subject, e.g., to allow localized delivery of the
drug to occur. Exemplary therapeutic agents include
chemotherapeutic agents such as doxorubicin (adriamycin),
gemcitabine (gemzar), daunorubicin, procarbazine, mitomycin,
cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU),
vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel
(taxotere), aldesleukin, asparaginase, busulfan, carboplatin,
paltin derivatives, cladribine, camptothecin,
CPT-1,10-hydroxy-7-ethylcamptothecin (SN38), dacarbazine, S--I
capecitabine, ftorafur, 5'deoxyfluorouridine, UFT, eniluracil,
deoxycytidine, 5-azacyto sine, 5-azadeoxycyto sine, allopurinol,
2-chloroadeno sine, trimetrexate, aminopterin,
methylene-10-deazaminopterin (MDAM), oxaplatin, picoplatin,
tetraplatin, satraplatin, platinum-DACH, ormaplatin, CI-973,
JM-216, and analogs thereof, epirubicin, etoposide phosphate,
9-aminocamptothecin, 10,11-methylenedioxycamptothecin, karenitecin,
9-nitrocamptothecin, TAS103, vindesine, L-phenylalanine mustard,
ifosphamidemefosphamide, perfosfamide, trophosphamide carmustine,
semustine, epothilones A-E, tomudex, 6-mercaptopurine,
6-thioguanine, amsacrine, etoposide phosphate, karenitecin,
acyclovir, valacyclovir, ganciclovir, amantadine, rimantadine,
lamivudine, zidovudine, bevacizumab, trastuzumab, rituximab,
5-Fluorouracil, and combinations thereof.
[0156] Suitable non-genetic therapeutic agents for use in
connection with the present invention may be selected, for example,
from one or more of the following: (a) anti-thrombotic agents such
as heparin, heparin derivatives, urokinase, clopidogrel, and PPack
(dextrophenylalanine proline arginine chloromethylketone); (b)
anti-inflammatory agents such as dexamethasone, prednisolone,
corticosterone, budesonide, estrogen, sulfasalazine and mesalamine;
(c) antineoplastic/antiproliferative/anti-miotic agents such as
paclitaxel, 5-fluorouracil, cisplatin, vinblastine, vincristine,
epothilones, endostatin, angiostatin, angiopeptin, monoclonal
antibodies capable of blocking smooth muscle cell proliferation,
and thymidine kinase inhibitors; (d) anesthetic agents such as
lidocaine, bupivacaine and ropivacaine; (e) anti-coagulants such as
D-Phe-Pro-Arg chloromethyl ketone, an RGD peptide-containing
compound, heparin, hirudin, antithrombin compounds, platelet
receptor antagonists, anti-thrombin antibodies, anti-platelet
receptor antibodies, aspirin, prostaglandin inhibitors, platelet
inhibitors and tick antiplatelet peptides; (f) vascular cell growth
promoters such as growth factors, transcriptional activators, and
translational promotors; (g) vascular cell growth inhibitors such
as growth factor inhibitors, growth factor receptor antagonists,
transcriptional repressors, translational repressors, replication
inhibitors, inhibitory antibodies, antibodies directed against
growth factors, bifunctional molecules consisting of a growth
factor and a cytotoxin, bifunctional molecules consisting of an
antibody and a cytotoxin; (h) protein kinase and tyrosine kinase
inhibitors (e.g., tyrphostins, genistein, quinoxalines); (i)
prostacyclin analogs; (j) cholesterol-lowering agents; (k)
angiopoietins; (l) antimicrobial agents such as triclosan,
cephalosporins, aminoglycosides and nitrofurantoin; (m) cytotoxic
agents, cytostatic agents and cell proliferation affectors; (n)
vasodilating agents; (o) agents that interfere with endogenous
vasoactive mechanisms; (p) inhibitors of leukocyte recruitment,
such as monoclonal antibodies; (q) cytokines; (r) hormones; (s)
inhibitors of HSP 90 protein (i.e., Heat Shock Protein, which is a
molecular chaperone or housekeeping protein and is needed for the
stability and function of other client proteins/signal transduction
proteins responsible for growth and survival of cells) including
geldanamycin, (t) smooth muscle relaxants such as alpha receptor
antagonists (e.g., doxazosin, tamsulosin, terazosin, prazosin and
alfuzosin), calcium channel blockers (e.g., verapimil, diltiazem,
nifedipine, nicardipine, nimodipine and bepridil), beta receptor
agonists (e.g., dobutamine and salmeterol), beta receptor
antagonists (e.g., atenolol, metaprolol and butoxamine),
angiotensin-II receptor antagonists (e.g., losartan, valsartan,
irbesartan, candesartan, eprosartan and telmisartan), and
antispasmodic/anticholinergic drugs (e.g., oxybutynin chloride,
flavoxate, tolterodine, hyoscyamine sulfate, diclomine), (u) bARKct
inhibitors, (v) phospholamban inhibitors, (w) Serca 2 gene/protein,
(x) immune response modifiers including aminoquizolines, for
instance, imidazoquinolines such as resiquimod and imiquimod, (y)
human apolioproteins (e.g., AI, AII, AIII, AIV, AV, etc.), (z)
selective estrogen receptor modulators (SERMs) such as raloxifene,
lasofoxifene, arzoxifene, miproxifene, ospemifene, PKS 3741, MF 101
and SR 16234, (aa) PPAR agonists such as rosiglitazone,
pioglitazone, netoglitazone, fenofibrate, bexaotene, metaglidasen,
rivoglitazone and tesaglitazar, (bb) prostaglandin E agonists such
as alprostadil or ONO 8815Ly, (cc) thrombin receptor activating
peptide (TRAP), (dd) vasopeptidase inhibitors including benazepril,
fosinopril, lisinopril, quinapril, ramipril, imidapril, delapril,
moexipril and spirapril, (ee) thymosin beta 4, and (ff)
phospholipids including phosphorylcholine, phosphatidylinositol and
phosphatidylcholine.
[0157] Preferred non-genetic therapeutic agents include taxanes
such as paclitaxel (including particulate forms thereof, for
instance, protein-bound paclitaxel particles such as albumin-bound
paclitaxel nanoparticles, e.g., ABRAXANE), sirolimus, everolimus,
tacrolimus, zotarolimus, Epo D, dexamethasone, estradiol,
halofuginone, cilostazole, geldanamycin, ABT-578 (Abbott
Laboratories), trapidil, liprostin, Actinomcin D, Resten-NG, Ap-17,
abciximab, clopidogrel, Ridogrel, beta-blockers, bARKct inhibitors,
phospholamban inhibitors, Serca 2 gene/protein, imiquimod, human
apolioproteins (e.g., AI-AV), growth factors (e.g., VEGF-2), as
well derivatives of the forgoing, among others.
[0158] Suitable genetic therapeutic agents for use in connection
with the present invention include anti-sense DNA and RNA as well
as DNA coding for the various proteins (as well as the proteins
themselves) and may be selected, for example, from one or more of
the following: (a) anti-sense RNA, (b) tRNA or rRNA to replace
defective or deficient endogenous molecules, (c) angiogenic and
other factors including growth factors such as acidic and basic
fibroblast growth factors, vascular endothelial growth factor,
endothelial mitogenic growth factors, epidermal growth factor,
transforming growth factor .alpha. and .beta., platelet-derived
endothelial growth factor, platelet-derived growth factor, tumor
necrosis factor .alpha., hepatocyte growth factor and insulin-like
growth factor, (d) cell cycle inhibitors including CD inhibitors,
and (e) thymidine kinase ("TK") and other agents useful for
interfering with cell proliferation. Also of interest is DNA
encoding for the family of bone morphogenic proteins ("BMP's"),
including BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1),
BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, and
BMP-16. Currently preferred BMP's are any of BMP-2, BMP-3, BMP-4,
BMP-5, BMP-6 and BMP-7. These dimeric proteins can be provided as
homodimers, heterodimers, or combinations thereof, alone or
together with other molecules. Alternatively, or in addition,
molecules capable of inducing an upstream or downstream effect of a
BMP can be provided. Such molecules include any of the "hedgehog"
proteins, or the DNA's encoding them.
[0159] Vectors for delivery of genetic therapeutic agents include
viral vectors such as adenoviruses, gutted adenoviruses,
adeno-associated virus, retroviruses, alpha virus (Semliki Forest,
Sindbis, etc.), lentiviruses, herpes simplex virus, replication
competent viruses (e.g., ONYX-015) and hybrid vectors; and
non-viral vectors such as artificial chromosomes and
mini-chromosomes, plasmid DNA vectors (e.g., pCOR), cationic
polymers (e.g., polyethyleneimine, polyethyleneimine (PEI)), graft
copolymers (e.g., polyether-PEI and polyethylene oxide-PEI),
neutral polymers such as polyvinylpyrrolidone (PVP), SP1017
(SUPRATEK), lipids such as cationic lipids, liposomes, lipoplexes,
nanoparticles, or microparticles, with and without targeting
sequences such as the protein transduction domain (PTD).
[0160] Cells for use in conjunction with the present invention
include cells of human origin (autologous or allogeneic), including
whole bone marrow, bone marrow derived mono-nuclear cells,
progenitor cells (e.g., endothelial progenitor cells), stem cells
(e.g., mesenchymal, hematopoietic, neuronal), pluripotent stem
cells, fibroblasts, myoblasts, satellite cells, pericytes,
cardiomyocytes, skeletal myocytes or macrophage, or from an animal,
bacterial or fungal source (xenogeneic), which can be genetically
engineered, if desired, to deliver proteins of interest.
[0161] Further therapeutic agents, not necessarily exclusive of
those listed above, have been identified as candidates for vascular
treatment regimens, for example, as agents targeting restenosis
(anti-restenotic agents). Suitable agents may be selected, for
example, from one or more of the following: (a) Ca-channel blockers
including benzothiazapines such as diltiazem and clentiazem,
dihydropyridines such as nifedipine, amlodipine and nicardapine,
and phenylalkylamines such as verapamil, (b) serotonin pathway
modulators including: 5-HT antagonists such as ketanserin and
naftidrofuryl, as well as 5-HT uptake inhibitors such as
fluoxetine, (c) cyclic nucleotide pathway agents including
phosphodiesterase inhibitors such as cilostazole and dipyridamole,
adenylate/Guanylate cyclase stimulants such as forskolin, as well
as adenosine analogs, (d) catecholamine modulators including
.alpha.-antagonists such as prazosin and bunazosine,
.beta.-antagonists such as propranolol and
.alpha./.beta.-antagonists such as labetalol and carvedilol, (e)
endothelin receptor antagonists such as bosentan, sitaxsentan
sodium, atrasentan, endonentan, (f) nitric oxide donors/releasing
molecules including organic nitrates/nitrites such as
nitroglycerin, isosorbide dinitrate and amyl nitrite, inorganic
nitroso compounds such as sodium nitroprusside, sydnonimines such
as molsidomine and linsidomine, nonoates such as diazenium diolates
and NO adducts of alkanediamines, S-nitroso compounds including low
molecular weight compounds (e.g., S-nitroso derivatives of
captopril, glutathione and N-acetyl penicillamine) and high
molecular weight compounds (e.g., S-nitroso derivatives of
proteins, peptides, oligosaccharides, polysaccharides, synthetic
polymers/oligomers and natural polymers/oligomers), as well as
C-nitroso-compounds, O-nitroso-compounds, N-nitroso-compounds and
L-arginine, (g) Angiotensin Converting Enzyme (ACE) inhibitors such
as cilazapril, fosinopril and enalapril, (h) ATII-receptor
antagonists such as saralasin and losartin, (i) platelet adhesion
inhibitors such as albumin and polyethylene oxide, (j) platelet
aggregation inhibitors including cilostazole, aspirin and
thienopyridine (ticlopidine, clopidogrel) and GP IIb/IIIa
inhibitors such as abciximab, epitifibatide and tirofiban, (k)
coagulation pathway modulators including heparinoids such as
heparin, low molecular weight heparin, dextran sulfate and
.beta.-cyclodextrin tetradecasulfate, thrombin inhibitors such as
hirudin, hirulog, PPACK(D-phe-L-propyl-L-arg-chloromethylketone)
and argatroban, FXa inhibitors such as antistatin and TAP (tick
anticoagulant peptide), Vitamin K inhibitors such as warfarin, as
well as activated protein C, (l) cyclooxygenase pathway inhibitors
such as aspirin, ibuprofen, flurbiprofen, indomethacin and
sulfinpyrazone, (m) natural and synthetic corticosteroids such as
dexamethasone, prednisolone, methprednisolone and hydrocortisone,
(n) lipoxygenase pathway inhibitors such as nordihydroguairetic
acid and caffeic acid, (o) leukotriene receptor antagonists, (p)
antagonists of E- and P-selectins, (q) inhibitors of VCAM-1 and
ICAM-1 interactions, (r) prostaglandins and analogs thereof
including prostaglandins such as PGE1 and PGI2 and prostacyclin
analogs such as ciprostene, epoprostenol, carbacyclin, iloprost and
beraprost, (s) macrophage activation preventers including
bisphosphonates, (t) HMG-CoA reductase inhibitors such as
lovastatin, pravastatin, atorvastatin, fluvastatin, simvastatin and
cerivastatin, (u) fish oils and omega-3-fatty acids, (v)
free-radical scavengers/antioxidants such as probucol, vitamins C
and E, ebselen, trans-retinoic acid and SOD (orgotein), SOD mimics,
verteporfin, rostaporfin, AGI 1067, and M 40419, (w) agents
affecting various growth factors including FGF pathway agents such
as bFGF antibodies and chimeric fusion proteins, PDGF receptor
antagonists such as trapidil, IGF pathway agents including
somatostatin analogs such as angiopeptin and ocreotide, TGF-.beta.
pathway agents such as polyanionic agents (heparin, fucoidin),
decorin, and TGF-.beta. antibodies, EGF pathway agents such as EGF
antibodies, receptor antagonists and chimeric fusion proteins,
TNF-.alpha. pathway agents such as thalidomide and analogs thereof,
Thromboxane A2 (TXA2) pathway modulators such as sulotroban,
vapiprost, dazoxiben and ridogrel, as well as protein tyrosine
kinase inhibitors such as tyrphostin, genistein and quinoxaline
derivatives, (x) matrix metalloprotease (MMP) pathway inhibitors
such as marimastat, ilomastat, metastat, batimastat, pentosan
polysulfate, rebimastat, incyclinide, apratastat, PG 116800, RO
1130830 or ABT 518, (y) cell motility inhibitors such as
cytochalasin B, (z) antiproliferative/antineoplastic agents
including antimetabolites such as purine analogs (e.g.,
6-mercaptopurine or cladribine, which is a chlorinated purine
nucleoside analog), pyrimidine analogs (e.g., cytarabine and
5-fluorouracil) and methotrexate, nitrogen mustards, alkyl
sulfonates, ethylenimines, antibiotics (e.g., daunorubicin,
doxorubicin), nitrosoureas, cisplatin, agents affecting microtubule
dynamics (e.g., vinblastine, vincristine, colchicine, Epo D,
paclitaxel and epothilone), caspase activators, proteasome
inhibitors, angiogenesis inhibitors (e.g., endostatin, angiostatin
and squalamine), rapamycin (sirolimus) and its analogs (e.g.,
everolimus, tacrolimus, zotarolimus, etc.), cerivastatin,
flavopiridol and suramin, (aa) matrix deposition/organization
pathway inhibitors such as halofuginone or other quinazolinone
derivatives, pirfenidone and tranilast, (bb) endothelialization
facilitators such as VEGF and RGD peptide, (cc) blood rheology
modulators such as pentoxifylline and (dd) glucose cross-link
breakers such as alagebrium chloride (ALT-711).
[0162] Numerous additional therapeutics for the practice of the
present invention may be selected from suitable therapeutic agents
disclosed in U.S. Pat. No. 5,733,925 to Kunz.
