U.S. patent application number 17/693262 was filed with the patent office on 2022-07-07 for cross-linked polymer modified nanoparticles.
This patent application is currently assigned to Oregon Health & Science University. The applicant listed for this patent is Oregon Health & Science University, PDX Pharmaceuticals, Inc.. Invention is credited to David Castro, Joe William Gray, Jingga Morry, Worapol Ngamcherdtrakul, Wassana Yantasee.
Application Number | 20220211878 17/693262 |
Document ID | / |
Family ID | 1000006193532 |
Filed Date | 2022-07-07 |
United States Patent
Application |
20220211878 |
Kind Code |
A1 |
Yantasee; Wassana ; et
al. |
July 7, 2022 |
CROSS-LINKED POLYMER MODIFIED NANOPARTICLES
Abstract
Disclosed herein are nanoconstructs comprising a nanoparticle,
coated with additional agents such as cationic polymers,
stabilizers, targeting molecules, labels, oligonucleotides and
small molecules. These constructs may be used to deliver compounds
to treat solid tumors and to diagnose cancer and other diseases.
Further disclosed are methods of making such compounds and use of
such compounds to treat or diagnose human disease.
Inventors: |
Yantasee; Wassana; (Lake
Oswego, OR) ; Ngamcherdtrakul; Worapol; (Portland,
OR) ; Morry; Jingga; (Portland, OR) ; Castro;
David; (Portland, OR) ; Gray; Joe William;
(Lake Oswego, OR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Oregon Health & Science University
PDX Pharmaceuticals, Inc. |
Portland
Portland |
OR
OR |
US
US |
|
|
Assignee: |
Oregon Health & Science
University
Portland
OR
PDX Pharmaceuticals, Inc.
Portland
OR
|
Family ID: |
1000006193532 |
Appl. No.: |
17/693262 |
Filed: |
March 11, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17398954 |
Aug 10, 2021 |
11305024 |
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17693262 |
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15429971 |
Feb 10, 2017 |
11207428 |
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17398954 |
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PCT/US2016/022655 |
Mar 16, 2016 |
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15429971 |
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62133913 |
Mar 16, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B82Y 30/00 20130101;
B82Y 5/00 20130101; A61K 47/6923 20170801; A61K 47/59 20170801;
A61K 47/6849 20170801; A61K 49/0093 20130101; A61K 49/1875
20130101; A61K 47/60 20170801; A61K 47/6929 20170801; A61K 38/16
20130101; A61K 49/1857 20130101; A61K 31/713 20130101; A61K 47/6855
20170801; A61K 47/551 20170801; A61K 49/186 20130101 |
International
Class: |
A61K 49/00 20060101
A61K049/00; A61K 31/713 20060101 A61K031/713; A61K 49/18 20060101
A61K049/18; A61K 47/60 20060101 A61K047/60; A61K 47/59 20060101
A61K047/59; A61K 47/69 20060101 A61K047/69; A61K 47/68 20060101
A61K047/68; A61K 47/55 20060101 A61K047/55; A61K 38/16 20060101
A61K038/16 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with the support of the United
States government under the terms of grant numbers RO1GM089918, and
R41DK094571, as well as contract number HHSN261201300078 C awarded
by the National Institutes of Health. The United States government
has certain rights in this invention.
Claims
1. A composition comprising: a plurality of multilayer
nanoconstructs each comprising: a silica nanoparticle, a silicon
nanoparticle, a silver nanoparticle, or a carbon nanotube
nanoparticle; polyethylenimine (PEI) bound to an exterior surface
of the nanoparticle; and polyethylene glycol (PEG) bound to the PEI
and/or to the nanoparticle; and a protein or peptide loaded on the
nanoconstructs, wherein the nanoconstructs loaded with the protein
or peptide have a hydrodynamic size Z-average diameter of about 200
nm or less.
2. The composition of claim 1, wherein the nanoconstructs loaded
with the protein or peptide have a polydispersity index (PDI) of no
more than about 0.37.
3. The composition of claim 1, wherein the PDI of the
nanoconstructs loaded with the protein or peptide is about 0.2.
4. The composition of claim 1, wherein the nanoparticle is a silica
nanoparticle.
5. The composition of claim 4, wherein the silica nanoparticle is a
mesoporous silica nanoparticle (MSNP).
6. The composition of claim 1, wherein the nanoparticle is a
silicon nanoparticle.
7. The composition of claim 1, wherein the nanoparticle is a silver
nanoparticle.
8. The composition of claim 1, wherein the nanoparticle is a carbon
nanotube.
9. The composition of claim 1, wherein the plurality of
nanoparticles consists essentially of materials selected from the
group consisting of: silica, silicon, silver, carbon nanotubes, and
combinations of two or more thereof.
10. The composition of claim 1, wherein the PEI of the
nanoconstructs is from about 10% to about 30% by weight of the
nanoconstructs.
11. The composition of claim 1, wherein the PEI is
cross-linked.
12. The composition of claim 11, wherein the PEI is cross-linked
using a cleavable cross-linker.
13. The composition of claim 1, wherein the PEI has an average size
of 1.8 kDa to 25 kDa.
14. The composition of claim 1, wherein the protein or peptide is
conjugated to the functional PEG.
15. The composition of claim 1, wherein the protein or peptide is
electrostatically bound to the PEI.
16. The composition of claim 1, wherein the nanoparticles are from
about 10 nm to about 90 nm in diameter.
17. The composition of claim 1, wherein the hydrodynamic size
Z-average diameter of the nanoconstructs is about 10 nm to about
200 nm.
18. The composition of claim 1, wherein the hydrodynamic size
Z-average diameter of the nanoconstructs is about 50 nm to about
200 nm.
19. The composition of claim 1, wherein the hydrodynamic size
Z-average diameter of the nanoconstructs is about 90 nm to about
120 nm.
20. The composition of claim 1, wherein protein or peptide is a
therapeutic agent.
21. The composition of claim 20, wherein therapeutic agent
comprises an antibody or an antigen-binding fragment thereof.
22. The composition of claim 21, wherein therapeutic antibody
comprises a monoclonal antibody.
23. The composition of claim 1, wherein the protein or peptide
comprises an antibody or an antigen-binding fragment thereof.
24. The composition of claim 1, wherein the protein or peptide
comprises a scFv antibody, a monoclonal antibody, an affibody, an
aptamer, a peptide, a ligand, or small targeting molecule.
25. The composition of claim 1, wherein the protein or peptide
comprises a cytokine.
26. The composition of claim 1, wherein the hydrodynamic diameter
of the nanoconstructs loaded with the protein or peptide is about
50 nm to about 100 nm.
27. The composition of claim 1, wherein the nanoconstructs further
comprise a small molecule.
28. The composition of claim 27, wherein the small molecule
comprises: a chemotherapeutic agent, an oligonucleotide, a small
molecule inhibitor, or a label.
29. The composition of claim 1, wherein the plurality of
nanoconstructs comprises a targeting agent.
30. The composition of claim 1, wherein the protein or peptide is a
targeting agent.
31. The composition of claim 1, further comprising trehalose.
32. The composition of claim 31, wherein the trehalose is from
about 1% to about 10% by weight of the plurality of
nanoconstructs.
33. The composition of claim 1, further comprising a label attached
to the nanoconstructs.
34. The composition of claim 33, wherein the label comprises at
least one of a lanthanide, a fluorescent dye, a gold nanoparticle,
a quantum dot, a PET tracer, or a MRI contrast agent.
35. A method of labeling a target, comprising contacting the
composition of claim 37 with the target under conditions to bind
the nanoconstruct to the target.
36. The method of claim 35, wherein the contacting occurs ex
vivo.
37. The method of claim 35, wherein the contacting occurs in vivo
in a subject.
38. The method of claim 35, wherein the label comprises at least
one of a lanthanide, a fluorescent dye, a gold nanoparticle, a
quantum dot, a PET tracer, or a MRI contrast agent.
39. The method of claim 35, further comprising: quantifying the
amount of target by detecting the label after the nanoconstruct
binds to the target.
40. The method of claim 35, further comprising administering the
labeled nanoconstruct to a subject and detecting the location of
the labeled nanoconstruct after the administering.
41. A method of delivering a protein or peptide to a site in a
human or other mammalian subject, the method comprising
administering an effective amount of the composition of claim 1 to
the human or other mammalian subject under conditions to deliver
the nanoconstruct to the site.
42. The method of claim 41, wherein the site is a cell.
43. The method of claim 42, wherein the nanoconstruct is
administered under conditions that the nanoconstruct is
internalized by the cell.
44. The method of claim 41, wherein the site is a tumor cell.
45. The method of claim 41, wherein the protein or peptide is
capable of modulating expression of a target protein.
46. The method of claim 41, wherein the subject is diagnosed with
cancer or diagnosed with or is at risk for fibrosis or
inflammation, and the effective amount is a therapeutically
effective amount.
47. The method of claim 41, wherein the nanoconstruct further
comprises a targeting agent.
48. The method of claim 41, wherein administration of the
composition reduces reactive oxygen species, bioavailable copper,
and/or NOX expression level in the subject or modulates an adverse
effect of one or more cytokines.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This is a continuation of co-pending U.S. patent application
Ser. No. 17/398,954, filed Aug. 10, 2021; which is a continuation
of U.S. patent application Ser. No. 15/429,971, filed Feb. 10,
2017, and issued as U.S. Pat. No. 11,207,428 on Dec. 28, 2021;
which is a continuation of PCT/US2016/022655, filed Mar. 16, 2016;
which claims priority to and the benefit of the earlier filing date
of U.S. Provisional Application No. 62/133,913, filed Mar. 16,
2015. Each of these earlier related applications is incorporated by
reference herein in its entirety.
BACKGROUND
[0003] A wide variety of molecular architectures have been
developed for the treatment of human pathologies, yet the inability
to effectively deliver such molecules to their intended biological
target has hindered their implementation as therapeutic modalities.
Oligonucleotides, proteins, antibodies and antigen-binding
fragments thereof, peptides, and small molecules alike have often
exhibited poor penetration through biological barriers, or
stability in metabolically active systems. In spite of the often
promising in vitro characteristics of compounds within these
classes, their inability to elicit therapeutic phenotypes has
inspired attempts to enhance their delivery and stability in
vivo.
[0004] For instance, RNA therapeutics represent a promising class
of compounds for modulating gene expression. RNA oligonucleotides,
such as small interfering RNA (siRNA) and micro RNA (miRNA), while
often effective in vitro, may experience short circulation
half-life and difficulty penetrating extracellular and
intracellular barriers. The same phenomenon has been observed for
peptidic therapeutics, such as small proteins, antibodies and
antigen-binding fragments thereof, and peptides, as well as a
variety of small molecules.
[0005] To address these challenges, a variety of particle-based
technologies have been developed with the aim of producing agents
capable of improving the biological delivery and stability of the
above molecules. For instance, a wide range of organic and
inorganic nanoparticle materials such as viral-capsids,
cyclodextrin, cationic polymers, gold nanoparticles, peptides (R.
Kanasty et al., 2013; D. Haussecker, 2012) and mesoporous silica
nanoparticles (MSNP) (C. Argyo et al., 2013; F. Tang et al., 2012)
have been evaluated as siRNA carriers with the potential to
increase siRNA half-life in the blood, allow escape from the
reticuloendothelial system, and enhance tumor specific cellular
uptake. However, none of the nanoconstructs have achieved a
desirable therapeutic index in clinics for treating solid tumors at
sites other than the liver.
[0006] Attempts to improve tumor accumulation have exploited
passive delivery using the enhanced permeability and retention
(EPR) effect of tumors (H. Maeda et al., 2000). It was found that
attachment of cationic polymers including
polyethylenimine-cyclodextrin (J. Shen et al., 2014) and
poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) (D. Lin et al.,
2013) increased cellular uptake of mesoporous silica nanoparticles.
While promising, significant anti-tumor activity in vivo has not
been reported for these constructs (J. Shen et al., 2014, D. Lin et
al., 2013).
[0007] There is therefore an unmet need for nanoparticle constructs
capable of improving the delivery and stability of therapeutic
compounds in vivo, such as oligonucleotides (e.g., siRNA or miRNA),
small proteins, peptides, and small molecules.
SUMMARY OF THE INVENTION
[0008] In one aspect, the invention features a nanoconstruct that
contains a cationic polymer bound to an exterior surface of a
nanoparticle, wherein the cationic polymer is cross-linked. The
nanoconstruct may further include a stabilizer bound to the
cationic polymer or nanoparticle, for instance, to prevents
aggregation of the nanoconstruct in solution. In some embodiments,
the nanoparticle is mesoporous, such as a mesoporous silica
nanoparticle. The nanoparticle may be a silica nanoparticle, a
silicon nanoparticle, an iron oxide nanoparticle, a gold
nanoparticle, a silver nanoparticle, or a carbon nanotube, e.g., a
mesoporous silica nanoparticle or an iron oxide nanoparticle.
[0009] The nanoconstruct may have a hydrodynamic diameter of from
about 10 to about 200 nm. In some embodiments, the nanoparticle has
a diameter of 5 to 90 nm. In some embodiments, the exterior surface
of the nanoparticle includes thiol, amine, carboxylate, or
phosphonate functional groups.
[0010] In some embodiments, the cationic polymer is from about 5%
to about 30% by weight of the nanoconstruct. In some embodiments,
the cationic polymer is from about 10% to about 25% by weight of
the nanoconstruct. The cationic polymer may be polyethylenimine
(PEI), chitosan, polypropyleneimine, polylysine, polyamidoamine,
poly(allylamine), poly(diallyldimethylammonium chloride),
poly(N-isopropyl acrylamide-co-acrylamide), poly(N-isopropyl
acrylamide-co-acrylic acid), diethylaminoethyl-dextran,
poly-(N-ethyl-vinylpyridinium bromide), poly(dimethylamino)ethyl
methacrylate, and/or poly(ethylene
glycol)-co-poly(trimethylaminoethylmethacrylate chloride). In some
embodiments, the polyethylenimine has a molecular weight of from
about 0.8 kDa to about 10 kDa. The cationic polymer may be
cross-linked by reacting cationic polymer on the surface of the
nanoparticle with a cross-linker in the presence of cationic
polymer in solution, e.g., to prevent or reduce aggregation of
nanoconstructs.
[0011] In some embodiments, the stabilizer is from about 1% to
about 30% by weight of the nanoconstruct. For instance, the
stabilizer may be from about 5% to about 25% by weight of the
nanoconstruct. The stabilizer may be polyethylene glycol (PEG),
dextran, polysialic acid, hyaluronic acid (HA), polyvinyl
pyrrolidone (PVP), polyvinyl alcohol (PVA), and polyacrylamide
(PAM). The polyethylene glycol may have a molecular weight of from
about 1 kDa to about 20 kDa.
[0012] In some embodiments, the nanoconstruct includes at least one
type of oligonucleotide, e.g., siRNA, miRNA, miRNA mimic, or
antisense oligomer, electrostatically bound to the cationic
polymer. In some embodiments, the at least one type of
oligonucleotide is siRNA, e.g., that targets one or more genes
selected from the group consisting of HER2, AKT1, AKT2, AKT3,
EPS8L1, GRB7, AR, Myc, VEGF, VEGF-R1, RTP801, proNGF, Keratin K6A,
Bcl-2, PLK1, LMP2, LMP7, MECL1, RRM2, PKN3, Survivin, HIF1 .alpha.,
Furin, KSP, eiF-4E, p53, .beta.-catenin, ApoB, PCSK9, HSP47, CFTR,
CTGF, SNALP, RSV nucleocapsids, CD47, PD-L1, and CTLA-4. The at
least one type of oligonucleotide may be from about 1% to about 15%
by weight of the nanoconstruct. In some embodiments, the at least
one type of oligonucleotide is from about 1% to about 5% by weight
of the nanoconstruct. The at least one type of oligonucleotide may
include two or more different siRNAs loaded onto the
nanoconstruct.
[0013] In some embodiments, the nanoconstruct further includes a
small molecule or a protein, e.g., a cytokine. In some embodiments,
the small molecule or protein is from about 0.5% to about 30% by
weight of the nanoconstruct. In some embodiments, the small
molecule is a chemotherapeutic agent, small molecule inhibitor, or
a polypeptide. In some embodiments, the small molecule is a label.
In some embodiments, the label is a lanthanide, a fluorescent dye,
a gold nanoparticle, a quantum dot, a positron emission tomography
(PET) tracer, or a magnetic resonance imaging (MRI) contrast
agent.
[0014] The nanoconstruct may also include a targeting agent, such
as an antibody, a scFv antibody, an affibody, an aptamer, a
peptide, or small targeting molecule. In some embodiments, the
targeting agent is from about 0.1% to about 10% by weight of the
nanoconstruct, e.g., from about 0.3% to about 5% by weight of the
nanoconstruct. In some embodiments, the small targeting molecule is
a carbohydrate or ligand.
[0015] In some embodiments, the nanoconstruct is lyophilized, for
instance, with a sugar, such as trehalose, or other lyoprotectant.
The sugar, e.g., trehalose, or other lyoprotectant may be from
about 1% to about 10% by weight of the nanoconstruct.
[0016] The invention also provides a pharmaceutical composition
including an effective amount of a nanoconstruct of the invention
and a pharmaceutically acceptable carrier.
[0017] In another aspect, the invention features a method of
delivering an agent to a site in a human or other mammalian
subject. The method may include administering an effective amount
of a nanoconstruct of the invention containing the agent to the
human or other mammalian subject. The administration is performed
under conditions to deliver the nanoconstruct to the site, such as
a cell or tumor. In some embodiments, the nanoconstruct is
administered under conditions that the nanoconstruct is
internalized by the cell. The nanoconstruct may be administered
subcutaneously, topically, systemically, intravesically, orally,
intratumorally, or intraperitoneally.
[0018] The subject is, for example, suffering from a disease or
condition characterized by over-expression of one or more genes
relative to expression of the one or more genes in a healthy
subject. In some embodiments, the disease or condition is AMD,
macular edema, chronic optic nerve atrophy, pachyonychia
congenital, chronic lymphocytic leukemia, metastatic lymphoma,
metastatic cancer, solid tumors, acute kidney injury, delayed graft
function, familia adenomatous polyposis, hypercholesterolemia,
liver fibrosis, cystic fibrosis, dermal scarring, Ebola infection,
RSV infection, or inflammation. In some embodiments, the
nanoconstruct is administered in an amount sufficient to treat the
subject having the disease or condition.
[0019] In some embodiments, the agent is a label, such as a
lanthanide, a gold nanoparticle, a quantum dot, a fluorescent dye,
a PET tracer, or a MRI contrast agent.
[0020] In some embodiments, the agent is a therapeutic agent, such
as a nucleic acid capable of modulating expression of a target
protein. The nucleic acid may be a siRNA, miRNA, miRNA mimic, or
antisense oligomer. In some embodiments, expression of the target
gene is reduced. In some embodiments, the therapeutic agent is a
chemotherapeutic agent, a small molecule inhibitor, an antibody, a
peptide, and/or a cytokine.
[0021] In some embodiments, the subject is diagnosed with cancer.
In some embodiments, the effective amount is a therapeutically
effective amount. In some embodiments, the cancer is resistant to a
monoclonal antibody or a small molecule inhibitor. The therapeutic
agent may be an oligonucleotide that targets expression of a
protein inhibited by the monoclonal antibody or the small molecule
inhibitor.
[0022] In some embodiments, the subject is diagnosed with or is at
risk for fibrosis or inflammation. In some embodiments, the
nanoconstruct reduces reactive oxygen species, bioavailable copper,
and/or NOX expression level in the subject. In some embodiments,
the agent is administered in an amount sufficient to reduce tumor
migration or inflammation in the human or other mammalian subject.
In some embodiments, the nanoconstruct modulates an adverse effect
of one or more cytokines.
[0023] In another aspect, the invention features a method of making
a nanoconstruct including providing a nanoparticle coated with a
cationic polymer and cross-linking the cationic polymer to make the
nanoconstruct. The nanoparticle may be a silica nanoparticle, a
silicon nanoparticle, an iron oxide nanoparticle, a gold
nanoparticle, a silver nanoparticle, or a carbon nanotube, e.g., a
mesoporous silica nanoparticle or an iron oxide nanoparticle. In
some embodiments, the cationic polymer is cross-linked in the
presence of free cationic polymer. In some embodiments, the
cationic polymer is polyethylenimine, chitosan, polypropyleneimine,
polylysine, polyamidoamine, poly(allylamine),
poly(diallyldimethylammonium chloride), poly(N-isopropyl
acrylamide-co-acrylamide), poly(N-isopropyl acrylamide-co-acrylic
acid), diethylaminoethyl-dextran, poly-(N-ethyl-vinylpyridinium
bromide), poly(dimethylamino)ethyl methacrylate, and/or
poly(ethylene glycol)-co-poly(trimethylaminoethylmethacrylate
chloride). The polyethylenimine may have a molecular weight of from
about 0.8 kDa to about 10 kDa.
[0024] The cationic polymer may be cross-linked using
dithiobis[succinimidyl propionate] (DSP),
3,3'-dithiobis(sulfosuccinimidyl propionate (DTSSP), or dimethyl
3,3'-dithiobispropionimidate (DTBP). For instance, in some
embodiments, the cationic polymer is cross-linked using DSP.
[0025] In some embodiments, the method includes attaching a
stabilizer to the nanoconstruct. The stabilizer may be selected
from the group consisting of polyethylene glycol, dextran,
polysialic acid, HA, PVP, PVA, and PAM. In some embodiments, the
polyethylene glycol has a molecular weight of from about 1 kDa to
about 20 kDa. In some embodiments, the method includes incubating
maleimide-polyethylene glycol-N-hydroxysuccinimidyl ester
(Mal-PEG-NHS) with the nanoconstruct at a weight ratio of from
about 0.5:1 to about 5:1.
[0026] In some embodiments, the method further includes attaching a
targeting agent to the nanoconstruct, e.g., to the nanoparticles,
cationic polymer, or stabilizer. The method may include admixing
the nanoconstruct with at least one type of oligonucleotide, e.g.,
a siRNA, miRNA, miRNA mimic, or antisense oligomer, that binds
noncovalently to the cationic polymer.
[0027] In some embodiments, the method includes admixing a small
molecule or protein with the nanoparticle or the nanoconstruct so
that the small molecule or protein binds to the nanoconstruct,
e.g., to the nanoparticles, cationic polymer, or stabilizer. The
small molecule or protein may be a chemotherapeutic agent, a label,
a peptide, and/or a cytokine.
[0028] In some embodiments, the method includes lyophilizing the
nanoconstruct, e.g., with a sugar, e.g., trehalose, or other
lyoprotectant.
[0029] In another aspect, the invention features a method of
labeling a target by contacting a nanoconstruct of the invention
with the target under conditions to bind the nanoconstruct to the
target. In some embodiments, the target is a cell or protein. The
nanoconstruct may be internalized by the cell. In some embodiments,
the nanoconstruct binds to the exterior of the cell. The
nanoparticle of the nanoconstruct is, for example, a silica
nanoparticle, a silicon nanoparticle, an iron oxide nanoparticle, a
gold nanoparticle, a silver nanoparticle, or a carbon nanotube,
e.g., a mesoporous silica nanoparticle or an iron oxide
nanoparticle.
[0030] In some embodiments, the label is a lanthanide, a
fluorescent dye, a gold nanoparticle, a quantum dot, a PET tracer,
or a MRI contrast agent. The method may include quantifying the
amount of target by detecting the label after the nanoconstruct
binds to the target.
[0031] In some embodiments, the method includes administering the
labeled target to a subject and detecting the location of the
target after the labeled target is administered. In some
embodiments, the nanoconstruct further includes a therapeutic
agent. In some embodiments, the detecting is by fluorescence,
magnetic resonance, or PET.
[0032] In another aspect, the invention features a multilayer
nanoconstruct that contains a mesoporous silica nanoparticle
between about 10 nm to about 90 nm in diameter and a cationic
polymer electrostatically bound to an exterior surface of the
mesoporous silica nanoparticle. The cationic polymer is
cross-linked. The nanoconstruct additionally contains a stabilizer
covalently attached to an amine of the cationic polymer, as well as
a targeting agent covalently attached to the stabilizer. The
stabilizer may prevent aggregation of the nanoconstruct in
solution.
[0033] In some embodiments, the cationic polymer is cross-linked
with a cleavable bond. In some embodiments, the mesoporous silica
nanoparticle is about 30 nm to about 60 nm in diameter. In some
embodiments, the mesoporous silica nanoparticle is an antioxidant.
In some embodiments, the hydrodynamic size of the nanoconstruct is
about 80 nm to about 200 nm, e.g., from about 90 nm to about 120
nm.
[0034] The nanoconstruct may include at least one type of
oligonucleotide electrostatically bound to the cationic polymer. In
some embodiments, the at least one type of oligonucleotide is a
siRNA, miRNA, miRNA mimic, or antisense oligomer. In some
embodiments, the mass ratio of the mesoporous silica nanoparticle
to the at least one type of oligonucleotide is about 10:1 to about
100:1. The at least one type of oligonucleotide may be a siRNA that
silences expression of PLK1, AKT1/BCL2, HER2, EPS8L1, and HSP47.
For instance, the at least one type of oligonucleotide may be
miR-342-5p.
