U.S. patent application number 17/331012 was filed with the patent office on 2021-11-11 for unimolecular nanoparticles for efficient delivery of therapeutic rna.
The applicant listed for this patent is Wisconsin Alumni Research Foundation. Invention is credited to Guojun CHEN, Shaoqin GONG.
Application Number | 20210346309 17/331012 |
Document ID | / |
Family ID | 1000005724693 |
Filed Date | 2021-11-11 |
United States Patent
Application |
20210346309 |
Kind Code |
A1 |
GONG; Shaoqin ; et
al. |
November 11, 2021 |
UNIMOLECULAR NANOPARTICLES FOR EFFICIENT DELIVERY OF THERAPEUTIC
RNA
Abstract
Provided are a unimolecular nanoparticle, a composition thereof,
and methods of use thereof, and includes 1) a dendritic polymer
having a molecular weight of about 500-120,000 Da and terminating
in hydroxyl, amino or carboxylic acid groups; 2) cationic polymers
attached to at least a majority of the terminating groups of the
dendritic polymer via a pH-sensitive linker, wherein each cationic
polymer comprises a polymeric backbone attached to cationic
functional groups and to weakly basic groups by disulfide bonds,
wherein the molar ratio of cationic functional groups to weakly
basic groups ranges from 1:1-5:1, and has a molecular weight from
about 1,000-5,000 Da; and 3) poly(ethylene glycol) attached to a
plurality of cationic polymers and having a terminal group selected
from a targeting ligand, OH, O-alkyl, NH.sub.2 , biotin, or a dye,
wherein the terminal group of at least one poly(ethylene glycol) is
having a molecular weight of about 1,000-15,000 Da.
Inventors: |
GONG; Shaoqin; (Middleton,
WI) ; CHEN; Guojun; (Madison, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wisconsin Alumni Research Foundation |
Madison |
WI |
US |
|
|
Family ID: |
1000005724693 |
Appl. No.: |
17/331012 |
Filed: |
May 26, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15819424 |
Nov 21, 2017 |
11058644 |
|
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17331012 |
|
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62426004 |
Nov 23, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/5192 20130101;
A61K 31/713 20130101; A61K 9/5146 20130101; A61P 35/00
20180101 |
International
Class: |
A61K 9/51 20060101
A61K009/51; A61K 31/713 20060101 A61K031/713; A61P 35/00 20060101
A61P035/00 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This technology was made with government support under
CA166178 awarded by the National Institutes of Health. The
government has certain rights in the technology.
Claims
1. A unimolecular nanoparticle and pharmaceutically acceptable
salts thereof, wherein the unimolecular nanoparticle comprises a
dendritic polymer core, cationic polymers, and outer poly(ethylene
glycol) blocks, wherein the cationic polymers link the dendritic
polymer core to the outer poly(ethylene glycol) blocks; wherein:
the dendritic polymer having a molecular weight of about 500 to
about 120,000 Da and terminating in hydroxyl, amino or carboxylic
acid groups; the cationic polymers attached to at least 50% of the
terminating groups of the dendritic polymer via a pH-sensitive
linker, wherein each cationic polymer comprises: a polymeric
backbone, cationic functional groups, weakly basic groups, and
disulfide bonds, wherein: the polymeric backbone is attached to the
cationic functional groups and the weakly basic groups by the
disulfide bonds, the molar ratio of the cationic functional groups
to the weakly basic groups ranges from 1:1 to 5:1, and the cationic
polymer has a molecular weight from about 1,000 to about 5,000 Da;
and the outer poly(ethylene glycol) blocks are attached to a
plurality of the cationic polymers and have a terminal group
selected from the group consisting of a targeting ligand, OH,
O-alkyl, NH.sub.2, biotin, and a dye, wherein the outer
poly(ethylene glycol) has a molecular weight of about 1,000 to
about 15,000 Da.
2. The unimolecular nanoparticle of claim 1, wherein the dendritic
polymer is a polyester or a poly(amido-amine).
3. The unimolecular nanoparticle of claim 1, wherein the dendritic
polymer is a hyper-branched polymer or a dendrimer.
4. The unimolecular nanoparticle of claim 1, wherein the dendritic
polymer has from 3 to 7 generations.
5. The unimolecular nanoparticle of claim 1, wherein the dendritic
polymer is a poly(amido-amine) dendrimer having 3 to 4
generations.
6. The unimolecular nanoparticle of claim 1, wherein the dendritic
polymer is a hyperbranched polyester having 3 to 4 generations.
7. The unimolecular nanoparticle of claim 1, wherein the
pH-sensitive linker comprises an imine, hydrazone, or cis-aconityl
group.
8. The unimolecular nanoparticle of claim 1, wherein the cationic
polymer comprises a polyamide backbone, disulfide linkers, amino
and/or ammonium salt groups, and imidazole and/or imidazolium salt
groups.
9. The unimolecular nanoparticle of claim 8, wherein the polyamide
backbone is selected from the group consisting of polyasparagine,
polyglutamine, polyornithine, and polylysine.
10. The unimolecular nanoparticle of claim 8, wherein the cationic
polymers comprise (C.sub.2-C.sub.6
alkylene)disulfide(C.sub.2-C.sub.6 alkyl)amino groups and/or salts
thereof, and (C.sub.2-C.sub.6 alkylene)disulfide(C.sub.2-C.sub.6
alkyl)aminocarbonylimidazole groups and/or salts thereof.
11. The unimolecular nanoparticle of claim 8, wherein the cationic
polymers comprise ethylene-disulfide-ethylamino groups and/or salts
thereof, and ethylene-disulfide-ethylaminocarbonylimidazole groups
and/or salts thereof.
12. The unimolecular nanoparticle of claim 1, wherein the targeting
ligand is a cofactor, carbohydrate, peptide, antibody, nanobody, or
aptamer.
13. The unimolecular nanoparticle of claim 1, wherein the targeting
ligand is selected from the group consisting of folic acid,
mannose, GE11, cRGD, KE108, octreotide, TAT cell penetrating
peptide, PSMA aptamer, TRC105, 7D12 nanobody, and CTB.
14. The unimolecular nanoparticle of claim 1 further comprising a
therapeutic RNA within the unimolecular nanoparticle.
15. The unimolecular nanoparticle of claim 14, wherein the
therapeutic RNA is an siRNA.
16. (canceled)
17. A composition comprising the unimolecular nanoparticle of claim
1 and a pharmaceutically acceptable carrier.
18. A method of preparing a unimolecular nanoparticle comprising
dispersing therapeutic RNA within the unimolecular nanoparticle of
claim 1.
19. A method of treating a cancer by administering an effective
amount of a unimolecular nanoparticle of claim 14, wherein the
therapeutic RNA inhibits expression of a gene necessary for
survival or growth of the cancer.
20. A kit comprising a package containing unimolecular nanoparticle
of claim 1, a package containing an effective amount of siRNA, and
directions for use of the kit.
21. A siRNA-loaded unimolecular nanoparticle comprising the
unimolecular nanoparticle of claim 1 and a siRNA, wherein the
loading of siRNA is about 10 wt % to about 20 wt %, based on the
total siRNA-loaded unimolecular nanoparticle weight.
22. A composition comprising the unimolecular nanoparticle and the
therapeutic RNA with the unimolecular nanoparticle of claim 14 and
a pharmaceutically acceptable carrier.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 15/819,424, filed Nov. 21, 2017, which claims the benefit of
and priority to U.S. Provisional Application No. 62/426,004 filed
Nov. 23, 2016, the contents of both of which are incorporated
herein by reference their entirety.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been filed electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Sep. 6, 2019, is named 032026-1364_SL.txt and is 12,534 bytes in
size.
FIELD
[0004] The present technology relates generally to the field of
siRNA drug delivery systems and methods of making and using such
systems. The compositions of such systems include a unimolecular
nanoparticle and siRNA drug(s).