[0163] Non-limiting examples of potentially suitable drugs include
anti-cancer agents, including, for example, docetaxel,
mitoxantrone, and mitoxantrone hydrochloride. In another
embodiment, the payload may be an anti-cancer drug such as
20-epi-1, 25 dihydroxyvitamin D3,4-ipomeanol, 5-ethynyluracil,
9-dihydrotaxol, abiraterone, acivicin, aclarubicin, acodazole
hydrochloride, acronine, acylfiilvene, adecypenol, adozelesin,
aldesleukin, all-tk antagonists, altretamine, ambamustine,
ambomycin, ametantrone acetate, amidox, amifostine,
aminoglutethimide, aminolevulinic acid, amrubicin, amsacrine,
anagrelide, anastrozole, andrographolide, angiogenesis inhibitors,
antagonist D, antagonist G, antarelix, anthramycin,
anti-dorsalizdng morphogenetic protein-1, antiestrogen,
antineoplaston, antisense oligonucleotides, aphidicolin glycinate,
apoptosis gene modulators, apoptosis regulators, apurinic acid,
ARA-CDP-DL-PTBA, arginine deaminase, asparaginase, asperlin,
asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2,
axinastatin 3, azacitidine, azasetron, azatoxin, azatyrosine,
azetepa, azotomycin, baccatin III derivatives, balanol, batimastat,
benzochlorins, benzodepa, benzoylstaurosporine, beta lactam
derivatives, beta-alethine, betaclamycin B, betulinic acid, BFGF
inhibitor, bicalutamide, bisantrene, bisantrene hydrochloride,
bisazuidinylspermine, bisnafide, bisnafide dimesylate, bistratene
A, bizelesin, bleomycin, bleomycin sulfate, BRC/ABL antagonists,
breflate, brequinar sodium, bropirimine, budotitane, busulfan,
buthionine sulfoximine, cactinomycin, calcipotriol, calphostin C,
calusterone, camptothecin derivatives, canarypox IL-2,
capecitabine, caraceraide, carbetimer, carboplatin,
carboxamide-amino-triazole, carboxyamidotriazole, carest M3,
carmustine, earn 700, cartilage derived inhibitor, carubicin
hydrochloride, carzelesin, casein kinase inhibitors,
castanosperrnine, cecropin B, cedefingol, cetrorelix, chlorambucil,
chlorins, chloroquinoxaline sulfonamide, cicaprost, cirolemycin,
cisplatin, cis-porphyrin, cladribine, clomifene analogs,
clotrimazole, collismycin A, collismycin B, combretastatin A4,
combretastatin analog, conagenin, crambescidin 816, crisnatol,
crisnatol mesylate, cryptophycin 8, cryptophycin A derivatives,
curacin A, cyclopentanthraquinones, cyclophosphamide, cycloplatam,
cypemycin, cytarabine, cytarabine ocfosfate, cytolytic factor,
cytostatin, dacarbazine, dacliximab, dactinomycin, daunorubicin
hydrochloride, decitabine, dehydrodidemnin B, deslorelin,
dexifosfamide, dexormaplatin, dexrazoxane, dexverapamil,
dezaguanine, dezaguanine mesylate, diaziquone, didemnin B, didox,
diethyhiorspermine, dihydro-5-azacytidine, dioxamycin, diphenyl
spiromustine, docetaxel, docosanol, dolasetron, doxifluridine,
doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene
citrate, dromostanolone propionate, dronabinol, duazomycin,
duocannycin SA, ebselen, ecomustine, edatrexate, edelfosine,
edrecolomab, eflomithine, eflomithine hydrochloride, elemene,
elsarnitrucin, emitefur, enloplatin, enpromate, epipropidine,
epirubicin, epirubicin hydrochloride, epristeride, erbulozole,
erythrocyte gene therapy vector system, esorubicin hydrochloride,
estramustine, estramustine analog, estramustine phosphate sodium,
estrogen agonists, estrogen antagonists, etanidazole, etoposide,
etoposide phosphate, etoprine, exemestane, fadrozole, fadrozole
hydrochloride, fazarabine, fenretinide, filgrastim, finasteride,
flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine,
fludarabine phosphate, fluorodaunorunicin hydrochloride,
fluorouracil, fluorocitabine, forfenimex, formestane, fosquidone,
fostriecin, fostriecin sodium, fotemustine, gadolinium texaphyrin,
gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors,
gemcitabine, gemcitabine hydrochloride, glutathione inhibitors,
hepsulfam, heregulin, hexamethylene bisacetamide, hydroxyurea,
hypericin, ibandronic acid, idarubicin, idarubicin hydrochloride,
idoxifene, idramantone, ifosfamide, ihnofosine, ilomastat,
imidazoacridones, imiquimod, immunostimulant peptides, insulin-like
growth factor-1 receptor inhibitor, interferon agonists, interferon
alpha-2A, interferon alpha-2B, interferon alpha-N1, interferon
alpha-N3, interferon beta-IA, interferon gamma-IB, interferons,
interleukins, iobenguane, iododoxorubicin, iproplatm, irinotecan,
irinotecan hydrochloride, iroplact, irsogladine, isobengazole,
isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F,
lamellarin-N triacetate, lanreotide, lanreotide acetate,
leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,
leukemia inhibiting factor, leukocyte alpha interferon, leuprolide
acetate, leuprolide/estrogen/progesterone, leuprorelin, levamisole,
liarozole, liarozole hydrochloride, linear polyamine analog,
lipophilic disaccharide peptide, lipophilic platinum compounds,
lissoclinamide, lobaplatin, lombricine, lometrexol, lometrexol
sodium, lomustine, lonidamine, losoxantrone, losoxantrone
hydrochloride, lovastatin, loxoribine, lurtotecan, lutetium
texaphyrin lysofylline, lytic peptides, maitansine, mannostatin A,
marimastat, masoprocol, maspin, matrilysin inhibitors, matrix
metalloproteinase inhibitors, maytansine, mechlorethamine
hydrochloride, megestrol acetate, melengestrol acetate, melphalan,
menogaril, merbarone, mercaptopurine, meterelin, methioninase,
methotrexate, methotrexate sodium, metoclopramide, metoprine,
meturedepa, microalgal protein kinase C uihibitors, MIF inhibitor,
mifepristone, miltefosine, mirimostim, mismatched double stranded
RNA, mitindomide, mitocarcin, mitocromin, mitogillin, mitoguazone,
mitolactol, mitomalcin, mitomycin, mitomycin analogs, mitonafide,
mitosper, mitotane, mitotoxin fibroblast growth factor-saporin,
mitoxantrone, mitoxantrone hydrochloride, mofarotene, molgramostim,
monoclonal antibody, human chorionic gonadotrophin, monophosphoryl
lipid a/myobacterium cell wall SK, mopidamol, multiple drug
resistance gene inhibitor, multiple tumor suppressor 1-based
therapy, mustard anticancer agent, mycaperoxide B, mycobacterial
cell wall extract, mycophenolic acid, myriaporone,
n-acetyldinaline, nafarelin, nagrestip, naloxone/pentazocine,
napavin, naphterpin, nartograstim, nedaplatin, nemorubicin,
neridronic acid, neutral endopeptidase, nilutamide, nisamycin,
nitric oxide modulators, nitroxide antioxidant, nitrullyn,
nocodazole, nogalamycin, n-substituted benzamides,
O6-benzylguanine, octreotide, okicenone, oligonucleotides,
onapristone, ondansetron, oracin, oral cytokine inducer,
ormaplatin, osaterone, oxaliplatin, oxaunomycin, oxisuran,
paclitaxel, paclitaxel analogs, paclitaxel derivatives, palauamine,
palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene,
parabactin, pazelliptine, pegaspargase, peldesine, peliomycin,
pentamustine, pentosan polysulfate sodium, pentostatin, pentrozole,
peplomycin sulfate, perflubron, perfosfamide, perillyl alcohol,
phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil,
pilocarpine hydrochloride, pipobroman, piposulfan, pirarubicin,
piritrexim, piroxantrone hydrochloride, placetin A, placetin B,
plasminogen activator inhibitor, platinum complex, platinum
compounds, platinum-triamine complex, plicamycin, plomestane,
porfimer sodium, porfiromycin, prednimustine, procarbazine
hydrochloride, propyl bis-acridone, prostaglandin J2, prostatic
carcinoma antiandrogen, proteasome inhibitors, protein A-based
immune modulator, protein kinase C inhibitor, protein tyrosine
phosphatase inhibitors, purine nucleoside phosphorylase inhibitors,
puromycin, puromycin hydrochloride, purpurins, pyrazorurin,
pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene
conjugate, RAF antagonists, raltitrexed, ramosetron, RAS farnesyl
protein transferase inhibitors, RAS inhibitors, RAS-GAP inhibitor,
retelliptine demethylated, rhenium RE 186 etidronate, rhizoxin,
riboprine, ribozymes, RH retinamide, RNAi, rogletimide, rohitukine,
romurtide, roquinimex, rubiginone B1, ruboxyl, safingol, safingol
hydrochloride, saintopin, sarcnu, sarcophytol A, sargramostim, SDI1
mimetics, semustine, senescence derived inhibitor 1, sense
oligonucleotides, signal transduction inhibitors, signal
transduction modulators, simtrazene, single chain antigen binding
protein, sizofuran, sobuzoxane, sodium borocaptate, sodium
phenylacetate, solverol, somatomedin binding protein, sonermin,
sparfosafe sodium, sparfosic acid, sparsomycin, spicamycin D,
spirogermanium hydrochloride, spiromustine, spiroplatin,
splenopentin, spongistatin 1, squalamine, stem cell inhibitor,
stem-cell division inhibitors, stipiamide, streptonigrin,
streptozocin, stromelysin inhibitors, sulfinosine, sulofenur,
superactive vasoactive intestinal peptide antagonist, suradista,
suramin, swainsonine, synthetic glycosaminoglycans, talisomycin,
tallimustine, tamoxifen methiodide, tauromustine, tazarotene,
tecogalan sodium, tegafur, tellurapyrylium, telomerase inhibitors,
teloxantrone hydrochloride, temoporfin, temozolomide, teniposide,
teroxirone, testolactone, tetrachlorodecaoxide, tetrazomine,
thaliblastine, thalidomide, thiamiprine, thiocoraline, thioguanine,
thiotepa, thrombopoietin, thrombopoietin mimetic, thymalfasin,
thymopoietin receptor agonist, thymotrinan, thyroid stimulating
hormone, tiazofurin, tin ethyl etiopurpurin, tirapazamine,
titanocene dichloride, topotecan hydrochloride, topsentin,
toremifene, toremifene citrate, totipotent stem cell factor,
translation inhibitors, trestolone acetate, tretinoin,
triacetyluridine, triciribine, triciribine phosphate, trimetrexate,
trimetrexate glucuronate, triptorelin, tropisetron, tubulozole
hydrochloride, turosteride, tyrosine kinase inhibitors,
tyrphostins, UBC inhibitors, ubenimex, uracil mustard, uredepa,
urogenital sinus-derived growth inhibitory factor, urokinase
receptor antagonists, vapreotide, variolin B, velaresol, veramine,
verdins, verteporfin, vinblastine sulfate, vincristine sulfate,
vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate
sulfate, vinleurosine sulfate, vinorelbine, vinorelbine tartrate,
vinrosidine sulfate, vinxaltine, vinzolidine sulfate, vitaxin,
vorozole, zanoterone, zeniplatin, zilascorb, zinostatin, zinostatin
stimalamer, or zorubicin hydrochloride.
[0164] In another embodiment, the nanoparticles of the invention
can be used to treat vulnerable plaque in a subject in need
thereof. In particular, the payload associated with, i.e.,
encapsulated within, the nanoparticle of the invention is a
biologically active agent used to stabilize a vulnerable plaque.
Such agents are described in U.S. Pat. No. 7,008,411, which is
incorporated herein by reference in its entirety.
[0165] In another embodiment, the nanoparticles of the invention
can be used to treat restenosis or atherosclerosis in a subject in
need thereof. Restenosis is the reobstruction of an artery
following interventional procedures such as balloon angioplasty or
stenting.
[0166] Additional examples of potentially suitable drugs include
for delivery by the nanoparticles of the invention include
doxorubicin, 2-aminochromone (U-86983, Upjohn and Pharmacia)
(U-86), cytarabine, vincristine, dalteparin sodium, cyclosporine A,
colchicines, etoposide, sirolimus, paclitaxel, ceramide,
cilostazol, clodronate, pamidronate, alendronate, ISA-13-1,
AG-1295, AGL-2043, dexamethasone, everolimus, ABT-578, tacrolimus
(FK506), estradiol, lantrunculinD, cytochalasin A, dexamethasone,
zotarolimus, angiopeptin, bisphosphonates, estrogen, angiopeptin,
ROCK inhibitors, PDGF inhibitors, MMP inhibitors, statins, and well
as combinations of these therapies (e.g., a combination of
zotarolimus and dexamethasone), as well as any therapeutic
disclosed in Circ Res 2003 Apr. 18; 92(7):e62-9. Epub 2003 Mar. 27;
J Pharm Sci 1998 October; 87(10):1229-34; Int J Nanomedicine 2007;
2(2):143-61; and Atherosclerosis 2002 February; 160(2):259-71,
which are incorporated herein by reference in their entirety.
[0167] In one embodiment, therapeutic or biologically active agents
may be released by the nanoparticles of the invention to induce
therapeutic angiogenesis, which refers to the processes of causing
or inducing angiogenesis and arteriogenesis, either downstream, or
away from the vulnerable plaque. Arteriogenesis is the enlargement
of pre-existing collateral vessels. Collateral vessels allow blood
to flow from a well-perfused region of the vessel into an ischemic
region (from above an occlusion to downstream from the occlusion).
Angiogenesis is the promotion or causation of the formation of new
blood vessels downstream from the ischemic region. Having more
blood vessels (e.g., capillaries) below the occlusion may provide
for less pressure drop to perfuse areas with severe narrowing
caused by a thrombus. In the event that an occlusive thrombus
occurs in a vulnerable plaque, the myocardium perfused by the
affected artery is salvaged. Representative therapeutic or
biologically active agents include, but are not limited to,
proteins such as vascular endothelial growth factor (VEGF) in any
of its multiple isoforms, fibroblast growth factors, monocyte
chemoatractant protein 1 (MCP-1), transforming growth factor alpha
(TGF-alpha), transforming growth factor beta (TGF-beta) in any of
its multiple isoforms, DEL-1, insulin like growth factors (IGF),
placental growth factor (PLGF), hepatocyte growth factor (HGF),
prostaglandin E1 (PG-E1), prostaglandin E2 (PG-E2), tumor necrosis
factor alpha (THF-alpha), granulocyte stimulating growth factor
(G-CSF), granulocyte macrophage colony-stimulating growth factor
(GM-CSF), angiogenin, follistatin, and proliferin, genes encoding
these proteins, cells transfected with these genes, pro-angiogenic
peptides such as PR39 and PR11, and pro-angiogenic small molecules
such as nicotine. The nanoparticles of the invention may also
include lipid lowering agents (e.g., hydroxy-methylglutaryl
coenzyme A (HMG CoA) reductase inhibitors, statins, niacin, bile
acid resins, and fibrates), antioxidants (e.g., vitamin E
(.alpha.-tocopherol), vitamin C, and .beta.-carotene supplements),
extracellular matrix synthesis promoters, inhibitors of plaque
inflammation and extracellular degradation, estradiol drug classes
and its derivatives.
[0168] Other therapeutic agents to be delivered in accordance with
the present invention include, but are not limited to, nucleic
acids (e.g., siRNA, RNAi, and microRNA agents), proteins (e.g.
antibodies), peptides, lipids, carbohydrates, hormones, metals,
radioactive elements and compounds, vaccines, immunological agents,
etc., and/or combinations thereof. In some embodiments, the agent
to be delivered is an agent useful in the treatment of cancer
(e.g., prostate cancer).
[0169] In one embodiment, the nanoparticles of this invention will
contain nucleic acids such as siRNA. Preferably, the siRNA molecule
has a length from about 10-50 or more nucleotides. More preferably,
the siRNA molecule has a length from about 15-45 nucleotides. Even
more preferably, the siRNA molecule has a length from about 19-40
nucleotides. Even more preferably, the siRNA molecule has a length
of from about 21-23 nucleotides.
[0170] The siRNA of the invention preferably mediates RNAi against
a target mRNA. The siRNA molecule can be designed such that every
residue is complementary to a residue in the target molecule.
Alternatively, one or more substitutions can be made within the
molecule to increase stability and/or enhance processing activity
of said molecule. Substitutions can be made within the strand or
can be made to residues at the ends of the strand.
[0171] The target mRNA cleavage reaction guided by siRNAs is
sequence specific. In general, siRNA containing a nucleotide
sequence identical to a portion of the target gene are preferred
for inhibition. However, 100% sequence identity between the siRNA
and the target gene is not required to practice the present
invention. Sequence variations can be tolerated including those
that might be expected due to genetic mutation, strain
polymorphism, or evolutionary divergence. For example, siRNA
sequences with insertions, deletions, and single point mutations
relative to the target sequence have also been found to be
effective for inhibition. Alternatively, siRNA sequences with
nucleotide analog substitutions or insertions can be effective for
inhibition.