[0035] In some embodiments, the at least one type of
oligonucleotide includes two or more different siRNAs loaded onto
the nanoconstruct, such as two or more different siRNAs loaded onto
the nanoconstruct that target different tumor genes. In some
embodiments, the two or more different siRNAs loaded onto the
nanoconstruct are selected from siPLK1, siAKT1/BCL2, siHER2,
siEPS8L1, or siHSP47.
[0036] In some embodiments, the stabilizer protects the at least
one type of oligonucleotide from serum enzymatic degradation for at
least 24 hours.
[0037] The mesoporous silica nanoparticle may be porous. For
instance, the pores may be from about 1 nm to about 6 nm in
diameter. In some embodiments, the pore has a first opening at a
first location on an exterior surface of the mesoporous silica
nanoparticle and a second different opening at a second location on
the exterior surface of the mesoporous silica nanoparticle.
[0038] In some embodiments, the nanoconstruct includes at least one
label, such as a lanthanide or fluorescent dye. In some
embodiments, the label is attached to both an inner surface of the
pore and the exterior surface of the mesoporous silica
nanoparticle. In some embodiments, the label is bound to the
cationic polymer.
[0039] The nanoconstruct may include a small molecule. In some
embodiments, the small molecule is attached on an inside of a pore.
The small molecule may be attached to the exterior surface of the
mesoporous silica nanoparticle. In some embodiments, the small
molecule is attached to the cationic polymer. In some embodiments,
the small molecule is about 0.5% to about 30% by weight of the
mesoporous silica nanoparticle. In some embodiments, the small
molecule is a chemotherapeutic agent, such as doxorubicin,
paclitaxel, docetaxel, cisplatin, carboplatin, rapamycin, or
camptothecin.
[0040] In some embodiments, the cationic polymer is about 5% to
about 40% by weight of the mesoporous silica nanoparticle. The
cationic polymer may be polyethylenimine, such as branched
polyethylenimine. In some embodiments, the polyethylenimine is
about 0.8 kDa to about 10 kDa. In some embodiments, the
polyethylenimine is about 5% to about 40% by weight of the
mesoporous silica nanoparticle.
[0041] In some embodiments, the stabilizer is between about 5 to
about 40% by weight of the mesoporous silica nanoparticle. The
stabilizer may be polyethylene glycol, such as polyethylene glycol
that is from about 1 kDa to about 20 kDa. In some embodiments, the
polyethylene glycol is about 5% to about 40% by weight of the
mesoporous silica nanoparticle.
[0042] In some embodiments, the nanoconstruct does not trigger
cytokine release from peripheral blood mononuclear cells.
[0043] In some embodiments, the targeting agent is about 0.5% to
about 10% by weight of the mesoporous silica nanoparticle. The
targeting agent may be a monoclonal antibody, a scFv antibody, an
aptamer, a peptide, or small targeting molecule agent. For
instance, the monoclonal antibody may be an anti-HER2 antibody,
anti-EGFR antibody, anti-CD20 antibody, anti-VEGF-A antibody,
anti-CD33 antibody, anti-CD52 antibody, or anti-TNF.alpha.
antibody. In some embodiments, the targeting agent is a therapeutic
agent, such as an anti-HER2 antibody.
[0044] In some embodiments, the nanoconstruct is lyophilized, e.g.,
with trehalose. In some embodiments, the trehalose is from about 5%
to about 10% by weight of the mesoporous silica nanoparticle.
[0045] In another aspect, the invention features a method of making
a nanoconstruct. The method may include combining a first
surfactant with a second different surfactant to form a first
mixture and adding a silica precursor to the first mixture to form
a second mixture. This can result in the synthesis of a mesoporous
nanoparticle. The method may include removing the first and second
surfactants from the mesoporous silica nanoparticle and coating an
exterior surface of the mesoporous silica nanoparticle with a
cationic polymer to form a mesoporous silica nanoparticle-cationic
polymer. The method may include cross-linking the cationic polymer
in the presence of free cationic polymer, conjugating a stabilizer
to an amine of the cationic polymer to form a mesoporous silica
nanoparticle-cationic polymer-stabilizer, and conjugating a
targeting agent to a maleimide group of the stabilizer to form the
nanoconstruct.
[0046] In some embodiments, the first surfactant is
cetyltrimethylammonium chloride. In some embodiments, the second
different surfactant is triethanolamine.
[0047] The first mixture may be heated prior to adding the silica
precursor. In some embodiments, the second mixture is heated prior
to recovering the mesoporous silica nanoparticles.
[0048] In some embodiments, the method includes adding
organosilanes to the second mixture after the solution is
heated.
[0049] In some embodiments, coating the cationic polymer on the
exterior surface of the mesoporous silica nanoparticle includes
mixing the cationic polymer with the mesoporous silica nanoparticle
in the presence of a solvent to form a third mixture. In some
embodiments, the mass ratio of the cationic polymer to the
mesoporous silica nanoparticle is about 1:1 to about 1:4. In some
embodiments, the cationic polymer is polyethylenimine. In some
embodiments, the polyethylenimine is about 0.8 kDa to about 10 kDa.
In some embodiments, the cationic polymer is cross-linked using DSP
(Dithiobis[succinimidyl propionate], DTSSP
(3,3'-dithiobis(sulfosuccinimidyl propionate), or DTBP (dimethyl
3,3'-dithiobispropionimidate). In some embodiments, the cationic
polymer is cross-linked using dithiobis succinimidyl
propionate.
[0050] In some embodiments, the stabilizer is added to the
mesoporous silica nanoparticle-cationic polymer in the presence of
a PBS buffer in an amount from about 5:1 to about 1:1 of the
mesoporous silica nanoparticle. In some embodiments, the stabilizer
is polyethylene glycol. In some embodiments, the polyethylene
glycol is about 1 kDa to about 20 kDa. In some embodiments, the
polyethylene glycol is about 5 kDa.
[0051] In some embodiments, the targeting agent is a monoclonal
antibody, a scFv antibody, an aptamer, a peptide, or a small
molecule targeting agent.
[0052] In some embodiments, the method includes admixing a
mesoporous silica nanoparticle-cationic
polymer-stabilizer-targeting agent construct with at least one type
of oligonucleotide.
[0053] In some embodiments, the at least one type of
oligonucleotide electrostatically binds to the nanoconstruct.
[0054] In some embodiments, the at least one type of
oligonucleotide is siRNA, miRNA, miRNA mimics, DNA, or an antisense
oligomer. In some embodiments, the at least one type of
oligonucleotide is siPLK1, siAKT1/BCL2, siHER2, siEPS8L1, or
siHSP47. In some embodiments, the at least one type of
oligonucleotide is miR-342-5p. In some embodiments, the
oligonucleotide is admixed with the mesoporous silica
nanoparticle-cationic polymer-stabilizer-targeting agent construct
at a mass ratio of nanoparticle per oligonucleotide of about 10:1
to about 100:1. In some embodiments, the at least one type of
oligonucleotide is siRNA. In some embodiments, the siRNA is against
HER2 (siHER2). In some embodiments, the siRNA is admixed with the
mesoporous silica nanoparticle-cationic
polymer-stabilizer-targeting agent construct at a mass ratio of
nanoparticle per oligonucleotide of about 25:1 to about 50:1.
[0055] In some embodiments, the method includes combining the first
mixture with a small molecule prior to coating the mesoporous
silica nanoparticle with the cationic polymer. In other
embodiments, the method includes combining the first mixture with a
small molecule after coating the mesoporous silica nanoparticle
with the cationic polymer. In some embodiments, the small molecule
is a chemotherapeutic agent. In some embodiments, the
chemotherapeutic agent is doxorubicin, paclitaxel, docetaxel,
cisplatin, carboplatin, rapamycin, or camptothecin.
[0056] In some embodiments, the method includes admixing at least
one label. In some embodiments, the label is added to the first
mixture. In some embodiments, the label is added to the mesoporous
silica nanoparticle. In some embodiments, the label is added to the
mesoporous silica nanoparticle-cationic polymer. In some
embodiments, the label is added to the mesoporous silica
nanoparticle-cationic polymer-stabilizer. In some embodiments, the
label is a lanthanide. In other embodiments, the label is a
fluorescent dye. In some embodiments, the fluorescent dye is
conjugated to both an inner surface of the pore and the exterior
surface of the mesoporous silica nanoparticle using an amine-NHS
ester reaction.
[0057] In some embodiments, the method includes lyophilizing the
nanoconstruct, e.g., with trehalose. The amount of the trehalose
may be 1-10% by weight of the mesoporous silica nanoparticle.
[0058] In another aspect, the invention features a method of
treating cancer in a human or other mammalian subject by
administering an effective amount of a nanoconstruct that contains
a mesoporous silica nanoparticle between about 30 nm to about 90 nm
in diameter and a cross-linked cationic polymer electrostatically
bound to an exterior surface of the mesoporous silica nanoparticle.
The nanoconstruct may contain a stabilizer covalently attached to
an amine of the cationic polymer, a targeting agent covalently
attached to the stabilizer, and at least one type of
oligonucleotide electrostatically bound to the cationic polymer on
the exterior surface of the mesoporous silica nanoparticle. The at
least one type of oligonucleotide may be protected by the
stabilizer, e.g., from enzymatic degradation.
[0059] In some embodiments, the cancer is resistant to a monoclonal
antibody, and the at least one type of oligonucleotide may target
the same gene as the monoclonal antibody. In some embodiments, the
cancer is resistant to a small molecule inhibitor, and the at least
one type of oligonucleotide may target the same gene as the small
molecule inhibitor. In some embodiments, the cancer is HER2+, and
may be resistant to trastuzumab and/or lapatinib. In some
embodiments, one dose of the nanoconstruct reduces the HER2 protein
levels by at least 40%. In some embodiments, the at least one type
of oligonucleotide can target two or more genes. In some
embodiments, the at least one type of oligonucleotide is a siRNA
duplex against both AKT1 and BCL2.
[0060] In some embodiments, the mesoporous silica nanoparticle is
an antioxidant. The mesoporous silica nanoparticle may be
therapeutic. In some embodiments, the at least one type of
oligonucleotide is a siRNA, miRNA, miRNA mimic, or antisense
oligomer. For instance, delivery of the siRNA on the nanoconstruct
may increase the rate of cancer cell death by at least 15% over
delivery of the siRNA with a transfection reagent.
[0061] In some embodiments, the nanoconstruct includes a
chemotherapeutic agent. In some embodiments, the addition of the
chemotherapeutic agent does not negatively impact the gene
silencing efficacy of the at least one type of oligonucleotide. In
some embodiments, co-delivery of the siRNA and the chemotherapeutic
agent improves cancer cell death caused by chemotherapeutic agents
by at least 10%.
[0062] In another aspect, the invention features a method of
diagnosing cancer in a mammalian subject. The method includes
administering a nanoconstruct that contains a porous mesoporous
silica nanoparticle between about 10 nm to about 80 nm in diameter
and a cross-linked cationic polymer electrostatically bound to an
exterior surface of the mesoporous silica nanoparticle. The
nanoconstruct may contain a stabilizer covalently attached to an
amine of the cationic polymer, a targeting agent covalently
attached to the stabilizer, and a label attached to the
nanoconstruct. The use of the label allows imaging of tumors and
quantification of tumor proteins targeted by the targeting
agent.
[0063] In some embodiments, the exterior surface of the mesoporous
silica nanoparticle includes thiol, amine, or phosphonate
functional groups. The label, e.g., a fluorescent dye or a
lanthanide, may be attached to functional groups on the surface of
the mesoporous silica nanoparticle.
[0064] In some embodiments, the nanoconstruct enables tumor
detection by MRI. In some embodiments, the label is gadolinium. The
label may be attached on both an inner surface of the pore in the
mesoporous silica nanoparticle and on the exterior surface of the
mesoporous silica nanoparticle. In some embodiments, the label is
attached to the cationic polymer electrostatically bound to the
exterior surface of the mesoporous silica nanoparticle. In some
embodiments, the targeting agent is a monoclonal antibody, a scFv
antibody, an aptamer, a peptide, or a small targeting molecule
agent. In some embodiments, the targeting agent is a monoclonal
antibody. In some embodiments, the targeting agent is an anti-HER2
antibody, anti-EGFR antibody, anti-CD20 antibody, anti-VEGF-A
antibody, anti-CD33 antibody, anti-CD52 antibody, or
anti-TNF.alpha. antibody.
[0065] In another aspect, the invention features a method of
characterizing targeted protein in a tissue specimen. The method
includes staining the targeted protein with a nanoconstruct
containing a mesoporous silica nanoparticle between about 10 nm to
about 80 nm in diameter and a cross-linked a cationic polymer
electrostatically bound to an exterior surface of the mesoporous
silica nanoparticle. The nanoconstruct may contain a stabilizer
covalently attached to an amine of the cationic polymer, a
targeting agent covalently attached to the stabilizer, and a label
attached to the mesoporous silica nanoparticle. The label allows an
amount of the targeted protein to be quantified, e.g., by
imaging.
[0066] In some embodiments, the exterior surface of the mesoporous
silica nanoparticle includes thiol, amine, or phosphonate
functional groups. The label may be attached to the functional
groups on the exterior surface of the mesoporous silica
nanoparticle. In some embodiments, the label is attached to the
cationic polymer on the exterior surface of the mesoporous silica
nanoparticle. In some embodiments, the label is lanthanide, such as
gadolinium, or a fluorescent dye. The targeted protein may be
quantified by mass spectrometry.
[0067] In another aspect, the invention features a method of
treating cancer, inflammation, and fibrosis in a human or other
mammalian subject. The method includes administering an effective
amount of a nanoconstruct containing a porous mesoporous silica
nanoparticle between about 30 nm to about 90 nm in diameter and a
cross-linked cationic polymer electrostatically bound to an
exterior surface of the mesoporous silica nanoparticle. The
nanoconstruct contains a stabilizer covalently attached to an amine
of the cationic polymer and at least one type of oligonucleotide
electrostatically bound to the cationic polymer on the exterior
surface of the mesoporous silica nanoparticle via electrostatic
interaction. The at least one type of oligonucleotide may be
protected by the stabilizer, e.g., from enzymatic degradation.
[0068] The nanoconstruct may be an antioxidant. In some
embodiments, the nanoconstruct reduces reactive oxygen species and
NOX4 expression level. The nanoconstruct may modulate the adverse
effect of cytokines. In some embodiments, use of the nanoconstruct
decreases a fibrotic marker. In some embodiments, the fibrotic
marker is COL I or alpha-SMA. The at least one type of
oligonucleotide may be a siRNA, miRNA, miRNA mimic, or an antisense
oligomer, such as a siRNA against HSP47 (siHSP47).
[0069] In some embodiments, the mesoporous silica nanoparticle
carrier is therapeutic. The nanoconstruct may include a small
molecule inhibitor selected from dasatinib, imatinib, and
nilotinib.
[0070] In some embodiments, the small molecule inhibitor is
attached to both an inner surface of the pore in the mesoporous
silica nanoparticle and the exterior surface of the mesoporous
silica nanoparticle. The small molecule inhibitor may be attached
to the cationic polymer. In some embodiments, the nanoconstruct
delivers the small molecule inhibitor to target cells. In some
embodiments, the nanoconstruct is administered subcutaneously. In
some embodiments, the nanoconstruct is administered topically.
[0071] For any of the above aspects, the nanoconstruct may include
a stabilizer, e.g., PEG, and a targeting agent, e.g., an antibody.
In these embodiments, the nanoparticles is, for example, mesoporous
silica or iron oxide, and cationic polymer is, for example, PEI.
Such nanoconstructs may further include an oligonucleotide, a small
molecule, and/or a label.
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] FIGS. 1A-1C show (FIG. 1A) TEM images of mesoporous silica
nanoparticle (MSNP) cores of various sizes (FIG. 1B) schematic of
the surface modification of the MSNP, and (FIG. 1C) the
hydrodynamic size distribution of the nanoconstructs made from MSNP
cores shown in (FIG. 1A) and surface modified as illustrated in
(FIG. 1B). Unless specified otherwise, MSNP was a S-47 core,
polyethylenimine (PEI) was 10 kDa and cross-linked, PEG was 5 kDa,
and antibody was trastuzumab (T) throughout this application.
[0073] FIGS. 2A-2B show (FIG. 2A) the hydrodynamic size profile of
MSNP modified as in FIG. 1 and synthesized under different
crosslinking conditions and (FIG. 2B) a graph depicting the
buffering capacity of three nanoconstructs with cross-linked
1.8-kDa PEI (T-NP.sup.1.8C), non-cross-linked 10-kDa PEI
(T-NP.sup.10), and cross-linked 10-kDa PEI (T-NP.sup.10C) measured
in 150 mM NaCl. The cross-linking condition was "0.1 mg/ml DSP+2 mg
PEI" as shown in FIG. 2A.
[0074] FIGS. 3A-3B show (FIG. 3A) the luciferase silencing efficacy
of nanoconstructs made from 5-kDa PEG but with varied PEG loading
conditions; 1:1 mass ratio of Mal-PEG-NHS:MSNP by adding
Mal-PEG-NHS as dry powder directly to the MSNP-PEI suspension in
PBS and stirring for 90-120 min or 5:1 mass ratio by first
dissolving Mal-PEG-NHS in PBS prior to overnight mixing with
MSNP-PEI suspension in PBS, and (FIG. 3B) the hydrodynamic size
profile of MSNP modified as in FIG. 1B with PEG of various
molecular weights (loaded as dry Mal-PEG-NHS at 1:1 mass ratio)
[0075] FIGS. 4A-4B are a series of charts showing the impact of
varied mesoporous silica nanoparticle (MSNP) per trastuzumab mass
ratio during synthesis on (FIG. 4A) cell viability, and (FIG. 4B)
cellular uptake by BT474 cells.
[0076] FIGS. 5A-5B are charts depicting the silencing of luciferase
in LM2-4luc+/H2N upon treatment with 30 nM siLUC on
trastuzumab-conjugated nanoconstruct (T-NP) at a NP/siRNA mass
ratio of (FIG. 5A) 25 and (FIG. 5B) 50 measured at 48 hours
post-transfection.
[0077] FIGS. 6A-6E show (FIG. 6A) the amount of intact siHER2 that
survived enzymatic degradation in human serum as measured by gel
electrophoresis, (FIG. 6B) quantification of corresponding intact
siHER2, (FIG. 6C) the effect of PEG on siRNA protection from the
enzymatic degradation (vs. MSNP-PEI and free siRNA), (FIG. 6D) the
effect of PEG on preventing nanoconstructs from aggregating into
larger sizes upon siRNA loading, and (FIG. 6E) nanoconstructs
produced by an optimized PEG loading condition. Method optimization
has been performed to reduce the amount of Mal-PEG-NHS usage from a
5:1 Mal-PEG-NHS:MSNP mass ratio to 1:1 as shown in FIG. 3A; the
resulting material could still protect siRNA (FIG. 6E) in the same
manner as that using a higher PEG ratio (5:1) (FIGS. 6A-6B).
[0078] FIGS. 7A-7B shows that (FIG. 7A) nanoconstruct (T-siRNA-NP)
has much smaller (desirable) hydrodynamic size and (FIG. 7B) better
silencing efficacy than the PEI-siRNA polyplex counterparts
(without MSNP core). N/P ratio is defined as the molar ratio of
polymer nitrogen (N) to oligonucleotide phosphate (P).
[0079] FIGS. 8A-8I show the cellular uptake of siSCR-NP by various
cell lines. FIG. 8A is a chart illustrating the % cellular uptake
of trastuzumab(T)-siSCR-NP by BT474 breast cancer cells (HER2+).
FIG. 8B is a chart illustrating the % cellular uptake of
trastuzumab(T)-siSCR-NP by SKBR3 breast cancer cells (HER2+). FIG.
8C is a chart illustrating the % cellular uptake of
trastuzumab(T)-siSCR-NP by MCF7 (HER2-) breast cancer cells. A
negative control antibody, rituximab (R), demonstrated specificity
for the trastuzumab-conjugated nanoconstruct counterpart. FIG. 8D
shows a western blot confirming the HER2 content of these 3 cell
lines. FIG. 8E shows the extent of cellular internalization of
dye-tagged siSCR nanoconstructs by BT474 cells. FIG. 8F shows the
extent of cellular internalization of dye-tagged siSCR
nanoconstructs by SKBR3 cells. FIG. 8G shows the extent of cellular
internalization of dye-tagged siSCR nanoconstructs by MCF7 cells.
FIGS. 8H-8I shows that when changing from trastuzumab to a
different antibody (e.g. anti-EGFR-antibody or cetuximab or "C"),
the material (C-siSCR-NP.sup.10C) could target (H) cells with high
EGFR expression. FIG. 8I shows higher cellular uptake of
C-siSCR-NP.sup.10C nanoconstructs by EGFR+ cells as compared to
cells with no EGFR expression.
[0080] FIGS. 9A-9D show the HER2 silencing efficacy and cancer cell
killing properties of siHER2 nanoconstructs. FIG. 9A is a graph
depicting HER2 knockdown by siHER2-nanoconstructs (T-NP.sup.10c) as
reduced HER2 protein expression per cell of three HER2+ cells at 72
hours post-treatment with siHER2 vs. siSCR (60 nM) on T-NP.sup.10c.
FIG. 9B is a graph showing reduced HER2 expression at the mRNA
level (48 hours post-treatment with siHER2 vs. siSCR), FIG. 9C is a
graph showing the functional outcome of increased apoptotic
activity (4 days post-treatment with siHER2 vs. siSCR). FIG. 9D is
a graph showing reduced cell viability of BT474 cells (4 days
post-treatment with siHER2 vs. siSCR).
[0081] FIG. 10 shows that T-siHER2-NP.sup.10C treatment resulted in
lower viability of HER2+ breast cancer cells but had little impact
on HER2- breast cells and non-breast cells (4 days post
transfection); with inset showing HER2 levels of the cells as
measured by Western blot.
[0082] FIG. 11 shows reduced HER2 protein expression (by
immunofluorescent staining) in BT474 upon treatment with
T-siSCR-NP.sup.10C (due to trastuzumab) and T-siHER2-NP.sup.10C
(due to siHER2 and trastuzumab). #1-4 represent replicates.
[0083] FIGS. 12A-12C show HER2 silencing efficacy of
siHER2-nanoconstructs. FIG. 12A is a graph showing HER2 protein
reduction in three HER2+ cell lines upon treatment with 60 nM
siHER2 vs. siSCR on nanoconstructs with cross-linked 1.8-kDa PEI.
FIG. 12B is a graph showing HER2 protein reduction in three
HER2.sup.+ cell lines upon treatment with 120 nM siHER2 vs. siSCR
on nanoconstructs with cross-linked 1.8-kDa PEI. FIG. 12C is a
graph showing HER2 protein reduction in three HER2+ cell lines upon
treatment with 60 nM siHER2 vs. siSCR on the commercial
transfection agent, DharmaFECT.TM.. The same treatment condition
was utilized as that used in FIG. 9A.
[0084] FIGS. 13A-13C show the effect of trastuzumab and
siHER2-nanoconstructs on cell viability. FIG. 13A is a graph
showing BT474-R to be resistant to trastuzumab, compared to
parental BT474. FIG. 13B is a graph showing the ability of
T-siHER2-NP.sup.10C to achieve the same response in BT474-R and
BT474 with respect to siHER2 action, while FIG. 13C is a graph
showing that trastuzumab on a nanoconstruct without siHER2
(T-siSCR-NP.sup.10C) elicits less of a response in the resistant
BT474-R cells. 60 nM siRNA was used, and cell viability was
measured 5 days post-treatment with media replenished overnight
after treatment.
[0085] FIGS. 14A-14C show the blood compatibility of
siHER2-nanoconstructs. FIG. 14A shows excellent blood compatibility
of siHER2-nanoconstructs (T-siHER2-NP.sup.1.8C and
T-siHER2-NP.sup.10C) with no significant increase in hemolysis over
vehicle controls (saline and PBS) or an FDA-approved
nanoparticle-drug benchmark (Abraxane). FIG. 14B is a graph showing
no significant increase in clotting time over vehicle controls
(saline and PBS) or FDA-approved nanoparticle-drug benchmarks
(Abraxane or Feraheme). FIG. 14C is a graph showing no significant
increase in platelet aggregation over vehicle controls (saline and
PBS) or FDA-approved nanoparticle-drug benchmarks (Abraxane or
Feraheme). 1.times. is anticipated human blood level, and 5.times.
is 5-fold of that level.
[0086] FIGS. 15A-15F show cytokine induction in peripheral blood
mononuclear cells following nanoconstruct treatment and endotoxin
test of the nanoconstruct. FIG. 15A is a chart showing IFN-.alpha.
induction in peripheral blood mononuclear cells following 24-hour
exposure with T-siHER2-NP.sup.1.8C, T-NP.sup.10C,
T-siHER2-NP.sup.10C, Abraxane, or Feraheme. FIG. 15B is a chart
showing IL-1.beta. induction in peripheral blood mononuclear cells
following 24-hour exposure with T-siHER2-NP.sup.1.8C, T-NP.sup.10C,
T-siHER2-NP.sup.10C, Abraxane, or Feraheme. FIG. 15C is a chart
showing IL-6 induction in peripheral blood mononuclear cells
following 24-hour exposure with T-siHER2-NP.sup.1.8C, T-NP.sup.10C,
T-siHER2-NP.sup.10C, Abraxane, or Feraheme. FIG. 15D is a chart
showing TNF-.alpha. induction in peripheral blood mononuclear cells
following 24-hour exposure with T-siHER2-NP.sup.1.8C, T-NP.sup.10C,
T-siHER2-NP1c1c, Abraxane, or Feraheme. FIG. 15E is an image
recorded from the LAL gel-clot assay on the nanoconstructs
(T-siHER2-NP.sup.10C) at 5.times. concentration. FIG. 15F is an
image recorded from the LAL gel-clot assay on Abraxane at 5.times.
concentration. Both are negative for endotoxin according to the
manufacturer's protocol.