BACKGROUND
[0005] Specific silencing of target genes using short interfering
RNA (siRNA) is of significant interest for the treatment of cancers
and other disease. siRNA molecules are double-stranded short chain
oligonucleotides that post-transcriptionally regulate protein
synthesis by sequence-specific matching with mRNA molecules,
thereby resulting in specific silencing of target genes. Currently,
several dozen potential siRNA therapies are undergoing clinical
trials. However, due to their negatively charged nature, limited
chemical stability, short plasma elimination half-life, and off
target effect, naked siRNA molecules show poor therapeutic
efficacy. Various viral and non-viral delivery systems have been
developed to improve the efficacy of siRNA therapy. Although viral
vectors provide high transfection efficiency, concerns associated
with insertional mutagenesis, immunogenicity, and cytotoxicity
limit their use. Non-viral delivery systems potentially offer a
safer and cheaper alternative to viral vectors. Currently there is
no clinically available method of siRNA delivery suitable for gene
silencing for treatment of diseases such as cancer.
SUMMARY
[0006] In one aspect, the present technology provides a
unimolecular nanoparticle that includes 1) a dendritic polymer
having a molecular weight of about 500 to about 120,000 Da and
terminating in hydroxyl, amino or carboxylic acid groups; 2)
cationic polymers attached to at least a majority of the
terminating groups of the dendritic polymer via a pH-sensitive
linker, wherein each cationic polymer comprises a polymeric
backbone attached to cationic functional groups and to weakly basic
groups by disulfide bonds, wherein the molar ratio of cationic
functional groups to weakly basic groups ranges from 1:1 to 5:1,
and has a molecular weight from about 1,000 to about 5,000 Da; and
3) poly(ethylene glycol) attached to a plurality of cationic
polymers and having a terminal group selected from a targeting
ligand, OH, O-alkyl, NH.sub.2, biotin, or a dye, wherein the
poly(ethylene glycol) has a molecular weight of about 1,000 to
about 15,000 Da.
[0007] In some embodiments of the unimolecular nanoparticle, the
dendritic polymer is a polyester or a poly(amido-amine). The
dendritic polymer may be a hyper-branched polymer or a dendrimer.
The dendritic polymer may have from 3-7 generations. In some
embodiments, the dendritic polymer is a poly(amido-amine) dendrimer
having 3 to 4 generations. In other embodiments, the dendritic
polymer is a hyperbranched polyester having 3 to 4 generations.
[0008] In some embodiments of the unimolecular nanoparticle, the
pH-sensitive linker contains an imine, hydrazone or cis-aconityl
group.
[0009] In some embodiments of the unimolecular nanoparticle, each
cationic polymer includes a polyamide backbone, disulfide linkers,
amino and/or ammonium groups, and imidazole groups. The polyamide
backbone may be, e.g., a polyasparagine, polyglutamine,
polyornithine, or polylysine. In some embodiments, the cationic
polymers comprises moieties selected from the group consisting of a
(C2-6 alkylene)disulfide(C2-C6 alkyl)amino group, a (C2-6
alkylene)disulfide(C2-C6 alkyl)aminocarbonylimidazole group, and
salts thereof. In certain embodiments, the cationic polymers
comprise moieties selected from the group consisting of
ethylene-disulfide-ethylamino group,
ethylene-disulfide-ethylaminocarbonylimidazole group and salts
thereof.
[0010] In some embodiments of the unimolecular nanoparticle, the
targeting ligand is a cofactor, carbohydrate, peptide, antibody,
nanobody, or aptamer. For example, the targeting ligand may be
selected from the group consisting of folic acid, mannose, GE11,
cRGD, KE108, octreotide, TAT cell penetrating peptide, P SMA
aptamer, TRC105, 7D12 nanobody, CTB.
[0011] In some embodiments, the unimolecular nanoparticle includes
a therapeutic RNA within the nanoparticle. The therapeutic RNA may
be an siRNA. The loading of the siRNA may be about 1 to about 20 wt
% of the unimolecular nanoparticle.
[0012] In some embodiments of the unimolecular nanoparticle, the
dendritic polymer is a hyperbranched polyester having 3-4
generations and a molecular weight of about 3,600 to about 7,400
Da; the pH-sensitive linker is a benzylimine; each cationic polymer
has a polyasparagine backbone attached to an
ethylene-disulfide-ethylamino or
ethylene-disulfide-ethylaminocarbonylimidazole group or salt
thereof, and the molar ratio of the amino to imidazole functional
groups is from 1:1 to 5:1; and the molecular weight of the PEG is
about 1,000 to about 15,000 Da.
[0013] In another aspect, the present technology provides a method
of preparing a unimolecular nanoparticle comprising dispersing
therapeutic RNA within any unimolecular nanoparticle described
herein.
[0014] In another aspect, the present technology provides
compositions comprising a unimolecular nanoparticle as described
herein and a pharmaceutically acceptable carrier.
[0015] In one aspect, the present technology provides methods of
treating a cancer by administering an effective amount of a
unimolecular nanoparticle as described herein loaded with a
therapeutic RNA, wherein the therapeutic RNA inhibits expression of
a gene necessary for survival or growth of the cancer. In some
embodiments of the method, therapeutic RNA is siRNA having a length
of 19 base pairs (bps) to 25 bps.
[0016] In another aspect, the present technology provides a kit
comprising a package containing a unimolecular nanoparticle as
described herein and a package containing an effective amount of
therapeutic siRNA and directions for use of the kit.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows a schematic diagram of the cellular uptake of
siRNA-complexed unimolecular NPs and the subcellular release of
siRNA from the siRNA-complexed NPs into the cytosol. pH/redox
dual-sensitive unimolecular NPs with excellent endosomal/lysosomal
escape and intracellular siRNA decomplexation capabilities for
efficient targeted delivery of siRNA.
[0018] FIGS. 2A-2C show .sup.1H NMR spectra of
P(BLA-NCA)-PEG-OCH.sub.3 (FIG. 2A), P(BLA-NCA)-PEG-Mal (FIG. 2B),
and H40-CHO (FIG. 2C).
[0019] FIGS. 3A-3D show .sup.1H NMR spectra of
H40-P(BLA-NCA)-PEG-OCH.sub.3NIal (FIG. 3A),
H40-P(Asp-AED)-PEG-OCH.sub.3/Mal (FIG. 3B),
H40-P(Asp-AED-ICA)-PEG-OCH.sub.3/Mal (FIG. 3D), and
H40-P(Asp-AED-ICA)-PEG-OCH.sub.3/Cy5/GE11 (FIG. 3D).
[0020] FIG. 4A shows gel retardation assays of siRNA/NPs with
various N/P ratios. FIG. 4B shows transmission electron microscopy
(TEM) images of the siRNA-complexed NPs (N/P=10). FIG. 4C shows in
vitro siRNA release from siRNA-complexed NPs in different buffers
at 37.degree. C. Data represent mean.+-.SD (n=3).
[0021] FIG. 5 shows an assessment of the endosomal/lysosomal escape
of siRNA-complexed NPs in MDA-MB-468 cells after 2 h incubation.
Endosomes/lysosomes stained with Lysotracker. siRNA was labeled
with Cy5.5. The nuclei were stained with Hoechst. Scale bar: 20
.mu.m.
[0022] FIG. 6 shows z-stack images for the assessment of the
endosomal/lysosomal escape of siRNA-complexed NPs in MDA-MB-468
cells. Scale bar: 2 .mu.m.
[0023] FIG. 7 shows the in vitro cellular uptake analysis.
Fluorescence images of MDA-MB-468 TNBC cells incubated with pure
medium (control), Cy5-labeled non-targeted (without GE11
conjugation) NPs, Cy5-labeled targeted (GE11-conjugated) NPs with a
blocking dose (2 .mu.m) of GE11 (i.e., blocking), and Cy5-labeled
targeted NPs at 37.degree. C. for 2 h (NP concentration: 100
.mu.g/mL). Targeted NPs significantly enhanced the cellular uptake
in EGFR-overexpressing TNBC cells. Scale bar: 50 .mu.m.
[0024] FIGS. 8A-B show an in vitro assessment of gene silencing
efficiency using (in FIG. 8A) fluorescence microscope and (in FIG.