[0172] Moreover, not all positions of an siRNA contribute equally
to target recognition. Mismatches in the center of the siRNA are
most critical and essentially abolish target RNA cleavage. In
contrast, the 3' nucleotides of the siRNA do not contribute
significantly to specificity of the target recognition. Generally,
residues at the 3' end of the siRNA sequence which is complementary
to the target RNA (e.g., the guide sequence) are not critical for
target RNA cleavage.
[0173] Sequence identity may readily be determined by sequence
comparison and alignment algorithms known in the art. To determine
the percent identity of two nucleic acid sequences (or of two amino
acid sequences), the sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in the first sequence or
second sequence for optimal alignment). The nucleotides (or amino
acid residues) at corresponding nucleotide (or amino acid)
positions are then compared. When a position in the first sequence
is occupied by the same residue as the corresponding position in
the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % homology=# of identical positions/total # of
positions.times.100), optionally penalizing the score for the
number of gaps introduced and/or length of gaps introduced.
[0174] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In one embodiment, the alignment generated
over a certain portion of the sequence aligned having sufficient
identity but not over portions having low degree of identity (i.e.,
a local alignment). A preferred, non-limiting example of a local
alignment algorithm utilized for the comparison of sequences is the
algorithm of Karlin and Altschul (1990) Proc. NatL Acad. Sci. USA
87:2264-68, modified as in Karlin and Altschul (1993) Proc. NatL
Acad. Sci. USA 90:5873. Such an algorithm is incorporated into the
BLAST programs (version 2.0) of Altschul, et al. (1990) J Mol.
Biol. 215:403-10.
[0175] In another embodiment, the alignment is optimized by
introducing appropriate gaps and percent identity is determined
over the length of the aligned sequences (i.e., a gapped
alignment). To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al.,
(1997) Nucleic Acids Res. 25(17):3389. In another embodiment, the
alignment is optimized by introducing appropriate gaps and percent
identity is determined over the entire length of the sequences
aligned (i.e., a global alignment). A preferred, non-limiting
example of a mathematical algorithm utilized for the global
comparison of sequences is the algorithm of Myers and Miller,
CABIOS (1989). Such an algorithm is incorporated into the ALIGN
program (version 2.0) which is part of the GCG sequence alignment
software package. When utilizing the ALIGN program for comparing
amino acid sequences, a PAM 120 weight residue table, a gap length
penalty of 12, and a gap penalty of 4 can be used.
[0176] Greater than 90% sequence identity, e.g., 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity,
between the siRNA and the portion of the target mRNA is preferred.
Alternatively, the siRNA may be defined functionally as a
nucleotide sequence (or oligonucleotide sequence) that is capable
of hybridizing with a portion of the target mRNA transcript (e.g.,
400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or
70.degree. C. hybridization for 12-16 hours; followed by washing).
Additional hybridization conditions include hybridization at
70.degree. C. in 1.times.SSC or 50.degree. C. in 1.times.SSC, 50%
formamide followed by washing at 70.degree. C. in 0.3.times.SSC or
hybridization at 70.degree. C. in 4.times.SSC or 50.degree. C. in
4.times.SSC, 50% formamide followed by washing at 67.degree. C. in
1.times.SSC. The hybridization temperature for hybrids anticipated
to be less than 50 base pairs in length should be 5-10.degree. C.
less than the melting temperature (Tm) of the hybrid, where Tm is
determined according to the following equations. For hybrids less
than 18 base pairs in length, Tm(.degree. C.)=2(# of A+T bases)+4(#
of G+C bases). For hybrids between 18 and 49 base pairs in length,
Tm(.degree. C.)=81.5+16.6 (log.sub.10[Na+])+0.41 (% G+C)-(600/N),
where N is the number of bases in the hybrid, and [Na+] is the
concentration of sodium ions in the hybridization buffer ([Na+] for
1.times.SSC=0.165 M). Additional examples of stringency conditions
for polynucleotide hybridization are provided in Sambrook, J., E.
F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., chapters 9 and 11, and Current Protocols in Molecular
Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons,
Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.
The length of the identical nucleotide sequences may be at least
about or about equal to 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35,
37, 40, 42, 45, 47 or 50 bases.
[0177] In one embodiment, the siRNA molecules of the present
invention are modified to improve stability in serum or in growth
medium for cell cultures. In order to enhance the stability, the
3'-residues may be stabilized against degradation, e.g., they may
be selected such that they consist of purine nucleotides,
particularly adenosine or guanosine nucleotides. Alternatively,
substitution of pyrimidine nucleotides by modified analogues, e.g.,
substitution of uridine by 2'-deoxythymidine is tolerated and does
not affect the efficiency of RNA interference. For example, the
absence of a 2'hydroxyl may significantly enhance the nuclease
resistance of the siRNAs in tissue culture medium.
[0178] In another embodiment of the present invention the siRNA
molecule may contain at least one modified nucleotide analogue. The
nucleotide analogues may be located at positions where the
target-specific activity, e.g., the RNAi mediating activity is not
substantially effected, e.g., in a region at the 5'-end and/or the
3'-end of the RNA molecule. Particularly, the ends may be
stabilized by incorporating modified nucleotide analogues.
[0179] Nucleotide analogues include sugar- and/or backbone-modified
ribonucleotides (i.e., include modifications to the phosphate-sugar
backbone). For example, the phosphodiester linkages of natural RNA
may be modified to include at least one of a nitrogen or sulfur
heteroatom. In preferred backbone-modified ribonucleotides the
phosphoester group connecting to adjacent ribonucleotides is
replaced by a modified group, e.g., of phosphothioate group. In
preferred sugar modified ribonucleotides, the 2'OH-group is
replaced by a group selected from H, OR, R, halo, SH, SR, NH.sub.2,
NHR, NR.sub.2 or NO.sub.2, wherein R is C.sub.1-C.sub.6 alkyl,
alkenyl or alkynyl and halo is F, Cl, Br or I.
[0180] Nucleotide analogues also include nucleobase-modified
ribonucleotides, i.e., ribonucleotides, containing at least one
non-naturally occurring nucleobase instead of a naturally occurring
nucleobase. Bases may be modified to block the activity of
adenosine deaminase. Exemplary modified nucleobases include, but
are not limited to, uridine and/or cytidine modified at the
5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine;
adenosine and/or guanosines modified at the 8 position, e.g.,
8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-
and N-alkylated nucleotides, e.g., N6-methyl adenosine are
suitable. It should be noted that the above modifications may be
combined.
[0181] RNA may be produced enzymatically or by partial/total
organic synthesis, any modified ribonucleotide can be introduced by
in vitro enzymatic or organic synthesis. In one embodiment, an
siRNA is prepared chemically. Methods of synthesizing RNA molecules
are known in the art, in particular, the chemical synthesis methods
as described in Verina and Eckstein (1998), Annul Rev. Biochem.
67:99. In another embodiment, an siRNA is prepared enzymatically.
For example, an siRNA can be prepared by enzymatic processing of a
long, double-stranded RNA having sufficient complementarity to the
desired target mRNA. Processing of long RNA can be accomplished in
vitro, for example, using appropriate cellular lysates and siRNAs
can be subsequently purified by gel electrophoresis or gel
filtration. siRNA can then be denatured according to art-recognized
methodologies. In an exemplary embodiment, siRNA can be purified
from a mixture by extraction with a solvent or resin,
precipitation, electrophoresis, chromatography, or a combination
thereof. Alternatively, the siRNA may be used with no or a minimum
of purification to avoid losses due to sample processing.
[0182] Alternatively, the siRNAs can also be prepared by enzymatic
transcription from synthetic DNA templates or from DNA plasmids
isolated from recombinant bacteria. Typically, phage RNA
polymerases are used such as T7, T3 or SP6 RNA polyimerase
(Milligan and Uhlenbeck (1989) Methods EnzynioL 180:51-62). The RNA
may be dried for storage or dissolved in an aqueous solution. The
solution may contain buffers or salts to inhibit annealing, and/or
promote stabilization of the double strands.
[0183] Commercially available design tools and kits, such as those
available from Ambion, Inc. (Austin, Tex.), and the Whitehead
Institute of Biomedical Research at MIT (Cambridge, Mass.) allow
for the design and production of siRNA. By way of example, a
desired mRNA sequence can be entered into a sequence program that
will generate sense and antisense target strand sequences. These
sequences can then be entered into a program that determines the
sense and antisense siRNA oligonucleotide templates. The programs
can also be used to add, e.g., hairpin inserts or Ti promoter
primer sequences. Kits also can then be employed to build siRNA
expression cassettes.
[0184] In various embodiments, siRNAs are synthesized in vivo, in
situ, and in vitro. Endogenous RNA polymerase of the cell may
mediate transcription in vivo or in situ, or cloned RNA polymerase
can be used for transcription in vivo or in vitro. For
transcription from a transgene in vivo or an expression construct,
a regulatory region (e.g., promoter, enhancer, silencer, splice
donor and acceptor, polyadenylation) may be used to transcribe the
siRNAs. Inhibition may be targeted by specific transcription in an
organ, tissue, or cell type; stimulation of an environmental
condition (e.g., infection, stress, temperature, chemical
inducers); and/or engineering transcription at a developmental
stage or age. A transgenic organism that expresses siRNAs from a
recombinant construct may be produced by introducing the construct
into a zygote, an embryonic stem cell, or another multipotent cell
derived from the appropriate organism.
[0185] In one embodiment, the target mRNA of the invention
specifies the amino acid sequence of at least one protein such as a
cellular protein (e.g., a nuclear, cytoplasmic, transmembrane, or
membrane-associated protein). In another embodiment, the target
mRNA of the invention specifies the amino acid sequence of an
extracellular protein (e.g., an extracellular matrix protein or
secreted protein). As used herein, the phrase "specifies the amino
acid sequence" of a protein means that the mRNA sequence is
translated into the amino acid sequence according to the rules of
the genetic code. The following classes of proteins are listed for
illustrative purposes: developmental proteins (e.g., adhesion
molecules, cyclin kinase inhibitors, Wnt family members, Pax family
members, Winged helix family members, Hox family members,
cytokines/lymphokines and their receptors, growth/differentiation
factors and their receptors, neurotransmitters and their
receptors); oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2,
BCL6, CBFA2. CBL, CSFIR, ERBA, ERBB, EBRB2, ERBB2, ERBB3, ETSI,
ETSI, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2,
MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM 1, PML, RET, SRC, TALI, TCL3,
and YES); tumor suppressor proteins (e.g., APC, BRCA1, BRCA2,
MADH4, MCC, NF 1, NF2, RB 1, TP53, and WTI); and enzymes (e.g., ACC
synthases and oxidases, ACP desaturases and hydroxylases,
ADPglucose pyrophorylases, acetylases and deacetylases, ATPases,
alcohol dehydrogenases, amylases, amyloglucosidases, catalases,
cellulases, chalcone synthases, chitinases, cyclooxygenases,
decarboxylases, dextrinases, DNA and RNA polymerases,
galactosidases, glucanases, glucose oxidases, granule-bound starch
synthases, GTPases, helicases, hemicellulases, integrases,
inulinases, invertases, isomerases, kinases, lactases, lipases,
lipoxygenases, lysozymes, nopaline synthases, octopine synthases,
pectinesterases, peroxidases, phosphatases, phospholipases,
phosphorylases, phytases, plant growth regulator synthases,
polygalacturonases, proteinases and peptidases, pullanases,
recombinases, reverse transcriptases, RUBISCOs, topoisomerases, and
xylanases), proteins involved in tumor growth (including
vascularization) or in metastatic activity or potential, including
cell surface receptors and ligands as well as secreted proteins,
cell cycle regulatory, gene regulatory, and apoptosis regulatory
proteins, immune response, inflammation, complement, or clotting
regulatory proteins.
[0186] As used herein, the term "oncogene" refers to a gene which
stimulates cell growth and, when its level of expression in the
cell is reduced, the rate of cell growth is reduced or the cell
becomes quiescent. In the context of the present invention,
oncogenes include intracellular proteins, as well as extracellular
growth factors which may stimulate cell proliferation through
autocrine or paracrine function. Examples of human oncogenes
against which siRNA and morpholino constructs can designed include
c-myc, c-myb, mdm2, PKA-I (protein kinase A type I), Abl-1, Bcl2,
Ras, c-Raf kinase, CDC25 phosphatases, cyclins, cyclin dependent
kinases (cdks), telomerase, PDGF/sis, erb-B, fos, jun, mos, and
src, to name but a few. In the context of the present invention,
oncogenes also include a fusion gene resulted from chromosomal
translocation, for example, the Bcr/Abl fusion oncogene.
[0187] Further proteins include cyclin dependent kinases, c-myb,
c-myc, proliferating cell nuclear antigen (PCNA), transforming
growth factor-beta (TGF-beta), and transcription factors nuclear
factor kappaB (NF-.kappa.B), E2F, HER-2/neu, PKA, TGF-alpha, EGFR,
TGF-beta, IGFIR, P12, MDM2, BRCA, Bcl-2, VEGF, MDR, ferritin,
transferrin receptor, IRE, C-fos, HSP27, C-raf and metallothionein
genes.
[0188] The siRNA employed in the present invention can be directed
against the synthesis of one or more proteins. Additionally or
alternatively, there can be more than one siRNA directed against a
protein, e.g., duplicate siRNA or siRNA that correspond to
overlapping or non-overlapping target sequences against the same
target protein. Accordingly, in one embodiment two, three, four or
any plurality of siRNAs against the same target mRNA can be
included in the nanoparticles of the invention. Additionally,
several siRNAs directed against several proteins can be employed.
Alternatively, the siRNA can be directed against structural or
regulatory RNA molecules that do not code for proteins.
[0189] In a preferred aspect of the invention, the target mRNA
molecule of the invention specifies the amino acid sequence of a
protein associated with a pathological condition. For example, the
protein may be a pathogen-associated protein (e.g., a viral protein
involved in immunosuppression or immunoavoidance of the host,
replication of the pathogen, transmission of the pathogen, or
maintenance of the infection), or a host protein which facilitates
entry of the pathogen into the host, drug metabolism by the
pathogen or host, replication or integration of the pathogen's
genome, establishment or spread of infection in the host, or
assembly of the next generation of pathogen. Alternatively, the
protein may be a tumor-associated protein or an autoimmune
disease-associated protein.
[0190] In one embodiment, the target mRNA molecule of the invention
specifies the amino acid sequence of an endogenous protein (i.e. a
protein present in the genome of a cell or organism). In another
embodiment, the target mRNA molecule of the invention specifies the
amino acid sequence of a heterologous protein expressed in a
recombinant cell or a genetically altered organism. In another
embodiment, the target mRNA molecule of the invention specifies the
amino acid sequence of a protein encoded by a transgene (i.e., a
gene construct inserted at an ectopic site in the genome of the
cell). In yet another embodiment, the target mRNA molecule of the
invention specifies the amino acid sequence of a protein encoded by
a pathogen genome which is capable of infecting a cell or an
organism from which the cell is derived.
[0191] By inhibiting the expression of such proteins, valuable
information regarding the function of said proteins and therapeutic
benefits which may be obtained from said inhibition may be
obtained.