[0087] FIGS. 16A-16B show representative immunofluorescence images
of HCC1954 tumor tissues collected from mice (n=4/group) at 4 days
post i.v. injection with one dose of T-NP.sup.10C loaded with
siHER2 or siSCR (1.25 mg/kg siRNA, NP/siRNA of 50) or PBS control
(A) and quantitative HER2 levels of the tumor tissues (B).
[0088] FIGS. 17A-17D are a series of graphs showing tumor growth in
mice bearing tumor xenografts following nanoconstruct treatment.
FIG. 17A is a graph showing tumor growth in mice bearing orthotopic
HCC1954 tumor xenografts receiving multiple doses (i.v., time
points indicated by arrows) of T-NP.sup.10C loaded with siHER2 or
siSCR (1.25 mg/kg siRNA, NP/siRNA of 50) or PBS control
(n=5/group). FIG. 17B is a graph showing tumor growth in mice
bearing orthotopic HCC1954 tumor xenografts receiving multiple
doses of trastuzumab (10 mg/kg, i.p.) or saline (n=7/group) at time
points indicated by arrows. FIG. 17C is a graph showing tumor
growth in mice bearing orthotopic HCC1954 tumor xenografts
receiving multiple doses of trastuzumab (5 mg/kg, i.v.) or saline
(n=5/group) at time points indicated by arrows. FIG. 17D is a graph
showing tumor growth in mice bearing orthotopic HCC1954 tumor
xenografts receiving multiple doses of trastuzumab (5 mg/kg, i.v.)
plus paclitaxel (3.1 mg/kg, i.v.) or saline (n=9/group) at time
points indicated by arrows.
[0089] FIG. 18 is a graph showing tumor growth in mice bearing
orthotopic HCC1954 tumor xenografts (n=11/group) post i.v.
injection with multiple doses of T-NP.sup.10 (O-87 core, not
cross-linked) loaded with siHER2 or siSCR (2.5 mg/kg siRNA,
NP/siRNA of 50) or PBS control at the time points indicated by the
arrows.
[0090] FIG. 19 is a graph depicting tumor growth in mice bearing
orthotopic HCC1954 xenografts treated with siHER2-NP.sup.10C (no
antibody) (n=8), T-siSCR-NP.sup.10C (n=7), or saline control (n=5)
with the arrows indicating the days of i.v. injection. siHER2 or
siSCR dose of 1.25 mg/kg and NP/siRNA ratio of 50.
[0091] FIGS. 20A-20B show tumor growth in mice bearing
trastuzumab-resistant tumor xenografts following nanoconstruct
treatment. FIG. 20A is a graph showing BT474-TRgf to be resistant
to trastuzumab in vitro compared to parental BT474. FIG. 20B is a
graph showing tumor growth in mice bearing BT474-TRgf xenografts
(n=5-7/group) post i.v. injection with saline, trastuzumab (2.5
mg/kg, twice weekly) or trastuzumab-conjugated nanoconstruct (T-NP)
loaded with siHER2 or siSCR at the time points indicated by arrows.
Black indicates 1.25 mg siRNA/kg, and gray indicates 2.5 mg
siRNA/kg.
[0092] FIGS. 21A-21E show the purity and specificity of HER2 scFV,
and size distribution, and luciferase silencing efficacy of
nanoconstructs containing HER2 scFV, trastuzumab (T), or folic acid
(FA). FIG. 21A shows a gel indicating comparable purity of three
batches of HER2 scFv. FIG. 21B is a diagram confirming the presence
of the HER2 scFv using Anti-6.times.HisTag antibody. FIG. 21C shows
specificity of HER2 scFv for HER2+ cells (BT474, SKBR3, and JIMT1)
over HER2- cells (MCF7 and MDAMB468). FIG. 21D shows the
hydrodynamic sizes of HER2 scFV conjugated NP (scFV-NP), folic acid
conjugated NP (FA-NP), and trastuzumab-conjugated NP (T-NP), all
loaded with same content of siHER2. FIG. 21E shows the luciferase
silencing efficacy of nanoconstructs having three different
targeting agents as in FIG. 21D. For HER2 scFV loading, 1 and 4% by
weight of MSNP was used during synthesis. For FA, 25% by weight as
Mal-PEG(5 kDa)-FA was used during synthesis.
[0093] FIGS. 22A-22D show the size, zeta potential, luciferase
silencing efficacy and cell viability effects of lyophilized and
freshly prepared nanoconstructs. FIG. 22A is a chart showing the
hydrodynamic diameter of lyophilized nanoconstructs (NP or T-NP)
with varied amounts of trehalose (TL) compared to freshly made
nanoconstructs (Fresh) from the same batch. FIG. 22B is a chart
showing the zeta potential of lyophilized nanoconstructs (NP or
T-NP) with varied amounts of trehalose (TL) compared to freshly
made nanoconstructs (Fresh) from the same batch. FIG. 22C is a
chart showing the silencing efficacy of lyophilized nanoconstructs
(NP or T-NP) with varied amounts of trehalose (TL) compared to
freshly made nanoconstructs (Fresh) from the same batch. FIG. 22D
is a chart showing the cancer cell-killing efficacy of lyophilized
nanoconstructs (NP or T-NP) with varied amounts of trehalose (TL)
compared to freshly made nanoconstructs (Fresh) from the same
batch. Data indicate 5% TL (by weight of nanoconstruct) as the best
condition for preserving all properties of the fresh material.
(A-B) no siRNA, (C) with 30 nM siLUC vs. siSCR, (D) with 60 nM
siHER2 vs. siSCR.
[0094] FIGS. 23A-23E show the size, zeta potential, siRNA loading
properties, luciferase silencing efficacy, and cancer cell-killing
properties of lyophilized trastuzumab-conjugated nanoconstructs
(T-NP) stored at various temperatures and time relative to those of
freshly prepared nanoconstructs from the same batch. FIG. 23A is a
chart showing the relative hydrodynamic diameter of lyophilized
T-NP with 5% trehalose (TL) and stored at various temperatures.
FIG. 23B is a chart showing the relative zeta potential of
lyophilized T-NP with 5% trehalose (TL) and stored at various
temperatures. FIG. 23C is a chart showing relative siRNA loading of
lyophilized T-NP with 5% trehalose (TL) and stored at various
temperatures. FIG. 23D is a chart showing relative silencing
efficacy of lyophilized T-NP with 5% trehalose (TL) and stored at
various temperatures. FIG. 23E is a chart showing relative cancer
cell-killing efficacy of lyophilized T-NP with 5% trehalose (TL)
and stored at various temperatures. Data indicate -20.degree. C. as
the best storage temperature for preserving all properties of the
fresh material for at least 24 weeks (6 months). (A-D): 30 nM siLUC
and siLUC/NP of 50; (E): 60 nM siHER2 and siHER2/NP of 50.
[0095] FIGS. 24A-24B are charts showing the viability of BT474 in
response to 30 nM (each) of various siRNA treatments (A) and
confirming protein knockdown in HCC38 cells by the dual-targeting
siRNA (B) (siAKT1/BCL2; one strand targets AKT1 gene, and the other
targets BCL2 gene).
[0096] FIG. 25 is a series of graphs depicting dose response
evaluation four days post-treatment with 30 nM siRNA targeting
HER2, PLK1, and AKT1/BCL2 in BT474, HCC1569, HCC1954, and JIMT1
cell lines.
[0097] FIG. 26 is a graph depicting the effect of siRNA targeting
of HER2, PLK1, and AKT1/BCL2 (delivered with commercial
transfection agent, DharmaFECT.TM.) on the viability of different
breast cancer subtypes and non-target organ cells.
[0098] FIGS. 27A-27B are graphs showing enhanced treatment
specificity to HER2+ breast cancer cell (HCC1954) compared to a
non-tumorigenic breast epithelial cell line (MCF10A) with siPLK1 or
miR342-5p delivered by the trastuzumab-conjugated nanoconstruct
(T-NP.sup.10C) (A), as well as the effect of delivery of siPLK1 or
miR342-5p by commercial DharmaFECT.TM., which exhibit poorer
treatment specificity to cancer cells over non-tumorigenic cells.
All experiments performed with 30 nM siRNA or miRNA (B).
[0099] FIGS. 28A-28B are charts showing the ability of
nanoconstructs to load and deliver GALA pore-forming peptide, which
enhances endosomal escape of the siRNA-nanoconstruct, promoting
luciferase silencing activity (A), as well as the effect of
co-delivery of trastuzumab (T), paclitaxel (PTX), and siRNA
(siHER2) loaded on nanoconstructs and administered to JIMT1 cancer
cells (B). (A): 15 nM siRNA, NP/siRNA 50, activity measured 2 days
post-treatment. (B): 30 nM siRNA, NP/siRNA 50, viability measured 5
days post-treatment.
[0100] FIG. 29 is a chart showing that the loading of
chemotherapeutics (paclitaxel, PTX, or doxorubicin, DOX) on the
nanoconstructs did not significantly impair silencing efficacy of
siRNA against luciferase (siLUC) (30 nM siRNA, NP/siRNA 50,
activity measured 5 days post-treatment).
[0101] FIG. 30 is a chart showing the HCC1954 tumor growth
inhibition effect of nanoconstructs loaded with both siHER2 and
paclitaxel (T-siHER2-NP(PTX)) over those loaded with siSCR and
paclitaxel (T-siSCR-NP(PTX)) or free drug counterparts
(trastuzumab+paclitaxel). Arrows indicate injections (1.25 mg
siRNA/kg, NP/siRNA 50).
[0102] FIGS. 31A-31D. FIGS. 31A-31B are a series of graphs showing
nanoconstructs containing PEI and PEG (MSNP-PEI-PEG and loaded with
non-targeting siSCR) could reduce intracellular ROS activity of
primary dermal fibroblast with a potency similar to that of NAC
(A), as well as the antioxidant properties (DPPH scavenging) of the
material (measured in a cell free system) were attributed to the
MSNP core rather than PEI or PEG (B). FIG. 31C is a graph showing
that the nanoconstructs could reduce protein expressions of
pro-fibrotic genes (HSP47, NOX4) and fibrotic markers (COL I and
alpha-SMA) without harming cells in an in vitro fibrosis model
(TGF-beta stimulated dermal fibroblast). FIG. 31D is a graph
showing that the nanoconstructs could reduce mRNA levels of NOX4,
COL I and alpha-SMA in a second in vitro fibrosis model (bleomycin
treated fibroblast). Data from Morry et al. 2015.
[0103] FIGS. 32A-32G show the experimental design and results of
assays conducted to probe the ability of nanoconstructs to treat
skin fibrotic disease. FIG. 32A is a schematic showing the study
design of a series of experiments aimed at determining the
effectiveness of siHSP47-nanoconstruct (MSNP-PEI-PEG) for treating
skin fibrotic disease in vivo (bleomycin stimulated skin fibrosis
in mice). FIG. 32B is a series of representative images showing
that the siHSP47-NP could reduce dermal thickening of mice caused
by bleomycin treatment. FIG. 32C is a chart showing the ability of
siHSP47-NP to reduce dermal thickening of mice caused by bleomycin
treatment. FIG. 32D is a chart showing the ability of siHSP47-NP to
reduce the expression of the fibrotic marker HSP47. FIG. 32E is a
chart showing the ability of siHSP47-NP to reduce the expression of
the fibrotic marker NOX4. FIG. 32F is a chart showing the ability
of siHSP47-NP to reduce the expression of the fibrotic marker
alpha-SMA. FIG. 32G is a chart showing the ability of siHSP47-NP to
reduce the expression of the fibrotic marker COL I. Some (albeit
less) reduction effects were observed with siSCR-NP due to the
antioxidant properties of the MSNP core.
[0104] FIGS. 33A-33D are graphs showing the cellular effects of
siPLK1-NP treatment. FIG. 33A is a graph showing that siPLK1-NP
treatment of LM2-4luc+/H2N cells could reduce PLK1 expression at
the mRNA level. FIG. 33B is a graph showing that siPLK1-NP
treatment can reduce PLK1 protein expression at the protein level.
FIG. 33C is a graph showing the ability of siPLK1-NP to increase
G2/M cell cycle phase distribution with a potency similar to that
of PLK1 inhibitor (BI2536). FIG. 33D is a graph showing the ability
of siPLK1-NP to decrease cancer cell viability. siRNA dose of 50 nM
and NP/siRNA of 50, BI2536 dose of 10 nM, treatment time: 24 hr for
(FIG. 33A), 48 hr for (FIG. 33B), 24 hr for (FIG. 33C) and 5 days
for (FIG. 33D).
[0105] FIGS. 34A-34H show the effect of nanoconstruct treatment on
LM2-4luc+H2N cancer cells. FIG. 34A is a graph showing that the
nanoconstruct treatment of LM2-4luc+/H2N cancer cells could reduce
intracellular ROS level with a potency similar to that of 2 mM NAC
and 5 .mu.M DPI. FIG. 34B is a graph showing that nanoconstruct
treatment of LM2-4luc+/H2N cancer cells does not cause cell death
at the concentrations used. FIG. 34C is a graph showing the ability
of nanoconstruct treatment of LM2-luc+/H2N cancer cells to reduce
NOX4 mRNA expression with a potency similar to that of 20 mM NAC.
FIG. 34D is a series of images showing that the nanoconstructs
(loaded with siSCR, DY677-siSCR, or siPLK1) could reduce cancer
cell migration in wound healing assays. FIG. 34E is a chart showing
that nanoconstructs loaded with siSCR or siPLK1 could reduce cancer
cell migration more effectively than siRNA delivered with
DharmaFECT.TM.. FIG. 34F is a series of images showing that the
nanoconstructs (loaded with siSCR) could inhibit cancer cell
invasion in FITC-gelatin degradation assays with a potency similar
to that of 5 .mu.M DPI. FIG. 34G is a chart showing that
nanoconstructs loaded with siSCR could inhibit cancer cell invasion
in FITC-gelatin degradation assays with a potency similar to that
of 5 .mu.M DPI. FIG. 34H is a chart showing that nanoconstructs
loaded with siSCR could inhibit cancer cell invasion in
Matrigel-coated Boyden chamber assays with a potency similar to the
DPI but greater than that of 2-10 mM NAC. A siRNA dose of 50 nM and
a NP/siRNA ratio of 50 by mass were used throughout.
[0106] FIGS. 35A-35H show the experimental design and results of
assays conducted to determine the therapeutic effects of
trastuzumab-conjugated nanoconstructs and loaded with siPLK1
(T-siPLK1-NP) in a metastatic cancer mouse model. FIG. 35A is a
schematic showing the design of short and long term studies aimed
at understanding whether T-siPLK1-NP can treat metastatic HER2+
breast cancer (LM2-4luc+/H2N) in vivo. FIG. 35B is a chart showing
the results of the Short-term study (n=5/group). T-siPLK1-NP could
reduce the incidence rate of cancer in various organs of mice and
total tumor burden after 6 doses of treatment. Some reduction of
the incidence rate was also observed with T-siSCR-NP. FIG. 35C is a
chart showing that T-siPLK1-NP could also reduce in vivo imaging
signals (IVIS) of the cancer in the thorax region (lung) of the
mice. FIG. 35D is a graph showing that T-siPLK1-NP could reduce
lung weight of mice bearing cancer to the level similar to that of
normal mice (without cancer). FIG. 35E is a graph showing that
T-siPLK1-NP could reduce PLK1 mRNA expression levels of the human
cancer presiding in the lungs of mice. Long-term study (n=8/group):
FIG. 35F is a chart showing that T-siPLK1-NP could reduce IVIS
signals of the cancer in the thorax region (lung) of the mice. FIG.
35G is a chart showing the extended survival of mice from the same
study due to T-siPLK1-NP treatment. FIG. 35H is a series of images
showing the ability of T-siPLK1-NP to reduce cancer lesions in the
lung tissues as shown by representative H&E images and
anti-human vimentin staining. Treatments as specified; vertical
lines in (C) and (F) represent dosing days.
[0107] FIG. 36 shows ceruloplasmin activity (a biomarker of
bioavailable copper) of the serum from the short-term study in
FIGS. 35A-35H (n=5/group, serum collected at sacrifice). "Saline"
represent to tumor mice with saline treatment; "Normal" represent
normal mice without tumors.
[0108] FIGS. 37A-37B show a series of lanthanides that were loaded
inside the pores of MSNP nanoparticles in various amounts (FIG.
37A), as well as a series of fluorescence micrographs demonstrating
specific staining of HER2+ cells and not HER2- cells with T-NPs
containing Dylight550 (FIG. 37B).
[0109] FIGS. 38A-38C show the size, luciferase silencing efficacy,
and T2 relaxivity of iron oxide-containing nanoconstructs. FIG. 38A
shows the hydrodynamic size distribution of nanoconstructs
containing an iron oxide nanoparticle core (ION, Feraheme) modified
with 10-kDa PEI, prepared using 4:1 by mass of ION:PEI (called F1)
or 3:1 of ION:PEI (called F2), 5-kDa PEG, and conjugated with
trastuzumab (T-F2). FIG. 38B is a graph showing the luciferase
silencing efficacy of the three materials with a siRNA dose of 50
nM and NP/siRNA of 10. FIG. 38C is a chart showing the enhanced T2
relaxivity of the F2 and T-F2 over Feraheme, measured by T2 MRI
(small animal Bruker BioSpin 11.75T MRI instrument) indicating that
the materials can be used as MRI contrast agents.
REFERENCE TO SEQUENCE LISTING
[0110] The nucleic acid sequences described herein are shown using
standard letter abbreviations, as defined in 37 C.F.R. .sctn.
1.822. Only one strand of each nucleic acid sequence is shown, but
the complementary strand is understood as included in embodiments
where it would be appropriate. A computer readable text file,
entitled "2MU0877.txt (Sequence Listing.txt)" created on or about
Jan. 27, 2022, with a file size of 4 KB, contains the sequence
listing for this application and is hereby incorporated by
reference in its entirety.
DETAILED DESCRIPTION
[0111] Described herein are nanoconstructs for the treatment or
diagnosis of disease including cancer, inflammation and fibrosis.
The nanoconstruct contains a nanoparticle, such as a mesoporous
silica nanoparticles (MSNP), a gold nanoparticle, a silver
nanoparticle, an iron oxide nanoparticle, or a carbon nanotube,
optionally loaded with a variety of additional agents including,
but not limited to, cationic polymers, stabilizers, targeting
agents, small molecules or proteins, labels, and/or
oligonucleotides. Combinations of various additional agents are
also contemplated. For example, the nanoconstruct includes a
cationic polymer, stabilizer, targeting agent, and small molecule,
label, and/or oligonucleotide. Nanoconstructs may also include more
than one type of cationic polymer, stabilizer, targeting agent,
small molecule, protein, label, and/or oligonucleotide. For
example, nanoconstructs may include multiple, different
oligonucleotides and/or small molecules or proteins that act on the
same or different targets. The use of such additional agents may
provide an additive or synergistic effect for disease treatment
when delivered together on a nanoconstruct.
Nanoparticles
[0112] Nanoparticles useful with the compositions and methods of
the invention include, without limitation, mesoporous silica
nanoparticles (e.g., MSNPs), iron oxide nanoparticles, silver
nanoparticles, gold nanoparticles, and carbon nanotubes.
Nanoparticles may or may not be porous. Exemplary sizes for the
nanoparticle cores are from about 5 nm to about 200 nm, about 5 to
about 20 nm, about 30 nm to about 100 nm, about 30 nm to about 80
nm, about 30 nm to about 60 nm, about 40 nm to about 80 nm, about
70 nm to about 90 nm, or about 5 nm, about 10 nm, about 20 nm,
about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm,
about 80 nm, about 90 nm, or about 100 nm. Generally, the
nanoparticle cores are spherical, although other shapes, such as
rods and discs, may also be used.
[0113] Additional components may be attached to nanoparticles by
various mechanisms, covalently or noncovalently. For example,
cationic polymers may be attached to nanoparticles by charge, e.g.,
for silica or iron oxide nanoparticles. Alternatively, the surfaces
of the nanoparticles may be altered to include reactive moieties
for conjugation to cationic polymers and/or other components, or
the cationic polymers or other components may include a moiety that
binds to the nanoparticles. For example, nanoparticle cores such as
silica, silicon, gold, iron oxide, and silver nanoparticles, as
well as carbon nanotubes, may be modified with reactive moieties
such as thiols, phosphonate, carboxylate, and amines prior to
attachment with cationic polymers and other components. Cationic
polymers and other components may be modified to include these or
other moieties, including but not limited to, maleimide, N-hydroxy
succinimidyl (NHS) esters, or azides, prior to binding to the
nanoparticle cores. Components may be attached directly to
nanoparticles, either on the surface or within pores if
present.
Cationic Polymers
[0114] In some embodiments, nanoparticles, such as MSNPs, are
coated with cationic polymers or other compounds. The cationic
polymer may bind to the surface of the nanoparticle using any
appropriate means. In some embodiments, the cationic polymer binds
to the nanoparticle via electrostatic interaction. The cationic
polymer may be any polymer with a positive charge, such as, but not
limited to, polyethylenimine (PEI) (see, e.g., Example III),
polyamidoamine, poly(allylamine), poly(diallyldimethylammonium
chloride), chitosan, poly(N-isopropyl acrylamide-co-acrylamide),
poly(N-isopropyl acrylamide-co-acrylic acid), poly(L-lysine),
diethylaminoethyl-dextran, poly-(N-ethyl-vinylpyridinium bromide),
poly(dimethylamino)ethyl methacrylate), or poly(ethylene
glycol)-co-poly(trimethylaminoethylmethacrylate chloride). Other
cationic polymers will be apparent to those of skill in the art,
and may be found, for example, in Polymer Handbook, 4th Edition,
Edited by: Brandrup, E. H. Immergut, and E. A. Grukle; John Wiley
& Sons, 2003).
[0115] The cationic polymers may be linear or branched. In some
embodiments, the cationic polymers may range in size from about 500
Da to 25 kDa and may be branched or linear. For example, branched
PEI with an average size of 1.8 kDa to 10 kDa may be loaded onto
the nanoparticle core. The ratio of cationic polymer to
nanoparticle may be varied depending on the desired result. The
cationic polymer may be present from 1 to 50 wt. % of polymer per
nanoconstruct, e.g., 5 to 40, 10 to 30%, 20 to 30%, 5 to 15%, 5 to
20%, 5 to 25%, 5 to 30%, 10 to 20%, 10 to 25%, or 25 to 40%, e.g.,
about 5, 10, 15, 20, 25, 30, or 35%. For example, as shown in
Example III, PEI per MSNP at a weight ratio of 1:4 during initial
coating and at a weight ratio of 1:4 during cross-linking resulted
in 14 wt. % PEI per nanoconstruct if 10-kDa PEI was used or 16 wt.
% if 1.8-kDa PEI was used (see, e.g., Table 5).
[0116] Endolysosomal escape and gene silencing efficacy is
increased by increasing buffering capacity of the nanoparticles.
Cross-linking the cationic polymers yields greater buffering
capacity as shown in FIG. 2B. In some embodiments, the cationic
polymers may be cross-linked, e.g., with a cleavable disulfide
bond, pre or post coating on the nanoparticle. In some embodiments,
the attached cationic polymers were cross-linked after binding to
the nanoparticles, e.g., MSNP, using, for example, DSP
(dithiobis[succinimidyl propionate]), DTSSP
(3,3'-dithiobis(sulfosuccinimidyl propionate), and DTBP (dimethyl
3,3'-dithiobispropionimidate). The cross-linking may occur in the
absence or presence of free PEI in the solution as shown in Example
III and FIG. 2A. In other embodiments, the cationic polymers may
not be cross-linked.
Stabilizers
[0117] A stabilizer may be conjugated to the nanoparticle and/or
the cationic polymer, e.g., by any appropriate means. In some
embodiments, a stabilizer is conjugated to an amine or other
reactive group of a cross-linked cationic polymer coated on the
nanoparticle (e.g., a MSNP). Exemplary stabilizers include, but are
not limited to, polyethylene glycol (PEG), dextran, polysialic
acid, hyaluronic acid (HA), polyvinyl pyrrolidone (PVP), polyvinyl
alcohol (PVA), and polyacrylamide (PAM).