8B) flow cytometry. GFP-expressing MDA-MA-468 cells treated with
pure medium (control), siRNA-complexed non-targeted NPs (siRNA-NT),
siRNA-complexed targeted NPs (siRNA-T), and siRNA-complexed RNAiMAX
(siRNA-RNAiMAX) for 24 h (40 nM of GFP-siRNA). FIG. 8C shows cell
viability analysis for MDA-MA-468 cells treated with pure medium
(control), siRNA-NT, siRNA-T, siRNA-RNAiMAX, and pure RNAiMAX for
24 h (40 nM of GFP-siRNA). All values are presented as a mean.+-.SD
(n=5); **: p<0.01; NS: not significant. Scale bar: 100
.mu.m.
[0025] FIG. 9 shows a schematic of an illustrative embodiment of an
siRNA-complexed unimolecular nanoparticle (NP) of the present
technology including a core comprised of H40 polyester
hyperbranched polymer attached to cationic polymers, which in turn
are attached to hydrophilic PEG segments and terminating in various
functional groups, including dye(s) and the targeting ligand GE11.
siRNA molecules partition within the NPs by electrostatic
interactions.
DETAILED DESCRIPTION
[0026] The following terms are used throughout as defined below.
All other terms and phrases used herein have their ordinary
meanings as one of skill in the art would understand.
[0027] As used herein and in the appended claims, singular articles
such as "a" and "an" and "the" and similar referents in the context
of describing the elements (especially in the context of the
following claims) are to be construed to cover both the singular
and the plural, unless otherwise indicated herein or clearly
contradicted by context.
[0028] As used herein, "about" will be understood by persons of
ordinary skill in the art and will vary to some extent depending
upon the context in which it is used. If there are uses of the term
which are not clear to persons of ordinary skill in the art, given
the context in which it is used, "about" will mean up to plus or
minus 10% of the particular term.
[0029] "Molecular weight" as used herein with respect to polymers
refers to weight average molecular weights (Mw) and can be
determined by techniques well known in the art including gel
permeation chromatography (GPC). GPC analysis can be performed, for
example, on a D6000M column calibrated with poly(methyl
methacrylate) (PMMA) using triple detectors including a refractive
index (RI) detector, a viscometer detector, and a light scattering
detector, and dimethylformamide as the eluent.
[0030] The terms "cancer," "neoplasm," "tumor," "malignancy" and
"carcinoma," used interchangeably herein, refer to cells or tissues
that exhibit an aberrant growth phenotype characterized by a
significant loss of control of cell proliferation. The methods and
compositions of this disclosure apply to malignant, pre-metastatic,
metastatic, and non-metastatic cells.
[0031] The term "therapeutic RNA" refers to single strand or duplex
RNA that modulates (e.g., silences, reduces, or inhibits)
expression of a target gene, e.g., by mediating the degradation of
mRNAs which are complementary to the sequence of the interfering
RNA, by providing an RNA that is absent or expressed at a lower
level in a subject having a particular disease or condition
relative to its levels in a subject that does not have the same
disease or condition. Examples of therapeutic RNAs include siRNA
and miRNA.
[0032] The term "small-interfering RNA" or "siRNA" refers to
double-stranded RNA (i.e., duplex RNA) that modulates (e.g.,
silences, reduces, or inhibits) expression of a target gene, e.g.,
by mediating the degradation of mRNAs which are complementary to
the sequence of the siRNA. Typically, siRNA has complete identity
or complementarity to the corresponding RNA sequence of its target
mRNA. siRNA includes RNA of having 15-60, 15-50, 15-50, or 15-40
(duplex) nucleotides in length, more typically about, 15-30, 15-25
or 19-25 (duplex) nucleotides in length, and may be 20-24, 21-22 or
21-23 (duplex) nucleotides in length. siRNA duplexes may include 3'
overhangs of 1, 2, 3, or 4 nucleotides and/or 5' phosphate termini.
The siRNA can be chemically synthesized or may be encoded by a
plasmid (e.g., transcribed as sequences that automatically fold
into duplexes with hairpin loops). siRNA can also be generated by
cleavage of longer dsRNA (e.g., dsRNA greater than about 25
nucleotides in length) with the E. coli RNase III or Dicer. These
enzymes process the dsRNA into biologically active siRNA (see,
e.g., Yang et al., PNAS USA 99: 9942-7 (2002); Calegari et al.,
PNAS USA 99: 14236 (2002); Byrom et al., Ambion TechNotes 10(1):
4-6 (2003); Kawasaki et al., Nucleic Acids Res. 31: 981-7 (2003);
Knight and Bass, Science 293: 2269-71 (2001); and Robertson et al.,
J. Biol. Chem. 243: 82 (1968)). Preferably, dsRNA are at least 50
nucleotides to about 100, 200, 300, 400 or 500 nucleotides in
length. A dsRNA may be as long as 1000, 1500, 2000, 5000
nucleotides in length, or longer. The dsRNA can encode for an
entire gene transcript or a partial gene transcript.
[0033] The phrase "inhibits expression of a gene" refers to the
ability of a therapeutic RNA, such as an siRNA, of the technology
to silence, reduce, or inhibit expression of a target gene (e.g.,
VEGF, EphA2, protein kinase N3 (PKN3), etc.). To examine the extent
of gene silencing, a test sample (e.g., a biological sample from
organism of interest expressing the target gene or a sample of
cells in culture expressing the target gene) is contacted with an
siRNA that silences, reduces, or inhibits expression of the target
gene. Expression of the target gene in the test sample is compared
to expression of the target gene in a control sample (e.g., a
biological sample from organism of interest expressing the target
gene or a sample of cells in culture expressing the target gene)
that is not contacted with the siRNA. Control samples (i.e.,
samples expressing the target gene) are assigned a value of 100%.
Silencing, inhibition, or reduction of expression of a target gene
is achieved when the value of the test sample relative to the
control sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%,
55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 10%, O% or a range between
and including any two of the foregoing values. Suitable assays
include, e.g., examination of mRNA levels using techniques known to
those of skill in the art such as dot blots, northern blots, in
situ hybridization, ELISA, immunoprecipitation, enzyme function, as
well as phenotypic assays known to those of skill in the art.
[0034] The phrase "a targeted receptor" refers to a receptor
expressed by a cell that is capable of binding a cell targeting
ligand. The cell targeting ligand may be a "tumor cell targeting
ligand." The receptor may be expressed on the surface of the cell.
The receptor may be a transmembrane receptor. Examples of such
targeted receptors include EGFR, .alpha..sub.v.beta..sub.3
integrin, somatostatin receptor, folate receptor, prostate-specific
membrane antigen, CD105, mannose receptor, and GM1 ganglioside.
[0035] The phrase "tumor cell targeting ligand" refers to a ligand
that binds to "a targeted receptor" unique to or overexpressed by a
cancer cell. The ligands may be capable of binding due to
preferential expression of a receptor for the ligand, accessible
for ligand binding, on the cancer cells. Examples of such ligands
include GE11 peptide, cRGD ((cyclo (RGDfC)), KE108 peptide,
octreotide, folic acid, prostate-specific membrane antigen (PSMA)
aptamer, TRC105, a human/murine chimeric IgG1 monoclonal antibody,
7D12 nanobody, mannose, and cholera toxin B (CTB). Additional
examples of such ligands include Rituximab, Trastuzumab,
Bevacizumab, Alemtuzumab, Panitumumab, RGD, DARPins, RNA aptamers,
DNA aptamers, analogs of folic acid and other folate
receptor-binding molecules, lectins, other vitamins, peptide
ligands identified from library screens, tumor-specific peptides,
tumor-specific aptamers, tumor-specific carbohydrates,
tumor-specific monoclonal or polyclonal antibodies, Fab or scFv
(i.e., a single chain variable region) fragments of antibodies such
as, for example, an Fab fragment of an antibody directed to EphA2
or other proteins specifically expressed or uniquely accessible on
metastatic cancer cells, small organic molecules derived from
combinatorial libraries, growth factors, such as EGF, FGF, insulin,
and insulin-like growth factors, and homologous polypeptides,
somatostatin and its analogs, transferrin, lipoprotein complexes,
bile salts, selecting, steroid hormones, Arg-Gly-Asp containing
peptides, retinoids, various galectins, .delta.-opioid receptor
ligands, cholecystokinin A receptor ligands, ligands specific for
angiotensin AT1 or AT2 receptors, peroxisome proliferator-activated
receptor .gamma. y ligands, .beta.-lactam antibiotics, small
organic molecules including antimicrobial drugs, and other
molecules that bind specifically to a receptor preferentially
expressed on the surface of tumor cells or on an infectious
organism, or fragments of any of these molecules.