[0192] In one embodiment, the nanoparticles of this invention
comprises one or more siRNA molecules to silence a PDGF beta gene,
Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, INK
gene, RAF gene, Erkl/2 gene, PCNA(p21) gene, MYB gene, JIJN gene,
FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A
gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC
gene, Skp2 gene, kinesin spindle protein gene, Bcr-Abl gene, Stat3
gene, cSrc gene, PKC gene, Bax gene, Bcl-2 gene, EGFR gene, VEGF
gene, myc gene, NF.kappa.B gene, STAT3 gene, survivin gene,
Her2/Neu gene, topoisomerase I gene, PLK1 gene, protein kinase 3
gene, CD31 gene, IGF-1 gene, topoisomerase II alpha gene, mutations
in the p73 gene, mutations in the p21 (WAF 1/CIP 1) gene, mutations
in the p27(KIP1) gene, mutations in the PPM1D gene, mutations in
the RAS gene, mutations in the caveolin I gene, mutations in the
MIB I gene, mutations in the MTAI gene, mutations in the M68 gene,
mutations in tumor suppressor genes, mutations in the p53 tumor
suppressor gene, mutations in the p53 family member DN-p63,
mutations in the pRb tumor suppressor gene, mutations in the APC1
tumor suppressor gene, mutations in the BRCA1 tumor suppressor
gene, mutations in the PTEN tumor suppressor gene, mLL fusiongene,
BCRIABL fusion gene, TEL/AML1 fusion gene, EWS/FLI1 fusion gene,
TLS/FUS1 fusion gene, PAX3/FKHR fusion gene, AML1/ETO fusion gene,
alpha v-integrin gene, Fit-i receptor gene, tubulin gene, Human
Papilloma Virus gene, a gene required for Human Papilloma Virus
replication, Human Immunodeficiency Virus gene, a gene required for
Human Immunodeficiency Virus replication, Hepatitis A Virus gene, a
gene required for Hepatitis A Virus replication, Hepatitis B Virus
gene, a gene required for Hepatitis B Virus replication, Hepatitis
C Virus gene, a gene required for Hepatitis C Virus replication,
Hepatitis D Virus gene, a gene required for Hepatitis D Virus
replication, Hepatitis E Virus gene, a gene required for Hepatitis
B Virus replication, Hepatitis F Virus gene, a gene required for
Hepatitis F Virus replication, Hepatitis G Virus gene, a gene
required for Hepatitis G Virus replication, Hepatitis H Virus gene,
a gene required for Hepatitis H Virus replication, Respiratory
Syncytial Virus gene, a gene that is required for Respiratory
Syncytial Virus replication, Herpes Simplex Virus gene, a gene that
is required for Herpes Simplex Virus replication, herpes
Cytomegalovirus gene, a gene that is required for herpes
Cytomegalovirus replication, herpes Epstein Barr Virus gene, a gene
that is required for herpes Epstein Barr Virus replication,
Kaposi's Sarcoma-associated Herpes Virus gene, a gene that is
required for Kaposi's Sarcoma-associated Herpes Virus replication,
JC Virus gene, human gene that is required for JC Virus
replication, myxovirus gene, a gene that is required for myxovirus
gene replication, rhinovirus gene, a gene that is required for
rhinovirus replication, coronavirus gene, a gene that is required
for coronavirus replication, West Nile Virus gene, a gene that is
required for West Nile Virus replication, St. Louis Encephalitis
gene, a gene that is required for St. Louis Encephalitis
replication, Tick-borne encephalitis virus gene, a gene that is
required for Tick-borne encephalitis virus replication, Murray
Valley encephalitis virus gene, a gene that is required for Murray
Valley encephalitis virus replication, dengue virus gene, a gene
that is required for dengue virus gene replication, Simian Virus 40
gene, a gene that is required for Simian Virus 40 replication,
Human T Cell Lymphotropic Virus gene, a gene that is required for
Human T Cell Lymphotropic Virus replication, Moloney-Murine
Leukemia Virus gene, a gene that is required for Moloney-Murine
Leukemia Virus replication, encephalomyocarditis virus gene, a gene
that is required for encephalomyocarditis virus replication,
measles virus gene, a gene that is required for measles virus
replication, Vericella zoster virus gene, a gene that is required
for Vericella zoster virus replication, adenovirus gene, a gene
that is required for adenovirus replication, yellow fever virus
gene, a gene that is required for yellow fever virus replication,
poliovirus gene, a gene that is required for poliovirus
replication, poxvirus gene, a gene that is required for poxvirus
replication, plasmodium gene, a gene that is required for
plasmodium gene replication, Mycobacterium ulcerans gene, a gene
that is required for Mycobacterium ulcerans replication,
Mycobacterium tuberculosis gene, a gene that is required for
Mycobacterium tuberculosis replication, Mycobacterium leprae
gene,-185-a gene that is required for Mycobacterium leprae
replication, Staphylococcus aureus gene, a gene that is required
for Staphylococcus aureus replication, Streptococcus pneumoniae
gene, a gene that is required for Streptococcus pneumoniae
replication, Streptococcus pyogenes gene, a gene that is required
for Streptococcus pyogenes replication, Chlamydia pneumoniae gene,
a gene that is required for Chlamydia pneumoniae replication,
Mycoplasma pneumoniae gene, a gene that is required for Mycoplasma
pneumoniae replication, an integrin gene, a selectin gene,
complement system gene, chemokine gene, chemokine receptor gene,
GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIG gene,
Pro-Platelet Basic Protein gene, MIP-11 gene, MIP-1J gene, RANTES
gene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene,
CMBKR3 gene, CMBKR5v, AIF-1 gene, 1-309 gene, a gene to a component
of an ion channel, a gene to a neurotransmitter receptor, a gene to
a neurotransmitter ligand, amyloid-family gene, presenilin gene, HD
gene, DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene,
SCA7 gene, SCA8 gene, allele gene found in LOH cells, or one allele
gene of a polymorphic gene. Examples of relevant siRNA molecules to
silence genes and methods of making siRNA molecules can be found
from commercial sources such as Dharmacon or from the following
patent applications: US2005017667, WO2006066158, WO2006078278, U.S.
Pat. No. 7,056,704, U.S. Pat. No. 7,078,196, U.S. Pat. No.
5,898,031, U.S. Pat. No. 6,107,094, EP 1144623, and EU 1144623, all
of which are incorporated by reference in their entireties. While a
number of specific gene silencing targets are listed, this list is
merely illustrative and other siRNA molecules could also be used
with the nanoparticles of this invention.
[0193] In one embodiment, the nanoparticles of this invention
comprise an siRNA molecule having RNAi activity against an RNA,
wherein the siRNA molecule comprises a sequence complementary to
any RNA having coding or non-encoding sequence, such as those
sequences referred to by GenBank Accession Nos. described in Table
V of PCT/US03/05028 (International PCT Publication No. WO 03/4654)
or otherwise known in the art.
[0194] In one embodiment, the nanoparticles of this invention
comprise an siRNA molecule which silences the vascular endothelial
growth factor gene. In another embodiment, the nanoparticles of
this invention comprise an siRNA molecule which silences the
vascular endothelial growth factor receptor gene.
[0195] In another embodiment, the nanoparticles of this invention
comprise an siRNA molecule, wherein the sequence of the siRNA
molecule is complementary to tumor-related targets, including, but
not limited to, hypoxia-inducible factor-1 (HIF-1), which is found
in human metastatic prostate PC3-M cancer cells (Mol. Carcinog.
2008 Jan. 31 [Epub ahead of print]); the HIF-1 downstream target
gene (Mol. Carcinog. 2008 Jan. 31 [Epub ahead of print]),
mitogen-activated protein kinases (MAPKs), hepatocyte growth factor
(HGF), interleukin 12p70 (IL12), glucocorticoid-induced tumor
necrosis factor receptor (GITR), intercellular adhesion molecule 1
(ICAM-1), neurotrophin-3 (NT-3), interleukin 17 (IL17), interleukin
18 binding protein a (IL18 Bpa) and epithelial-neutrophil
activating peptide (ENA78) (see, e.g., "Cytokine profiling of
prostatic fluid from cancerous prostate glands identifies cytokines
associated with extent of tumor and inflammation", The Prostate
Early view Published Online: 24 Mar. 2008); PSMA (see, e.g.,
"Cell-Surface labeling and internalization by a fluorescent
inhibitor of prostate-specific membrane antigen" The Prostate Early
view Published Online: 24 Mar. 2008); Androgen receptor (AR),
keratin, epithelial membrane antigen, EGF receptor, and E cadherin
(see, e.g., "Characterization of PacMetUT1, a recently isolated
human prostate cancer cell line"); peroxisomes
proliferators-activated receptor .gamma. (PPAR.gamma.; see e.g.,
The Prostate Volume 68, Issue 6, Date: 1 May 2008, Pages: 588-598);
the receptor for advanced glycation end products (RAGE) and the
advanced glycation end products (AGE), (see, e.g., "V domain of
RAGE interacts with AGEs on prostate carcinoma cells" The Prostate
Early view Published Online: 26 Feb. 2008); the receptor tyrosine
kinase erb-B2 (Her2/neu), hepatocyte growth factor receptor (Met),
transforming growth factor-beta 1 receptor (TGF.beta.R1), nuclear
factor kappa B (NF.kappa.B), Jagged-1, Sonic hedgehog (Shh), Matrix
metalloproteinases (MMPs, esp. MMP-7), Endothelin receptor type A
(ET.sub.A), Endothelin-1 (ET-1), Nuclear receptor subfamily 3,
group C, member 1 (NR3C1), Nuclear receptor co-activator 1 (NCOA1),
NCOA2, NCOA3, E1A binding protein p300 (EP300), CREB binding
protein (CREBBP), Cyclin G associated kinase (GAK), Gelsolin(GSN),
Aldo-keto reductase family 1, member C1 (AKR1C1), AKR1C2, AKR1C3,
Neurotensin(NTS), Enolase 2(ENO2), Chromogranin B (CHGB,
secretogranin 1), Secretagogin (SCGN, or EF-hand calcium binding
protein), Dopa decarboxylase(DDC, or aromatic L-amino acid
decarboxylase), steroid receptor co-activator-1 (SRC-1), SRC-2
(a.k.a. TIF2), SRC-3 (a.k.a. AIB-1) (see, e.g., "Longitudinal
analysis of androgen deprivation of prostate cancer cells
identifies pathways to androgen independence" The Prostate Early
view Published Online: 26 Feb. 2008); estrogen receptors
(ER.alpha., ER.beta. or GPR30) (see, e.g., The Prostate Volume 68,
Issue 5, Pages 508-516); the melanoma cell adhesion molecule (MCAM)
(see, e.g., The Prostate Volume 68, Issue 4, Pages 418-426;
angiogenic factors (such as vascular endothelial growth factor
(VEGF) and erythropoietin), glucose transporters (such as GLUT1),
BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) (see,
e.g., The Prostate Volume 68, Issue 3, Pages 336-343); types 1 and
2 5.alpha.-reductase (see, e.g., The Journal of Urology Volume 179,
Issue 4, Pages 1235-1242); ERG and ETV1, prostate-specific antigen
(PSA), prostate-specific membrane antigen (PSMA), prostate stem
cell antigen (PSCA), .alpha.-Methylacyl coenzyme A racemase
(AMACR), PCA3.sup.DD3 glutathione-S-transferase, pi 1 (GSTP1), p16,
ADP-ribosylation factor (ARF), O-6-methylguanine-DNA
methyltransferase (MGMT), human telomerase reverse transcriptase
(hTERT), early prostate cancer antigen (EPCA), human kallikrein 2
(HK2) and hepsin (see, e.g., The Journal of Urology Volume 178,
Issue 6, Pages 2252-2259); bromodomain containing 2 (BRD2),
eukaryotic translation initiation factor 4 gamma, 1 (eIF4G1),
ribosomal protein L13a (RPL13a), and ribosomal protein L22 (RPL22)
(see, e.g., N Engl J Med 353 (2005), p. 1224); HER2/neu, Derlin-1,
ERBB2, AKT, cyclooxygenase-2 (COX-2), PSMD3, CRKRS, PERLD1, and
C17ORF37, PPP4C, PARN, ATP6V0C, C16orf14, GBL, HAGH, ITFG3,
MGC13114, MRPS34, NDUFB10, NMRAL1, NTHL1, NUBP2, POLR3K, RNPS1,
STUB1, TBL3, and USP7. All of the references described herein are
incorporated herein by reference in their entireties.
[0196] Thus, in one embodiment, the invention comprises a
nanoparticle comprising a targeting moiety (e.g., CREKA, an
aptamer, or affibody), a biodegradable polymer, a stealth polymer,
and an siRNA molecule. In one embodiment, the invention comprises a
nanoparticle comprising a targeting moiety (e.g., CREKA, an
aptamer, or affibody), a biodegradable polymer, a stealth
component, and an siRNA molecule that silences the vascular
endothelial growth factor gene. In one embodiment, the invention
comprises a nanoparticle comprising a targeting moiety (e.g.,
CREKA, an aptamer, or affibody), a biodegradable polymer, a stealth
component, and an siRNA molecule that silences the vascular
endothelial growth factor receptor gene. In another embodiment, the
invention comprises a nanoparticle comprising a targeting moiety
(e.g., CREKA, an aptamer, or affibody), PLGA, polyethylene glycol,
and an siRNA molecule. In one embodiment, the invention comprises a
nanoparticle comprising a targeting moiety (e.g., CREKA, an
aptamer, or affibody), a biodegradable polymer, a stealth
component, and an siRNA molecule wherein the nanoparticle can
selectively accumulate in the prostate or in the vascular
endothelial tissue surrounding a cancer. In one embodiment, the
invention comprises a nanoparticle comprising a targeting moiety
(e.g., CREKA, an aptamer, or affibody), a biodegradable polymer, a
stealth component, and an siRNA molecule wherein the nanoparticle
can selectively accumulate in the prostate or in the vascular
endothelial tissue surrounding a cancer and wherein the
nanoparticle can be endocytosed by a PSMA expressing cell.
[0197] In another embodiment, the siRNA that is incorporated into
the nanoparticle of the invention are those that treat prostate
cancer, such as those disclosed in U.S. application Ser. No.
11/021,159 (siRNA sequence is complementary to SEQ ID No.8:
gaaggccagu uguauggac), and U.S. application Ser. No. 11/349,473
(discloses siRNAs that bind to a region from nucleotide 3023 to
3727 of SEQ ID No. 1). Both of these references are incorporated
herein by reference in their entirety.
[0198] In another embodiment, the therapeutic agents of the
nanoparticles of the invention include RNAs that can be used to
treat cancer, such as anti-sense mRNAs and microRNAs. Examples of
microRNAs that can be used as therapeutic agents for the treatment
of cancer include those disclosed in Nature 435 (7043): 828-833;
Nature 435 (7043): 839-843; and Nature 435 (7043): 834-838, all of
which are incorporated herein by reference in their entireties.
[0199] In one embodiment, the invention specifically excludes
nanoparticles containing iron oxide. In one embodiment, blood
clotting does not occur at the location where the nanoparticle
accumulates.
[0200] In one embodiment, the therapeutic agents used in
conjunction with the nanoparticles of the invention include one or
more agents useful for the treatment of restenosis. Examples of
such agents include, but are not limited to, everolimus,
paclitaxel, zotarolimus, pioglitazone, BO-653, rosiglitazone,
sirolimus, dexamethasone, rapamycin, tacrolimus, biophosphonates,
estrogen, angiopeptin, statin, PDGF inhibitors, ROCK inhibitors,
MMP inhibitors, and 2-CdA. In a certain embodiment, the therapeutic
agents useful for the treatment of restenosis are zotarolimus and
dexamethasone, including combinations of zotarolimus and
dexamethasone.
[0201] In a preferred embodiment, the nanoparticles of the
invention can be delivered to or near a vulnerable plaque using a
medical device such as a needle catheter, drug eluding stent or
stent graft. Such devices are well known in the art, and are
described, for example, in U.S. Pat. No. 7,008,411, which is
incorporated herein by reference in its entirety. In one
embodiment, a drug eluting stent and/or needle catheter may be
implanted at the region of vessel occlusion that may be upstream
from a vulnerable plaque region. A medical device, such as a drug
eluting stent, needle catheter, or stent graft may be used to treat
the occlusive atherosclerosis (i.e., non-vulnerable plaque) while
releasing the nanoparticle of the invention to treat a vulnerable
plaque region distal or downstream to the occlusive plaque. The
nanoparticle may be released slowly over time.
[0202] The nanoparticles of the invention can also be delivered to
a subject in need thereof using the Genie.TM. balloon catheter
available from Acrostak (http://www.acrostak.com/genie_en.htm). The
nanoparticles of the invention can also be delivered to a subject
in need thereof using delivery devices that have been developed for
endovascular local gene transfer such as passive diffusion devices
(e.g., double-occlusion balloon, spiral balloon), pressure-driven
diffusion devices (e.g., microporous balloon, balloon-in-balloon
devices, double-layer channeled perfusion balloon devices,
infusion-sleeve catheters, hydrogel-coated balloons), and
mechanically or electrically enhanced devices (e.g., needle
injection catheter, iontophoretic electric current-enhanced
balloons, stent-based system), or any other delivery system
disclosed in Radiology 2003; 228:36-49, or Int J Nanomedicine 2007;
2(2):143-61, which are incorporated herein by reference in their
entirety.
[0203] Once the inventive conjugates have been prepared, they may
be combined with pharmaceutical acceptable carriers to form a
pharmaceutical composition, according to another aspect of the
invention. As would be appreciated by one of skill in this art, the
carriers may be chosen based on the route of administration as
described below, the location of the target issue, the drug being
delivered, the time course of delivery of the drug, etc.