[0118] Stabilizers may have multiple chemically reactive groups,
e.g., for attachment to the nanoparticle, cationic polymer, and/or
other component. For example (e.g., Example IV), reactive
stabilizer, e.g., PEG, derivatives may have two electrophilic
moieties, such as maleimide-PEG-N-hydroxysuccinimidyl ester
(Mal-PEG-NHS), which contains both a Michael acceptor and an
activated ester. The stabilizer, e.g., PEG, used in conjunction
with the compositions and methods of the invention generally has a
molecular weight ranging between 500 Da-40 kDa, e.g., 2-10 kDa as
shown in FIG. 3A. With 5-kDa PEG, the material is optimal in terms
of size and PEG loading content (FIG. 3A). The stabilizer may be
present from 1 to 50 wt. % of stabilizer per nanoconstruct, e.g., 5
to 30 wt. %, 10 to 20%, 10 to 25%, 5 to 15%, 5 to 20%, 5 to 25%, or
1 to 10%, e.g., about 5, 10, 15, 20, 25, 35, 40 or 45%. As shown in
Example IV, Mal-PEG(5-kDa)-NHS per MSNP is used at a weight ratio
of 1:1 to 5:1 during the synthesis (FIG. 3B), which results in
about 6-23% of PEG per nanoconstruct (see Table 5).
Labeling Agents
[0119] In some embodiments, the nanoconstruct may be labeled, e.g.,
with a lanthanide or fluorescent dye, e.g., as shown in Example
XXIV and FIG. 37. A label may be any substance capable of aiding a
machine, detector, sensor, device, column, or enhanced or
unenhanced human eye from differentiating a labeled composition
from unlabeled compositions. Examples of labels include, but are
not limited to, radioactive isotopes (e.g., PET tracers), dyes,
stains, quantum dots, gold nanoparticles, enzymes, nonradioactive
metals (e.g., MRI contrast agents), magnets, biotin, protein tags,
any antibody epitope, or any combination thereof. Exemplary
fluorescent dyes include, but are not limited to, FITC, RITC,
Cy.TM. dyes, amine-reactive Dylight.RTM. dyes, and amine-reactive
Alexa Fluor.RTM. dyes. In some embodiments, lanthanides can be
loaded onto hydroxyl, thiol, amine or phosphonate groups of
nanoparticles, e.g., MSNPs, by covalent bonding or adsorption,
e.g., as shown in FIG. 37A. Lanthanides can facilitate sample
detection with high sensitivity and resolution, e.g., by mass
spectrometry (FIG. 37A), while fluorescent dyes permit sample
quantification by fluorescent imaging techniques, e.g., as shown in
FIG. 37B. Nanoconstructs containing lanthanides such as gadolinium
can also serve as MRI contrast agents for imaging disease
sites.
[0120] In some embodiments, the labels, such as fluorescent dyes,
may be loaded inside the pores of nanoparticles, e.g., amine-MSNPs
via nucleophilic acyl substitution, e.g., between one or more
nanoparticle-bound amines and an activated ester moiety (such as an
NHS ester) appended to a fluorescent dye. Such labels produce
nanoconstructs for fluorescence imaging applications as shown in
FIG. 37B). Such a label may be added prior to or after loading of
the cationic polymer and/or stabilizer. In further embodiments, the
label may be attached to the cationic polymer, stabilizer, or other
component prior to or after their attachment to the nanoparticle by
any appropriate means.
Targeting Agents
[0121] The nanoconstruct may be delivered specifically or
non-specifically. Nanoconstructs may be delivered to a site, e.g.,
for therapy, analysis, or diagnosis, such as a cell or tissue. The
site may be in vivo or ex vivo. In some embodiments, the
nanoconstructs may be delivered to tumors via the leaky vasculature
of tumors. In some embodiments, the affinity of the cell or tissue,
e.g., tumors, for cationic particles may permit nanoconstructs
endowed with a positive charge due to the presence of cationic
polymers to accumulate at the site, e.g., tumors and disease sites
(see, e.g., FIG. 19 showing efficacy of siHER2-nanoparticle without
targeting agent in tumor xenografts in mice). In other embodiments,
the nanoconstructs may further include a targeting agent, e.g., for
specific delivery of the nanoconstructs to sites such as tumors
(see, e.g., FIGS. 16, 17, and 20 showing the efficacy of
trastuzumab-conjugated siHER2-nanoparticle in tumor xenografts in
mice). Targeting agents may be used to target a site and optionally
to aid or induce internalization into a cell.
[0122] Exemplary targeting agents include, but are not limited to,
monoclonal antibodies, single chain variable fragment (scFv)
antibodies, other antigen binding fragments of antibodies,
aptamers, small targeting molecules (e.g., ligands that bind to
cell surface receptors such as N-acetylgalactosamine, mannose,
transferrin, and folic acid), aptamers, carbohydrates, and peptides
that have binding affinity to a cell or tissue, e.g., a tumor. The
targeting agents may be attached to the nanoparticles, cationic
polymer, or stabilizer by any appropriate means. In some
embodiments, the targeting agents are trastuzumab (FIGS. 4 and 8),
cetuximab (FIG. 8I), or HER2 scFV (FIGS. 21A-21E), which may be
attached to a stabilizer, e.g., PEG, that is already attached to
the nanoparticles. In some embodiments, the targeting agents, such
as folic acid and transferrin, are first attached to a stabilizer,
e.g., PEG, prior to attachment on the nanoparticle (FIGS. 21D-21E
for folic acid (FA) conjugated nanoparticle). In further
embodiments, the targeting agents, such as monoclonal antibodies,
may have a therapeutic effect (see, e.g., FIGS. 9, 11, and 28).
Exemplary monoclonal antibodies include, but are not limited to,
anti-HER2 antibody, anti-EGFR antibody, anti-CD20 antibody,
anti-VEGF-A antibody, anti-CD33 antibody, anti-CD52 antibody, and
anti-TNF.alpha. antibody, such as alemtuzumab, bevacizumab,
cetuximab, gemtuzumab, panitumumab, rituximab, infliximab,
tositumomab, pertuzumab, and trastuzumab.
[0123] The targeting agents may be attached to the nanoconstructs
by any means, and suitable conjugation chemistries are known in the
art and described herein. In some embodiments (e.g., Example V),
the targeting agent is thiolated and subsequently conjugated with
Mal-PEG-PEI-MSNP via a thiol-maleimide reaction. In some
embodiments (e.g., Example V), the targeting agents are first
attached to the PEG stabilizer (e.g., FA or transferrin on PEG-NHS,
commercially available) prior to conjugation to the nanoparticle by
reaction of an NHS ester and an amine. The targeting agent may be
present from 0.1 to 10 wt. % of targeting agent per nanoconstruct,
e.g., 0.1 to 1 % or 1 to 5%, e.g., 1 to 10% for antibody or 0.1 to
2% for scFV, e.g., about 1, 2,3, 4, 5, 6, 7, 8, or 9%. For example,
the MSNP per trastuzumab may be at the weight ratio of 50:1 to 1:1
(as shown in FIGS. 4A-4B) during the synthesis, resulting in 0.5 to
6 wt. % of trastuzumab per nanoconstruct (Table 5 showing 3 wt. %
antibody if 10:1 ratio is used). In another example, 1-4 wt. % of
HER2 scFV per MSNP during the synthesis results in 0.3-0.5 wt. % of
HER2 scFV per nanoconstruct.
Small Molecules and Proteins
[0124] In some embodiments, the nanoconstruct may be loaded with
small molecules, proteins (i.e., other than targeting agents), or
other therapeutic agents. Small molecules are molecules, typically
with a molecular weight less than about 1000 Daltons, or in some
embodiments, less than about 500 Daltons, wherein the molecule is
capable of modulating, to some measurable extent, an activity of a
target molecule. Exemplary small molecules such as peptides, small
molecule inhibitors, chemotherapeutics, and other drugs may
increase the therapeutic effects of the nanoconstruct, e.g., as
shown in FIGS. 28-30. The small molecules may be located on the
exterior of the nanoparticles, e.g., on PEI, and/or within the
pores of the nanoparticle core, e.g., MSNP. Small molecules, such
as small molecule inhibitors and other chemotherapeutic agents, may
be selected based on the efficacy and specificity, e.g., in killing
cancer cells over non-target cells. In addition to small molecules,
small proteins such as cytokines with molecular weights less than
about 50 kDa can also be loaded on the nanoparticle in the same
manner as small molecules.
[0125] In some embodiments, chemotherapeutic agents such as
paclitaxel, docetaxel, and doxorubicin may be loaded on
nanoconstructs by hydrogen bonding with nanoparticle, e.g., MSNP,
surface moieties (e.g., hydroxyl or silanol) and/or cationic
polymer, e.g., PEI. Hydrophobic drugs (e.g., paclitaxel and
docetaxel) can be loaded on the nanoconstructs during or after
cationic polymer coating, e.g., with PEI in ethanol, while
hydrophilic drugs (e.g., doxorubicin) can be loaded on the
nanoconstructs after stabilizer coating, e.g., PEG, in PBS. The
small molecule may be present from 0.01 to 50 wt. % of small
molecule per nanoparticle, e.g., 0.1 to 30%, 1 to 30%, 1 to 20%, 1
to 10%, 1 to 5%, 0.1 to 1%, 0.5 to 5%, or 0.5 to 10%. For example,
small molecule per MSNP is used at a weight ratio of 1:10 to 1:1,
which results in 0.1 to 30% by weight of drug per
nanoconstruct.
[0126] Exemplary chemotherapeutic agents include, but are not
limited to, methotrexate; plicamycin (mithramycin); mitotane;
mercaptopolylysine; pyrimidine analogs such as fluorouracil;
anthracyclic antibiotics such as doxorubicin; maytansinoids such as
ansamitocin; D-arabinosyl nucleosides, such as arabinosyl adenine;
alkylating agents such as PAM, I-PAM, altretamine, procarbazine,
busulfan, dacarbazine, temozolomide, thiotepa and dacarbazine;
purine antagonists such as mercaptopurine; actinomycins such as
dactinomycin; mitomycins such as mitomycin C; anti-steroids such as
aminoglutethimide; anti-microtubules such as estramustine and
vinblastine; anti-androgens such as flutamide; GnRH analogs such as
leuprolide; megestrol acetate; estrogen receptor antagonists such
as tamoxifen; amsacrine (m-AMSA); asparaginase (I-asparaginase);
topoisomerase inhibitors such as etoposide (VP-16); cytokines such
as interferon .alpha.-2a and interferon .alpha.-2b; podophyllotoxin
derivatives such as teniposide (VM-26); arabinosyl cytosine;
nitrogen mustards such as chlorambucil, cyclophosphamide,
uramustine, ifosfamide, melphalan, bendamustine, mustine, and
melphalan; nitrosoureas such as carmustine, fotemustine, lomustine,
streptozocin, and semustine; platinum based anti-neoplastic agents
such as carboplatin, cisplatin, triplatin tetranitrate and
oxaliplatin; folate antimetabolites such as pemetrexed,
raltitrexed, edatrexate, denopterin and cladribine; nucleoside
analogs such as gemcitabine; purine nucleoside antimetabolites such
as clofarabine; antimetabolites such as
(N-((5-(((1,4-dihydro-2-methyl-4-oxo-6-quinazolinyl) methyl)
methylamino)-2-thienyl)carbonyl)-L-glutamic acid); glutamine
antagonists such as 6-diazo-5-oxo-L-norleucine; purine analogs such
as fludarabine and thioguanine; prodrugs such as capecitabine and
methyl aminolevulinate; mitotic inhibitors such as vincristine,
vinorelbine, and vindesine; anthracyclines such as daunorubicin,
doxorubicin, epirubicin, valrubicin, and idarubicin;
anthracenediones such as mitoxantrone; glycopeptide antibiotics
such as bleomycin; TNF inhibitors such as etanercept;
aminolevulinic acid; tyrosine kinase inhibitors such as dasatinib,
imatinib, nilotinib, lapatinib, neratinib, and erlotinib; epidermal
growth factor receptor inhibitors such as gefitinib; protein kinase
inhibitors such as sunitinib and vandetanib; platelet reducing
agents such as anagrelide; proteasome inhibitors such as
bortezomib; denileukin diftitox; pentostatin; pegaspargase;
alagebrium (3-phenacyl-4,5-dimethylthiazolium; aminophylline;
muramyl tripeptide; and mifamurtide. Other therapeutic agents are
known in the art.
Oligonucleotides
[0127] In some embodiments one or more oligonucleotides may be
attached to the nanoconstruct including, but not limited to, siRNA,
miRNA, miRNA mimics, or antisense oligomers. Typically, the
oligonucleotides will be capable of altering, e.g., reducing,
expression of a target protein, e.g., by RNAi or antisense effect.
Alternatively, the oligonucleotide may act as a probe in a cell or
tissue of interest. Exemplary oligonucleotides include, but are not
limited to, oligonucleotides that silence the expression of PLK1,
AKT1/BCL2, HER2, EPS8L1, or HSP47 such as siPLK1, siAKT1/BCL2,
siHER2, siEPS8L1, siHSP47, or miR-342-5P (see, e.g., Example XII
and XX). Other targets are described herein. The oligonucleotide
may be attached by any means. In some embodiments, the negatively
charged siRNA is attached to the positively charged cationic
polymer on the nanoparticle, e.g., MSNP, using an electrostatic
interaction. The oligonucleotides may target one or more genes
expressed in a cancer cell, such as one or more genes encoding a
protein that promotes cell growth, tumor vascularization, or escape
from apoptosis. In some embodiments, a single oligonucleotide may
target a plurality of genes with varying potency. In other
embodiments, a plurality of oligonucleotides may target a single
gene. In further embodiments, a plurality of oligonucleotides may
target a plurality of genes.
[0128] Oligonucleotides may be present from about 1% to 10% by
weight, e.g., about 2% to about 4% by weight. For example, MSNP per
siRNA (NP/siRNA) is used at the weight ratio ranging between about
10:1 to about 100:1 during the binding process, achieving complete
binding. Optimal gene knock down efficacy is achieved at NP/siRNA
of 25-50 as shown in FIGS. 5A-5B. For example, the MSNP-PEI-PEG
(with or without a targeting agent) may be loaded with one or more
siRNA sequences directed towards the target genes such as, but not
limited to, siHER2, siAKT (isoforms 1, 2, 3), siBCL2, siAKT1/BCL2
(an siRNA duplex in which one strand can knock down AKT1 and the
other can knock down BCL2, Table 1), siPLK1, siGRB7, and siEPS8L1.
The screenings of these siRNA are shown in FIGS. 24-26. The
nanoparticles may also be loaded with miRNA such as miR-342-5p
under the same conditions with siRNA (FIG. 27A).
[0129] Exemplary siRNA sequences used herein are siHER2,
siAKT1/BCL2, siRNA control designated siSCR, and a siRNA against
luciferase designated siLUC. Specific sequences are shown in Table
1.
TABLE-US-00001 Table 1 Exemplary siRNA sequences SiRNA siRNA
sequence siHER2 Sense: 5' CACGUUUGAGUCCAUGCCCAAUU 3' (SEQ ID NO. 1)
Antisense: 5' UUGGGCAUGGACUCAAACGUGUU 3' (SEQ ID NO. 2) siLUC
Sense: 5' CGGAUUACCAGGGAUUUCAtt 3' (SEQ ID NO. 3) Antisense: 5'
UGAAAUCCCUGGUAAUCCGtt 3' (SEQ ID NO. 4) siSCR Sense: 5'
UGGUUUACAUGUCGACUAA 3' (SEQ ID NO. 5) Antisense: 5'
UUAGUCGACAUGUAAACCA 3' (SEQ ID NO. 6) siAKT1/ Anti-Akt1: BCL2 5'
AUUCAGUUUCACAUUGCUUGGUGAC 3' (SEQ ID NO. 7) Anti-Bcl2: 5'
GUCACCAAGAACUGUGACACAGAAGGG 3' (SEQ ID NO. 8)
[0130] The exemplary siHER2 was selected by measuring the efficacy
and specificity with which it reduced HER2 mRNA levels and growth
in HER2.sup.+ cell lines but not HER2.sup.- cell lines. The dose of
the siHER2 required to inhibit cell growth by 50% (GI50) was <5
nM in 19 out of 20 HER2+ cell lines (14 out of 20 cells did not
respond to 30 .mu.g/ml trastuzumab).
[0131] The exemplary siHER2 selected was bound to
MSNP-PEI-PEG-Trastuzumab (T-NP) to create a nanoconstruct. The
construct increases siRNA protection against enzymatic degradation
in blood (FIGS. 6A-6E), enhances tumor-specific cellular uptake
(FIGS. 8A-8I), and achieves over 80% of HER2 knockdown efficacy
(FIG. 9A). The optimized nanoconstruct produced apoptotic death in
HER2 positive (HER2+) breast cancer cells grown in vitro (FIG.
9C-D), but not in HER2 negative (HER2-) breast cancer or non-breast
cells (FIG. 10). As seen in FIGS. 16A-16B, one dose of the
siHER2-nanoparticles reduced HER2 protein levels by 60% in
trastuzumab-resistant HCC1954 xenografts in mice and multiple
intravenous doses administered over 3 weeks significantly inhibited
tumor growth (p<0.004) (FIGS. 17A-17D). The material also had a
therapeutic impact in another tumor model shown to be resistant to
trastuzumab (FIG. 20).
Nanoconstruct Synthesis
[0132] Components may be bound to nanoparticles or other components
by any means including covalent and electrostatic binding. Various
conjugation chemistries are known in the art and described herein.
In some embodiments, one or more of the components are bound to the
surface of the nanoparticles. In other embodiments, one or more of
the components are bound within the pores of the nanoparticle
(e.g., MSNP). In further embodiments, one or more of the components
are bound to each other. For example, in some embodiments, a
targeting agent may be covalently bound to a stabilizer, which is
covalently bound to the cationic polymer (e.g., via an amine),
which is in turn electrostatically bound to the exterior of the
nanoparticle, and a small molecule is bound to the interior surface
of a pore. In some embodiments, the pore has a first opening at a
first location on the exterior surface of the nanoparticle (e.g.,
MSNP) and a second, different opening at a second location on the
exterior surface of the nanoparticle. Additional components may be
bound anywhere along the length of the inside of the pore.
[0133] While nanoparticles, such as MSNPs, may be acquired
commercially or created by any method, in some embodiments MSNPs
are formed by combining a first surfactant with a second, different
surfactant to form a first mixture, heating up the first mixture
and adding a silica precursor to the first mixture to form a second
mixture, holding the temperature for a period of time to generate
MSNPs, and recovering the MSNPs by centrifugation. Surfactants can
be removed by mixing the MSNP in acidic solvent under reflux
conditions. In some embodiments, the first mixture may be heated
prior to adding the silica precursor. In other embodiments, the
first mixture may be at room temperature and the second mixture may
be heated. The resulting MSNPs may have uniform or non-uniform
particle size with high porosity, e.g., as shown in FIG. 1A.
[0134] For example, to form uniform MSNPs, cetyltrimethylammonium
chloride (CTAC) may be combined with triethanolamine (TEA) in
water, and heated to 95.degree. C., while tetraethyl orthosilicate
is added as shown in Example I. Variation of the amount of TEA
while holding the amount of CTAC constant can be used to alter the
size of the resulting MSNPs. In some embodiments, the amount of TEA
is between about 100 to about 600 .mu.L, about 200 to about 450
.mu.L, or about 200 to about 350 .mu.L. Non-uniform MSNPs may be
created using a strong base, such as NaOH. For example,
cetyltrimethyl ammonium bromide (CTAB) may be used as the
surfactant and NaOH may be used as the base catalyst as shown in
Example II.
[0135] Iron oxide nanoparticles can be purchased (e.g., Feraheme)
or synthesized, e.g., as described in Example XXV. Gold and silver
nanoparticles can be synthesized following various published
protocols or purchased from vendors such as Sigma Aldrich, Nanocs,
nanoComposix. Carbon nanotubes can be synthesized following various
published protocols or purchased from vendors such as Sigma
Aldrich, US Research nanomaterial, and American Elements.
[0136] In some embodiments, functional groups such as, but not
limited to, thiol, amine, carboxylate, or phosphonate may be added
to the nanoparticles, e.g., MSNPs, during synthesis through the use
of one or more reagents, e.g., organosilanes such as, but not
limited to, (3-aminopropyl)triethoxysilane and
(3-aminopropyl)trimethoxysilane). Organosilanes may be added before
or after the surfactants are removed from the MSNPs. Analogous
reagents and other organic reagents, such as glutathione,
mercaptopropionic acid, DMSA, PEG-thiol, oleic acid, and dextran
may be employed to modify iron oxide nanoparticles, silver
nanoparticles, gold nanoparticles, and carbon nanotubes.
Functionalized nanoparticles can also be purchased directly, e.g.,
carbon nanotubes having surface modified with carboxylic acid,
amide, polyaminobenzene sulfonic acid, octadecylamine, and PEG can
be purchased from Sigma Aldrich.
[0137] The resulting nanoparticles, e.g., MSNPs after surface
modification, may be of any appropriate size, e.g., from about 20
nm to about 200 nm, about 30 nm to about 100 nm, about 40 nm to
about 200 nm, about 50 nm to about 200 nm, about 30 nm to about 80
nm, 40 nm to about 80 nm, about 30 nm, about 40 nm, about 30 nm to
about 60 nm, about 50 nm, or about 60 nm. Exemplary hydrodynamic
sizes are shown in FIGS. 1C, 2A, 3A, and 21D for final
nanoconstructs with MSNP cores and FIG. 38A for final
nanoconstructs with iron oxide cores.
Nanoconstruct Formulations and Methods of Use
[0138] Nanoconstructs may be formulated, as is known in the art,
for therapeutic, diagnostic, or research use. Typically, such
formulations for therapy or diagnosis include the nanoconstructs
suspended in a pharmaceutically acceptable carrier. Nanoconstructs
may be employed for in vivo or ex vivo use. Effects of the agents
contained in the nanoconstruct may occur intracellularly or
extracellularly. In particular, nanoconstructs may be employed to
deliver oligonucleotides to cells to modulate, e.g., reduce,
expression of a gene. Nanoconstructs may also be employed to
delivery labels or other agents intracellularly or within an
extracellular site.
[0139] The nanoconstructs may be used immediately upon formulation
or may be stored. In some embodiments, the nanoconstructs may be
lyophilized into dry states using a lyoprotectant, such as a sugar
like trehalose. Optimal trehalose and lyophilization conditions may
preserve the nanoconstruct in terms of particle size and charge
(FIGS. 22A-B) and efficacy in terms of gene knock down efficacy
(FIGS. 22C-D) compared to freshly made material. Nanoconstructs of
the invention are stable for at least 6 months when lyophilized
(FIGS. 23A-23E).
[0140] Effective amounts of a nanoconstruct for therapeutic
administration will be readily determined by those of ordinary
skill in the art, depending on clinical and patient-specific
factors.
[0141] These and other effective unit dosage amounts may be
administered in a single dose, or in the form of multiple daily,
weekly or monthly doses, for example in a dosing regimen of twice
per week for a 3 week cycle. In additional embodiments, dosages may
be administered in concert with other treatment regimens in any
appropriate dosage regimen depending on clinical and
patient-specific factors. The amount, timing and mode of delivery
of compositions of the invention comprising a disease treating
effective amount of a nanoconstruct will be routinely adjusted on
an individual basis, depending on such factors as weight, age,
gender, and condition of the individual, the acuteness of the
disease and/or related symptoms, whether the administration is
prophylactic or therapeutic, and on the basis of other factors
known to effect drug delivery, absorption, pharmacokinetics
including half-life, and efficacy.
[0142] Formulations of the invention will ordinarily be selected to
approximate a minimal dosing regimen that is necessary and
sufficient to substantially prevent or alleviate the symptoms of
the disease including cancer, fibrosis and inflammation in the
mammalian subject, including humans. Therapeutic dosage and
administration protocol will often include repeated dosing over a
course of several days or even one or more weeks or years. An
effective treatment regimen may also involve prophylactic dosage
administered on a day or multi-dose per day basis lasting over the
course of days, weeks, months or even years.
[0143] The compositions of the present invention may further
include a pharmaceutically acceptable carrier appropriate for the
particular mode of administration being employed. Dosage forms of
the compositions of the present invention include excipients
recognized in the art of pharmaceutical compounding as being
suitable for the preparation of dosage units as discussed above.
Such excipients include, without intended limitation, binders,
fillers, lubricants, emulsifiers, suspending agents, sweeteners,
flavorings, preservatives, buffers, wetting agents, disintegrants,
effervescent agents and other conventional excipients and additives
and/or other additives that may enhance stability, delivery,
absorption, half-life, efficacy, pharmacokinetics, and/or
pharmacodynamics, reduce adverse side effects, or provide other
advantages for pharmaceutical use.
[0144] Some nanoconstructs of the invention are designed for
parenteral administration, e.g. to be administered intravenously,
intramuscularly, intratumorally, subcutaneously, or
intraperitoneally, including aqueous and non-aqueous sterile
injectable solutions which, like many other contemplated
compositions of the invention, may optionally contain
anti-oxidants, buffers, bacteriostats and/or solutes which render
the formulation isotonic with the blood of the mammalian subject;
and aqueous and non-aqueous sterile suspensions which may include
suspending agents and/or thickening agents. The formulations may be
presented in unit-dose or multi-dose containers. Additional
compositions and formulations of the invention may include polymers
for extended release following parenteral administration. The
parenteral preparations may be solutions, dispersions or emulsions
suitable for such administration. The subject agents may also be
formulated into polymers for extended release following parenteral
administration. Pharmaceutically acceptable formulations and
ingredients will typically be sterile or readily sterilizable,
biologically inert, and easily administered. Such materials are
well known to those of ordinary skill in the pharmaceutical
compounding arts. Parenteral preparations typically contain
buffering agents and preservatives, and injectable fluids that are
pharmaceutically and physiologically acceptable such as water,
physiological saline, balanced salt solutions, aqueous dextrose,
glycerol or the like. Extemporaneous injection solutions, emulsions
and suspensions may be prepared from sterile powders, granules and
tablets of the kind previously described. Preferred unit dosage
formulations are those containing a daily dose or unit, daily
sub-dose, as described herein above, or an appropriate fraction
thereof, of the active ingredient(s).