[0036] In some embodiments, a cell penetrating peptide may also be
attached to one or more PEG terminal groups in addition to the
targeting ligand. A "cell penetrating peptide," also referred to as
a "protein transduction domain (PTD)," a "membrane translocating
sequence," and a "Trojan peptide", refers to a short peptide (e.g.,
from 4 to about 40 amino acids) that has the ability to translocate
across a cellular membrane to gain access to the interior of a cell
and to carry into the cells a variety of covalently and
noncovalently conjugated cargoes, including proteins,
oligonucleotides, and liposomes. They are typically highly cationic
and rich in arginine and lysine amino acids. Examples of such
peptides include TAT cell penetrating peptide (GRKKRRQRRRPQ (SEQ ID
NO: 1)); MAP (KLAL) KLALKLALKALKAALKLA (SEQ ID NO: 2); Penetratin
or Antenapedia PTD RQIKWFQNRRMKWKK (SEQ ID NO: 3); Penetratin-Arg:
RQIRIWFQNRRMRWRR (SEQ ID NO: 4); antitrypsin (358-374):
CSIPPEVKFNKPFVYLI (SEQ ID NO: 5); Temporin L:
FVQWFSKFLGRIL-NH.sub.2 (SEQ ID NO: 6); Maurocalcine:
GDC(acm)LPHLKLC (SEQ ID NO: 7); pVEC (Cadherin-5):
LLIILRRRIRKQAHAHSK (SEQ ID NO: 8); Calcitonin:
LGTYTQDFNKFHTFPQTAIGVGAP (SEQ ID NO: 9); Neurturin:
GAAEAAARVYDLGLRRLRQRRRLRRERVRA (SEQ ID NO: 10); Penetratin:
RQIKIWFQNRRMKWKKGG (SEQ ID NO: 11); TAT-HA2 Fusion Peptide:
RRRQRRKKRGGDIMGEWGNEIFGAIAGFLG (SEQ ID NO: 12); TAT (47-57)
YGRKKRRQRRR (SEQ ID NO: 13); SynB1 RGGRLSYSRRRFSTSTGR (SEQ ID NO:
14); SynB3 RRLSYSRRRF (SEQ ID NO: 15); PTD-4 PIRRRKKLRRL (SEQ ID
NO: 16); PTD-5 RRQRRTSKLMKR (SEQ ID NO: 17); FHV Coat-(35-49)
RRRRNRTRRNRRRVR (SEQ ID NO: 18); BMV Gag-(7-25) KMTRAQRRAAARRNRWTAR
(SEQ ID NO: 19); HTLV-II Rex-(4-16) TRRQRTRRARRNR (SEQ ID NO: 20);
HIV-1 Tat (48-60) or D-Tat GRKKRRQRRRPPQ (SEQ ID NO: 21); R9-Tat
GRRRRRRRRRPPQ (SEQ ID NO: 22); Transportan
GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 23) chimera; MAP
KLALKLALKLALALKLA (SEQ ID NO: 24); SBP or Human P1
MGLGLHLLVLAAALQGAWSQPKKKRKV (SEQ ID NO: 25); FBP
GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 26); MPG
ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya (SEQ ID NO: 27) (wherein cya is
cysteamine); MPG(ANLS) ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-cya (SEQ ID
NO: 28); Pep-1 or Pep-1-Cysteamine ac-KETWWETWWTEWSQPKKKRKV-cya
(SEQ ID NO: 29); Pep-2 ac-KETWFETWFTEWSQPKKKRKV-cya (SEQ ID NO:
30); Periodic sequences, Polyarginines RxN (4<N<17) (SEQ ID
NO: 31) chimera; Polylysines KxN (4<N<17) (SEQ ID NO: 32)
chimera; (RAca)6R (SEQ ID NO: 33); (RAbu)6R (SEQ ID NO: 34); (RG)6R
(SEQ ID NO: 35); (RM)6R (SEQ ID NO: 36); (RT)6R (SEQ ID NO: 37);
(RS)6R (SEQ ID NO: 38); R10 (SEQ ID NO: 39); (RA)6R (SEQ ID NO:
40); and R7 (SEQ ID NO: 41).
[0037] A "dye" refers to small organic molecules having a molecular
weight of 2000 Da or less or a protein which is able to emit light.
Non-limiting examples of dyes include fluorophores,
chemiluminescent or phosphorescent entities. For example, dyes
useful in the present technology include but are not limited to
cyanine dyes (e.g., Cy2, Cy3, Cy5, Cy5.5, Cy7, and sulfonated
versions thereof), fluorescein isothiocyanate (FITC), ALEXA
FLUOR.RTM. dyes (e.g., ALEXA FLUOR.RTM. 488, 546, or 633),
DYLIGHT.RTM. dyes (e.g., DYLIGHT.RTM. 350, 405, 488, 550, 594, 633,
650, 680, 755, or 800) or fluorescent proteins such as GFP (Green
Fluorescent Protein).
[0038] The present technology provides pharmaceutical compositions
and medicaments comprising any of one of the embodiments of the
siRNA delivery systems disclosed herein and a pharmaceutically
acceptable carrier or one or more excipients. The compositions may
be used in the methods and treatments described herein. In one
aspect the present technology provides a drug delivery system for
the prevention or treatment of cancer. The pharmaceutical
composition may include an effective amount of any of one of the
embodiments of the compositions disclosed herein. In any of the
above embodiments, the effective amount may be determined in
relation to a subject. "Effective amount" refers to the amount of
compound or composition required to produce a desired effect. One
example of an effective amount includes amounts or dosages that
yield acceptable toxicity and bioavailability levels for
therapeutic (pharmaceutical) use including, but not limited to, the
inhibition (i.e., slowing, halting or reversing) or treatment of
cancer in a subject. As used herein, a "subject" or "patient" is a
mammal, such as a cat, dog, rodent or primate. Typically the
subject is a human, and, preferably, a human at risk for or
suffering from cancer. The term "subject" and "patient" can be used
interchangeably. An effective amount of a therapeutic RNA, such as
an siRNA or a therapeutically effective amount of a siRNA is an
amount sufficient to produce the desired effect, e.g., a decrease
in the expression of a target sequence in comparison to the normal
expression level detected in the absence of the siRNA.
[0039] In one aspect, the present technology provides unimolecular
nanoparticles designed to deliver therapeutic RNA selectively to
tumor cells. The RNA is protected within the nanoparticles until it
reaches the cytoplasm of the targeted cell. The technology employs
a unique combination of pH sensitive and redox sensitive
functionality to release the RNA intact from the nanoparticles only
once the nanoparticles are within the targeted cells.
[0040] The present unimolecular nanoparticles include three
distinct polymeric domains: a dendritic polymer, which serves as
the core, cationic polymers attached to the terminal groups of the
dendritic polymer and PEG, attached to the terminal groups of the
cationic polymers. Thus, the unimolecular nanoparticle may be
described as a multi-arm star-like block copolymer. Therapeutic
RNA, such as siRNA or miRNA may be loaded into the unimolecular
nanoparticles described herein. While not wishing to be bound by
theory, it is believed that the therapeutic RNA is bound by
electrostatic interactions with the cationic polymers on the
interior of the nanoparticle.
[0041] The dendritic polymer has a molecular weight of about 500 to
about 120,000 Da and terminates in hydroxyl, amino or carboxylic
acid groups. The molecular weight of the dendritic polymer will
vary based on the type of polymer and number of generations
employed. Suitable molecular weights include about 500, about 1000,
about 2000, about 3000, about 4000, about 5000, about 6000, about
7000, about 8000, about 9000, about 10,000, about 15,000, about
20,000, about 30,000, about 40,000, about 50,000, about 75,000,
about 100,000, about 120,000 Da, or a range between and including
any two of the forgoing values. In some embodiments the molecular
weight of the dendritic polymer is about 1,000 to about 10,000 Da.