Methods of Treatment
[0204] In some embodiments, targeted particles in accordance with
the present invention may be used to treat, alleviate, ameliorate,
relieve, delay onset of, inhibit progression of, reduce severity
of, and/or reduce incidence of one or more symptoms or features of
a disease, disorder, and/or condition. In some embodiments,
inventive targeted particles may be used to treat cancer, e.g.,
breast cancer, and/or cancer cells, e.g., breast cancer cells.
[0205] The term "cancer" includes pre-malignant as well as
malignant cancers. Cancers include, but are not limited to,
prostate, gastric cancer, colorectal cancer, skin cancer, e.g.,
melanomas or basal cell carcinomas, lung cancer, cancers of the
head and neck, bronchus cancer, pancreatic cancer, urinary bladder
cancer, brain or central nervous system cancer, peripheral nervous
system cancer, esophageal cancer, cancer of the oral cavity or
pharynx, liver cancer, kidney cancer, testicular cancer, biliary
tract cancer, small bowel or appendix cancer, salivary gland
cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma,
chondrosarcoma, cancer of hematological tissues, and the like.
"Cancer cells" can be in the form of a tumor, exist alone within a
subject (e.g., leukemia cells), or be cell lines derived from a
cancer.
[0206] Cancer can be associated with a variety of physical
symptoms. Symptoms of cancer generally depend on the type and
location of the tumor. For example, lung cancer can cause coughing,
shortness of breath, and chest pain, while colon cancer often
causes diarrhea, constipation, and blood in the stool. However, to
give but a few examples, the following symptoms are often generally
associated with many cancers: fever, chills, night sweats, cough,
dyspnea, weight loss, loss of appetite, anorexia, nausea, vomiting,
diarrhea, anemia, jaundice, hepatomegaly, hemoptysis, fatigue,
malaise, cognitive dysfunction, depression, hormonal disturbances,
neutropenia, pain, non-healing sores, enlarged lymph nodes,
peripheral neuropathy, and sexual dysfunction.
[0207] In one aspect of the invention, a method for the treatment
of cancer (e.g. breast cancer) is provided. In some embodiments,
the treatment of cancer comprises administering a therapeutically
effective amount of inventive targeted particles to a subject in
need thereof, in such amounts and for such time as is necessary to
achieve the desired result. In certain embodiments of the present
invention a "therapeutically effective amount" of an inventive
targeted particle is that amount effective for treating,
alleviating, ameliorating, relieving, delaying onset of, inhibiting
progression of, reducing severity of, and/or reducing incidence of
one or more symptoms or features of cancer.
[0208] In one aspect of the invention, a method for administering
inventive compositions to a subject suffering from cancer (e.g.
breast cancer) is provided. In some embodiments, particles to a
subject in such amounts and for such time as is necessary to
achieve the desired result (i.e., treatment of cancer). In certain
embodiments of the present invention a "therapeutically effective
amount" of an inventive targeted particle is that amount effective
for treating, alleviating, ameliorating, relieving, delaying onset
of, inhibiting progression of, reducing severity of, and/or
reducing incidence of one or more symptoms or features of
cancer.
[0209] In other embodiments, the nanoparticles of the present
invention can be used to inhibit the growth of cancer cells, e.g.,
breast cancer cells. As used herein, the term "inhibits growth of
cancer cells" or "inhibiting growth of cancer cells" refers to any
slowing of the rate of cancer cell proliferation and/or migration,
arrest of cancer cell proliferation and/or migration, or killing of
cancer cells, such that the rate of cancer cell growth is reduced
in comparison with the observed or predicted rate of growth of an
untreated control cancer cell. The term "inhibits growth" can also
refer to a reduction in size or disappearance of a cancer cell or
tumor, as well as to a reduction in its metastatic potential.
Preferably, such an inhibition at the cellular level may reduce the
size, deter the growth, reduce the aggressiveness, or prevent or
inhibit metastasis of a cancer in a patient. Those skilled in the
art can readily determine, by any of a variety of suitable indicia,
whether cancer cell growth is inhibited.
[0210] Inhibition of cancer cell growth may be evidenced, for
example, by arrest of cancer cells in a particular phase of the
cell cycle, e.g., arrest at the G2/M phase of the cell cycle.
Inhibition of cancer cell growth can also be evidenced by direct or
indirect measurement of cancer cell or tumor size. In human cancer
patients, such measurements generally are made using well known
imaging methods such as magnetic resonance imaging, computerized
axial tomography and X-rays. Cancer cell growth can also be
determined indirectly, such as by determining the levels of
circulating carcinoembryonic antigen, prostate specific antigen or
other cancer-specific antigens that are correlated with cancer cell
growth. Inhibition of cancer growth is also generally correlated
with prolonged survival and/or increased health and well-being of
the subject.
[0211] The present invention is directed, in part, to the discovery
that a collagen IV alpha-2 chain related polypeptide can act as a
receptor for the CREKA tumor targeting peptide. Collagens are a
major component of the extracellular matrix (ECM), an
interconnected molecular network providing mechanical support for
cells and tissues and regulating biochemical and cellular processes
such as adhesion, migration, gene expression and differentiation
(see, e.g., U.S. Patent Application No. 2005/0048063, which is
incorporated herein by reference in its entirety). In higher
animals, at least 19 distinct collagen types differing in their
higher order structures and functions have been identified based on
the presence of the characteristic collagen triple-helix structure.
The collagens are sometimes categorized into the fibrillar and
nonfibrillar collagens. The fibrillar (interstitial) collagens
include types I, II, III, V and XI, while the nonfibrillar
collagens include types IV, VI, IX, X, XI, XII, XIV and XIII.
[0212] As a non-limiting example, a method of the invention for
treating cancer can be useful for treating breast cancer. Targeting
poly(amio acids) useful in the invention include those which
selectively target tumor vasculature and selectively bind
non-helical collagen. Targeting poly(amio acids) useful in the
invention also include those which selectively target to tumor
vasculature and selectively bind collagen IV, and those which
selectively target tumor vasculature and selectively bind denatured
collagen IV in preference to native collagen IV.
[0213] Inventive therapeutic protocols involve administering a
therapeutically effective amount of an inventive targeted particle
to a healthy individual (i.e., a subject who does not display any
symptoms of cancer and/or who has not been diagnosed with cancer).
For example, healthy individuals may be "immunized" with an
inventive targeted particle prior to development of cancer and/or
onset of symptoms of cancer; at risk individuals (e.g., patients
who have a family history of cancer; patients carrying one or more
genetic mutations associated with development of cancer; patients
having a genetic polymorphism associated with development of
cancer; patients infected by a virus associated with development of
cancer; patients with habits and/or lifestyles associated with
development of cancer; etc.) can be treated substantially
contemporaneously with (e.g., within 48 hours, within 24 hours, or
within 12 hours of) the onset of symptoms of cancer. Of course
individuals known to have cancer may receive inventive treatment at
any time.
[0214] In another aspect, the invention provides a method of
treating cardiovascular conditions in a subject in need thereof,
comprising administering to the subject an effective amount of the
controlled-release system of the invention. Such cardiovascular
conditions include, but are not limited to, restenosis and
vulnerable plaque. In one embodiment, the nanoparticles of this
invention are delivered locally to the coronary arteries, central
arteries, peripheral arteries, veins, and bile ducts. In another
embodiment, the nanoparticles of this invention are delivered
locally to the coronary arteries, central arteries, peripheral
arteries, veins, and bile ducts after the implantation of a stent
in such tissue in a patient for the treatment of restenosis. In
another embodiment, the nanoparticles of this invention are
administered to a patient undergoing a coronary angioplasty, a
peripheral angioplasty, a renal artery angioplasty, or a carotid
angioplasty in order to prevent resenosis.
[0215] In one embodiment, the nanoparticles of this invention pass
through the endothelial layer of a blood vessel due to plaque
damage of the endothelial tissue and bind to the basement
membrane.
[0216] In another aspect, the invention provides a method of
treating restenosis in a subject in need thereof, comprising
administering to the subject an effective amount of the
controlled-release system of the invention. In one embodiment, the
controlled-release system is locally administered to a designated
region of the blood vessel where the restenosis occurs. In still
another embodiment, the controlled-release system is administered
via a medical device. In yet another embodiment, the medical device
is a drug eluding stent, needle catheter, or stent graft. In
another embodiment, the invention provides a method of treating
restenosis in a subject in need thereof, comprising administering
to the subject an effective amount of the controlled-release system
of the invention wherein the controlled release system contains a
drug selected from the group consisting of everolimus, paclitaxel,
zotarolimus, pioglitazone, BO-653, rosiglitazone, sirolimus,
dexamethasone, rapamycin, tacrolimus, biophosphonates, estrogen,
angiopeptin, statin, PDGF inhibitors, ROCK inhibitors, MMP
inhibitors, and 2-CdA. In another embodiment, the invention
provides a method of treating restenosis in a subject in need
thereof, comprising administering to the subject an effective
amount of the controlled-release system of the invention wherein
the controlled release system contains two drugs selected from
everolimus, paclitaxel, zotarolimus, pioglitazone, BO-653,
rosiglitazone, sirolimus, dexamethasone, rapamycin, tacrolimus,
biophosphonates, estrogen, angiopeptin, statin, PDGF inhibitors,
ROCK inhibitors, MMP inhibitors, and 2-CdA. In another embodiment,
the invention provides a method of treating restenosis in a subject
in need thereof, comprising administering to the subject an
effective amount of the controlled-release system of the invention
wherein the controlled release system contains zotarolimus and
dexamethasone.
[0217] In one embodiment, the nanoparticles of this invention are
delivered locally to the coronary arteries, central arteries,
peripheral arteries, veins, and bile ducts. In another embodiment,
the nanoparticles of this invention are delivered locally to the
coronary arteries, central arteries, peripheral arteries, veins,
and bile ducts after the implantation of a stent in such tissue in
a patient for the treatment of restenosis. In another embodiment,
the nanoparticles of this invention are administered to a patient
undergoing a coronary angioplasty, a peripheral angioplasty, a
renal artery angioplasty, or a carotid angioplasty in order to
prevent resenosis. In another embodiment, the nanoparticles of this
invention are administered within 12 hours of a patient undergoing
a coronary angioplasty, a peripheral angioplasty, a renal artery
angioplasty, or a carotid angioplasty in order to prevent
resenosis. In another embodiment, the nanoparticles of this
invention are administered locally to a patient undergoing a
coronary angioplasty, a peripheral angioplasty, a renal artery
angioplasty, or a carotid angioplasty in order to prevent
resenosis.
Pharmaceutical Compositions
[0218] As used herein, the term "pharmaceutically acceptable
carrier" means a non-toxic, inert solid, semi-solid or liquid
filler, diluent, encapsulating material or formulation auxiliary of
any type. Remington's Pharmaceutical Sciences. Ed. by Gennaro, Mack
Publishing, Easton, Pa., 1995 discloses various carriers used in
formulating pharmaceutical compositions and known techniques for
the preparation thereof. Some examples of materials which can serve
as pharmaceutically acceptable carriers include, but are not
limited to, sugars such as lactose, glucose, and sucrose; starches
such as com starch and potato starch; cellulose and its derivatives
such as sodium carboxymethyl cellulose, ethyl cellulose, and
cellulose acetate; powdered tragacanth; malt; gelatin; talc;
excipients such as cocoa butter and suppository waxes; oils such as
peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil;
corn oil and soybean oil; glycols such as propylene glycol; esters
such as ethyl oleate and ethyl laurate; agar; detergents such as
TWEEN.TM. 80; buffering agents such as magnesium hydroxide and
aluminum hydroxide; alginic acid; pyrogen-free water; isotonic
saline; Ringer's solution; ethyl alcohol; and phosphate buffer
solutions, as well as other non-toxic compatible lubricants such as
sodium lauryl sulfate and magnesium stearate, as well as coloring
agents, releasing agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives and antioxidants can also be
present in the composition, according to the judgment of the
formulator. If filtration or other terminal sterilization methods
are not feasible, the formulations can be manufactured under
aseptic conditions.
[0219] The pharmaceutical compositions of this invention can be
administered to a patient by any means known in the art including
oral and parenteral routes. In certain embodiments parenteral
routes are desirable since they avoid contact with the digestive
enzymes that are found in the alimentary canal. According to such
embodiments, inventive compositions may be administered by
injection (e.g., intravenous, subcutaneous or intramuscular,
intraperitoneal injection), rectally, vaginally, topically (as by
powders, creams, ointments, or drops), or by inhalation (as by
sprays).
[0220] In one embodiment, the nanoparticles of the present
invention are administered to a subject in need thereof
systemically, e.g., by IV infusion or injection. In a particular
embodiment, the nanoparticles of the present invention are locally
administered to a subject in need thereof. As used herein, "local
administration" is when nanoparticles of the invention are brought
into contact with the blood vessel wall or vascular tissue through
a device (e.g., a stent).
[0221] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension, or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.,
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables. In one
embodiment, the inventive conjugate is suspended in a carrier fluid
comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v)
TWEEN.TM. 80. The injectable formulations can be sterilized, for
example, by filtration through a bacteria-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0222] Compositions for rectal or vaginal administration may be
suppositories which can be prepared by mixing the inventive
conjugate with suitable non-irritating excipients or carriers such
as cocoa butter, polyethylene glycol, or a suppository wax which
are solid at ambient temperature but liquid at body temperature and
therefore melt in the rectum or vaginal cavity and release the
inventive conjugate.
[0223] Dosage forms for topical or transdermal administration of an
inventive pharmaceutical composition include ointments, pastes,
creams, lotions, gels, powders, solutions, sprays, inhalants, or
patches. The inventive conjugate is admixed under sterile
conditions with a pharmaceutically acceptable carrier and any
needed preservatives or buffers as may be required. Ophthalmic
formulations, ear drops, and eye drops are also contemplated as
being within the scope of this invention. The ointments, pastes,
creams, and gels may contain, in addition to the inventive
conjugates of this invention, excipients such as animal and
vegetable fats, oils, waxes, paraffins, starch, tragacanth,
cellulose derivatives, polyethylene glycols, silicones, bentonites,
silicic acid, talc, and zinc oxide, or mixtures thereof.
Transdermal patches have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms
can be made by dissolving or dispensing the inventive conjugates in
a proper medium. Absorption enhancers can also be used to increase
the flux of the compound across the skin. The rate can be
controlled by either providing a rate controlling membrane or by
dispersing the inventive conjugates in a polymer matrix or gel.
[0224] Powders and sprays can contain, in addition to the inventive
conjugates of this invention, excipients such as lactose, talc,
silicic acid, aluminum hydroxide, calcium silicates, and polyamide
powder, or mixtures thereof. Sprays can additionally contain
customary propellants such as chlorofluorohydrocarbons.
[0225] When administered orally, the inventive nanoparticles can
be, but are not necessarily, encapsulated. A variety of suitable
encapsulation systems are known in the art ("Microcapsules and
Nanoparticles in Medicine and Pharmacy," Edited by Doubrow, M., CRC
Press, Boca Raton, 1992; Mathiowitz and Langer J. Control. Release
5:13, 1987; Mathiowitz et al. Reactive Polymers 6:275, 1987;
Mathiowitz et al. J. Appl. Polymer Sci. 35:755, 1988; Langer Ace.
Chem. Res. 33:94, 2000; Langer J. Control. Release 62:7, 1999;
Uhrich et al. Chem. Rev. 99:3181, 1999; Zhou et al. J. Control.
Release 75:27, 2001; and Hanes et al. Pharm. Biotechnol. 6:389,
1995). The inventive conjugates may be encapsulated within
biodegradable polymeric microspheres or liposomes. Examples of
natural and synthetic polymers useful in the preparation of
biodegradable microspheres include carbohydrates such as alginate,
cellulose, polyhydroxyalkanoates, polyamides, polyphosphazenes,
polypropylfumarates, polyethers, polyacetals, polycyanoacry lates,
biodegradable polyurethanes, polycarbonates, polyanhydrides,
polyhydroxyacids, poly(ortho esters), and other biodegradable
polyesters. Examples of lipids useful in liposome production
include phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides.