[0145] In some embodiments, the topical carrier used to deliver the
nanoconstruct is an emulsion, gel or ointment. In other
embodiments, the therapeutic compounds as described herein may be
formulated in a spray formulation.
[0146] Emulsions, such as creams and lotions are a dispersed system
comprising at least two immiscible phases, one phase dispersed in
the other as droplets ranging in diameter from 0.1 .mu.m to 100
.mu.m. An emulsifying agent is typically included to improve
stability. When water is the dispersed phase and an oil is the
dispersion medium, the emulsion is termed a water-in-oil emulsion.
When an oil is dispersed as droplets throughout the aqueous phase
as droplets, the emulsion is termed an oil-in-water emulsion.
Emulsions, such as creams and lotions that can be used as topical
carriers and their preparation are disclosed in Remington: The
Science and Practice of Pharmacy (Loyd V. Allen 22.sup.nd ed.
2012), hereby incorporated herein by reference.
[0147] Ointments may be homogeneous, viscous, semi-solid
preparation, most commonly a greasy, thick oil (oil 80%-water 20%)
with a high viscosity. The ointment can be used as an emollient or
for the application of active ingredients to the skin for
protective, therapeutic, or prophylactic purposes where a degree of
occlusion is desired.
[0148] A cream is an emulsion of oil and water in approximately
equal proportions. It penetrates the stratum corneum outer layer of
skin quite well. Cream is generally thinner than ointment, and
maintains its shape when removed from its container.
[0149] The vehicle of an ointment/cream is known as the ointment
base. The choice of a base depends upon the clinical indication for
the ointment. The different types of ointment bases include, but
are not limited to: hydrocarbon bases, e.g. hard paraffin, soft
paraffin, microcrystalline wax and ceresine; absorption bases, e.g.
wool fat, beeswax; Water soluble bases, e.g., macrogols 200, 300,
and 400; Emulsifying bases, e.g. emulsifying wax, Vegetable oils,
e.g. olive oil, coconut oil, sesame oil, almond oil and peanut oil.
The therapeutic compounds are dispersed in the base and later get
divided after the drug penetrates into the wound. Ointments/creams
can be formulated incorporating hydrophobic, hydrophilic, or
water-emulsifying bases to provide preparations that are
immiscible, miscible, or emulsifiable with skin secretions. They
can also be derived from fatty hydrocarbon, absorption,
water-removable, or water-soluble bases. For example, a
cream/ointment base can contain the active agent, white petrolatum,
water, allantoin, EDTA, Stearyl alcohol, Brij 721, Brij 72,
methylcelluloses, isopropyl myristate, Sorbitan monooleate,
Polyoxyl 40 stearate, butylated hydroxytoluene, propylene glycol,
methylparaben, propylparaben, deionized water to 100%, and buffer
to neutral pH among other ingredients.
[0150] In another embodiment, the topical carrier used to deliver a
compound of the invention is a gel, for example, a two-phase gel or
a single-phase gel. Gels are semisolid systems consisting of
suspensions of small inorganic particles or large organic molecules
interpenetrated by a liquid. When the gel mass comprises a network
of small discrete inorganic particles, it is classified as a
two-phase gel. In some embodiments the liquid may be water or
another aqueous media and the gel mass is defined as a hydrogel.
Hydrogels can include, but are not limited to, alginates,
polyacrylates, polyalkylene oxides, and/or poly N-vinyl
pyrrolidone. The hydrogel may also be amorphous, i.e. a viscous gel
as opposed to a solid such as a formulation of
carboxymethylcellulose containing a humectant such as propylene
glycol or glycerin. Exemplary amorphous hydrogels include, but are
not limited to, maltodextra-beta glucan, acemannan,
carboxymethylcellulose, pectin, xanthan gum, collagen, keratin and
honey.
[0151] Nanoconstructs may be packaged into biodegradable capsules
for oral administration. Alternatively, a nanoconstruct suspension
may be installed inside the bladder. This is similar to
intravesical chemotherapy, in which the drug administered to the
bladder will come into direct contact with cancer cells in the
bladder lining.
[0152] The invention disclosed herein will also be understood to
encompass diagnostic compositions for diagnosing the risk level,
presence, severity, or treatment indicia of, or otherwise managing
diseases including, but not limited to, neoplastic diseases by
contacting a labeled (e.g., isotopically labeled, fluorescent
labeled or otherwise labeled to permit detection of the labeled
compound using conventional methods) nanoconstruct to a mammalian
subject (e.g., to a cell, tissue, organ, or individual) at risk or
presenting with one or more symptom(s) of a cell proliferation
disease, such as a cancer, and thereafter detecting the presence,
location, metabolism, and/or binding state (e.g., detecting binding
to an unlabeled binding partner involved in malignant cell receptor
physiology/metabolism) of the labeled compound using any of a broad
array of known assays and labeling/detection methods. In exemplary
embodiments, a nanoconstruct is isotopically-labeled by having one
or more atoms replaced by an atom having a different atomic mass or
mass number. Examples of isotopes that can be incorporated into the
disclosed compounds include isotopes of hydrogen, carbon, nitrogen,
oxygen, phosphorous, fluorine and chlorine, such as .sup.2H,
.sup.3H, .sup.13C, .sup.14C, .sup.15N, .sup.18O, .sup.17O,
.sup.31p, .sup.32P, .sup.35S, .sup.18F, and .sup.36Cl,
respectively. The isotopically-labeled compound is then
administered to an individual or other subject and subsequently
detected as described above, yielding useful diagnostic and/or
therapeutic management data, according to conventional
techniques.
[0153] Nanoconstructs may include various therapeutic moieties,
such as targeting agents, oligonucleotides, and/or small molecules,
and may have intrinsic therapeutic properties. For example, in some
embodiments the nanoconstruct (e.g., a nanoconstruct containing a
MSNP) may be an antioxidant. The antioxidant nanoconstruct can
deliver siRNA that can reduce pro-fibrotic genes such as HSP47 and
NOX4 as shown in FIGS. 31A-31D, decreasing the adverse effects of
pro-inflammatory cytokines such as TGF-beta. This has proven to be
effective at decreasing fibrotic markers such as COL 1 and
alpha-SMA in vitro and in mouse models of fibrotic disease.
Suitable effective unit dosage amounts of the active compounds for
administration to mammalian subjects, including humans, can be
readily determined by one of skill in the art. For example, for
siRNA, the amount may range from about 0.01 to about 1 mg siRNA/kg,
about 0.1 to about 0.75 mg siRNA/kg, about 0.1 to about 1 mg
siRNA/kg per dose and any subset thereof.
[0154] Nanoconstructs containing a lanthanide and/or a fluorescent
dye may be used as fluorescent, mass spectrometry, PET, and/or MRI
probes for both in vitro and in vivo applications. In vitro, such
nanoconstructs can be used to detect or quantify target proteins in
tissue specimens or cells by immunofluorescence, flow cytometry, or
mass cytometry (see, e.g., Example XXIV). In these applications,
the targeting agents on the nanoconstruct will bind specifically
with the protein receptors on the cells (e.g., EGFR or HER2 on
cancer cells). In vivo, the nanoconstructs labeled with PET
tracers, gadolinium chelates, or containing an iron oxide
nanoparticle core can be used as PET or MRI contrast agents (see,
e.g., Example XXVI) to detect diseases (e.g., cancer or
inflammation). Additionally, nanoconstructs can be transfected into
cells of interest (e.g., stem cells or immune cells) before such
cells are infused to patients. This allows monitoring of these
nanoconstruct-contained cells over time by non-invasive methods,
such as fluorescence imaging, PET, and MRI.
[0155] Nanoconstructs made from iron oxide, gold, and silver
nanoparticles can be used in hyperthermia treatment. Alternating
magnetic fields and irradiation have been shown to heat up such
nanoparticles, increasing temperature of tumors (e.g., up to
40.degree. C.) in which the nanoconstructs are accumulated. This
can enhance cargo release and/or induce cancer cell death.
[0156] The intrinsic properties of the nanoconstructs may have
therapeutic benefits for cancer treatment. For example, in some
embodiments, the antioxidant property of the nanoconstruct (from
MSNP) may modulate reactive oxygen species in cancer cells,
resulting in decreased migration and invasion of cancer cells (see,
e.g., FIGS. 34A-34H). This effect was also confirmed in a mouse
model of metastasis, as tumor spreading was observed to be
attenuated in nanoconstruct-treated mice (see, e.g., FIGS.
35A-35H).
[0157] Additionally, amine groups, e.g., from PEI on
nanoconstructs, are known to be highly effective chelators of
copper. Copper is believed to be a cofactor for angiogenesis and
hence metastasis in cancer (Brewer et al., 2000). Thus, the
PEI-nanoconstruct may provide a therapeutic benefit similar to that
observed for tetrathiomolybdate (TM, a copper chelator) in its
clinical trial on metastatic solid tumor patients (Brewer et al,
2000). In this trial, five out of six TM-treated patients had mild
copper deficiency (e.g. as assessed on the basis of a lower serum
concentration of ceruloplasmin, a biomarker for total body copper
status) and achieved stable disease. We also observed reduced serum
ceruloplasmin in agreement with attenuated cancer metastasis and
tumor burden with our nanoconstruct (see, e.g., Example XXIII).
[0158] In addition to cancer and fibrosis, the nanoconstructs are
capable of delivering oligonucleotides to cells to alter gene
expression (e.g., via RNA interference) and can thus also be used
to treat other diseases involving aberrant gene expression.
Exemplary siRNAs that can be incorporated into nanoconstructs of
the invention may target genes listed in Table 2, e.g., to treat
the corresponding diseases shown therein.
TABLE-US-00002 TABLE 2 Exemplary diseases that can be treated by
gene modulation Category Diseases siRNA target Ophthalmology AMD
VEGF, RTP801, VEGF-R1 macular edema VEGF, RTP801 chronic optic
nerve proNGF atrophy Genetic pachyonychia Keratin K6A disorder
congenita (genetic disease) Oncology chronic lymphocytic Bcl-2
leukemia metastatic lymphoma PLK1, LMP2, LMP7, MECL1 solid tumors
HER2, AKT1, AKT1, Bcl-2, AR, Myc, EGFR, Grb7, EPS8L1, RRM2, PKN3,
Survivin, HIF1a, Furin, KSP, VEGF, eiF-4E Inflammation acute kidney
injury p53 delayed graft function p53 familia adenomatous b-catenin
polyposis Metabolic Hypercholestrolemia ApoB, PCSK9 disease
Fibrosis liver fibrosis HSP47 cystic fibrosis CFTR Dermatology
dermal scarring and CTGF, HSP47 fibrosis Viral Infection Ebola
infection SNALP RSV infection RSV nucleocapsids Immunotherapy
Cancer CD47, PD-L1, CTLA-4
[0159] The nanoconstructs may be used in the treatment of cancer,
e.g., breast cancer. In some embodiments, the tumors may be
resistant to monoclonal antibodies, small molecule inhibitors,
and/or other chemotherapeutic agents, but still respond to siRNA
delivered by nanoconstructs. In further embodiments, the siRNA
targets the same genes/proteins that monoclonal antibodies and
small molecule inhibitors target, but gene/protein suppression with
siRNA is superior to that of antibodies or small molecule
inhibitors and overcomes cancer resistance to the antibodies and
small molecule inhibitors (see, e.g., Example XIII).
[0160] In some embodiments, more than one therapeutic agent may be
used to increase effectiveness of the nanoconstruct. For example,
FIGS. 28A-28B show the synergistic or additive effect of
co-delivery of trastuzumab (HER2 antibody), chemotherapeutic
paclitaxel (PTX), and siHER2 in terms of killing JIMT1, a multiple
drug resistant HER2+ breast cancer cell. In further embodiments, as
shown in FIG. 29, loading of chemotherapeutic agents such as
paclitaxel (PTX) or doxorubicin (DOX) does not negatively impact
the ability of siRNA on the same nanoconstruct to silence the
targeted genes. FIG. 30 shows the effective inhibition of HCC1954
tumor growth in mice upon the treatment with nanoconstruct
containing the three agents (T-siHER2-NP (PTX)).
[0161] As used herein, the terms "treat," treating," "treatment,"
"therapeutic" and the like refer to reducing or ameliorating a
disorder and/or symptoms associated therewith. It will be
appreciated that, although not precluded, treating a disorder or
condition does not require that the disorder, condition or symptoms
associated therewith be completely eliminated.
[0162] By "effective amount" is meant the amount of an agent
required to ameliorate the symptoms of a disease or arrest the
progression of a disease relative to an untreated subject or to
label a target, e.g., protein, cell, or tissue, sufficiently for
detection. The effective amount of an active therapeutic agent for
the treatment of a disease or injury varies depending upon the
manner of administration, the age, body weight, and general health
of the subject.
[0163] Various assays and model systems can be readily employed to
determine the therapeutic effectiveness in the treatment of cancer,
inflammation, fibrosis and other diseases. For example,
effectiveness may be demonstrated using a complete blood count
(CBC). The measurements taken in a CBC include a white blood cell
count (WBC), a red blood cell count (RBC), the red cell
distribution width, the hematocrit, and the amount of hemoglobin.
Some signs of cancer which are visible in a CBC include a low
hematocrit, a sharp decrease in the number of blood platelets, and
a low level of neutrophils. An effective amount of a composition of
the present invention may increase the levels measured in a
complete blood count by 10%, 20%, 30%, 50% or greater increase, up
to a 75-90%, or 95% or greater in comparison to individuals who
have not been treated by the compositions described herein. The
compositions as described herein may be effective in increasing all
or some parts of the complete blood count in comparison to those
treated with placebo. Effective amounts may also move the blood
protein of an individual towards the optimal category for each type
of protein.
[0164] Effectiveness in the treatment of neoplastic diseases may
also be determined by a number of methods such as, but not limited
to, microscopic examination of blood cells, bone marrow aspiration
and biopsy, cytogenetic analysis, biopsy, immunophenotyping, blood
chemistry studies, analysis of tumor biomarkers in blood, a
complete blood count, lymph node biopsy, peripheral blood smear,
visual analysis of a tumor or lesion, or any other method of
evaluating and/or diagnosing malignancies and tumor progression
known to those of skill in the art.
[0165] Effectiveness of the compositions and methods herein in the
treatment of cancer or other disease may be evaluated by screening
for markers in the blood depending on the specific cancer.
[0166] Within additional aspects of the invention, combinatorial
disease treating formulations and coordinate administration methods
are provided which employ an effective amount of a nanoconstruct
and one or more secondary or adjunctive agent(s) that is/are
combinatorially formulated or coordinately administered with the
nanoconstruct to yield a combined, multi-active disease treating
composition or coordinate treatment method.
[0167] Exemplary combinatorial formulations and coordinated
treatment methods in this context employ the nanoconstruct, in
combination with one or more secondary anti-tumor agent(s), or with
one or more adjunctive therapeutic agent(s) that is/are useful for
treatment or prophylaxis of the targeted (or associated) disease,
condition and/or symptom(s) in the selected combinatorial
formulation or coordinate treatment regimen. For most combinatorial
formulations and coordinate treatment methods of the invention, a
nanoconstruct is formulated, or coordinately administered, in
combination with one or more adjunctive therapeutic agent(s), to
yield a combined formulation or coordinate treatment method that is
combinatorially effective or coordinately useful to treat
neoplastic diseases and one or more symptom(s) of a secondary
disease or condition in the subject. Exemplary combinatorial
formulations and coordinate treatment methods in this context
employ a nanoconstruct, in combination with one or more secondary
or adjunctive therapeutic agents selected from, e.g.,
chemotherapeutic agents, anti-inflammatory agents, doxorubicin,
vitamin D3, cytarabine, daunorubicin, cyclophosphamide, gemtuzumab
ozogamicin, idarubicin, mercaptopurine, mitoxantrone, thioguanine,
aldesleukin, asparaginase, carboplatin, etoposide phosphate,
fludarabine, methotrexate, etoposide, dexamethasone, and choline
magnesium trisalicylate. In addition, secondary therapies can
include, but are not limited to, radiation treatment, hormone
therapy and surgery.
[0168] In exemplary embodiments, a nanoconstruct and another agent
will each be present in a disease treating/preventing amount (i.e.,
in singular dosage which will alone elicit a detectable alleviation
of symptoms in the subject). Alternatively, the combinatorial
formulation may comprise one or both nanoconstruct and another
agent (wherein the other agent is not a nanoconstruct) in
sub-therapeutic singular dosage amount(s), wherein the
combinatorial formulation comprising both agents features a
combined dosage of both agents that is collectively effective in
eliciting the desired response.
[0169] To practice coordinated administration methods of the
invention, a nanoconstruct may be administered, simultaneously or
sequentially, in a coordinated treatment protocol with one or more
of the adjunctive therapeutic agents contemplated herein. Thus, in
certain embodiments a compound is administered coordinately with
another agent and any other secondary or adjunctive therapeutic
agent contemplated herein, using separate formulations or a
combinatorial formulation as described above (i.e., comprising both
a nanoconstruct and another agent). This coordinate administration
may be done simultaneously or sequentially in either order, and
there may be a time period while only one or both (or all) active
therapeutic agents individually and/or collectively exert their
biological activities. In another embodiment, such coordinated
treatment methods are derived from protocols for the administration
of one or more chemotherapeutics.
[0170] The pharmaceutical compositions of the present invention may
be administered by any means that achieve their intended
therapeutic or prophylactic purpose. Suitable routes of
administration for the compositions of the invention include, but
are not limited to, conventional delivery routes, devices and
methods including injectable methods such as, but not limited to,
intravenous, intramuscular, intraperitoneal, intraspinal,
intrathecal, intracerebroventricular, intraarterial, subcutaneous
and intranasal routes. Additional means of administration comprise
topical application.
[0171] Aspects and applications of the invention presented here are
described in the drawings and detailed description of the
invention. Unless specifically noted, it is intended that the words
and phrases in the specification and the claims be given their
plain, ordinary, and accustomed meaning to those of ordinary skill
in the applicable arts.
[0172] In the following examples, and for the purposes of
explanation, numerous specific details are set forth in order to
provide a thorough understanding of the various aspects of the
invention. It will be understood, however, by those skilled in the
relevant arts, that the present invention may be practiced without
these specific details. In other instances, known structures and
devices are shown or discussed more generally in order to avoid
obscuring the invention. It should be noted that there are many
different and alternative configurations, devices and technologies
to which the disclosed inventions may be applied. The following
examples are illustrative of disclosed methods. In light of this
disclosure, those of skill in the art will recognize that
variations of these examples and other examples of the disclosed
method would be possible without undue experimentation.
EXAMPLES
Example I
Synthesis of Uniform Nanoparticle Cores
[0173] The sol-gel synthesis of mesoporous silica nanoparticle
cores (MSNPs) was modified from previous reports (L. Pan et al.,
2012; I. Slowing et al., 2007). For 47-nm MSNPs, nanoparticle (NP)
(S-47), 0.15 M cetyltrimethylammonium chloride (CTAC) and 350 .mu.L
of triethanolamine (TEA) were mixed in 125 mL of water at
95.degree. C. Then, 3 mL of tetraethyl orthosilicate (TEOS) was
added, and the mixture was stirred for one hour. All chemicals were
purchased from Sigma Aldrich, USA.
[0174] After mixing, the pellets were recovered from suspension by
centrifugation, washed with a copious amount of ethanol, and dried
overnight. The particles were then resuspended and refluxed in
acidic methanol (0.6 M HCl in methanol) overnight to remove CTAC
and TEA. Bare MSNPs were then washed with ethanol and dried in a
desiccator overnight. MSNP (dry) size was measured with TEM
(Phillips/FEI CM120/Biotwin TEM, Hillsboro, Oreg.) and hydrodynamic
size with Zetasizer (ZS-90/Malvern, Malvern, U.K.). Varying the
amount of TEA from 200-450 .mu.l per 125 mL of reaction solution
while holding the CTAC concentration constant at 0.15 M resulted in
MSNP of different particle sizes; e.g., from 61.+-.7 nm to 47.+-.4
nm and 34.+-.3 nm (dry size by TEM) as shown in FIG. 1A.
[0175] Multiple individual batches of mesoporous silica
nanoparticles were created to determine the reproducibility of
nanoparticle synthesis. As shown in Table 3, the hydrodynamic size
of the S-47 MSNP core is highly reproducible, e.g., with a relative
standard deviation (RSD) of 2.4% from 6 batches.
TABLE-US-00003 TABLE 3 Reproducibility of nanoparticle synthesis.
Hydrodynamic Size, Batch Z-average .+-. SD (nm) 1 61.1 .+-. 0.7 2
58.1 .+-. 0.6 3 59.7 .+-. 0.5 4 57.7 .+-. 0.9 5 60.8 .+-. 0.8 6
58.8 .+-. 0.3 Average 59.4 % Relative standard 2.4 deviation
Example II
Synthesis of Non-Uniform Nanoparticle Cores
[0176] Non-uniform MSNPs (O-87) were synthesized in the presence of
a strong base. 6 mM cetyltrimethyl ammonium bromide (CTAB) was
dissolved in 240 mL of aqueous solution of pH 11.0 (adjusted by 2 M
NaOH). When the temperature stabilized at 80.degree. C., 2.5 mL of
TEOS was added, and the reaction continued for 2 hours. After
mixing, the pellets were recovered from suspension by
centrifugation, washed with a copious amount of ethanol, and dried
overnight. The particles were then resuspended and refluxed in
acidic methanol (0.6 M HCl in methanol) overnight to remove CTAB.
Bare MSNPs were then washed with ethanol and dried in a desiccator
overnight. MSNP (dry) size was measured with TEM (Phillips/FEI
CM120/Biotwin TEM, Hillsboro, Oreg.) and hydrodynamic size with
Zetasizer (ZS-90/Malvern, Malvern, U.K.). These non-uniform
nanoparticle cores were 87.+-.14 nm in size (see, e.g., FIG.
1A).
[0177] MSNPs prepared from Examples I and II were then coated
layer-by-layer with cross-linked or non-cross-linked PEI, PEG, and
targeting antibody. The nanoconstructs were then loaded with siRNA
cargo as entailed in Examples III-VI. The characterization of
compositions, size, and charge of the nanoconstructs are summarized
in Example VII. FIG. 1B provides a schematic illustration of such
surface decoration, and FIG. 1C shows the size of various
nanoconstructs using different MSNP cores; all of which were
surface modified with 10-kDa PEI (cross-linked for S-34, S-47, and
S-61 MSNPs; non-cross-linked for O-87 MSNP), 5-kDa PEG, trastuzumab
(HER2 antibody), and siRNA, respectively. FIG. 10 indicates that
S-47 core yielded the most desirable hydrodynamic size of the final
nanoconstruct.
Example III
Polyethylenimine (PEI) Attachment to MSNPs and Cross-Linking
Methods
[0178] MSNPs prepared as described in Examples I and II were coated
with PEI by shaking 10 mg MSNPs and 2.5 mg PEI in ethanol solution
for 3 hours at room temperature. Next, the PEI-MSNP, was pelleted
down. The material then underwent PEG attachment in Example IV or
PEI cross-linking first prior to PEG attachment. For the PEI
cross-linking method, the PEI-MSNP was re-suspended in ethanol
solution containing 0 or 2.0 mg PEI (10 kDa) and 0.1-0.5 mg/ml DSP
(DSP; dithiobis [succinimidyl propionate]; Lomant's Reagent) as a
crosslinker. The solution was shaken for another 40 minutes. The
particles were pelleted down, washed, and resuspended in PBS (pH
7.2). The hydrodynamic sizes of materials made under these various
cross-linking conditions are shown in FIG. 2A, in which using 0.1
mg/ml DSP and 2 mg PEI during cross-linking yielded the most
desirable size (similar to no cross-linker), while cross-linking
alone without introducing 2 mg PEI causes aggregates due to
inter-particle cross-linking.
Example IV
Polyethylene Glycol (PEG) Attachment to MSNPs
[0179] Mal-PEG-NHS was used to attach PEG on the nanoparticles (via
an NHS ester). For PEG loading, 50 mg of mal-PEG-NHS was conjugated
to the primary amine of PEI-MSNP (10 mg as MSNP) from Example III
(using either cross-linked or non-cross-linked PEI-MSNP) in PBS
buffer under shaking (20 hr, RT).