The core of the unimolecular nanoparticle may be a dendrimer such
as a poly(amido-amine) (PAMAM) dendrimer having from 3 to 7
generations (e.g., 3, 4, 5, 6, or 7 generations or a range between
and including any two of the foregoing values) or a hyperbranched
polymer such as a polyester hyperbranched polymer (e.g., Boltorn
H30 and H40, which are prepared from 2,2-bis(methylol)propionic
acid). PAMAM will be understood to refer to a polymer having a
C.sub.2-C.sub.4 a, w-diamine initiator and C.sub.3-C.sub.4 acrylate
and diamine building blocks for each subsequent generation.
Typically the building blocks are C.sub.2 1,2-diamines and C.sub.3
acrylates (not counting the methyl ester carbon, which serves as a
temporary protecting group). In some embodiments, the PAMAM
dendrimer has from 3 to 4 generations. In some embodiments, the
dendritic polymer is a hyperbranched polyester having 3 to 4
generations. The number of generations will determine the number of
arms available for attachment to the cationic polymers. Although
not every arm of the dendritic polymer must terminate in amino,
hydroxyl, carboxylic acid groups, the majority of arms of the
dendritic polymer do, e.g., more than 50%, more than 60%, more than
70%, more than 80% or more than 90% of arms of the dendritic
polymer terminate in amino, hydroxyl, or carboxylic acid groups. In
some embodiments, e.g., where the dendritic polymer is PAMAM, all
of the arms terminate in amino groups, hydroxyl groups, or
carboxylic acid groups.
[0042] The cationic polymers of the unimolecular nanoparticle link
the core dendritic polymer to the outer poly(ethylene glycol) (PEG)
blocks. The cationic polymers of the unimolecular nanoparticles are
attached to at least a majority of the terminating groups of the
dendritic polymer via a pH-sensitive linker. The pH-sensitive
linker includes a functional group which is readily hydrolyzed upon
a change from alkaline pH to acid pH. In some embodiments the
pH-sensitive linker will be stable at the pH of blood (about 7.4)
and extracellular space in tissue, but hydrolyze at the lower pH of
the endosome or lysosome (about 5.5-6.5). Suitable pH-sensitive
linkers include imine (e.g., benzylimine), hydrazone and
cis-aconityl linkers. While not wishing to be bound by theory,
hydrolysis of the pH-sensitive linker is intended to release the
block cationic-PEG copolymer from the dendritic core upon a change
in pH from alkaline to acid.
[0043] Each cationic polymer is made up of a polymeric backbone
attached to cationic functional groups and to weakly basic groups
by redox-sensitive linkers that include disulfide bonds. The
polymeric backbone may be a polyamide backbone such as a found in
peptides and proteins. In some embodiments the polyamide is a
polyasparagine, polyglutamine, polyornithine, or polylysine. The
cationic functional groups may be functional groups having a pka of
at least about 8 (e.g., a pka of 8, 8.5, 9, 9.5, 10, 10.5, 11 ora
range between and including any two of the foregoing values).
Suitable groups include primary, secondary and tertiary amines,
amidines, and guanidines. It will be understood that the cationic
functional groups may be attached to the sidechains of the
polyamide backbone. For example aspartic acid and glutamic acid
side chains may be derivatized with disulfides formed from
aminoalkylenethiols: (polyamide
backbone)-CH.sub.2CH.sub.2-C(O)NH-(C1-6 alkylene)-S--S-(C1-C6
alkylene)-NH.sub.2), or (polyamide backbone)-CH.sub.2-C(O)NH-(C1-6
alkylene)-S--S-(C1-C6 alkylene)-NH.sub.2). When derivatized in this
fashion, it will be understood that the polyaspartic acid or
polyglutamic acid are now a polyasparagine or a polyglutamine,
respectively. Similarly, polyornithine and polylysine may be
attached to cationic functional groups through suitably
functionalized species such as
carboxy-alkylene-disulfide-alkylene-amino groups, e.g., (polyamide
backbone)-CH.sub.2CH.sub.2-CHNH--C(O)-(C1-6 alkylene)-S--S-(C1-C6
alkylene)-NH.sub.2), or (polyamide
backbone)-CH.sub.2CH.sub.2CH.sub.2NH--C(O)-(C1-6
alkylene)-S--S-(C1-C6 alkylene)-NH.sub.2).
[0044] Weakly basic groups useful in the unimolecular nanoparticles
may have a pKa between about 5.5 and about 7.0, e.g., a pKa of 5.5,
5.75, 6, 6.25, 6.5, 6.75, 7, or a range between and including any
two of the foregoing values. In some embodiments, the weakly basic
group is imidazole or pyridinyl. In certain embodiments, the molar
ratio of cationic functional groups to weakly basic groups ranges
from 1 to 5; in others it is 2 to 4. Suitable molar ratios include
about 1, about 2, about 3, about 4, and about 5 or a range between
and including any two of the foregoing values.
[0045] In certain embodiments, the cationic polymer has a molecular
weight from about 1,000 to about 5,000 Da; in others it is about
1,500 to about 4,000 Da. Suitable molecular weights for the
cationic polymers include about 1,000, about 1,500, about 2,000,
about 2,500, about 3,000, about 3,500, about 4,000, about 4,500,
about 5,000 or a range between and including any two of the
foregoing values.
[0046] In some embodiments, each cationic polymer comprises a
polyamide backbone, disulfide linkers, amino groups, and imidazole
groups. In some embodiments, the cationic polymers comprise
moieties selected from the group consisting of (C2-6
alkylene)disulfide(C2-C6 alkyl)amino group, (C2-6
alkylene)disulfide(C2-C6 alkyl)aminocarbonylimidazole group, and
salts thereof. the cationic polymers comprise moieties selected
from the group consisting of ethylene-disulfide-ethylamino group,
ethylene-disulfide-ethylaminocarbonylimidazole group and salts
thereof.
[0047] PEG is a hydrophilic polymer that forms the outer layer of
the unimolecular nanoparticle. The PEG polymeric blocks are
attached to a plurality of the cationic polymers.
[0048] Each arm of the PEG terminates in one of various groups
selected from a targeting ligand, OH, O-(C1-C6)alkyl, NH.sub.2,
biotin or a dye. In some embodiments the PEG terminates in OH or
O-(C1-C6)alkyl, and in still others the PEG terminates in in an
OC.sub.1-3 alkyl group. In still other embodiments, the PEG
terminates in a targeting ligand. The targeting ligand may be
selected from the group consisting of a cofactor, carbohydrate,
peptide, antibody, nanobody, or aptamer. In other embodiments, the
targeting ligand is selected from the group consisting of folic
acid, mannose, GE11, cRGD, KE108, octreotide, TAT cell penetrating
peptide, PSMA aptamer, TRC105, 7D12 nanobody, and CTB.
[0049] Typically each arm of the PEG has 23 to 340 repeat units or
a molecular weight of about 1,000 to about 15,000 Da. Suitable
molecular weights for each PEG block of the unimolecular
nanoparticle include about 1,000, about 1,500, about 2,000, about
2,500, about 3,000, about 4,000, about 5,0000, about 7,500, about
10,000, or about 15,000 Da, or a range between and including any
two of the foregoing values.
[0050] In another aspect, the unimolecular nanoparticle includes a
therapeutic RNA within the nanoparticle, such as an siRNA. In some
embodiments, the loading of the siRNA is about 1 to about 20 wt %
of the unimolecular nanoparticle. For example, the loading of the
siRNA may be about 1 wt %, 2 wt %, 3 wt %, 4 wt %, 5 wt %, 6 wt %,
7 wt %, 8 wt %, 9 wt %, 10 wt %, 11 wt %, 12 wt %, 13 wt %, 14 wt
%, 15 wt %, 16 wt %, 17 wt %, 18 wt %, 19 wt %, 20 wt % ora range
between and including any two of the foregoing values.