[0226] Pharmaceutical compositions for oral administration can be
liquid or solid. Liquid dosage forms suitable for oral
administration of inventive compositions include pharmaceutically
acceptable emulsions, microemulsions, solutions, suspensions,
syrups, and elixirs. In addition to an encapsulated or
unencapsulated conjugate, the liquid dosage forms may contain inert
diluents commonly used in the art such as, for example, water or
other solvents, solubilizing agents and emulsifiers such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethylformamide, oils (in particular, cottonseed, groundnut,
corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid
esters of sorbitan, and mixtures thereof. Besides inert diluents,
the oral compositions can also include adjuvants, wetting agents,
emulsifying and suspending agents, sweetening, flavoring, and
perfuming agents. As used herein, the term "adjuvant" refers to any
compound which is a nonspecific modulator of the immune response.
In certain embodiments, the adjuvant stimulates the immune
response. Any adjuvant may be used in accordance with the present
invention. A large number of adjuvant compounds is known in the art
(Allison Dev. Biol. Stand. 92:3-11, 1998; Unkeless et al. Annu.
Rev. Immunol. 6:251-281, 1998; and Phillips et al. Vaccine
10:151-158, 1992).
[0227] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the encapsulated or unencapsulated conjugate is mixed with at least
one inert, pharmaceutically acceptable excipient or carrier such as
sodium citrate or dicalcium phosphate and/or (a) fillers or
extenders such as starches, lactose, sucrose, glucose, mannitol,
and silicic acid, (b) binders such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,
sucrose, and acacia, (c) humectants such as glycerol, (d)
disintegrating agents such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate, (e) solution retarding agents such as paraffin, (f)
absorption accelerators such as quaternary ammonium compounds, (g)
wetting agents such as, for example, cetyl alcohol and glycerol
monostearate, (h) absorbents such as kaolin and bentonite clay, and
(i) lubricants such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof. In the case of capsules, tablets, and pills, the dosage
form may also comprise buffering agents.
[0228] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the pharmaceutical formulating art.
[0229] It will be appreciated that the exact dosage of the targeted
particle is chosen by the individual physician in view of the
patient to be treated, in general, dosage and administration are
adjusted to provide an effective amount of the targeted particle to
the patient being treated. As used herein, the "effective amount"
of an targeted particle refers to the amount necessary to elicit
the desired biological response. As will be appreciated by those of
ordinary skill in this art, the effective amount of targeted
particle may vary depending on such factors as the desired
biological endpoint, the drug to be delivered, the target tissue,
the route of administration, etc. For example, the effective amount
of targeted particle containing an anti-cancer drug might be the
amount that results in a reduction in tumor size by a desired
amount over a desired period of time. Additional factors which may
be taken into account include the severity of the disease state;
age, weight and gender of the patient being treated; diet, time and
frequency of administration; drug combinations; reaction
sensitivities; and tolerance/response to therapy.
[0230] The nanoparticles of the invention may be formulated in
dosage unit form for ease of administration and uniformity of
dosage. The expression "dosage unit form" as used herein refers to
a physically discrete unit of nanoparticle appropriate for the
patient to be treated. It will be understood, however, that the
total daily usage of the compositions of the present invention will
be decided by the attending physician within the scope of sound
medical judgment. For any nanoparticle, the therapeutically
effective dose can be estimated initially either in cell culture
assays or in animal models, usually mice, rabbits, dogs, or pigs.
The animal model is also used to achieve a desirable concentration
range and route of administration. Such information can then be
used to determine useful doses and routes for administration in
humans. Therapeutic efficacy and toxicity of nanoparticles can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., ED.sub.50 (the dose is
therapeutically effective in 50% of the population) and LD.sub.50
(the dose is lethal to 50% of the population). The dose ratio of
toxic to therapeutic effects is the therapeutic index, and it can
be expressed as the ratio, LD.sub.50/ED.sub.50. Pharmaceutical
compositions which exhibit large therapeutic indices may be useful
in some embodiments. The data obtained from cell culture assays and
animal studies can be used in formulating a range of dosage for
human use.
[0231] The present invention also provides any of the
above-mentioned compositions in kits, optionally with instructions
for administering any of the compositions described herein by any
suitable technique as previously described, for example, orally,
intravenously, pump or implantable delivery device, or via another
known route of drug delivery. "Instructions" can define a component
of promotion, and typically involve written instructions on or
associated with packaging of compositions of the invention.
Instructions also can include any oral or electronic instructions
provided in any manner.
[0232] The "kit" typically defines a package including any one or a
combination of the compositions of the invention and the
instructions, but can also include the composition of the invention
and instructions of any form that are provided in connection with
the composition in a manner such that a clinical professional will
clearly recognize that the instructions are to be associated with
the specific composition.
[0233] The kits described herein may also contain one or more
containers, which may contain the inventive composition and other
ingredients as previously described. The kits also may contain
instructions for mixing, diluting, and/or administrating the
compositions of the invention in some cases. The kits also can
include other containers with one or more solvents, surfactants,
preservative and/or diluents (e.g., normal saline (0.9% NaCl), or
5% dextrose) as well as containers for mixing, diluting or
administering the components in a sample or to a subject in need of
such treatment.
[0234] The compositions of the kit may be provided as any suitable
form, for example, as liquid solutions or as dried powders. When
the composition provided is a dry powder, the composition may be
reconstituted by the addition of a suitable solvent, which may also
be provided. In embodiments where liquid forms of the composition
are used, the liquid form may be concentrated or ready to use. The
solvent will depend on the nanoparticle and the mode of use or
administration. Suitable solvents for drug compositions are well
known, for example as previously described, and are available in
the literature. The solvent will depend on the nanoparticle and the
mode of use or administration.
[0235] The invention also involves, in another aspect, promotion of
the administration of any of the nanoparticle described herein. In
some embodiments, one or more compositions of the invention are
promoted for the prevention or treatment of various diseases such
as those described herein via administration of any one of the
compositions of the present invention. As used herein, "promoted"
includes all methods of doing business including methods of
education, hospital and other clinical instruction, pharmaceutical
industry activity including pharmaceutical sales, and any
advertising or other promotional activity including written, oral
and electronic communication of any form, associated with
compositions of the invention.
[0236] The following examples are intended to illustrate certain
embodiments of the present invention, but do not exemplify the full
scope of the invention.
EXAMPLES
[0237] The invention is further illustrated by the following
examples. The examples should not be construed as further
limiting.
Example 1
Amphiphilic Nanoparticle with Aptamer
[0238] In one embodiment, the A10 RNA aptamer which binds to the
Prostate Specific Membrane Antigen (PSMA) on the surface of
prostate cancer cells is conjugated to DSPE
(1,2-Distearoyl-sn-glycero-3-phosphoethanolamine)-PEG-COOH using
EDC/NHS chemistry with a conjugate concentration of 0.7 mg/mL. 0.21
mg of this DSPE-PEG-aptamer bioconjugate is mixed with 0.07 mg
lecithin in 2 mL aqueous solution containing 4% ethanol. 1 mg
poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) is dissolved in
1 mL tetrahydrofuran (THF) solvent, to which 5% docetaxel of the
mass of PLGA is added. This PLGA solution is then mixed with the
aqueous solution of lecithin/DSPE-PEG-Aptamer. These mixtures are
vortexed for 3 minutes, followed by stirring for 2 hours. In order
to remove all organic solvents, these mixtures are then dialyzed
for another 4 hours against PBS buffer. This procedure would yield
nanoparticles targeting to prostate cancer cells expressing PSMA
antigens.
[0239] In a second embodiment, poly(D,L-lactic-co-glycolic acid)
(PLGA) is used as a polymeric core, lecithin monolayer (.about.2.5
nm) as a lipid shell, poly(ethylene glycol) (PEG) as a stealth
material, and the A10 RNA aptamer were used to develop targeted
PLGA-Lecithin-PEG nanoparticles (NPs). Particle size could be tuned
within the range from 40 nm to 500 nm, accompanied with a surface
zeta potential ranging from -80 mV to -30 mV. Using docetaxel (a
widely used chemotherapeutics for cancers) as a model small
molecule hydrophobic drug, the PLGA-Lecithin-PEG NP had drug
encapsulation efficiency around 65% as contrast to 19% for the
conventional PLGA-b-PEG diblock copolymer NP. In addition, less
than 20% drugs were released from the NP during the first 6 hours,
which holds broad promise for clinical applications. Both in vitro
and in vivo results demonstrated that the attached RNA aptamer
effectively targeted PLGA-Lecithin-PEG NPs to prostate cancer cells
which express PSMA antigen on their plasma membrane, such as LNCaP
cells. FIG. 4 shows a schematic illustration of amphiphilic
compound assisted polymeric nanoparticles for targeted drug
delivery. FIGS. 5A and 5B show size and zeta-potential stabilities
of nanoparticles prepared according to this example. FIGS. 6 and 7
demonstrate drug encapsulation efficiency of a lipid assisted
polymeric nanoparticle as compared with a non-lipid assisted
polymeric nanoparticle. FIG. 8 shows a drug release profile for a
nanoparticle prepared according to this example.
[0240] Encapsulation efficiency is determined by taking a known
amount of DNA, encapsulating it into a nanoparticle, removing any
unencapsulated DNA by filtration, lysing the nanoparticle, then
detecting the amount of DNA that was encapsulated by measuring its
absorbance of light at 260 nm. The encapsulation efficiency is
calculated by taking the amount of DNA that was encapsulated, then
dividing it by the amount of DNA that we began with. Stated
alternatively, it is the fraction of initial DNA that is
successfully encapsulated.
[0241] Zeta potential is determined by Quasi-elastic laser light
scattering with a ZetaPALS dynamic light scattering detector
(Brookhaven Instruments Corporation, Holtsville, N.Y.; 15 mW laser,
incident beam=676 nm).
Example 2
Amphiphilic Nanoparticle with CREKA
[0242] The peptide CREKA is conjugated to DSPE-PEG-Maleimide before
formulating nanoparticles using the protocol of Example 1. This
peptide will target the delivery and uptake of the nanoparticles to
extracellular basement membranes which are exposed under the leaky
endothelial layer covering atherosclerotic plaques.
Example 3
Amphiphilic Nanoparticle with AXYLZZLN
[0243] The peptide AXYLZZLN, or conservative variants or
peptidomimetics thereof, wherein X and Z are variable amino acids,
can be conjugated to DSPE-PEG-Maleimide before formulating
nanoparticles using the protocol of Example 1. This peptide will
target the delivery and uptake of the nanoparticles to
extracellular basement membranes which are exposed under the leaky
endothelial layer covering atherosclerotic plaques.
Example 4
Anti-Her2/AKERC-Targeted Nanoparticle
Triblock Polymer Synthesis
[0244] Maleimide terminal poly(D,L-lactide)-block-poly(ethylene
glycol) (PLA-PEG-MAL) (Mw.about.10 kDa determined by GPC) was
synthesized by ring opening polymerization. Carboxylic acid
terminal poly(D,L-lactide) and/or poly(lactic-co-glycolic acid) was
purchased from the DURECT corporation (Pelham, Ala.). Bifunctional
PEG (HO-PEG-MAL) was purchased from Nektar Therapeutics (San
Carlos, Calif.). Cysteine end terminal affibody was purchased from
Affibody.RTM. (Sweden). All other reagents were purchased from
Sigma Aldrich.
[0245] Maleimide-poly(ethylene glycol)-block-poly(.sub.D,L-lactic
acid) (MAL-PEG-PLA), COOH-poly(ethylene
glycol)-block-poly(.sub.D,L-lactic acid) (COOH-PEG-PLA), and
methoxypoly(ethylene glycol)-block-poly(.sub.D,L-lactic acid)
(mPEG-PLA) were synthesized by ring opening polymerization in
anhydrous toluene using tin(II) 2-ethylhexanoate as catalyst.
General procedure for syntheses of the copolymers is as follows:
.sub.D,L-Lactide (1.6 g, 11.1 mmol) and MAL-PEG.sub.3500-OH (0.085
mmol) or COOH-PEG.sub.3500-OH (0.085 mmol) in anhydrous toluene (10
mL) was heated to reflux temperature (ca. 120.degree. C.), after
which the polymerization was initiated by adding tin(II)
2-ethylhexanoate (20 mg). After stirring for 9 h with reflux, the
reaction mixture was cooled to room temperature. To this solution
was added cold water (10 mL) and then resulting suspension was
stirred vigorously at room temperature for 30 min to hydrolyze
unreacted lactide monomers. The resulting mixture was transferred
to separate funnel containing CHCL.sub.3 (50 mL) and water (30 mL).
After layer separation, organic layer was collected, dried using
anhydrous MgSO.sub.4, filtered, and concentrated under reduced
vacuum. Then, hexane was added to the concentrated solution to
precipitate polymer product. Pure MAL-PEG.sub.3500-PLA or
COOH-PEG3500-PLA was collected as a white solid. 111PEG2000-PLA was
also prepared by same procedure above. Both copolymers were
characterized by 'H-NMR (400 MHz, Bruker Advance DPX 400) and gel
permeation chromatography (GPC) (Waters Co, Milford, Mass., USA).
Alternatively, the conjugation of PLGA or PLA and PEG was achieved
in the presence of 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide
hydrochloride) (EDC) and N-hydroxysuccinimide (NHS). Briefly, PLGA
particles were dissolved in acetonitrile. The carboxylic end of
PLGA was activated by mixing with NHS and EDC at a molar ratio of
COOH to EDC and NHS and stir overnight at room temperature. The
excess EDC and NHS in the solution were quenched by adding
2-mercaptoethanol. The NHS activated PLGA was purified by
precipitation in a solution containing ethyl ether and methanol,
and followed by centrifugation at 3000 g for 10 minutes. To
conjugate the amine end of NH2-PEG-MAL with the NHS-activated PLGA,
both polymers mixed at a molar ratio of 1:1.3
(PLGA-NHS:NH2-PEG-MAL) at room temperature overnight. The resulting
PLGA-PEG-MAL copolymer was purified by precipitation in ethyl
ether-methanol solution. The conjugation of the maleimide end of
MAL-PEG-PLGA and the free end thiol of affibody.
[0246] Nanoparticles were formed by precipitating the triblock
copolymer in water. Briefly, the triblock polymer was dissolved in
acetonitrile, and then mixed slowly with water. The nanoparticles
formed instantly upon mixing. The residual acetonitrile in the
suspension was evaporated by continuously stirring the suspension
at room temperature for 4 hrs. Alternatively, polymeric
nanoparticles were formed in a first step as above and subsequently
functionalized with affibody in aqueous solution.
Anti-Her2 Affibody Targeted Polymeric Nanoparticles
[0247] The synthesis of a multi-block polymer is initiated by
conjugation of functionalized biodegradable polyesters with
chemical groups such as, but not limited to, maleimide or
carboxylic acid for easy conjugation to one end of thiol, amine or
similarly functionalized polyethers. The conjugation of polymer to
the affibody will be performed in organic solvents such as but not
limited to dimethyl sulfoxide, dichloromethane, acetonitrile,
chloroform, dimethylformamide, tetrahydrofuran, and acetone or in
aqueous buffer including phosphate buffers and Tris buffers. The
other free end of the polyether would be functionalized with
chemical groups for conjugation to a library of targeting molecules
such as affibodies, and its derivatives. The affibody may be
conjugated through functional group including but not limited to
thiol, amine, carboxylates, hydroxyls, aldehydes, ketones and
photoreactions. The conjugation reaction between the targeting
molecules and the poly-ester-ether copolymer is achieved by adding
the affibody molecules solublized in an organic solvent or aqueous
solution. Following each of the two conjugation reactions,
unconjugated macromers are washed away by precipitating the polymer
of interest in solvents such as but not limited to ethyl ether,
hexane, methanol and ethanol. Alternatively, the nanopartciles
conjugated to affibody in aqueous solution are washed using
distilled water and ultracentrifuge membranes. Biodegradable and
biocompatible polymer poly(lactide-co-glycolide) (PLGA)/PLA and
polyethylene glycol (PEG) can be used as a model for the block
copolymer of poly(ester-ether). In a representative embodiment, the
human epidermal growth factor receptor 2 (HER-2/neu, also known as
erbB-2) can be used for breast or ovarian specific targeting using
an anti-HER-2 Affibody as the targeting molecule to cancer cells.
Carboxylic acid modified PLGA (PLGA-COOH) or PLA can be conjugated
to the amine modified heterobifunctional PEG (NH2-PEG-Maleimide)
and form a copolymer of PLGA-PEG-COOH. By using a C-end terminal
cysteine modified Anti-Her2 affibody (HS-Affibody). A triblock
copolymer of PLGA/PLA-PEG-Affibody can be obtained by conjugating
the maleimide end of PEG and free thiol functional group on the
affibody. The multiblock polymer can also be useful for imaging and
diagnostic applications. In such embodiment, a photo-sensitive or
environmental-responsible compound will be linked to the multiblock
polymer.