[0180] PEG:MSNP ratios. To reduce the usage of expensive
Mal-PEG-NHS, Mal-PEG-NHS can be first dissolved in DMF or added as
a dry powder directly to the PEI-MSNP suspension in PBS to limit
the hydrolysis of NHS and enhance the PEG loading efficacy and
reduce the reaction time. This reduces the Mal-PEG-NHS by 5 fold to
a 1:1 weight ratio of Mal-PEG-NHS per MSNP and reduces the reaction
time from 20 hr to 1.5 hr. For example, 10 mg of PEI-MSNP were
re-suspended in 1 ml PBS. Then, 10 mg of Mal-PEG-NHS (as a powder)
were added into the PEI-MSNP solution. The mixture was then
vortexed for one minute and sonicated for five minutes to ensure
re-suspension. The mixture was then further shaken for 1.5 hours.
The resulting nanoconstructs were subsequently recovered by
centrifugation and washed as aforementioned. This dry powder method
avoids the use of potentially harmful DMF solvent and hence is
preferable. It yields similar PEG loading (e.g., about 18%, Table
5) and siRNA protection (FIG. 6E) as those using 5:1 of Mal-PEG-NHS
per MSNP ratio (see FIGS. 6A-C). The luciferase silencing efficacy
using these two materials for delivering siRNA against luciferase
(siLUC) (method outlined in Example VIII) is also the same (FIG.
3A).
[0181] Molecular weights of PEG. Different molecular weights of PEG
were also optimized. PEGs with different molecular weights (2, 5,
7.5 and 10 kDa) were tested. The role of PEG is to provide a
stealth layer to the nanoparticle, hence higher density of PEG
loading (brush-like conformation) is more desirable than lower
density of PEG loading (random coil conformation). FIG. 3B shows
that 2-kDa PEG is not sufficient to prevent siRNA-nanoconstructs
from aggregating into larger particles. A PEG of 5-kDa and above
appears to prevent aggregation effectively. The amounts of PEG
loaded on nanoconstructs with 5-, 7.5- and 10-kDa were quantified
with the TGA to be 18%, 10% and 12% by mass, respectively. On a
molar basis, 5-kDa PEG has around three times higher loading on the
nanoconstructs than the 7.5-kDa or 10-kDa counterparts. Low molar
loading of higher-MW PEGs suggests the PEG conformation is not
brush-like at higher-MW PEG chains. 5-kDa PEG provided the highest
PEG loading, suggesting an optimal brush-like stealth condition for
our nanoconstructs and was used throughout the studies.
Example V
Conjugation of Targeting Agents (Antibody, Antibody Fragment, Folic
Acid) on MSNP-PEI-PEG
[0182] Antibody. Antibody conjugation of MSNP-PEI-PEG utilized a
thiol-maleimide reaction modified from the literature (P.
Yousefpour et al., 2011, M. N. Koopaei et al., 2011). First,
antibody (e.g., trastuzumab (T), cetuximab (C), or rituximab (R))
was thiolated with Traut's reagent (2-iminothiolane) in PBS (pH
8.0) under shaking with 50-fold molar excess of Traut's reagent for
2 hours and then purified by Zeba spin column--MW-40,000 (Thermo
Fisher Scientific). Thiolated antibody was then mixed with
MSNP-PEI-PEG at an antibody:nanoparticle mass ratio of 1:1 to 1:50.
The reaction was completed overnight at 4.degree. C. under shaking
conditions (300 rpm). The material was pelleted down, resuspended
in PBS, and washed with a copious amount of PBS. FIG. 4A shows the
cell killing effect (method outlined in Example XII) upon
delivering siHER2 (siRNA against HER2) with T-NP having different
wt. % of trastuzumab. FIG. 4B shows the degree of cellular uptake
(method outlined in Example XI) of T-NP having different wt. % of
trastuzumab in BT474 (HER2+) cell line. It can be seen that
antibody:nanoparticle mass ratio of 1:20 and 1:10 can equally
enhance cellular uptake of T-NP, but cell killing started to reduce
if too little antibody was used. For optimal cost and efficacy
trade-off, 1:10 ratio was selected and used throughout unless
specified otherwise.
[0183] Antibody fragment (scFV). Antibody fragment can be
conjugated to the nanoparticle in a similar manner with antibody.
Bacteria expressing a HER2 scFv clone (Poul et al., 2000) were
grown overnight followed by expansion on the second day and
stimulation with IPTG (thiogalactopyranoside) to produce scFv
protein. The culture was subsequently pooled, and periplasmic bound
scFv was extracted and purified on a Ni-NTA column according to
manufacturer's protocol. Concentration of scFv was determined using
a Nanodrop UV-Vis spectrometer, and purity was verified by gel
electrophoresis. FIG. 21A shows the successful purification of
HER2-scFv as indicated by the specific 25 kD band of scFv in the
purified sample compared to the non-purified and wash eluent. FIG.
21B shows additional verification of scFv identity by probing with
an antibody that specifically recognizes the 6.times. histidine tag
present at the c-terminal of the scFv molecule. FIG. 21C shows HER2
scFV to be specific to HER2+ cells (BT474, SKBR3, JIMT1) over HER2-
cells (MCF7 and MDAMB468). The scFv are attached to the end of the
PEG chain on nanoconstructs by coupling cysteine of the scFv to the
maleimide group on MSNP-PEI-PEG. Protein characterization indicated
0.4 wt. % of scFV per nanoparticle. FIG. 21D shows the size of HER2
scFV conjugated nanoparticles loaded with 3 wt. % siRNA; the size
was measured in PBS. FIG. 21E shows the luciferase silencing
efficacy (experiment outlined in Example VIII) of nanoparticles
loaded with different amount of scFV in solution (1% or 4%, both of
which resulted in 0.4 wt. % loading of scFv per nanoparticle).
[0184] Folic acid. NHS ester-PEG-Folic acid (FA) was obtained
commercially from Nanocs. NHS-PEG-FA was attached on the
nanoparticles (via an NHS ester) in a similar manner as Example IV.
2.5 mg of NHS-PEG(5 kD)-FA was conjugated to the primary amine of
cross-linked PEI-MSNP (10 mg) from Example III in PBS buffer under
shaking (1.5 hours, RT). NHS-PEG(5 kD) (5-20 mg) was added to
further stabilize (bind with) the remaining amine active surface
for another hour. FIG. 21D shows the hydrodynamic size of
FA-siHER2-NP, and FIG. 21E shows the luciferase silencing efficacy
of the material.
Example VI
siRNA Loading on Targeting Agent Conjugated MSNP-PEI-PEG or
MSNP-PEI-PEG
[0185] The loading of siRNA was achieved by mixing
trastuzumab-conjugated MSNP-PEI-PEG (designated as T-NP) or
MSNP-PEI-PEG (NP) and siRNA at nanoparticle/siRNA (NP/siRNA) mass
ratio of 10 to 100 in PBS solution (0.5 to 1 hour, room temp, 200
rpm shaking), which resulted in complete binding (no siRNA left in
the supernatant) as monitored by fluorescent method and gel
electrophoresis (see Example VII). Likewise, the loading of siRNA
on HER2 scFV-NP and FA-NP were performed in a similar manner.
Example VII
Characterization of Nanoconstructs
[0186] After surface modification, the nanoconstructs had a
hydrodynamic size of .about.100 nm for the three uniform-sized core
materials (S-34, S-47, S-61) and 200 nm for the non-uniform-sized
core material (O-87) in water as shown in Table 4 and the size
distribution histograms are shown in FIG. 1C. All materials are
positively charged due to the PEI. All materials have fairly narrow
size distribution (PDI about 0.2) with the exception of S-34. The
PEI and PEG loadings were analyzed by thermogravimetric analysis
(TGA Q50, TA Instruments, DE). Pierce Micro BCA kit (Thermo Fisher
Scientific) was used to quantify the antibody or scFV loading on
the nanoconstructs by analyzing for the remaining (unbound)
antibodies in the supernatant. Likewise, fluorescent analysis of
the remaining Dylight677-tagged siRNA in the supernatant was used
to quantify siRNA loading, and was confirmed by gel
electrophoresis. The composition of two representative materials
are summarized in Table 5.
TABLE-US-00004 TABLE 4 TEM size, hydrodynamic size, and zeta
potential of six different nanoconstructs. Hydrodynamic Material
MSNP core size (DLS) Zeta (MSNP size (nm) Surface Size charge core)
by TEM.sup.(a) modification.sup.(b) (nm).sup.(c) PDI.sup.(d) (mV)
O-87 87 .+-. 14 T-NP.sup.10 214 .+-. 22 0.22 22 .+-. 0.5 S-61 61
.+-. 7 T-NP.sup.10 113 .+-. 1.0 0.20 18 .+-. 0.4 T-NP.sup.10C 131
.+-. 0.3 0.20 19 .+-. 3.7 S-47 47 .+-. 4 T-NP.sup.10C 117 .+-. 0.5
0.19 25 .+-. 0.1 T-NP.sup.1.8C 117 .+-. 2.4 0.20 19 .+-. 4.0 S-34
34 .+-. 3 T-NP.sup.10C 133 .+-. 4.1 0.37 19 .+-. 4.0 .sup.(a)Core
size measured in dry state, average size of 50 particles.
.sup.(b)"10" stands for 10-kDa PEI; "1.8C" and "10C" stand for
cross-linked 1.8-kDa and cross-linked 10-kDa PEI, respectively. All
PEI-MSNP were then conjugated with 5-kDa PEG, and trastuzumab (T).
.sup.(c)Average of three measurements; the z-average diameter and
polydispersity index (PDI) values were defined according to
International Standard on DLS (ISO13321). .sup.(d)PDI ranges from 0
to 1; smaller number indicates narrower size distribution; e.g.,
PDI < 0.05 is considered monodisperse (one size only), while PDI
> 0.5 indicates a broad distribution of particle sizes.
TABLE-US-00005 TABLE 5 Composition of T-siRNA-NP (all reported as
percent by mass of nanoparticle) % PEI % PEG % Anti- NP/siRNA mass
Surface (by (by body (by ratio (fluorescent Material modification
TGA) TGA) BCA) method) S-47 T-NP.sup.10C 13.5 18.2 3 Complete at
S-47 T-NP.sup.1.8C 15.9 6.1 3 NP/siRNA of 10 and above
Example VIII
Gene Knockdown Efficacy of siRNA on Various Nanoconstructs
[0187] The LM2-4luc+/H2N cell line (over-expressing luciferase and
HER2; J. M. du Manoir et al., 2006) was used for initial assessment
of the nanoparticles for gene silencing efficacy when delivering
siRNA against luciferase (siLUC). Cells were plated at 3000
cells/well in a 96-well plate. One day after seeding, cells were
treated with siLUC-nanoparticles. The nanoparticles were loaded
with siLUC at NP/siRNA ratio of 25 or 50 by mass. They were applied
to each well at a fixed dose of 30 nM siLUC. The commercially
available transfection agent, DharmaFECT.TM. (Dharmacon, Lafayette,
Colo.), with the same siLUC dose served as a positive control.
Non-targeting or scrambled siRNA (siSCR) was used throughout as a
negative control. After overnight incubation (.about.20 hours),
cells were washed once to remove the nanoconstructs and replenished
with a complete media. At 48 hours post-treatment, cells were lysed
and analyzed for luciferase activity by the Luciferase Glow Assay
Kit (Thermo Fisher Scientific, Waltham, Mass.) and protein
concentration by the BCA protein assay kit (Thermo Fisher
Scientific), following manufacturer's protocols. Luciferase
activity of the lysate was normalized with the corresponding
protein concentration in the same well and reported as a percentage
of the untreated or siSCR controls. All treatments were performed
with replicates.
[0188] NP/siRNA ratio. We compared different nanoconstructs (four
core sizes, loaded with PEI of 1.8-kDa or 10-kDa, cross-linked or
not cross-linked) for luciferase silencing efficacy as shown in
FIG. 5A and B for NP/siRNA of 25 and 50, respectively. Silencing
efficacy is defined as the reduced luciferase level due to the
siLUC treatment vs. the siSCR control. The materials with NP/siRNA
of 50 offered better gene silencing efficacy (per same dose of
siLUC) (FIG. 5B) than those with NP/siRNA of 25 (FIG. 5A) due to
the greater number of nanoparticles to which the cells were
exposed. Materials with an NP/siRNA mass ratio of 50 were then used
throughout unless specified otherwise.
[0189] Core size. Without cross-linking, smaller particles had
reduced silencing efficacy compared to larger particles (e.g., see
S-61 vs. O-87, both were modified with 10-kDa PEI, designated as
T-NP.sup.10 in FIG. 5).
[0190] Cross-linking. In FIG. 5, cross-linking indeed increased the
silencing efficacy (e.g., T-NP.sup.10C vs. T-NP.sup.10 on S-61).
The smaller core size (S-34) resulted in aggregation (FIG. 1C) and
less silencing efficacy (FIG. 5A). The best hydrodynamic size (FIG.
1C) and best silencing efficacy (FIG. 5) was achieved with S-47,
modified with 10-kDa-PEI and with cross-linking (see T-NP.sup.10C
on S-47) and is used throughout and referred to as "T-NP" unless
specified otherwise.
[0191] In addition, when compared to the PEI-siRNA polyplex
(without MSNP core), T-NP was superior in terms of size and
luciferase silencing efficacy, as shown in FIG. 7A and B,
respectively.
Example IX
Buffering Capacity of Cross-Linked-PEI Nanoconstructs
[0192] Internalized nanoparticles ultimately end up in perinuclear
lysosomal vesicles. siRNAs must escape from these vesicles to the
cytosol to silence expression. Increasing the buffering capacity of
small nanoparticles in order to increase siRNA endosomal release
based on the proton sponge effect principle was achieved by PEI
cross-linking.
[0193] As outlined in Example III, the optimal PEI cross-linking
condition was 0.1 mg/ml of DSP with 2 mg (free) PEI added to the
binding solution. For measuring buffering capacity of cross-linked
and non-cross-linked materials, the nanoparticles were suspended at
0.2 mg/mL in 150 mM NaCl (pH 9, pH adjusted with 0.05 M NaOH). Upon
stabilization at pH 9.0, 5 .mu.L of 0.05 M HCl was added and the
solution was continuously stirred. When reaching steady state, the
pH was recorded and the acid was added again. The process was
repeated until the pH plateaued at around 3.0. The solution pH was
then reported as a function of the amount of acid added.
[0194] FIG. 2B shows the buffering capacities of nanoparticles with
cross-linked 1.8-kDa PEI (T-NP.sup.1.8C), cross-linked 10-kDa PEI
(T-NP.sup.10C), and non-cross-linked 10-kDa PEI (T-NP.sup.10) in
150 mM NaCl. As shown in FIG. 2B, the nanoparticles had buffering
capacity in the order of
T-NP.sup.10C>T-NP.sup.10>T-NP.sup.1.8C. The cross-linking of
the PEI on the nanoconstruct creates more secondary and tertiary
amines yielding greater buffering capacity than primary amines, and
promoting higher endosomal escape of siRNA based on proton sponge
effect theory. This agrees well with the superior gene silencing
efficacy of the cross-linked materials outlined in Example
VIII.
Example X
Serum Protection Assay
[0195] siRNA-nanoparticles were incubated with 50 v/v % human serum
in PBS for a specified period of time (0, 0.5, 1, 2, 4, 8, 24, and
48 hours) at 37.degree. C. under continuous shaking. At the end of
each time point, the sample was mixed with proteinase K (200
.mu.g/mL) and frozen at -80.degree. C. to stop the enzymatic
reaction. For the analysis, samples were thawed and mixed with 1.0
wt. % SDS in order to release siRNA from the nanoparticles. The
sample was then mixed with an equal amount of 2.times. loading
buffer and loaded into a 15% TBE-urea gel (BioRad). The gel ran at
100 V for the first 20 minutes and 150 V for another hour. The gel
was then stained with SyBR Gold (Life Technologies) following the
manufacturer's protocol and viewed in a UV chamber. The band
intensity was analyzed by ImageJ software (National Institutes of
Health, Bethesda, Md.). The fraction of intact siRNA was reported
as a function of time that the siRNA-nanoparticles were in 50%
serum. These results were compared to those obtained for free
siHER2 without nanoparticles.
[0196] FIG. 6A shows the amount of intact siHER2 that survived
enzymatic degradation as measured by gel electrophoresis. The
corresponding siHER2 quantification based on the band intensity and
location is shown in FIG. 6B. Without the nanoparticles, naked
siHER2 was degraded within 0.5 hour (observed as bands shifted
toward lower molecular weight), and its half-life was about 1 hour,
in agreement with previous reports for other siRNAs (A. Mantei et
al., 2008; D. M. Dykxhoorn et al., 2006). T-siHER2-NP.sup.1.8C
fully protected siHER2 at least 8 hours, while T-siHER2-NP.sup.10C
fully protected siHER2 at least 24 hours. The siRNA on both
nanoparticle platforms experienced much less degradation than on
the cyclodextrin-based nanoparticle that went to clinical trials
and showed antitumor efficacy (M. E. Davis, 2009). The siRNA on
such material experienced 50% degradation within 12 hours, and 70%
within 24 hours under 50% serum conditions (D. W. Bartlett et al.,
2007).
[0197] The higher siRNA protection for T-siHER2-NP.sup.10C compared
to T-siHER2-NP.sup.1.8C is likely due to the higher PEG content of
T-siHER2-NP.sup.10C, e.g., 18.2% vs. 6.1% (Table 5). PEG provides a
steric blocking effect (Z. Zhang et al., 2007; R. Gref, 2000) that
reduces enzymatic degradation of siRNA (S. Mao et al., 2006). In a
separate experiment (FIG. 6C), without PEG, siRNA on PEI-MSNP
degraded faster than naked siRNA since positively charged PEI
recruited more negatively charged enzymes to degrade siRNA. In
addition, without steric PEG, significant aggregation of siRNA
loaded nanoconstruct was observed (FIG. 6D). The steric effect of
PEG also reduces binding of blood proteins to the nanoparticles (R.
Gref, 2000).
Example XI
Cellular Uptake Analysis
[0198] Cells were harvested and resuspended in 1 million cells/150
.mu.L/tube. Each tube was mixed with 150 .mu.L of siSCR (tagged
with Alexa-488) nanoconstructs in PBS (containing 100 .mu.g
nanoparticle). Upon nanoconstruct addition, cells were placed on a
rocker in the cell incubator (37.degree. C., 5% CO.sub.2) for 0.5
or 2.0 hours. Cells were then washed (centrifuge at 115 g, 5 min)
with 1 mL FACS buffer (1.times. Phosphate Buffered Saline (Ca/Mg++
free)+1 mM EDTA+25 mM HEPES pH 7.0+1% FBS (Heat-Inactivated) three
times. Cells were then resuspended in 550 .mu.L of FACS buffer,
transferred to a 5-mL tube, and kept on ice until analysis. For
cells stained with free antibody (for gating purpose), antibody
labeling was performed on ice and under rocking conditions. Cells
were stained with primary antibody (trastuzumab or rituximab: 2
.mu.g per tube) for an hour, washed with PBS, stained with
secondary antibody (Anti-human Alexa 488: 2 .mu.g per tube) for 45
minutes, then washed 2 times with PBS, and re-suspended in 550
.mu.L of FACS buffer before analysis. All tubes were
counter-stained for cellular DNA with 2 .mu.L of 5 mM DRAQ5 (Cell
Signaling) for 15 minutes on ice. All tubes (except
antibody-labeled cells for gating purpose) were then incubated with
500 .mu.L of Trypan Blue (0.4% in PBS) to quench fluorescence
outside of the cells, and subjected to flow cytometry analysis.
10,000 events (cells) were analyzed for each sample. The intensity
was processed with FlowJo software (NIH, Bethesda, Md.).
[0199] The specificity with which trastuzumab-conjugated
nanoconstructs were taken up by cells that over-express the HER2
protein was assessed. The nanoconstruct contained a scrambled siRNA
and conjugated with trastuzumab (designated hereafter as
T-siSCR-NP.sup.1.8C and T-siSCR-NP.sup.10C) or with rituximab
targeting CD20 (designated as R-siSCR-NP.sup.1.8C and
R-siSCR-NP.sup.10C). Cellular uptake of T-siSCR-NP.sup.10C and
T-siSCR-NP.sup.1.8C in HER2+ breast cancer cells, BT474 and SKBR3,
and the HER2- cell line MCF-7 was measured at 0.5 or 2.0 hours post
exposure to the nanoconstructs. The siSCR was tagged with the
fluorescent reporter, Alexa 488, for these experiments to enable
quantitative analysis of siSCR uptake. R-siSCR-NP.sup.10C and
R-siSCR-NP.sup.1.8C served as a negative control since BT474, SKBR3
and MCF-7 cells do not over-express CD20. The amount of Alexa
488-tagged siSCR inside individual cells was measured using flow
cytometry. FIG. 8A-C shows that T-siSCR-NP.sup.10C were taken up
effectively (>90%) into HER2+ cells (BT474 and SKBR3), but not
HER2- cells (MCF7) and that uptake increased by extending the
exposure time from 0.5 hr to 2 hr. Furthermore, uptake of
T-siSCR-NP.sup.10C was greater than T-siSCR-NP.sup.1.8C.
R-siSCR-NP.sup.10C and R-siSCR-NP.sup.1.8C were not taken up
efficiently by any of the cell lines, indicating the ability of T
to promote cellular uptake to HER2+ cells. FIG. 8D illustrates HER2
protein expression in the three cell lines being evaluated. FIG.
8E-G show the average intensity of Alexa 488-tagged siSCR signal
per cell and the same trend can be observed. This confirms that
trastuzumab-conjugated nanoconstructs enter cells primarily by a
HER2-receptor mediated endocytosis mechanism and not by adsorptive
endocytosis of positively charged particles as reported for
PEI-MSNP (H. Zhang, et al., 2011).
[0200] As another example, cetuximab (anti-EGFR antibody) was used
as a targeting agent on the nanoconstruct, named C-NP, in a similar
manner as T-NP. C-NP also showed preferential uptake to EGFR+ cells
over EGFR- cells (FIGS. 8H-I).
Example XII
HER2 Protein Knockdown and Cell Viability
[0201] HER2+ breast cancer cells (BT474, SKBR3 and HCC1954) were
seeded in a 96-well plate for 24 hours prior to treatment.
Nanoconstructs were loaded with siHER2 or siSCR at NP/siRNA 50.
siRNA dose was fixed at 60 nM. Media was switched to complete media
after overnight incubation. Three days after treatment with
nanoconstructs, cells were fixed and analyzed for HER2 protein
expression by immunofluorescence. HER2 mRNA and .beta.-actin mRNA
levels were analyzed at 48 hours post-treatment using the
Quantigene 2.0 Reagent System (Panomics) following the
manufacturer's protocol. The HER2 mRNA level was then normalized
with .beta.-actin mRNA (housekeeping gene) and reported as the
percentage of the siSCR control. Cell viability and apoptosis were
analyzed four days post-treatment using the CellTiter-Glo.RTM.
Luminescent Assay (Promega) and the Caspase-Glo.RTM. 3/7 Assay
Systems (Promega), respectively. Caspase activity was normalized
with the cell viability. Both were reported as a percentage of the
untreated control.
[0202] The efficiency of T-siHER2-NP.sup.10C in inhibiting HER2
mRNA levels and HER2 protein expression in the HER2+ breast cancer
cell lines, BT474, SKBR3, and HCC1954 was assessed. As shown in
FIG. 9A, T-siHER2-NP.sup.10C reduced HER2 levels by 81-93% compared
to T-siSCR-NP.sup.10C. As shown in FIG. 9B, there was 44% reduction
in HER2 mRNA relative to siSCR control. The cleaved Caspase 3 and 7
assay for apoptotic markers shows that apoptotic activity was
three-fold greater after treatment with T-siHER2-NP.sup.10C than
with T-siSCR-NP.sup.10C (FIG. 9C). This is consistent with reduced
cell viability (using a cellular ATP level assay) shown in FIG.
9D.
[0203] Treatment with T-siSCR-NP.sup.10C reduced HER2 levels and
killed HER2+ breast cancer cells, indicating that the antibody (T)
on the nanoconstruct not only serves as a cell targeting agent but
also has therapeutic effect. FIG. 11, for example, shows that HER2
levels in BT474 were reduced by 41% with T-siSCR-NP.sup.10C (due to
T effect) and by 87% with the T-siHER2-NP.sup.10C (due to T and
siHER2 effect) compared to untreated controls. Likewise, FIG. 9D
shows that cell viability was reduced 59% by T-siSCR-NP.sup.10C and
86% by T-siHER2-NP.sup.10C.
[0204] Cell viability after treatment with T-siHER2-NP.sup.10C was
also measured in a panel of HER2+ breast cancer cells, HER2- breast
cells, and HER2- non-breast cells. FIG. 10 shows that treatment
with T-siHER2-NP.sup.10C greatly reduced viability of HER2+ breast
cancer cells (BT474, SKBR3, HCC1954 and JIMT-1), while having
little impact on HER2- breast cells (MCF-7, MDA-MB-231, MDA-MB-468,
MCF-10a), and HER2- non-breast cells (HepG2 and HEK-293). The cell
killing effect was dependent on the HER2 protein levels of the
cells (Inset of FIG. 10).
[0205] FIG. 9A shows that T-siHER2-NP.sup.10C was more effective
than T-siHER2-NP.sup.1.8C (FIG. 12A) at equivalent siRNA dose.