[0051] Any therapeutic RNA may be used in the present unimolecular
nanoparticle drug delivery systems. While not wishing to be bound
by theory, it is believed that the cationic polymers of the
unimolecular nanoparticle bind the therapeutic RNA via
electrostatic interactions between the negatively charged phosphate
backbone of the therapeutic RNA and the cationic functional groups
of the cationic polymers. Hence, loading of the therapeutic RNA is
independent of the base sequence of the RNA. Likewise, therapeutic
RNA of a variety of sequence lengths may be loaded into the
unimolecular nanoparticle. In some embodiments, the length of the
therapeutic RNA is 20, 21, 22, 23 or 24 bps or a range between and
including any two of the foregoing values. In certain embodiments,
the length of the therapeutic RNA is from about 21 to about 23
bps.
[0052] In some embodiments, the therapeutic RNA loaded in the
unimolecular nanoparticle is an siRNA that inhibits expression of a
gene necessary for survival or growth of a cancer. The gene
necessary for survival or growth of the cancer may be selected from
oncogenes, mutated tumor suppressor genes, and genes involved in
tumor progression and cell cycle progression. In certain
embodiments, the siRNAs interfere with transcription of genes for
VEGF, EphA2, protein kinase N3 (PKN3), PLK1, KSP, ribonucleotide
reductase regulatory subunit M2 (RRM2), gro-a, MDR-1, androgen
receptor (AR), acid ceramidase (AC), HIF1, CDK4, GATA2, and the
like. In some embodiments, the length of the siRNA is 20, 21, 22,
23 or 24 bps or a range between and including any two of the
foregoing values. In certain embodiments, the length of the siRNA
is from about 21 to about 23 bps.
[0053] The unimolecular nanoparticles may be prepared using
standard techniques. For example, a dendritic polymer in which most
or all of the surface arms terminate in amino, hydroxyl, or
carboxylic acid groups may be conjugated to the cationic polymers
via amide, ester, or ether groups. Typically, ester and amide
linkages are used for ease of formation. Likewise, the PEG blocks
may be attached to the cationic polymers via ester, amide or ether
groups. In some embodiments, the PEG has a hydroxy group on one end
and an alkoxy or carbonylalkoxy on the other. Standard coupling
conditions such as the use of tin catalysis or coupling agents or
active esters may be used to form the ester or amide bonds.
[0054] The unimolecular nanoparticles described herein may be used
to treat, inhibit or prevent cancer by administering an effective
amount of the unimolecular nanoparticle wherein the siRNA inhibits
expression of a gene necessary for survival or growth of the
cancer.
[0055] The compositions described herein can be formulated for
various routes of administration, for example, by parenteral,
rectal, nasal, vaginal administration, or via implanted reservoir.
Parenteral or systemic administration includes, but is not limited
to, subcutaneous, intravenous, intraperitoneal, and intramuscular
injections. The following dosage forms are given by way of example
and should not be construed as limiting the instant present
technology.
[0056] Injectable dosage forms generally include solutions or
aqueous suspensions which may be prepared using a suitable
dispersant or wetting agent and a suspending agent so long as such
agents do not interfere with formation of the nanoparticles
described herein. Injectable forms may be prepared with acceptable
solvents or vehicles including, but not limited to sterilized
water, Ringer's solution, 5% dextrose, or an isotonic aqueous
saline solution.
[0057] Besides those representative dosage forms described above,
pharmaceutically acceptable excipients and carriers are generally
known to those skilled in the art and are thus included in the
instant present technology. Such excipients and carriers are
described, for example, in "Remingtons Pharmaceutical Sciences"
Mack Pub. Co., New Jersey (1991), which is incorporated herein by
reference.
[0058] Specific dosages may be adjusted depending on conditions of
disease, the age, body weight, general health conditions, sex, and
diet of the subject, dose intervals, administration routes,
excretion rate, and combinations of drug conjugates. Any of the
above dosage forms containing effective amounts are well within the
bounds of routine experimentation and therefore, well within the
scope of the instant present technology. By way of example only,
such dosages may be used to administer effective amounts of the
siRNA drugs to the patient and may include about 0.1 mg/kg, about
0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about
0.75 mg/kg, about 1 mg/kg, about 1.25 mg/kg, about 1.5 mg/kg, or a
range between and including any two of the forgoing values. Such
amounts may be administered parenterally as described herein and
may take place over a period of time including but not limited to 5
minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2
hours, 3 hours, 5 hours, 10 hours, 12, hours, 15 hours, 20 hours,
24 hours or a range between and including any of the foregoing
values. The frequency of administration may vary, for example, once
per day, per 2 days, per 3 days, per week, per 10 days, per 2
weeks, or a range between and including any of the foregoing
frequencies. Alternatively, the compositions may be administered
once per day on 2, 3, 4, 5, 6 or 7 consecutive days. A complete
regimen may thus be completed in only a few days or over the course
of 1, 2, 3, 4 or more weeks.
[0059] The nanoparticles described herein may be prepared by
methods comprising dispersing the siRNA within the unimolecular
nanoparticle. The drug delivery systems include compositions
comprising unimolecular nanoparticles dispersed within a
pharmaceutically acceptable carrier or one or more excipients, and
an effective amount of anti-cancer siRNA dispersed within the
unimolecular nanoparticle. As used herein, "dispersed" means
distributed, in a generally uniform or in a non-uniform fashion. In
some embodiments, the siRNA is dispersed in a generally uniform
fashion within the nanoparticle. However, it will be understood
that nanoparticles with a non-uniform distribution of siRNA,
especially those with small variations in concentration of the
siRNA are within the scope of the present technology. The
anti-cancer siRNA may also be non-uniformly distributed within the
unimolecular nanoparticles.
[0060] In another aspect, the present technology provides kits
including the components needed to prepare any of the compositions
described herein. For example, a kit may include a package
containing a unimolecular nanoparticle and a package containing an
effective amount of siRNA and directions for use of the kit. In
such kits, the unimolecular nanoparticle may include any of those
described herein and any of the siRNAs described herein. In some
embodiments, the kits may include separate packages for the
unimolecular nanoparticles and siRNAs. The present kits allow the
user to prepare the drug delivery composition described herein by
dispersing the siRNA in the unimolecular nanoparticles.
[0061] The examples herein are provided to illustrate advantages of
the present technology and to further assist a person of ordinary
skill in the art with preparing or using the nanoparticle
compositions of the present technology. To the extent that the
compositions include ionizable components, salts such as
pharmaceutically acceptable salts of such components may also be
used. The examples herein are also presented in order to more fully
illustrate the preferred aspects of the present technology. The
examples should in no way be construed as limiting the scope of the
present technology, as defined by the appended claims. The examples
can include or incorporate any of the variations, aspects or
aspects of the present technology described above. The variations,
aspects or aspects described above may also further each include or
incorporate the variations of any or all other variations, aspects
or aspects of the present technology.
EXAMPLES
[0062] The present technology describes a pH/redox dual-sensitive
cationic unimolecular NP containing imidazole residues developed
for siRNA delivery (FIG. 1). The unimolecular NP was formed by a
multi-arm star block copolymer, H40-poly(aspartic
acid-(2-aminoethyl disulfide)-(4-imidazolecarboxylic
acid))-poly(ethylene glycol) (i.e., H40-P(Asp-AED-ICA)-PEG), in an
aqueous solution. Because of its covalent nature, the unimolecular
NP has excellent stability in vitro and in vivo. The cationic core
formed by P(Asp-AED-ICA) blocks was used for siRNA complexation
through electrostatic interactions while the PEG shell was used to
provide good water solubility and reduced opsonization of NPs
during blood circulation. NPs are taken up by cells through
endocytosis. The imidazole groups in the cationic segment have a
pKa of .about.6.0 and can thus absorb protons in the acidic
endocytic compartments (endosomes/lysosomes), leading to osmotic
swelling and endosome/lysosome-membrane disruption (i.e., the
proton sponge effect), thereby facilitating the endosomal/lysosomal
escape of the siRNA-complexed NPs. Moreover, siRNA molecules were
complexed within the NPs by an electrostatic interaction with a
cationic P(Asp-AED-ICA) block containing cleavable disulfide bonds.