[0248] The targeted nanoparticles are formed by precipitation of
the multi-block polymer in an aqueous environment. The nanoparticle
formulation system described here is compatible with high
throughput biological assays in order to test the nanoparticles
generated from the multi-block polymer. Alternatively, polymeric
nanoparticles can be formed by nanoprecipitation and subsequently
functionalized with the affibody in aqueous solution. It is
possible to control the density of affibody on the surface and to
optimize the formulation polymer/affibody for therapeutic
application.
AKERC Peptide Targeted Lipid-Polymer Nanoparticles
[0249] The peptide is first chemically conjugated to the
hydrophilic region of a lipid molecule. This conjugate is then
mixed with a certain ratio of unconjugated lipid molecule in an
aqueous solution containing one or more water-miscible solvents. In
a preferred embodiment, the amphiphilic lipid can be, but is not
limited to, one or a plurality of the following:
phosphatidylcholine, lipid A, cholesterol, dolichol, shingosine,
sphingomyelin, ceramide, cerebroside, sulfatide, glycosylceramide,
phytosphingosine, phosphatidylethanolamine, phosphatidylglycerol,
phosphatidylinositol, phosphatidylserine, cardiolipin, phophatidic
acid, and lysophophatides. The water miscible solvent can be, but
is not limited to: acetone, ethanol, methanol, and isopropyl
alcohol. Separately, a biodegradable polymeric material is mixed
with the agent or agents to be encapsulated in a water miscible or
partially water miscible organic solvent. In a preferred
embodiment, the biodegradable polymer can be, but is not limited to
one or a plurality of the following: poly(D,L-lactic acid),
poly(D,L-glycolic acid), poly(s-caprolactone), or their copolymers
at various molar ratios. The carried agent can be, but is not
limited to, one or a plurality of the following: therapeutic drugs,
imaging probes, or hydrophobic or lipophobic molecules for medical
use. The water miscible organic solvent can be but is not limited
to: acetone, ethanol, methanol, or isopropyl alcohol. The partially
water miscible organic solvent can be, but is not limited to:
acetonitrile, tetrahydrofuran, ethyl acetate, isopropyl alcohol,
isopropyl acetate, or dimethylformamide. The resulting polymer
solution is then added to the aqueous solution of conjugated and
unconjugated amphiphilic lipid to yield nanoparticles by the rapid
diffusion of the organic solvent into the water and evaporation of
the organic solvent.
[0250] In a preferred embodiment, the peptide AKERC, which binds to
collagen IV in the extracellular basement membranes, is conjugated
to DSPE-PEG-Maleimide (DSPE: 1,2
distearoyl-sn-glycero-3-phosphoethanolamine sodium salt) using
EDC/NHS chemistry with a conjugate concentration of 0.7 mg/mL. 0.21
mg of this DSPE-PEG-KAERC bioconjugate is mixed with 0.07 mg
lecithin in 2 mL aqueous solution containing 4% ethanol. 1 mg
poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) is dissolved in
1 mL acetonitrile (ACN) solvent, to which 5% docetaxel of the mass
of PLGA is added. This PLGA solution is then mixed with the aqueous
solution of lecithin/DSPE-PEG-KAERC. These mixtures are vortexed
for 3 minutes, followed by stirring for 2 hours. In order to remove
all organic solvents, these mixtures are then washed three times
using copious PBS buffer. This peptide will target the delivery and
uptake of the nanoparticles to extracellular basement membranes
which are exposed under the leaky endothelial layer covering
atherosclerotic plaques.
Example 5
CREKA-Targeted Nanoparticle
Lipid-PEG-CREKA Synthesis:
[0251] 1 mg CREKA peptide is dissolved in 0.2 mL PBS buffer
containing 10 mg dithiothreitol (DTT). The solution is incubated at
room temperature for 30 minutes before mixed with 1 mg
DSPE-PEG-Maleimide (DSPE: 1,2
distearoyl-sn-glycero-3-phosphoethanolamine sodium salt). These
mixtures are incubated at 4.degree. C. for 24 hr. In order to
remove DTT agent and the extra CREKA, these mixtures are then
dialyzed against PBS buffer for 48 hr.
CREKA-Targeted Nanoparticle Synthesis:
[0252] 0.03 mg of the DSPE-PEG-CREKA bioconjugate is mixed with
0.07 mg lecithin in 2 mL aqueous solution containing 4% ethanol. 1
mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) is dissolved
in 1 mL acetonitrile (ACN). This PLGA solution is then mixed with
the aqueous solution of lecithin/DSPE-PEG-CREKA. These mixtures are
vortexed for 3 minutes, followed by stirring for 2 hours. In order
to remove all organic solvents, these mixtures are then washed
three times using copious PBS buffer. For fluorescence imaging
purposes, 10% of the PLGA polymer is labeled with a fluorescent
probe such as Alexa Fluor 647.
CREKA-Targeted Nanoparticle with Therapeutic Agent:
[0253] For example, 0.03 mg DSPE-PEG bioconjugate is mixed with
0.07 mg lecithin in 2 mL aqueous solution containing 4% ethanol. 1
mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) is dissolved
in 1 mL acetonitrile solvent, to which 5% docetaxel of the mass of
PLGA is added. The lecithin/DSPE-PEG solution is first heated up to
65.degree. C. for 3 minutes. Then the PLGA solution is added to the
aqueous solution of lecithin/DSPE-PEG dropwise under gentle
stirring. These mixtures are vortexed for 3 minutes, followed by
stirring for 2 hours. In order to remove all organic solvents,
these mixtures are then dialyzed for another 3 hours against PBS
buffer. These procedures would yield lipid-polymer hybrid
nanoparticles with a diameter of about 50-60 nm and a zeta
potential of about -40 mV.
[0254] In another method, 0.036 mg DSPE-PEG-CREKA triblock compound
is mixed with 0.07 mg lecithin in 2 mL aqueous solution containing
4% ethanol. 1 mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100
kD) is dissolved in 1 mL acetonitrile solvent, to which 5%
docetaxel of the mass of PLGA is added. For fluorescence imaging
purpose, 10% of the PLGA polymer is labeled with a fluorescent
probe such as Alexa Fluor 647. The lecithin/DSPE-PEG solution is
first heated up to 65.degree. C. for 3 minutes. Then the PLGA
solution is added to the aqueous solution of lecithin/DSPE-PEG
dropwise under gentle stirring. These mixtures are vortexed for 3
minutes, followed by stirring for 2 hours. In order to remove all
organic solvents, these mixtures are then dialyzed for another 3
hours against PBS buffer. Alternatively, these mixtures can be
washed three times using copious PBS buffer to remove organic
solvents and any free molecules. These procedures would yield
CREKA-targeted lipid-polymer nanoparticles with specific a binding
affinity to extracellular basement membrane.
[0255] In another method, 0.03 mg DSPE-PEG-Maleimide bioconjugate
is mixed with 0.07 mg lecithin in 2 mL aqueous solution containing
4% ethanol. 1 mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100
kD) is dissolved in 1 mL acetonitrile solvent, to which 5%
docetaxel of the mass of PLGA is added. For fluorescence imaging
purpose, 10% of the PLGA polymer is labeled with a fluorescent
probe such as Alexa Fluor 647. The Lecithin/DSPE-PEG solution is
first heated up to 65.degree. C. for 3 minutes. Then the PLGA
solution is added to the aqueous solution of lecithin/DSPE-PEG
dropwise under gentle stirring. These mixtures are vortexed for 3
minutes, followed by stirring for 2 hours. In order to remove all
organic solvents, these mixtures are then dialyzed for another 3
hours against PBS buffer. Alternatively, these mixtures can be
washed three times using copious PBS buffer to remove organic
solvents and any free molecules. 0.03 mg CREKA peptide is dissolved
in 0.2 mL PBS buffer containing 1 mg dithiothreitol (DTT). The
solution is incubated at room temperature for 30 minutes before
mixed with 1 mg PLGA-Lipid-PEG-Maleimide nanoparticle. These
mixtures are incubated at 4.degree. C. for 24 hr. In order to
remove DTT agent and the extra CREKA, these mixtures washed three
times using PBS buffer. These procedures would yield CREKA-targeted
lipid-polymer nanoparticles with specific a binding affinity to
extracellular basement membrane.
[0256] In another method, 0.036 mg DSPE-PEG-CREKA triblock compound
can be mixed with 0.07 mg lecithin in 2 mL aqueous solution
containing 4% ethanol. 1 mg poly(D,L-lactic-co-glycolic acid)
(PLGA, Mw=100 kD) can be dissolved in 1 mL acetonitrile solvent, to
which 5% of a combination of dexamethasone and zotarolimus of the
mass of PLGA can be added.
[0257] In another method, 0.036 mg DSPE-PEG-CREKA triblock compound
can be mixed with 0.07 mg lecithin in 2 mL aqueous solution
containing 4% ethanol. 1 mg poly(D,L-lactic-co-glycolic acid)
(PLGA, Mw=100 kD) can be dissolved in 1 mL acetonitrile solvent, to
which 5% of everolimus of the mass of PLGA can be added.
[0258] In another method, 0.036 mg DSPE-PEG-CREKA triblock compound
can be mixed with 0.07 mg lecithin in 2 mL aqueous solution
containing 4% ethanol. 1 mg poly(D,L-lactic-co-glycolic acid)
(PLGA, Mw=100 kD) can be dissolved in 1 mL acetonitrile solvent, to
which 5% of paclitaxel of the mass of PLGA can be added.
ARYLQKLN-Targeted Nanoparticle Synthesis:
[0259] For ease of conjugation, a cystin amino acid is added to the
C-terminal or N-terminal of ARYLQKLN. The additional
procedures/methods follow those of CREKA-targeted nanoparticles
given immediately above.
CREKA-Targeted Nanoparticle Binding to Collagen-Coated Surface
[0260] To prepare a collagen IV-coated surface, 0.5 mL Collagen IV
acetic acid solution (10 .mu.g/mL) is spread to completely cover
the bottom of a glass well. After 1 hr incubation at 37.degree. C.,
the extra collagen is removed by washing the surface using copious
water. The quality of the coating is checked by atomic force
microscopy (AFM). The collagen coated surface is then incubated
with 1 mL CREKA-targeted nanoparticle aqueous solution (0.25 mg/mL)
for 30 minutes at room temperature. The extra nanoparticles are
washed by copious PBS buffer. Fluorescence microscopy is used to
image the sample, thereby identifying the binding efficiency of
CREKA-targeted nanoparticle to collagen coated surface.
CREKA-Targeted Nanoparticle Binding to Rat Basement Membrane Ex
Vivo:
[0261] A rat is sacrificed and its abdominal aorta is exposed in
situ. After washing the aorta with copious PBS buffer, a balloon
catheter is placed in the aorta. The balloon is inflated with 0.2
mL air and dragged through the aorta five times to injure the
endothelia layer. The proper amount of CREKA-targeted nanoparticles
are injected into the aorta and incubated for 30 minutes under
constant pressure. The extra nanoparticles are washed away using
copious PBS buffer. 10 mm abdominal aorta is sectioned and kept in
4% formaldehyde solution for histological imaging use.
[0262] Other peptides such as d-CREKA and CEAKR (a scrambled
sequence) are used as negative control to investigate the binding
specificity of CREKA-targeted nanoparticles to basement membrane.
d-CREKA and CEAKR replace CREKA and repeated in the above
experiments.
Example 6
Prophetic Example of Synthesis of PLGA-PEG-CREKA Macromolecule for
Preparation of CREKA-Targeted Nanoparticle
##STR00004##
[0263] Example 7
Local Delivery of Nanoparticle
[0264] As described herein, the nanoparticles of the invention can
be delivered to a subject using a variety of methods, such as
intravenous or local administration using, e.g., a balloon stent.
The following example demonstrates how to test the advantages of
locally delivering the nanoparticles of the invention.
Pre-Interventional Procedures
[0265] Animal Monitoring and Examination
[0266] Upon arrival at the Testing Facility and until sacrifice,
the animals will be monitored and observed at least once a day. A
physical examination of all animals entered in the study will be
done by the Facility Veterinarian or a trained employee during
acclimation, as per Testing Facility SOPs.
[0267] Fasting
[0268] Fasting will be conducted prior to induction of anesthesia.
Food, including any dietary supplements, will be withheld the
morning of the procedure. Water will not be withheld.
[0269] Anesthesia
[0270] Animals will be tranquilized with acepromazine administered
subcutaneously [SC]. Animal weight will be recorded. Anesthesia
induction will be achieved with propofol injected intravenously
[IV]. Upon induction of light anesthesia, the subject animal will
be intubated and supported with passive balloon ventilation.
Isoflurane in oxygen will be administered to maintain a surgical
plane of anesthesia. Fluid therapy will be achieved by saline
injections before surgery. Intravenous saline injection may be
performed to replace blood loss or to correct low systemic blood
pressure.
Anticoagulant Therapy
[0271] During the procedure an initial bolus of heparin (.about.70
IU/kg) will be given following cannulation of the carotid artery.
Additional heparin may be given if needed.
[0272] Animal Preparation
[0273] The animal will be placed in dorsal recumbency, and the hair
will be removed from the access areas. A rectal temperature probe
will be inserted, and the temperature will be monitored regularly.
The access site will be prepared with topical application of
chlorhexidine, 70% isopropyl alcohol and proviodine. The area will
then be appropriately draped to maintain a sterile field.
Denudation Procedures
[0274] Vascular Access
[0275] After induction of anesthesia, the left or right carotid
artery will be accessed with an incision made in the throat region.
An application of bupivacain on the carotid access site will be
performed to achieve local anesthesia and manage pain after
surgery. An arterial sheath will be introduced and advanced into
the artery.
[0276] Vessel Angiography
[0277] Before the first angiogram, 1 ml of nitroglycerine (0.5
mg/ml) IV will be given. For subsequent angiograms, more nitro can
be given if slow flow or spams are observed, as per operator
judgment and animal condition. The iliac artery will be
circumscribed (from the femoral to the internal iliac branch) and
Quantitative Angiography (QA) will be performed to document the
vessel size. Extra angiograms may be recorded at this point or
later on in the procedure at the discretion of the operator. In
such case, the operator will select a suitable angiogram for
analysis.
[0278] Balloon Injury
[0279] The appropriate balloon will be advanced over the guidewire
to traverse the distal portion of the pre-selected injury site. The
inflated balloon will then be retracted from the femoral artery
back into the aorta to enable denudation of the target vasculature.
The balloon will be deflated and re-advanced to traverse the target
injury site. At the operator's discretion, the inflation pressure
will be adjusted for each subsequent denudation pass, based on the
amount of force required to pull the balloon. If there is little
resistance to the pulling, the balloon inflation pressure will be
increased by an increment of one Atm. If there is too much
resistance, the balloon inflation pressure will be decreased by an
increment of one ATM. The third denudation will not be performed if
resistance is still present. The balloon will be deflated while it
is in the terminal descending aorta and the denudation procedure
repeated one more time (total 3 times denudation). Post-denudation
angiogram will be performed and TIMI flow will be assessed. Animals
with post-TIMI flow of zero or 1 will receive intra-arterial
infusion of nitroglycerine (at the discretion of the
interventionalist) to restore the flow to 2 or 3.
Test Article Delivery
[0280] The delivery catheter will be introduced into the artery
over the guide wire. The Genie balloon catheter will be
continuously inflated at a low pressure of 2 atm that allows for
distal and proximal occlusion of the vessel while simultaneously
forming a central drug depot. A continuous pressure of 2 atm is
maintained throughout application of the contrast agent. The total
volume injected will be recorded. Following treatment of the vessel
a final angiography will be taken and recorded, TIMI flow and QCA
will also be performed and documented.
Monitoring Procedures
[0281] Parameters including isoflurane level, blood oxygen
saturation, pulse rate, and temperature will be regularly monitored
and manually recorded and noted in the raw data for each
animal.
Closure
[0282] Following successful delivery and completion of angiography,
all catheters and the sheath will be removed from the animals and
the carotid artery will be ligated.
Necropsy
[0283] Upon completion of follow-up angiography the animals will be
kept deeply anesthetised before euthanasia with a rapid bolus of
pentobarbital.
[0284] Stented arteries along with proximal and distal non-stented
segments will then be dissected out rinsed and immersion-fixed in
neutral-buffered formalin and processed for histology.