However, FIG. 12B shows that doubling the dose of
T-siHER2-NP.sup.1.8C could reduce HER2 protein levels by 79-83% in
SKBR3 and HCC1954.
[0206] Overall, T-siHER2-NP.sup.10C demonstrated better HER2
knock-down and cancer cell killing efficacy than
T-siHER2-NP.sup.1.8C. Encouragingly, the T-siHER2-NP.sup.10C (FIG.
9A) outperformed commercially available DharmaFECT.TM. in all cell
lines (FIG. 12C).
Example XIII
Overcoming Drug Resistant Cancer with siHER2
[0207] The efficacy of T-siHER2-NP.sup.10C in intrinsically
trastuzumab-resistant HER2+ cell lines (HCC1954 and JIMT1), in a
parental HER2+ cell line (BT474) that responds to trastuzumab and
lapatinib, and in BT474-R, a derivative cell line that was made
lapatinib-resistant by long-term treatment with 1 .mu.M lapatinib
was also assessed. FIG. 13A shows that the BT474-R cells were much
less responsive to trastuzumab compared to parental BT474. However,
FIG. 13B shows that both trastuzumab-sensitive and resistant cell
lines responded similarly with respect to siHER2 action (see
T-siHER2-NP.sup.10C vs. T-siSCR-NP.sup.10C control). This indicates
that siHER2 on the nanoconstruct can overcome cancer resistance to
trastuzumab. Meanwhile, FIG. 13C shows that BT474-R was less
responsive to T-siSCR-NP.sup.10C than BT474, indicating that the
BT474-R cells were indeed resistant to trastuzumab (on the
nanoconstructs).
Example XIV
Blood Compatibility (Hemolysis, Coagulation, and Platelet
Aggregation)
[0208] The T-siHER2-NP.sup.1.8C and T-siHER2-NP.sup.10C were
assessed for hemolysis, thrombogenesis, and platelet aggregation,
and the results were benchmarked with FDA-approved nanoparticle
products: Abraxane (Paclitaxel-albumin nanoparticles) and Feraheme
(iron oxide nanoparticles used as a MRI contrast agent).
Nanoparticles were tested at 1.times. and 5.times. of the intended
human blood level. Studies of blood compatibility were performed
following or with minor modification from the Nanotechnology
Characterization Laboratory's (Frederick, Md.) published
protocols.
[0209] Hemolysis. Human blood was collected in the presence of
EDTA, and serum was removed. Red blood cells were suspended at
1.times.10.sup.9 cells per mL and exposed to nanoparticle (final
concentrations of 70 or 350 .mu.g/mL for 1.times. or 5.times.,
respectively) for 4 hours and 37.degree. C. Following
centrifugation, absorbance of hemoglobin in the supernatants (at
542 nm) was measured and used to quantify percent hemolysis.
Abraxane (Celgene) at 94 .mu.g/mL for 1 .times. and 470 .mu.g/mL
for 5.times. was used. As shown in FIG. 14A, T-siHER2-NP.sup.1.8C
and T-siHER2-NP.sup.10C did not cause hemolysis of red blood cells
at either dose, while complete blood lysis was achieved with 0.025%
Triton-.times. (the positive control).
[0210] Coagulation (thrombogenesis) assay. Platelet-poor plasma
(PPP) was obtained following a two-step centrifugation of isolated
blood (diluted in 3.2% sodium citrate, 1:10). After the first spin
at (2150 g, 10 min), the top portion of plasma (.about.75% of the
total volume) was collected without disturbing the plasma at the
bottom. The collected portion was centrifuged again at the same
speed for 10 minutes, and the top portion (.about.75% of the total
volume) was collected as PPP. Nanoconstructs were mixed with 0.15
mL PPP to the final concentration of 70 or 350 .mu.g/mL. The tubes
were incubated for 30 minutes at 37.degree. C. After 30-minute
incubation, 0.05 mL of APTT-xl reagent was added and incubated for
3 minutes in the Trinity Biotech KC-4 coagulation analyzer. After
which, 8.3 mM CaCl.sub.2 was added and the time until the onset of
coagulation was recorded. Abraxane at 1.times. and 5.times. doses
was used as a control. Likewise, Feraheme (AMAG Pharmaceuticals) at
102 .mu.g/ml for 1.times. and 510 .mu.g/ml for 5.times., was also
used in parallel. FIG. 14B shows that the nanoconstructs and
Abraxane did not affect the coagulation time of platelet poor
plasma since all took about 37 s. Only Feraheme prolonged the
coagulation time, in agreement with known side effects related to
abnormal clotting previously reported (M. H. Schwenk 2010).
[0211] Platelet aggregation assay. Platelet-rich plasma (PRP) was
obtained following centrifugation of isolated blood (diluted in
3.2% sodium citrate, 1:10). The isolated blood was centrifuged at
200 g for 20 minutes. The supernatant (which contains PRP) was
collected and maintained at room temperature prior to treatment.
Following a 1-min incubation at 37.degree. C. (baseline), reactions
were initiated by addition of nanoparticle (70 or 350 .mu.g/mL) or
collagen related peptide (CRP; 100 .mu.g/ml) and monitored for
three minutes for optical density via an aggregometer (Chrono-log
Corp). Abraxane at 1.times. and 5.times. as entailed above was also
used as a benchmark. FIG. 14C shows that the nanoparticles and
Abraxane did not trigger platelet aggregation while a collagen
related peptide used as a positive control triggered aggregation
immediately.
Example XV
Immune Response-Peripheral Blood Mononuclear Cell (PBMC) Cytokine
Release Assay
[0212] A PBMC cytokine release assay was conducted according to the
recommendations and method by the Nanotechnology Characterization
Lab (NCL) of NCl for immunological studies of nanoparticles. The in
vitro cell based assay evaluated cytokine production by PBMCs
(200,000 cells/well) following a 24-hour exposure to the test
materials. Test materials included nanoconstructs with and without
siHER2 to investigate the potential impact of siRNA mediated immune
response. Following incubation, cell culture supernatants were
collected and analyzed for IL-1.beta., IL-6, IFN-.alpha., and
TNF-.alpha. by a cytometry bead array (Milliplex Magnetic Bead)
following the manufacturer's protocol. Abraxane and Feraheme were
used as drug benchmarks since there is no siRNA based nanoconstruct
drug in the market.
[0213] The effect of T-siHER2-NP.sup.1.8C and T-siHER2-NP.sup.10C
on immune response was evaluated upon treating peripheral blood
mononuclear cells (PBMCs) isolated from human blood with these
nanoconstructs. PBMCs have been reported to respond to siRNA
transfection with a sequence-specific TLR 7/8 dependent induction
of IFN-.alpha. and TNF-.alpha. (R. Broering et al., 201; M.
Zamanian-Daryoush, 2008). The TLR7/8 agonist, R848, was used as a
direct positive control since TLR7 and TLR8 are located within the
endosomes (A. Chaturvedi, S. K. Pierce, 2009) where nanoconstructs
and siRNA are expected to reside. FIGS. 15A-F show that neither
T-siHER2-NP.sup.1.8C nor T-siHER2-NP.sup.10C increased the levels
of IL-6 and TNF-.alpha. at either the 1.times. or 5.times. level,
while Abraxane significantly increased both cytokines at the
5.times. level. Both nanoconstructs increased the levels of
IFN-.alpha. and IL-1.beta. somewhat, but not to the extent observed
for Abraxane for IL-1.beta. and Feraheme for IFN-.alpha.. The
immune response was not significantly different for nanoconstructs
with or without siRNA, suggesting that the response was not siRNA
specific. Lastly, the PBMC immunological response to
T-siHER2-NP.sup.10C was not significantly different than that to
T-siHER2-NP.sup.1.8C.
Example XVI
Endotoxin (LAL Gel-Clot) Assay of Nanoconstructs
[0214] To ensure sterility of our material production,
T-siHER2-NP.sup.1.8C and T-siHER2-NP.sup.10C were tested for
lipopolysaccharides or LPS (endotoxin), produced by gram-negative
bacterial contamination. About 35% of clinically relevant
nanoparticles have been found to carry this contaminant (R. M.
Crist et al., 2013). The two nanoconstructs were tested along with
Abraxane as an FDA drug benchmark. All were negative for endotoxin
(FIGS. 15E and F). This suggests an advantage of layer-by-layer
modification, as sequential washing steps promote sterilization of
the resulting product.
Example XVII
Mouse Tumor Models and In Vivo Efficacy Studies
[0215] In vivo gene silencing studies were performed in orthotopic
mouse tumor models; 4.times.10.sup.6 HCC1954 cells (unless
specified otherwise) were implanted into the mammary fat pads of
5-week-old SCID mice (Charles River, Wilmington, Mass.) and allowed
to grow to an average size of about 250 mm.sup.3. Mice were then
grouped and proceeded to receive a single injection (tail vein) of
the nanoconstructs (T-siHER2-NP.sup.10C or T-siSCR-NP.sup.10C, 1.25
mg/kg siRNA), or the PBS control. The tumors were harvested four
days after treatment and analyzed for HER2 protein expression by
immunofluorescence as shown in FIG. 16A and quantified in FIG. 16B.
FIG. 16B shows that the HER2 protein levels in the HCC1954 tumors
were reduced by 59% compared to saline control (p<0.0013) and by
47% compared to treatment with the T-siSCR-NP.sup.10C control
(p<0.015). It should be noted that 23% (p=0.27 vs. saline
control) of the HER2 reduction in the siSCR control is likely due
to trastuzumab on the nanoconstructs.
[0216] FIG. 17A shows that 5 intravenous tail vein injections of
T-siHER2-NP.sup.10C (1.25 mg siHER2/kg), over a period of three
weeks significantly inhibited tumor growth, while
T-siSCR-NP.sup.10C produced little effect. This response is
noteworthy since HCC1954 has been established as resistant to
cisplatin (I. Beyer et al., 2012), trastuzumab (I. Beyer et al.,
2012), and pertuzumab (F. Henjes, et al., 2012) in vitro and/or in
mice. HCC1954 resistance in vivo to trastuzumab and a
trastuzumab/paclitaxel combination was confirmed in our lab in
FIGS. 17B-D.
[0217] T-siHER2-NP.sup.10C (S-47 core, about 100 nm in size, FIG.
17A) showed better tumor growth inhibition efficacy than the
non-cross-linked larger particles (T-siRNA-NP.sup.10, with O-87
core, about 200 nm in size, FIG. 18) on the same HCC1954 tumor
model (n=11/group) even at half the dose of siRNA (i.e., 1.25 mg/kg
in FIG. 17A vs. 2.5 mg /kg in FIG. 18).
[0218] The nanoconstructs without trastuzumab (siHER2-NP.sup.10C
also inhibit tumor growth in the HCC1954 tumor model as shown in
FIG. 19. Tumor mice were treated with siHER2-NP (n=8), T-siSCR-NP
(n=7), or saline control (n=5) at the siHER2 or siSCR dose of 1.25
mg/kg and NP/siRNA ratio of 50 on days specified with arrows. This
is owed to the passive delivery, relying on enhanced permeability
and retention (EPR) effect of the tumors for the nanoconstructs,
followed by the uptake of positively charged nanoconstructs to
cancer cells, yielding tumor growth inhibition effect.
[0219] In another example, BT474-TRgf (BT474 variant developed to
be resistant to trastuzumab by serial passaging the cells in mice,
Francia et al., 2012) was used to evaluate the efficacy of
T-siHER2-NP. While the BT474-TRgf cells were resistant to
trastuzumab in vitro (FIG. 20A) and in a tumor mouse model (FIG.
20B), they responded to T-siHER2-NP treatment in the tumor model
(FIG. 20B). siHER2 dose was 1.25-2.5 mg/siRNA and 5 animals per
treatment group.
Example XVIII
Lyophilization of Nanoconstructs for Long Term Storage
[0220] Nanoconstruct suspensions (T-NP or NP) in 0.1 M Tris HCl (pH
7.4) were added to a solution of trehalose to achieve a final
concentration of 10 mg/mL MSNP and 0-25 weight % of trehalose per
nanoparticle. The well-mixed suspension was frozen slowly from room
temperature to -55.degree. C. in a freeze dryer. This was followed
by primary and secondary drying. Primary drying (-40.degree. C.,
100 .mu.Bar, 24 hr) was to sublimate the ice crystals formed during
the freezing step. Secondary drying (20.degree. C., 20 .mu.Bar, 12
hr) was to eliminate bound water molecules on the nanoconstruct
surface. The finished products were in the form of powder in sealed
and storable bottles.
[0221] All lyophilized nanoconstructs with trehalose content of
0-10% retained the average size and charge of the freshly made
nanoconstruct after 1 min of sonication as shown in FIG. 22A-B,
respectively. However, the 0% trehalose conditions yielded larger
size distribution (not shown). After binding with either siLUC or
siHER2, lyophilized materials still yielded very comparable
performance with freshly made material counterparts (from the same
batch) in terms of luciferase silencing ability (FIG. 22A, with
siLUC) and cell viability of BT474 cells (FIG. 4B, with siHER2).
Some exceptions are also evident in FIG. 22D. The 0% trehalose
conditions yielded T-NP that was more toxic to cells than freshly
made material. Hence, based on the size distribution and efficacy,
the 5% trehalose, the lowest additive that yielded the same
efficacy as the freshly made counterpart, was selected for future
lyophilization processes and long term storage evaluation.
[0222] Further, T-NP lyophilized with 5% trehalose and stored at
-20.degree. C. to 37.degree. C. were monitored for their properties
over a 24-week storage time. FIGS. 23A-23E shows that when stored
at -20.degree. C., T-NP maintained (A) hydrodynamic size, (B) zeta
potential, (C) siRNA loading, (D) luciferase silencing efficacy,
and (E) cancer cell killing efficacy similar to freshly made
nanoparticles (Fresh) from the same batch. Data indicates that
-20.degree. C. is the best storage temperature for preserving all
properties of the fresh material for at least 24 weeks (6 months).
Similar outcomes were achieved with NP without T.
Example XIX
Reproducibility and Scalability of Nanoconstruct Production
[0223] Many nanoparticle platforms are known to have problems with
batch-to-batch reproducibility--especially at a larger scale. MSNP
core production via sol-gel chemistry and layer-by-layer surface
modification affords very reproducible and scale-able production of
the nanoconstruct. Multiple individual batches of mesoporous silica
nanoparticles were created to determine reproducibility of
nanoparticle synthesis. As shown in Table 6, the size, charge, and
silencing efficacy were closely correlated with 2.4% relative
standard deviation (RSD) of particle sizes from 6 batches,
indicating that the material synthesis is highly reproducible.
Scaling-up from 300 mg/batch to 6 g/batch has been accomplished
(e.g., by simply increasing the reaction solution from 125 mL to
2.5 L during nanoparticle synthesis), which yielded similar
material properties with the smaller batches.
TABLE-US-00006 TABLE 6 Reproducibility of nanoconstruct synthesis
before and after surface modification. MSNP core T-NP.sup.10C
Hydrodynamic Hydrodynamic Zeta % Luc Size Z- size Z- Potential
silencing average .+-. average .+-. Average .+-. efficacy Batch SD
(nm) SD (nm) SD (mV) (vs. siSCR) 1 61.1 .+-. 0.7 115.8 .+-. 4.0
25.0 .+-. 0.1 75.7 .+-. 4.0% 2 58.1 .+-. 0.6 117.4 .+-. 0.5 24.9
.+-. 0.1 80.5 .+-. 2.8% 3 59.7 .+-. 0.5 114.5 .+-. 7.1 25.0 .+-.
0.1 76.1 .+-. 2.4% 4 57.7 .+-. 0.9 123.8 .+-. 3.3 25.0 .+-. 0.1
76.6 .+-. 3.9% 5 60.8 .+-. 0.8 113.2 .+-. 2.3 25.0 .+-. 0.1 76.2
.+-. 2.8% 6 58.8 .+-. 0.3 115.6 .+-. 1.3 25.0 .+-. 0.1 77.0 .+-.
2.2% Average 59.4 116.7 25.0 77.0 % Relative 2.4 3.2 0.2 2.3 SD
Example XX
Additional siRNA Targeting Genes Beyond HER2 for Treating
Cancer
[0224] The initial screen sought to assess the efficacy of
individual siRNAs in HER2.sup.+ breast cancer. SiRNA against AKT
(isoforms 1, 2, 3), BCL2, PLK1, GRB7, and EPS8L1, all were
benchmarked against the previously optimized siHER2 sequence. Four
cell lines were chosen, representing trastuzumab-resistant
(HCC1569, HCC1954, JIMT1) and sensitive (BT474) HER2+ breast
cancer. For each gene, the siRNA sequences were also varied
(Hs_AKT1_5 FlexiTube siRNA (SI002991450), Hs_AKT1_8 FlexiTube siRNA
(SI00287742), Hs_AKT1_10 FlexiTube siRNA (SI02757244) and
Hs_AKT1_11 FlexiTube siRNA (SI02758406) for AKT1 shown to be
efficacious by Qiagen) (Qiagen, Valencia, Calif., USA) as possible.
Representative data for BT474 is shown in FIG. 24A.
[0225] We also evaluated dual targeting siRNA against AKT1/BCL2
(e.g., one siRNA duplex has one strand targeting AKT1 and the other
strand targeting BCL2) (Table 1). FIGS. 24A-24B confirm the cell
killing efficacy (A) and protein knockdown activity (B) of the
dual-targeting siRNA.
[0226] In the four cell lines tested, none of the individual AKT1
or BCL2 siRNAs worked better than the siAKT1/BCL2 (dual-targeting
siRNA) or siPLK1. Therefore, the three siRNAs selected from the
initial screening are siHER2, siAKT1/BCL2, and siPLK1. FIG. 25
shows the cell viability 4 days post-exposure to the siRNAs at the
indicated dose range (0.01-30 nM). The siRNA targeting PLK1 proved
to be the most potent of all siRNAs in each respective cell line;
the IC.sub.50 (dose inhibiting 50% growth) of siPLK1 is about 0.2
nM for 3 out of 4 cell lines studied.
[0227] The cell panel was expanded to include triple negative
breast cancer, ER+, and non-breast cells. As shown in FIG. 26,
siPLK1 is still the most potent among the three siRNAs evaluated.
(FIG. 26). However, siPLK1 was toxic to non-target cells (e.g.,
non-targeted cells in FIG. 26) when delivered with commercial
DharmaFECT.TM.. Treatment specificity may be achieved for breast
cancer cells (HCC1954) over normal breast epithelial cells
(MCF-10A) when the siPLK1 is delivered with the nanoconstructs
(T-NP), compared to DharmaFECT.TM., as shown in FIGS. 27A and B,
respectively.
[0228] FIG. 27A also shows the ability of T-NP in delivering miRNA
mimics (miR342-5p, obtained from GE Dharmacon (Lafayette, Colo.,
USA) and eliciting killing of cancer cells but not MCF-10A.
Example XXI
Screening of siRNA in Combination with Paclitaxel
[0229] Cells were first treated with 30 nM siRNAs (and commercial
DharmaFECT.TM.) followed by 5 nM paclitaxel treatment 24 hours
later. Cell viability was determined 3 days later. As shown in
Table 7, the combination of siRNA with paclitaxel enhanced the
overall reduction in cell viability compared to paclitaxel alone
(Scramble 30 Paclitaxel, top row). The % enhancement by various
siRNAs with paclitaxel over paclitaxel alone was also summarized in
Table 7. In short, knocking down HER2 had the greatest impact on
paclitaxel toxicity in BT474 (having highest HER2 level). For other
cells, the best efficacy was achieved with paclitaxel+siPLK1. The
effect of paclitaxel combined with siAKT1/BCL2 or siEPS8L1 was also
significant. The best enhancement by siAKT1/BCL2 (38-41%) appeared
to be with basal cells, HCC1954, BT549, and HCC70. EPS8L1 is
involved in EGFR signaling and functions in cell migration. As a
result, efficacy of EPS8L1 was not restricted to HER2+ cells and
proved effective in the two triple negative cell lines tested (both
having high EGFR).
TABLE-US-00007 TABLE 7 Screening of siRNA targeting HER2, PLK1,
AKT1/BCL2 and EPS8L1 in combination with the chemotherapeutic drug
paclitaxel. Cell Viability (% of Scramble Control) Treatments HER2
Positive Triple Negative siRNA Luminal Basal Targets Paclitaxel
BT474 SKBR3 HCC1954 BT549 HCC70 Scramble + 58% 39% 55% 59% 70% HER2
- 35% 51% 68% 51% 76% + 26% 33% 49% 40% 49% Enhanced 32% 6% 6% 19%
21% Paclitaxel effect due to gene knockdown AKT1 + - 74% 77% 47%
54% 42% BCL2 + 39% 29% 17% 23% 29% Enhanced 19% 10% 38% 36% 41%
Paclitaxel effect due to gene knockdown PLK1 - 33% 14% 6% 15% 33% +
33% 13% 5% 13% 27% Enhanced 25% 26% 50% 46% 43% Paclitaxel effect
due to gene knockdown EPS8L1 - 37% 53% 57% 40% 65% + 27% 29% 35%
13% 35% Enhanced 31% 10% 20% 46% 35% Paclitaxel effect due to gene
knockdown
Example XXII
Delivery of Other Cargos by Nanoconstructs
[0230] Peptide delivery. FIG. 28A shows the ability of
nanoparticles to load and deliver small peptides into cells.
MSNP-PEI-PEG (non-cross-linked PEI) was loaded with GALA peptide
(MW of 3 kDa) by electrostatic interaction in the same manner as
siRNA. GALA was mixed with siRNA-nanoconstructs (13-130 nM GALA and
10 mg/L nanoconstructs) for 20 minutes in PBS (room temp, 300 rpm
shaking). GALA clearly enhanced silencing efficacy of the
nanoconstructs, owing to its ability as a pore-forming peptide to
enhance endosomal escape of the siRNA-nanoconstructs (Li et al.,
2004).
[0231] Co-delivery of antibody, siRNA, and chemotherapeutic. The
nanoconstructs (MSNP-PEI-PEG or NP) can also co-deliver antibody,
siRNA, and chemotherapeutic drugs to cancer cells. FIG. 28B shows
the effects of trastuzumab (T), siHER2, and/or paclitaxel (PTX)
loaded on the nanoconstruct in JIMT-1 cells. The combination of
paclitaxel and trastuzumab on the same nanoparticle was very
potent, which masked the effect of siHER2. Loading of PTX was
accomplished by mixing 3 mg of PTX to ethanol solution containing
10 mg MSNP-PEI during the cross-linking step described in Example
III. Doxorubicin (DOX) loading was performed by mixing 3 mg of DOX
with 10 mg of MSNP in water overnight. MSNP-Dox was then
centrifuged down and resuspended in ethanol for subsequent loading
of PEI, PEG, and trastuzumab as outlined in Example III-V. In order
to confirm that drug-loaded T-NP can still deliver siRNA and elicit
gene knockdown effectively. Drug-loaded NP (paclitaxel (PTX) or
doxorubicin (DOX)) with or without trastuzumab were evaluated for
ability to deliver siLUC and elicit luciferase gene knockdown. FIG.
29 shows that the drugs did not negatively impact the gene
knock-down efficacy of the materials. In addition, the combination
of paclitaxel, trastuzumab, and siHER2 shows greater efficacy in
inhibiting tumor growth in vivo than the free trastuzumab and
paclitaxel combination as shown in FIG. 30.
Example XXIII
Antioxidant and/or Copper Chelating Nanoconstruct for siRNA
Delivery
[0232] Another advantage of the MSNP based nanoconstruct is that
the MSNP can reduce reactive oxygen species (ROS) in cells
triggered by menadione as shown in FIG. 31A. The ROS reduction was
attributed to the MSNP core and not the cationic PEI-PEG coating.
This was confirmed by the finding (in a cell-free system) in FIG.
31B that bare MSNP core displayed higher DPPH free radical
scavenging ability than the MSNP coated with PEI-PEG layer. FIG.
31B also shows that free PEI had no ROS scavenging ability. Note
that DPPH is a stable free radical that is typically used to assess
the ROS scavenging ability of antioxidants. Due to the antioxidant
properties, the MSNP-PEI-PEG could reduce the effects of
pro-inflammatory cytokine TGF-beta by inhibiting NOX4, HSP47, COL
I, and alpha-SMA as shown in FIG. 31C. Likewise, FIG. 31D shows
that the nanoconstruct could reduce the pro-fibrotic markers such
as HSP47, COL I, and/or alpha-SMA, triggered by bleomycin. Bleo
fibroblast denotes murine dermal fibroblasts harvested from
bleomycin-induced scleroderma mouse model. Thus, the nanoconstruct
has the potential to treat fibrosis as was confirmed in a fibrotic
mouse model created by bleomycin treatment of mouse skin as shown
in FIGS. 32A-32G (Morry et al., 2015). Treatment schedule is
outlined in FIG. 32A. Reduction in skin fibrogenesis was observed
with siSCR-nanoconstruct treatment but was enhanced with the
siHSP47-nanoconstruct. This was evidenced by the reduction in skin
thickness (FIGS. 32B-C), HSP47 (D), NOX4 (E), alpha-SMA (F), and
COL I(G) compared to bleomycin treated mice.