The cationic segments were conjugated onto the hyperbranched
polymer (H40) via a pH-sensitive aromatic imine bond, which can be
hydrolyzed in the endosome/lysosome, but stays relatively stable at
physiological conditions (pH 7.4). Furthermore, once inside the
cells, it was expected that the pendant mercaptoethylamine group
(SH-CH.sub.2-CH.sub.2-NH.sub.2) would be cleaved from the
P(Asp-AED-ICA) block by highly concentrated GSH (2-10 mM) in the
cytosol. The GSH concentration in the cytosol is 100-1000 times
higher than that in bodily fluids, including blood and
extracellular milieu (2-20 .mu.M GSH) where the disulfide bonds are
stable. The enzyme, gamma-interferon-inducible lysosomal thiol
reductase (GILT in the endosomes/lysosomes), in combination with
cysteines, may also trigger the cleavage of disulfide bonds. Hence,
the pH/redox dual-sensitive characteristic of the NPs may
facilitate the release of siRNA from the NPs. The NPs were also
functionalized with GE11 peptide, which can efficiently bind to the
epidermal growth factor receptor (EGFR) to achieve active tumor
targeting. EGFR is one of the most common receptors overexpressed
in many types of cancer cells, including triple negative breast
cancers (TNBCs), ovarian cancers, pancreatic cancers, and so on.
These pH/redox dual-sensitive unimolecular NPs, with excellent
endosomal/lysosomal escape abilities, may be promising nanocarriers
for the targeted delivery of siRNA.
Example 1: Preparation of siRNA-Loaded Unimolecular
Nanoparticle
[0063] Materials. BOLTRON.RTM. H40 (a hyperbranched polyester with
64 hydroxyl terminal groups; M.sub.n: 2,833 Da) was kindly provided
by Perstorp Polyols Inc., USA, and purified by fractional
precipitation in acetone and tetrahydrofuran (THF). .beta.-Benzyl
l-aspartate N-carboxyanhydride (BLA-NCA) was prepared as previously
reported. See M. Prabaharan, J. J. Grailer, S. Pilla, D. A.
Steeber, S. Gong, Amphiphilic multi-arm-block copolymer conjugated
with doxorubicin via pH-sensitive hydrazone bond for tumor-targeted
drug delivery, Biomaterials, 30 (2009) 5757-5766. The
heterobifunctional poly(ethylene glycol) (PEG) derivatives,
methoxy-PEG-NH.sub.2 (OCH.sub.3-PEG-OH, M.sub.n=5 kDa) and
maleimide-PEG-NH.sub.2 (Mal-PEG-NH.sub.2, M.sub.n=5 kDa), were
purchased from JenKem Technology (Allen, Tex., USA). Cy5 dye was
obtained from Lumiprobe Corporation (Hallandale Beach, Fla., USA).
GE11 peptide (YHWYGYTPQNVIGGGGC) (SEQ ID NO: 42) was synthesized by
Tufts University Core Facility (Boston, MA, USA). GFP-siRNA-Cy5.5,
GFP-siRNA, dimethyl sulfoxide (DMSO), 2-carboxybenzylaldehyde,
2-aminoethyl disulfide, 4-imidazolecarboxylic acid, and stannous
(II) octoate (Sn(Oct)2) were purchased from Sigma-Aldrich (St.
Louis, Mo., USA). 4-Dimethylamino pyridine (DMAP) and
1,3-dicyclohexylcarbodiimide (DCC) were purchased from ACROS and
used without further purification. Other reagents, including
RNAiMAX, were purchased from Thermo Fisher Scientific (Fitchburg,
Wis., USA) and used as received unless otherwise stated.
[0064] Synthesis of H40-poly(aspartic acid-(2-aminoethyl
disulfide)-(4-imidazolecarboxylic acid))-poly(ethylene
glycol)-OCH.sub.3/Cy5/GE11 (i.e.,
H40-P(Asp-AED-ICA)-PEG-OCH.sub.3/Cy5/GE11)
[0065] Synthesis of poly(.beta.-benzyl l-aspartate
N-carboxyanhydride)-poly(ethylene glycol)-Mal (i.e.,
P(BLA-NCA)-PEG-Mal). P(BLA-NCA)-PEG-Mal was prepared by
ring-opening polymerization of BLA-NCA using NH.sub.2-PEG-Mal as
the macro-initiator. Briefly, BLA-NCA (53 mg), and NH.sub.2-PEG-Mal
(25 mg) were dissolved in D1VIF (5 mL). The reaction was carried
out at 55.degree. C. under argon for 48 h. The resulting mixture
was then added dropwise into a 10-fold volume of cold diethyl
ether. The precipitate was collected by filtration using a Buchner
funnel, washed with diethyl ether, and dried under vacuum. The
P(BLA-NCA)-PEG-OCH.sub.3 was synthesized following a similar method
using NH.sub.2-PEG-OCH.sub.3 instead.
[0066] Synthesis of H40-carboxybenzaldehyde (i.e., H40-CHO). H40-OH
(10 mg), 2-carboxybenzylaldehyde (82 mg), DCC (135 mg), and DMAP
(8.3 mg) were dissolved in anhydrous DMSO (3 mL). The solution was
stirred at room temperature under argon for 48 h. Thereafter, the
dicyclohexylurea was removed by filtration using a Buchner funnel.
The solution was collected and poured into a 10-fold volume of cold
diethyl ether. The precipitate was collected by filtration using a
Buchner funnel, washed with diethyl ether, and dried under
vacuum.
[0067] Synthesis of H40-poly(.beta.-benzyl l-aspartate
N-carboxyanhydride)-poly(ethylene glycol)-OCH.sub.3/Mal (i.e.,
H40-P(BLA-NCA)-PEG-OCH.sub.3/Mal). H40-CHO (5 mg),
P(BLA-NCA)-PEG-OCH.sub.3 (25 mg), and P(BLA-NCA)-PEG-Mal (8 mg)
were dissolved in DMSO. The reaction was conducted at room
temperature for 24 h. Thereafter, the resulting solution was
dialyzed (molecular weight cut-off: 15 kDa) against DMSO for the
first 24 h and DI water for another 24 h. The product was obtained
after lyophilization. The H40-P(BLA-NCA)-PEG-OCH.sub.3 was
synthesized following a similar method.
[0068] Synthesis of H40-poly(aspartic acid-(2-aminoethyl
disulfide))-poly(ethylene glycol)-OCH.sub.3/Mal (i.e.,
H40-P(Asp-AED)-PEG-OCH.sub.3/Mal). 2-Aminoethyl disulfide (13.1 mg)
and H40-P(BLA-NCA)-PEG-OCH.sub.3/Mal (20 mg) were dissolved in DMSO
(10 mL). The reaction was carried out at room temperature for 24 h.
Thereafter, the resulting solution was dialyzed (molecular weight
cut-off: 15 kDa) against DI water for 48 h. The product was
obtained after lyophilization. The H40-P(Asp-AED)-PEG-OCH.sub.3 was
synthesized following a similar method.
[0069] Synthesis of H40-poly(aspartic acid-(2-aminoethyl
disulfide)-(4-imidazolecarboxylic acid))-poly(ethylene
glycol)-OCH.sub.3/Mal (i.e., H40-P(Asp-AED-ICA)-PEG-OCH.sub.3/Mal).
4-Imidazolecarboxylic acid (2.2 mg),
H40-P(Asp-AED)-PEG-OCH.sub.3/Mal (20 mg), DCC (4.4 mg), and
N-hydroxysuccinimide (2.9 mg) were dissolved in DMSO (5 mL). The
reaction was carried out at room temperature for 24 h. Thereafter,
the resulting solution was dialyzed (molecular weight cut-off: 15
kDa) against DI water for 48 h. The product was obtained after
lyophilization. The H40-(Asp-AED-ICA)-PEG-OCH.sub.3 was synthesized
following a similar method.
[0070] Synthesis of H40-poly(aspartic acid-(2-aminoethyl
disulfide)-(4-imidazolecarboxylic acid))-poly(ethylene
glycol)-OCH.sub.3/GE11 (i.e.,
H40-(PAsp-AED-ICA)-PEG-OCH.sub.3/Cy5/GE11). Cy5-SH was first
prepared by a reaction between Cy5-NH.sub.2 and Traut's reagent.