Histopathology
[0285] The treated iliacs will be cut in 4 sections that will be
embedded in paraffin 4 separate blocks. For each block 2 adjacent
sections will be prepared and then 2 other adjacent section
.about.100.mu. deeper in the block. One section from each adjacent
pair will be stained with hematoxylin and eosin (H&E). The
remaining section of each adjacent pair will be left unstained and
sent for fluorescence analyses. The H&E stained section will be
analyzed by the Study Pathologist for the extent of vessel injury,
the presence of endothelium and other relevant observations. The
analysis will be reported as a narrative text including
representative images, with scoring of some parameters if deemed
appropriate.
Example 8
HER-2 Targeted Drug Encapsulated NanoParticles (FIGS. 21-24)
[0286] To develop HER-2 targeted drug encapsulated NPs, the
anti-HER-2 Affibody was conjugated to the thiol-reactive maleimide
end group of the PLA-PEG-Maleimide (PLA-PEG-Mal) copolymer through
a stable thioether bond and the targeting specificity and efficacy
was evaluated using fluorescent microscopy. Subsequently, we
encapsulated paclitaxel into the targeted polymeric NPs and
examined whether this system could increase the drug cytotoxicity
in HER-2 positive cell lines: SK-BR-3 and SK-OV-3.
Materials and Methods
[0287] Conjugation and Characterization of nanoparticle-Affibody
bioconjugates: PLA-PEG-Mal polymeric NPs were incubated under
stirring conditions with the Anti-HER-2 Affibody molecules (15 kDa)
at a molar ratio of Affibody:PLA-PEG-Mal of 5% to form a stable
bioconjugate. The NP-Affibody bioconjugates were purified to remove
free Affibody molecules using Amicon Ultra centrifuge device (100
kDa molecular weight size exclusion). Subsequently, the thioether
bond formation between the PLA-PEG-Mal NPs and the Affibody
molecules was characterized using proton nuclear magnetic resonance
(.sup.1H-NMR, 600 MHz, Bruker Advance). Additionally, the chemical
attachment of the fluorescent Affibody was confirmed using Ultra
Violet Imaging system (Kodak Electrophoresis Documentation and
Analysis System 120). The Affibody molecule was fluorescently
labeled with a red fluorescent probe, Alexa Fluor 532 (Invitrogen),
purified and subsequently conjugated to PLA-PEG-Mal polymeric NPs
at different molar ratios of Affibody:PLA-PEG-Mal ranging from 0 to
20% (molar ratio). Then the purified NP-Affibody bioconjugates
suspensions were imaged using a UV Kodak camera assisted with a red
filter to show the visible effect of the fluorescent Affibody
conjugated on non-fluorescent polymeric NPs.
[0288] Uptake assays of targeted and untargeted nanoparticles: The
human ovarian adenocarcinoma (SK-OV-3; ATCC), human breast
adenocarcinoma (SK-BR-3; ATCC), and human pancreatic adenocarcinoma
(Capan-1, ATCC) were the HER-2 positive cell lines used for
cytotoxicity and uptake efficacy studies of the NP-Affibody
bioconjugates. HER-2 positive cell lines were grown in chamber
slides (Cab-TekII, 8 wells; Nunc) within their growth medium
(Modified McCoy's 5a (ATCC) supplemented with 100 units/ml aqueous
penicillin G, 100 ug/ml streptomycin, and 10% FBS) to 70%
confluence in 24 h (i.e., 50,000 cells/cm.sup.2) in 5% CO.sub.2
incubator. On the day of the experiment, cells were washed with
pre-warmed PBS and incubated with pre-warmed phenol-red-reduced
OptiMEM media for 30 minutes, before adding 50 .mu.g of NPs or
NP-Affibody bioconjugates loaded with same amount of fluorescent
NBD dye. NP formulations were incubated with cells for 2 hours at
37.degree. C., washed with PBS three times, fixed with 4%
paraformaldehyde, blocked for 30 minutes at room temperature with
1% BSA/PBS, permeabilized with 0.01% Triton-X for 3 minutes,
counterstained with Alexa-Fluor Phalloidin-Rhodamine (cytoskeleton
staining), 4',6-diamidino-2-phenylindole (DAPI, nucleus staining),
mounted in fluorescence protecting imaging solution, and visualized
using fluorescent microscopy (DeltaVision system; Olympus IX71).
Digital images of green, red and blue fluorescence were acquired
along the z-axis at 0.2 .mu.m intervals using 60.times. oil
immersion objective and DAPI, FITC and Rhodamine filters
respectively. Images were overlaid, deconvoluted and 3D
reconstruction was performed using Softwork software for
acquisition and analysis.
[0289] In vitro cellular toxicity assay of paclitaxel encapsulated
into targeted and untargeted NPs: SK-BR-3 and SK-OV-3 were grown in
96-well plates at concentrations leading to 70% confluence in 24 h
(i.e., 50,000 cells/cm.sup.2). Defined amounts of paclitaxel were
encapsulated into targeted and non-targeted nanoparticles and
incubated with cell lines (5 ug Paclitaxel/well) in OptiMEM for two
hours. Next, cells were washed and fresh media was supplemented.
The cells were then allowed to grow for 72 hours and cell viability
was assessed colorimetrically with MTS reagents (Invitrogen).
Results
[0290] Development of targeted, controlled release drug delivering
NP-Affibody bioconjugates. We first synthesized a copolymer
comprised of a hydrophobic block, poly(.sub.D,Llactic acid), and a
hydrophilic block, poly(ethylene glycol) with a maleimide terminal
group (PLA-PEG-Mal). Then the copolymers form negatively charged
NPs with a core-shell structure in aqueous environment via the
nanoprecipitation method. The hydrophobic core of the NPs is
capable of carrying pharmaceuticals, especially poorly soluble
drugs. The hydrophilic shell not only provides a "stealth" layer,
together with the surface charge property (Zeta potential)=-10
mV.+-.5 mV), to improve the stability and the circulation half-time
of these drug delivering NPs, but also functional maleimide groups
for Affibody conjugation. Lack of protein adsorption in solutions
including 10%, 20% and 100% serum demonstrated the stability of NP
size (<100 nm). We also evaluated the freeze-drying process for
storing the nanoparticles in a dry state, as described previously.
We were able to reconstitute nanoparticles with a similar original
size after lyophilization, confirming the stability of this type of
carrier to this process.
[0291] The anti-HER-2 Affibody molecule was previously selected
against the extracellular domain of the HER-2 protein and further
modified by affinity maturation and dimerization. The anti-HER-2
Affibody is commercially available and has been shown to have high
binding specificity and affinity in vitro and in vivo as a targeted
imaging agent. Particle size and surface charge (Zeta potential) of
PLA-PEG-Mal NPs both with and without Affibody were characterized
using laser light scattering, ZetaPALS system and electron
microscopy (FIGS. 21A and 21B). The addition of Affibody molecules
on the surface of the NPs did not significantly affect the size,
size distribution and surface charge of the NPs (NP=70.+-.5 nm,
NP-Affibody 85.+-.5 nm). The chemical conjugation of the Affibody
molecules on the surface of the PLA-PEG-Mal NPs was confirmed using
UV imaging (FIGS. 21A and 21B) and proton nuclear magnetic
resonance spectroscopy in d-DMSO (.sup.1H-NMR) (FIG. 22C). To
visualize the presence of Affibody molecules on the NPs, we labeled
Affibody molecules with fluorescence probe, Alexa Fluor 532, and
subsequently conjugated them to the PLA-PEG-Mal NPs with different
molar ratios of Affibody:PLA-PEG-Mal (0, 1, 2, 5, 20%). The
NP-Affibody bioconjugates were then exposed under a UV lamp to
observe their fluorescence signals. No fluorescence signal was
observed from the NPs without fluorescently labeled Affibody,
however, the fluorescence intensity from those NPs with fluorescent
Affibody continuously enhances with the increase of
Affibody:PLA-PEG-Mal molar ratio. The .sup.1H-NMR spectrum of the
purified PLA-PEG-Affibody in d-DMSO showed the characteristic peaks
of PLA-PEG at chemical shift of .delta..about.1.4 ppm (--CH3 of the
PLA backbone), .delta..about.3.6 ppm of (--CH.sub.2 of the PEG
backbone) and .delta..about.5.2 ppm (--CH of the PLA backbone).
Additionally, we observed the characteristic peaks of the Affibody
molecule in the chemical shift region of .delta.=7-8 ppm that
represents the amide bonds (NH--CO) within the Affibody polypeptide
molecule. The NMR results suggest successful conjugation of the
Affibody on the surface of PLA-PEG-Mal NPs.
[0292] Efficient and specific receptor mediated internalization of
NP-Affibody bioconjugates. We next demonstrated the efficient
binding and internalization of targeted NP-Affibody bioconjugates
to HER-2 positive cancer cells using three cell lines: Capan-1,
SK-BR-3, and SK-OV-3 (see FIG. 22). After incubating NBD dye
encapsulated NP-Affibody bioconjugates with the cells for 2 hr at
37.degree. C. and removing the excess bioconjugates, we observed a
large amount of green dots in a punctuate pattern inside the
targeted cells, suggesting an efficient targeting and
internalization mechanism of the .about.80 nm NP-Affibody
bioconjugates to the HER-2 positive cells. In contrast, untargeted
PLA-PEG NPs were slightly taken up by the cell lines after the same
duration of incubation (FIG. 22). To minimize cell passage effect
on the observed results, this experiment was repeated four times
with different cell passages and all of them gave the same
observations. We also verified the cellular localization of the
NP-Affibody bioconjugates using a z-axis scanning fluorescent
microscopy and 3D image reconstitution. The rotated cross section
of the 3D reconstitution images of a SK-BR-3 cell demonstrated the
internalization of targeted NP-Affibody bioconjugates to the cell
(FIG. 23). Orlova et al. have shown the binding ability of
Anti-HER-2 Affibody within 1 hr using immunofluorescence method.
Our results are consistent with their findings and suggest a
receptor mediated endocytosis mechanism. Internalization through an
endocytosis mechanism has been previously described for anti-HER-2
monoclonal antibodies and is consistent with the kinetics of our
NPs entering the cells. Similarly, targeted drug delivery using RGD
peptide sequences to integrins has also shown efficient binding and
internalization in multiple types of cancers. In contrast, the
anti-HER-2 approach offers better cancer diseases specificity with
high affinity to HER-2 cell membrane receptors expressed in
multiple types of cancers.
[0293] In vitro cellular cytotoxicity assays using breast cancer
and ovarian cancer cells (MTS assays). We prepared targeted and
untargeted NPs (with and without paclitaxel) to evaluate their
differential cytotoxicity using in vitro cell viability assay (MTS
assays) with breast cancer and ovarian cancer cells (SK-BR-3;
SK-OV-3), which over-express the HER-2 cell membrane receptors. In
this study, we incubated various NP formulations with SK-BR-3 and
SK-OV-3 cancer cells for 2 hours in optimem, washed cells with PBS
to remove excess of NPs, and supplemented with fresh cell growth
medium. We further incubated the cells for 3 days before using MTS
assay to quantify cell viability which was normalized to that of
the cells in the absence of NPs. The results showed that drug
encapsulated targeted NPs had the highest cytotoxicity to both
SK-BR-3 and SK-OV-3 cell lines; cell viability was 70.+-.5% and
59.+-.5%, respectively (FIGS. 24A and 24B). The ANOVA test
indicated that the cell viability of targeted NPs differed
significantly from that of untargeted NPs (p<0.05). In contrast,
NPs without encapsulated drugs are not toxic to both cell lines.
These results are consistent with our previous studies using
targeted NP-aptamer bioconjugates to deliver drugs to prostate
cancer cells. Therefore, this NP-Affibody bioconjugate system holds
great potential to be used as a biocompatible and biodegradable
targeted drug delivery platform for multiple types of cancers
therapy. For a specific application, it would be feasible to tune
some parameters of the bioconjugates such as NP size, surface
charge and Affibody packing density to optimize the drug delivery
pharmacokinetics and its targeting efficiency.
[0294] FIG. 10 demonstrates a schematic illustration of a
CREKA-targeted PLGA-Lipid-PEG nanoparticle.
[0295] FIGS. 11A and 11B demonstrate that (A) CREKA-targeted
PLGA-Lipid-PEG nanoparticles effectively bind to collagen IV coated
surface. For fluorescence imaging purpose, fluorescent probe Alexa
647 was chemically conjugated to PLGA polymer; (B) a bare
(nontargeted) PLGA-Lipid-PEG nanoparticles rarely bind to collagen
IV coated surface.
[0296] FIGS. 12A and 12B demonstrate (A) H&E staining of normal
rat aorta; (B) H&E staining of balloon injured aorta;
endothelium layer was removed.
[0297] FIGS. 13A and 13B demonstrates CREKA-targeted PLGA-Lipid-PEG
nanoparticles effectively bind to balloon-injured rat aorta. The
nanoparticles were incubated with the aorta for 10 minutes. The
extra nanoparticles were washed away with copious PBS buffer. Then
the aorta was harvested, fixed and prepared for imaging. (A)
Fluorescence image of the aorta; (B) Overlay of fluorescence image
and phase image of the same aorta.
[0298] FIGS. 14A and 14B demonstrate that D-CREKA-targeted
PLGA-Lipid-PEG nanoparticles (D-form of amino acids) do not bind to
balloon-injured rat aorta. The nanoparticles were incubated with
the aorta for 10 minutes. The extra nanoparticles were washed away
with copious PBS buffer. Then the aorta was harvested, fixed and
prepared for imaging. (A) Fluorescence image of the aorta; (B)
Overlay of fluorescence image and phase image of the same
aorta.
[0299] FIGS. 15A and 15B demonstrate that scrambled peptide
CEAKR-targeted PLGA-Lipid-PEG nanoparticles do not bind to
balloon-injured rat aorta. The nanoparticles were incubated with
the aorta for 10 minutes. The extra nanoparticles were washed away
with copious PBS buffer. Then the aorta was harvested, fixed and
prepared for imaging. (A) Fluorescence image of the aorta; (B)
Overlay of fluorescence image and phase image of the same
aorta.
[0300] FIGS. 16A and 16B demonstrate that CREKA-targeted
PLGA-Lipid-PEG nanoparticles do not bind to a normal rat aorta. The
nanoparticles were incubated with the aorta for 10 minutes. The
extra nanoparticles were washed away with copious PBS buffer. Then
the aorta was harvested, fixed and prepared for imaging. (A)
Fluorescence image of the aorta; (B) Overlay of fluorescence image
and phase image of the same aorta.
[0301] FIG. 17 is a schematic illustration of CREKA-targeted
PLGA-Lipid-PEG nanoparticle
[0302] FIG. 18 demonstrates fluorescence images of
ARYLQKLN-targeted PLGA-Lipid-PEG nanoparticles incubating with
basement membrane proteins for 10 minutes: (A) PBS; (B) Collagen I;
(C) Collagen II; (D) Collagen IV; (E) Fibronectin; and (F)
vitronectin.
[0303] FIG. 19 demonstrates that ARYLQKLN-targeted PLGA-Lipid-PEG
nanoparticles bind to a balloon-injured rat aorta. The
nanoparticles were incubated with the aorta for 10 minutes. The
extra nanoparticles were washed away with copious PBS buffer. Then
the aorta was harvested, fixed and prepared for imaging. (A)
Fluorescence image of the aorta; (B) Overlay of fluorescence image
and phase image of the same aorta.
[0304] FIG. 20 demonstrates that ARYLQKLN-targeted PLGA-Lipid-PEG
nanoparticles do not bind to normal rat aorta. The nanoparticles
were incubated with the aorta for 10 minutes. The extra
nanoparticles were washed away with copious PBS buffer. Then the
aorta was harvested, fixed and prepared for imaging. (A)
Fluorescence image of the aorta; (B) Overlay of fluorescence image
and phase image of the same aorta.
[0305] For FIGS. 24A and 24B: Cell viability assay (MTS assay) to
evaluate the differential toxicity of targeted (Np-Affb) and
untargeted nanoparticles (Np) with and without encapsulated
paclitaxel (Ptxl). In this assay, the nanoparticle formulations
were incubated for 2 hours, cells were subsequently washed and
incubated in cell growth media to allow the effect of the drug on
the cell cycles before quantifying the nanoparticle formulations
toxicities against two cancer cell lines expressing HER-2 (SK-BR-3
and SK-OV-3).ANNOVA test "*" p<0.01; "**" p<0.05.
* * * * *
References