[0233] In addition, NOX4 was found to be overexpressed in the
majority of breast cancer cell lines, primary breast tumors, and
ovarian tumors. The overexpression of NOX4 in normal breast
epithelial cells could result in cellular senescence, resistance to
programmed cell death, and tumorigenic transformation of the cells
(K. A. Graham, 2010), establishing therapeutic utility of the MSNP
carrier as a NOX reducer in addition to the therapeutic agents it
delivers.
[0234] Antioxidant properties of nanoparticles coupled with siPLK1
can elicit treatment effects in breast cancer metastasis. FIGS.
33A-33D show in vitro PLK1 knockdown (A-B), G2/M cell cycle arrest
(C), and cancer cell killing (D) induced by siPLK1-NP. FIGS.
34A-34H show that the antioxidant property of nanoparticles can
scavenge ROS in cancer cells (A) without toxicity effect (B),
resulting in decreased NOX expression (C), decreased cancer cell
migration (in wound healing assay) (D-E), and decreased invasion of
cancer cells (F-H) that was more effective than an established
antioxidant NAC at 2-10 mM (H). The T-siSCR-NP nanoconstruct also
limited tumor spread in vivo as shown in FIG. 35B as cancer cells
were found primarily in the primary established site of this model
(lung), compared to untreated mice where tumor signals can be found
in other distant organs similar to metastasis sites of human breast
cancer. When combined with siPLK1, T-siPLK1-NP resulted in tumor
growth inhibition in lungs (FIGS. 35B-D, F and H) and subsequent
prolonged survival in mice beyond T-siSCR-NP treatment (G). PLK1
knock down in the tumors was also observed (FIG. 35E).
[0235] Amine groups, e.g., from PEI on nanoconstructs, are known to
be highly effective chelators of copper, which is a cofactor for
angiogenesis and hence metastasis in cancer (Brewer et al., 2000).
In agreement with our data, the lowest cancer metastasis and tumor
burden was achieved with T-siPLK1-NP (FIG. 35, short term study),
which agreed with the lowest serum ceruloplasmin (a biomarker for
total body copper status) in corresponding mice (FIG. 36).
Example XXIV
Lanthanide and Fluorescent Dye Loading on MSNPs
[0236] MSNPs containing phosphonate groups were prepared by adding
3-trihydroxysilylpropyl methylphosphonate in situ during the MSNP
synthesis described in Example I and Example II. Specifically, 500
mg of CTAB was dissolved in 240 ml of water (adjusted by 2 M NaOH).
After temperature stabilized at 80.degree. C., 2.5 mL of TEOS was
added in to solution under stirring condition. After 15 minutes,
635 .mu.L of 3-(trihydroxylsilyl) propyl methyl phosphonate was
added. The reaction continued for 2 hours, and the pellets were
recovered from suspension by centrifugation, washed with a copious
amount of ethanol, and dried overnight. The particles were then
resuspended and refluxed in acidic methanol (0.6 M HCl in methanol)
overnight to remove CTAB. The resulting phosphonate-MSNPs were then
washed with ethanol and dried in a desiccator overnight.
[0237] For lanthanide loading, 5 mg/ml of MSNP was suspended in
water. Then 0.01-100 mg/L lanthanides were mixed with the MSNP
suspension. The mixture was shaken for two hours. The material was
then washed with a copious amount of water, and the material was
dried. MSNP can undergo surface modifications (e.g., PEI, PEG,
antibody loading as aforementioned) and can be used as a
fluorescent probe or a probe for mass spectrometry.
[0238] For characterization, MSNP of known dry weight was mixed
with 10 M HNO.sub.3 for acid leaching of lanthanides. The leachate
was then subjected to a lanthanide assay with ICP-MS. The amount of
each lanthanide loaded on nanoparticle (as wt. %) is shown in FIG.
37A. FIG. 37B shows that the nanoconstruct can also be loaded with
fluorescent dyes (on PEI layer) and/or an antibody (such as an
anti-HER2 antibody), that recognizes HER2 proteins on the target
cells.
Example XXV
Nanoconstructs Made from Other Inorganic Cores (e.g., Iron Oxide
NPs)
[0239] As an alternative to silica, iron oxide (Fe.sub.3O4)
nanoparticles (ION) can be used. Surface modification of such
nanoparticles can be performed using similar methods to those
described for MSNP. For example, a particle called DMSA-ION can be
prepared. Specifically, ION were prepared by high temperature
reaction of tris(acetylacetonato)iron(III) in the present of
stabilizing surfactants--1,2-hexadecanediol, lauric acid and lauryl
amine--in benzyl ether. The adduct was purified with ethanol and
hexane to obtain precursor ION-lauric acid. The precursor
nanoparticles then underwent a ligand exchange reaction from
hydrophobic lauric acid to hydrophilic meso-2,3-dimercaptosuccinic
acid (DMSA) which renders the nanoparticles water-soluble. The
final DMSA-ION were magnetically purified with water, ethanol and
acetone. ION of <10 nm by TEM has been achieved (Yantasee et al,
2007).
[0240] At neutral pH, DMSA has a negative charge which allows the
ligand to electrostatically bind to positively-charged
polyethylenimine (PEI). PEI modification allows the nanoparticles
to bind with negative charged siRNA. Under low pH environment of
intracellular compartment, DMSA is protonated and release the bound
PEI which in turn releases the siRNA. Evaluation of ION based
nanoconstruct for delivery of antibody, siRNA, chemotherapeutic,
and/or dyes will be performed similar to MSNP nanoconstruct. Their
advantages are small size for easy tumor accumulation,
biocompatible, and MRI compatible.
[0241] In this example, iron oxide nanoparticles have been surface
modified in a similar manner as MSNP using aforementioned methods
with minor modification. Specifically, iron oxide nanoparticles
(commercially available and FDA approved Ferumoxytol or Feraheme)
were dispersed in ethanol solution and PEI (10 kDa) was added to
the solution at a concentration 25 or 33 wt. % of PEI (10 kDa) per
iron oxide. The mixture was subsequently shaken, centrifuged, and
washed twice with PBS. Dry mal-PEG(5-kDa)-NHS was then added at a
1:1 ratio by mass of iron oxide to PEI in PBS. The resulting
solution was shaken for two hours at room temperature, and was
subsequently washed in PBS. Targeting agent (trastuzumab, T) was
incorporated in the same manner as Example V. siRNA loading was
achieved by mixing the resulting nanoconstructs with siRNA for 1
hour prior to characterization. The final hydrodynamic size of the
nanoconstruct was approximately 50-100 nm in average as shown in
FIG. 38A for material prepared with 25 wt. % PEI (called F1) or 33
wt. % PEI (called F2). The nanoconstructs exhibited a
polydispersity index of less than 0.15 and a potential of about
10-20 mV as measured in water, which increased from -50 mV of the
ION (Feraheme).
Example XXVI
Iron Oxide Nanoconstructs for siRNA Delivery and MRI Contrast
Agent
[0242] Nanoconstructs containing iron oxide nanoparticles can be
used to deliver siRNA and achieve effective gene silencing. Iron
oxide nanoconstructs synthesized by the method of Example XXV were
loaded with 50 nM siLUC at a nanoconstruct per siLUC mass ratio of
10 and used to treat LM2-4luc+/H2N cells expressing luciferase
similar to Example VIII. Good luciferase knock down efficacy
(40-50%) was achieved as shown in FIG. 38B.
[0243] Iron oxide based nanoconstructs can serve as an MRI contrast
agent. T2 relaxivity was calculated by T2 MRI (small animal Bruker
BioSpin 11.75T MRI instrument (31 cm horizontal bore), Paravision
5.1 software) as increasing Fe concentrations in PBS. T2 values
were found using ImageJ software and plotted as 1/T2 (1/s) vs Fe
(mM) to yield relaxivity (R2 coefficient) as the slope (FIG. 38C).
FIG. 38C shows that the final nanoconstruct T-F2 (conjugated with
trastuzumab and loaded with siHER2) has 2-3 fold enhanced R2
coefficient, compared to unmodified ION (Ferumoxytol). Hence, the
iron oxide nanoconstruct has the potential to serve as a diagnostic
tool (MRI probe). In addition, since the materials are magnetic,
they can potentially be used for magnetically guided delivery of
therapeutics in vivo or in humans.
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[0312] The invention is further described in the following numbered
embodiments:
[0313] Embodiment 1. A multilayer nanoconstruct comprising a
cationic polymer bound to an exterior surface of a nanoparticle,
wherein the cationic polymer is cross-linked.
[0314] Embodiment 2. The nanoconstruct of embodiment 1, further
comprising a stabilizer bound to the cationic polymer or
nanoparticle, wherein the stabilizer prevents aggregation of the
nanoconstruct in solution.
[0315] Embodiment 3. The nanoconstruct of embodiment 1 or 2,
wherein the nanoparticle is mesoporous.
[0316] Embodiment 4. The nanoconstruct of any one of embodiments
1-3, wherein the nanoparticle is a silica nanoparticle, a silicon
nanoparticle, an iron oxide nanoparticle, a gold nanoparticle, a
silver nanoparticle, or a carbon nanotube.
[0317] Embodiment 5. The nanoconstruct of any one of embodiments
1-4, wherein the nanoconstruct has a hydrodynamic diameter of from
about 10 to about 200 nm.
[0318] Embodiment 6. The nanoconstruct of embodiment 5, wherein the
nanoparticle has a diameter of 5 to 90 nm.
[0319] Embodiment 7. The nanoconstruct of any one of embodiments
1-6, wherein the cationic polymer is from about 5% to about 30% by
weight of the nanoconstruct.
[0320] Embodiment 8. The nanoconstruct of embodiment 7, wherein the
cationic polymer is from about 10% to about 25% by weight of the
nanoconstruct.
[0321] Embodiment 9. The nanoconstruct of any one of embodiments
1-8, wherein the cationic polymer is selected from the group
consisting of polyethylenimine, chitosan, polypropyleneimine,
polylysine, polyamidoamine, poly(allylamine),
poly(diallyldimethylammonium chloride), poly(N-isopropyl
acrylamide-co-acrylamide), poly(N-isopropyl acrylamide-co-acrylic
acid), diethylaminoethyl-dextran, poly-(N-ethyl-vinylpyridinium
bromide), poly(dimethylamino)ethyl methacrylate, and/or
poly(ethylene glycol)-co-poly(trimethylaminoethylmethacrylate
chloride).
[0322] Embodiment 10. The nanoconstruct of embodiment 9, wherein
the cationic polymer is polyethylenimine having a molecular weight
of from about 0.8 kDa to about 10 kDa.
[0323] Embodiment 11. The nanoconstruct of any one of embodiments
1-10, wherein the cationic polymer is cross-linked by reacting
cationic polymer on the surface of the nanoparticle with a
cross-linker in the presence of cationic polymer in solution.
[0324] Embodiment 12. The nanoconstruct of any one of embodiments
2-11, wherein the stabilizer is from about 1% to about 30% by
weight of the nanoconstruct.
[0325] Embodiment 13. The nanoconstruct of embodiment 12, wherein
the stabilizer is from about 5% to about 25% by weight of the
nanoconstruct.
[0326] Embodiment 14. The nanoconstruct of any one of embodiments
2-13, wherein the stabilizer is selected from the group consisting
of polyethylene glycol, dextran, polysialic acid, hyaluronic acid
(HA), polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), and/or
polyacrylamide (PAM).
[0327] Embodiment 15. The nanoconstruct of embodiment 14, wherein
the stabilizer is polyethylene glycol having a molecular weight of
from about 1 kDa to about 20 kDa.
[0328] Embodiment 16. The nanoconstruct of any one of embodiments
1-15, further comprising at least one type of oligonucleotide
electrostatically bound to the cationic polymer.
[0329] Embodiment 17. The nanoconstruct of embodiment 16, wherein
the at least one type of oligonucleotide is a siRNA, miRNA, miRNA
mimic, or antisense oligomer.
[0330] Embodiment 18. The nanoconstruct of embodiment 16 or 17,
wherein the at least one type of oligonucleotide is siRNA.
[0331] Embodiment 19. The nanoconstruct of embodiment 18, wherein
the siRNA targets one or more genes selected from the group
consisting of HER2, AKT1, AKT2, AKT3, EPS8L1, GRB7, AR, Myc, VEGF,
VEGF-R1, RTP801, proNGF, Keratin K6A, Bcl-2, PLK1, LMP2, LMP7,
MECL1, RRM2, PKN3, Survivin, HIF1.alpha., Furin, KSP, eiF-4E, p53,
.beta.-catenin, ApoB, PCSK9, HSP47, CFTR, CTGF, SNALP, RSV
nucleocapsids, CD47, PD-L1, and CTLA-4.
[0332] Embodiment 20. The nanoconstruct of any one of embodiments
16-19, wherein the at least one type of oligonucleotide is from
about 1% to about 15% by weight of the nanoconstruct.
[0333] Embodiment 21. The nanoconstruct of embodiment 20, wherein
the at least one type of oligonucleotide is from about 1% to about
5% by weight of the nanoconstruct.
[0334] Embodiment 22. The nanoconstruct of any one of embodiments
16-21, wherein the at least one type of oligonucleotide comprises
two or more different siRNAs loaded onto the nanoconstruct.
[0335] Embodiment 23. The nanoconstruct of any one of embodiments
1-22, further comprising a small molecule or a protein.
[0336] Embodiment 24. The nanoconstruct of embodiment 23, wherein
the small molecule is from about 0.5% to about 30% by weight of the
nanoconstruct.
[0337] Embodiment 25. The nanoconstruct of embodiment 23 or 24,
wherein the small molecule is a chemotherapeutic agent, small
molecule inhibitor, or a polypeptide.
[0338] Embodiment 26. The nanoconstruct of embodiment 23 or 24,
wherein the small molecule is a label.
[0339] Embodiment 27. The nanoconstruct of embodiment 26, wherein
the label is a lanthanide, a fluorescent dye, a gold nanoparticle,
a quantum dot, a positron emission tomography (PET) tracer, and/or
a magnetic resonance imaging (MRI) contrast agent.
[0340] Embodiment 28. The nanoconstruct of embodiment 23, wherein
the protein is a cytokine.
[0341] Embodiment 29. The nanoconstruct of any one of embodiments
1-28, further comprising a targeting agent.
[0342] Embodiment 30. The nanoconstruct of embodiment 29, wherein
the targeting agent is an antibody, a scFv antibody, an affibody,
an aptamer, a peptide, and/or small targeting molecule.
[0343] Embodiment 31. The nanoconstruct of embodiment 29 or 30,
wherein the targeting agent is from about 0.1% to about 10% by
weight of the nanoconstruct.
[0344] Embodiment 32. The nanoconstruct of embodiment 31, wherein
the targeting agent is from about 0.3% to about 5% by weight of the
nanoconstruct.
[0345] Embodiment 33. The nanoconstruct of any one of embodiment
30-32, wherein the small targeting molecule is a carbohydrate or
ligand.
[0346] Embodiment 34. The nanoconstruct of any one of embodiments
1-33, wherein the nanoconstruct is lyophilized.
[0347] Embodiment 35. The nanoconstruct of embodiment 34, wherein
the nanoconstruct is lyophilized with trehalose.
[0348] Embodiment 36. The nanoconstruct of embodiment 35, wherein
the trehalose is from about 1% to about 10% by weight of the
nanoconstruct.
[0349] Embodiment 37. The nanoconstruct of any one of embodiments
1-36, wherein the exterior surface of the nanoparticle comprises
thiol, amine, carboxylate, and/or phosphonate functional
groups.
[0350] Embodiment 38. A method of delivering an agent to a site in
a human or other mammalian subject comprising administering an
effective amount of the nanoconstruct of any one of embodiments
1-37 comprising the agent to the human or other mammalian subject
under conditions to deliver the nanoconstruct to the site.
[0351] Embodiment 39. The method of embodiment 38, wherein the site
is a cell.
[0352] Embodiment 40. The method of embodiment 39, wherein the
nanoconstruct is administered under conditions that the
nanoconstruct is internalized by the cell.
[0353] Embodiment 41. The method of any one of embodiments 38-40,
wherein the subject is suffering from a disease or condition
characterized by over-expression of one or more genes relative to
expression of the one or more genes in a healthy subject.
[0354] Embodiment 42. The method of embodiment 41, wherein the
disease or condition is selected from the group consisting of AMD,
macular edema, chronic optic nerve atrophy, pachyonychia
congenital, chronic lymphocytic leukemia, metastatic lymphoma,
metastatic cancer, solid tumors, acute kidney injury, delayed graft
function, familia adenomatous polyposis, hypercholesterolemia,
liver fibrosis, cystic fibrosis, dermal scarring, Ebola infection,
RSV infection, and inflammation.
[0355] Embodiment 43. The method of embodiment 41 or 42, wherein
the nanoconstruct is administered in an amount sufficient to treat
the subject having the disease or condition.
[0356] Embodiment 44. The method of any one of embodiments 38-43,
wherein the agent is a label.
[0357] Embodiment 45. The method of embodiment 44, wherein the
label is a lanthanide, a fluorescent dye, a PET tracer, or a MRI
contrast agent.
[0358] Embodiment 46. The method of any one of embodiments 38-43,
wherein the agent is a therapeutic agent.
[0359] Embodiment 47. The method of embodiment 46, wherein the
therapeutic agent is a nucleic acid capable of modulating
expression of a target protein.
[0360] Embodiment 48. The method of embodiment 47, wherein the
nucleic acid is a siRNA, miRNA, miRNA mimic, or antisense oligomer,
and expression of the target protein is reduced.
[0361] Embodiment 49. The method of embodiment 46, wherein the
therapeutic agent is a chemotherapeutic agent, a small molecule
inhibitor, an antibody, a peptide, and/or a cytokine.
[0362] Embodiment 50. The method of any one of embodiments 38-49,
wherein the subject is diagnosed with cancer, and the effective
amount is a therapeutically effective amount.
[0363] Embodiment 51. The method of embodiment 50, wherein the
cancer is resistant to a monoclonal antibody or a small molecule
inhibitor.
[0364] Embodiment 52. The method of embodiment 51, wherein the
therapeutic agent is an oligonucleotide that targets expression of
a protein inhibited by the monoclonal antibody or the small
molecule inhibitor.
[0365] Embodiment 53. The method of any one of embodiments 38-52,
wherein the nanoconstruct further comprises a targeting agent.
[0366] Embodiment 54. The method of embodiment 53, wherein the
targeting agent is an antibody, a scFv antibody, an affibody, an
aptamer, a peptide, and/or small targeting molecule.
[0367] Embodiment 55. The method of any one of embodiments 38-54,
wherein the subject is diagnosed with or is at risk for fibrosis or
inflammation.
[0368] Embodiment 56. The method of any one of embodiments 38-55,
wherein the nanoconstruct reduces reactive oxygen species,
bioavailable copper, and/or NOX expression level in the
subject,
[0369] Embodiment 57. The method of any one of embodiments 38-56,
wherein the agent is administered in an amount sufficient to reduce
tumor migration or inflammation in the human or other mammalian
subject.
[0370] Embodiment 58. The method of any one of embodiments 38-57,
wherein the nanoconstruct modulates an adverse effect of one or
more cytokines.
[0371] Embodiment 59. The method of any one of embodiments 38-58,
wherein the nanoconstruct is administered subcutaneously,
topically, systemically, intravesically, orally, intratumorally, or
intraperitoneally.
[0372] Embodiment 60. A method of making a nanoconstruct
comprising: providing a nanoparticle coated with a cationic
polymer, and cross-linking the cationic polymer to make the
nanoconstruct.
[0373] Embodiment 61. The method of embodiment 60, wherein the
nanoparticle is a silica nanoparticle, a silicon nanoparticle, an
iron oxide nanoparticle, a gold nanoparticle, a silver
nanoparticle, or a carbon nanotube.
[0374] Embodiment 62. The method of embodiment 60 or 61, wherein
the cationic polymer is cross-linked in the presence of free
cationic polymer.
[0375] Embodiment 63. The method of embodiment 62, wherein the
cationic polymer is cross-linked in the presence of a
stoichiometric excess of the free cationic polymer.
[0376] Embodiment 64. The method of any one of embodiments 60-63,
wherein the cationic polymer is selected from the group consisting
of polyethylenimine, chitosan, polypropyleneimine, polylysine,
polyamidoamine, poly(allylamine), poly(diallyldimethylammonium
chloride), poly(N-isopropyl acrylamide-co-acrylamide),
poly(N-isopropyl acrylamide-co-acrylic acid),
diethylaminoethyl-dextran, poly-(N-ethyl-vinylpyridinium bromide),
poly(dimethylamino)ethyl methacrylate, and/or poly(ethylene
glycol)-co-poly(trimethylaminoethylmethacrylate chloride).
[0377] Embodiment 65. The method of embodiment 64, wherein the
cationic polymer is polyethylenimine having a molecular weight of
from about 0.8 kDa to about 10 kDa.
[0378] Embodiment 66. The method of any one of embodiments 60-65,
wherein the cationic polymer is cross-linked using
dithiobis[succinimidyl propionate] (DSP),
3,3'-dithiobis(sulfosuccinimidyl propionate (DTSSP), or dimethyl
3,3'-dithiobispropionimidate (DTBP).
[0379] Embodiment 67. The method of embodiment 66, wherein the
cationic polymer is cross-linked using DSP.
[0380] Embodiment 68. The method of any one of embodiments 60-67,
further comprising attaching a stabilizer to the nanoconstruct.
[0381] Embodiment 69. The method of embodiment 68, wherein the
stabilizer is selected from the group consisting of polyethylene
glycol, dextran, polysialic acid, HA, PVP, PVA, and PAM.
[0382] Embodiment 70. The method of embodiment 69, wherein the
stabilizer is polyethylene glycol having a molecular weight of from
about 1 kDa to about 20 kDa.
[0383] Embodiment 71. The method of embodiment 70, wherein the
method comprises incubating maleimide-polyethylene
glycol-N-hydroxysuccinimidyl ester (Mal-PEG-NHS) with the
nanoconstruct at a weight ratio of from about 0.5:1 to about
5:1.
[0384] Embodiment 72. The method of any one of embodiments 60-71,
further comprising attaching a targeting agent to the
nanoconstruct.
[0385] Embodiment 73. The method of any one of embodiments 60-72,
further comprising admixing the nanoconstruct with at least one
type of oligonucleotide that binds noncovalently to the cationic
polymer.
[0386] Embodiment 74. The method of embodiment 73, wherein the at
least one type of oligonucleotide is a siRNA, miRNA, miRNA mimic,
or antisense oligomer.
[0387] Embodiment 75. The method of any one of embodiments 60-74,
further comprising admixing a small molecule or protein with the
nanoparticle or the nanoconstruct so that the small molecule or
protein binds to the nanoconstruct.
[0388] Embodiment 76. The method of embodiment 75, wherein the
small molecule or protein is a chemotherapeutic agent, a label, a
peptide, and/or a cytokine.
[0389] Embodiment 77. The method of any one of embodiments 60-76,
further comprising lyophilizing the nanoconstruct.
[0390] Embodiment 78. The method of embodiment 77, wherein the
nanoconstruct is lyophilized with trehalose.
[0391] Embodiment 79. A method of labeling a target comprising
contacting the nanoconstruct of any one of embodiments 1-37 with
the target under conditions to bind the nanoconstruct to the
target.
[0392] Embodiment 80. The method of embodiment 79, wherein the
target is a cell or protein.
[0393] Embodiment 81. The method of embodiment 80, wherein the
nanoconstruct is internalized by the cell.
[0394] Embodiment 82. The method of embodiment 80 or 81, wherein
the nanoconstruct binds to the exterior of the cell.
[0395] Embodiment 83. The method of any one of embodiments 79-82,
wherein the label is a lanthanide, a fluorescent dye, a gold
nanoparticle, a quantum dot, a PET tracer, and/or a MRI contrast
agent.
[0396] Embodiment 84. The method of any one of embodiments 79-83,
further comprising quantifying the amount of target by detecting
the label after the nanoconstruct binds to the target.
[0397] Embodiment 85. The method of any one of embodiments 79-84,
further comprising administering the labeled target to a subject
and detecting the location of the target after the
administering.
[0398] Embodiment 86. The method of any one of embodiments 79-85,
wherein the nanoconstruct further comprises a therapeutic
agent.
[0399] Embodiment 87. The method of embodiment 84 or 85, wherein
the detecting is by fluorescence, magnetic resonance, or PET.
[0400] Embodiment 88. The method of any one of embodiments 79-87,
wherein the nanoparticles is an iron oxide nanoparticle.
Sequence CWU 1
1
8123RNAArtificial SequenceSynthetic Construct 1cacguuugag
uccaugccca auu 23223RNAArtificial SequenceSynthetic Construct
2uugggcaugg acucaaacgu guu 23321DNAArtificial SequenceSynthetic
Construct 3cggauuacca gggauuucat t 21421DNAArtificial
SequenceSynthetic Construct 4ugaaaucccu gguaauccgt t
21519RNAArtificial SequenceSynthetic Construct 5ugguuuacau
gucgacuaa 19619RNAArtificial SequenceSynthetic Construct
6uuagucgaca uguaaacca 19725RNAArtificial SequenceSynthetic
Construct 7auucaguuuc acauugcuug gugac 25827RNAArtificial
SequenceSynthetic Construct 8gucaccaaga acugugacac agaaggg 27
* * * * *