Briefly, Cy5-NH.sub.2 (0.3 mg) and Traut's reagent (0.51 mg) were
dissolved in DMSO. The solution was stirred at room temperature in
complete darkness for 4 h. H40-P(Asp-AED-ICA)-PEG-OCH.sub.3/Mal (20
mg) and GE11 (1.3 mg) were added into the above solution. After 24
h, the reaction solution was dialyzed (molecular weight cut-off: 15
kDa) against DI water for 48 h. The product was obtained after
lyophilization. The H40-P(Asp-AED-ICA)-PEG-OCH.sub.3/Cy5 and
H40-P(Asp-AED-ICA)-PEG-OCH.sub.3/GE11 were synthesized following a
similar method. Polymers H40-P(Asp-AED-ICA)-PEG-OCH.sub.3/Cy5 and
H40-P(Asp-AED-ICA)-PEG-OCH.sub.3/Cy5/GE11 were only used for the
cellular uptake analysis. For all other experiments,
H40-P(Asp-AED-ICA)-PEG-OCH.sub.3 and
H40-P(Asp-AED-ICA)-PEG-OCH.sub.3/GE11 were used.
[0071] Preparation of siRNA-Complexed Unimolecular NPs (i.e.,
siRNA-complexed NPs) and Gel Retardation Assay. To prepare
siRNA-complexed NPs, siRNA and H40-P(Asp-AED-ICA)-PEG were
dissolved in PBS and the solution was mixed for 30 min under gentle
shaking. The binding ability of siRNA to NPs was studied by agarose
gel electrophoresis. The siRNA-complexed NPs were prepared at
different N/P ratios (molar ratio of nitrogen in polymers to
phosphorus in siRNA: 2, 5, 7, 10, and 15). Electrophoresis was
carried out on 1% agarose gel in a TAE (Tris-acetate-EDTA) buffer
solution with a current of 100 V for 35 min. The final siRNA
concentration was 1 .mu.g per well. The retardation of the
complexes was visualized on a UV illuminator (Bio-Rad Baloratories,
Inc., Hercules, Calif., USA) to show the position of the complexed
siRNA band relative to that of naked siRNA.
[0072] Characterization. .sup.1H NMR spectra of all intermediate
and final polymer products were recorded on a Varian Mercury Plus
300 spectrometer in DMSO-d.sub.6 or CDCl.sub.3 at 25.degree. C.
Molecular weights (M.sub.n and M.sub.w) and polydispersity indices
(PDI) of the polymers were determined by a gel permeation
chromatography (GPC) system equipped with a refractive index
detector, a viscometer detector, and a light scattering detector
(Viscotek, USA). Fourier transform infrared (FT-IR) spectra were
recorded on a Bruker Tensor 27 FT-IR spectrometer. The morphologies
of the siRNA-complexed NPs were studied by dynamic light scattering
(DLS; ZetaSizer Nano ZS90, Malvern Instruments, USA; 0.5 mg/mL) and
transmission electron microscopy (TEM, FEI Tecnai G.sup.2 F30 TWIN
300 KV, E.A. Fischione Instruments, Inc. USA).
##STR00001##
[0073] Results and Discussion
[0074] Polymer Synthesis and siRNA Encapsulation. pH/redox
dual-sensitive multi-arm star block copolymer
H40-P(Asp-AED-ICA)-PEG-OCH.sub.3/Cy5/GE11 was synthesized as
outlined in Scheme 1. P(BLA-NCA)-PEG-OCH.sub.3 and
P(BLA-NCA)-PEG-Mal were first synthesized by ring-opening
polymerization of BLA-NCA using NH.sub.2-PEG-OCH.sub.3 and
NH.sub.2-PEG-Mal as the macro-initiators, respectively. Their
chemical structures were confirmed by .sup.1H NMR spectra as shown
in FIG. 2(A) and (B). The peaks at (e) 7.28-7.40 ppm and (d) 5.15
ppm were assigned to the protons in the benzyl and methylene groups
in the P(BLA-NCA) side chains, respectively. The signals labeled as
(c) at 2.6-2.8 ppm were ascribed to the methylene group of the side
chain that connects the main chain in the P(BLA-NCA) segment. The
peak located at (a) 3.57 ppm corresponded to the methylene protons
of the oxyethylene repeat units in the PEG segment. The Mal group
in the P(BLA-NCA)-PEG-Mal at 6.7 ppm was also observed. The number
of BLA-NCA repeat units in the polymers was calculated to be 20
based on the relative intensity ratio of the methylene proton (a)
of the PEG chain and the methylene proton (d) near the benzyl group
of the PBLA chain. The molecular weights of the
NH.sub.2-PEG-OCH.sub.3 and NH.sub.2-PEG-Mal polymers as measured by
GPC (Table 1) were 9,040 and 9,105 g/mol, respectively, which was
consistent with that determined by the .sup.1H NMR analyses.
[0075] The benzylaldehyde-functionalized H40 (H40-CHO) was prepared
by an esterification reaction. The chemical structure was also
confirmed by .sup.1H NMR spectrum (FIG. 2C). The peaks at 0.97-1.2
ppm and 3.8-4.1 ppm were assigned to the protons in H40. The peaks
at (a) 7.6-7.8 ppm corresponded to the protons in the phenyl group
of H40-CHO as labeled. Thereafter, P(BLA-NCA)-PEG-OCH.sub.3 and
P(BLA-NCA)-PEG-Mal (molar ratio: 3.1/1) polymers were then
conjugated to H40-CHO through imine bonds to form
H40-P(BLA-NCA)-PEG-OCH.sub.3/Mal. In the .sup.1H NMR spectrum shown
in FIG. 3(A), other than the proton peaks assigned to
P(BLA-NCA)-PEG-OCH.sub.3 and P(BLA-NCA)-PEG-Mal, proton peaks
ascribed to H40 were also observed. The GPC analyses further
demonstrated the formation of H40-P(BLA-NCA)-PEG-OCH.sub.3/Mal and
its molecular weight was measured to be 200,833 Da, which was
significantly larger than that of the linear NH.sub.2-PEG-OCH.sub.3
or NH.sub.2-PEG-Mal polymers. The average number of arms in the
H40-P(BLA-NCA)-PEG-OCH.sub.3/Mal was calculated to be 22 based on
the molecular weights of H40-P(BLA-NCA)-PEG-OCH.sub.3/Mal,
NH.sub.2-PEG-OCH.sub.3, and NH.sub.2-PEG-Mal. Thereafter, the
H40-P(BLA-NCA)-PEG-OCH.sub.3/Mal polymer underwent aminolysis by
using 2-aminoethyl disulfide (AED) to form water-soluble polymer
H40-P(Asp-AED)-PEG-OCH.sub.3/Mal. As shown in FIG. 3(B), the
absence of proton peaks at 7.28-7.40 ppm and 5.15 ppm, and the
presence of proton peaks at 2.71 and 3.17 ppm ascribed to the
protons in AED, demonstrated the formation of
H40-P(Asp-AED)-PEG-OCH.sub.3/Mal.
[0076] Imidazole groups were selectively conjugated to
H40-P(Asp-AED)-PEG-OCH.sub.3/Mal (molar ratio: 5/1) through an
amidization reaction for enhanced endosomal/lysosomal escape. The
characteristic proton peaks at (j) 7.23 and (k) 7.91 ppm for
imidazole groups were observed in FIG. 3 (C). In the last step,
GE11 peptide and Cy5 dye were conjugated to
H40-P(Asp-AED-ICA)-PEG-OCH.sub.3/Mal (molar ratio: 3/2/1) though a
Mal-SH reaction. Proton peaks assigned to the GE11 and Cy5
molecules, as labeled in FIG. 3(D), were also observed.
TABLE-US-00001 TABLE 1 GPC analyses of polymers. Polymers M.sub.n
(g/mol) PDI P(BLA-NCA)-PEG-OCH.sub.3 9,040 1.4 P(BLA-NCA)-PEG-Mal
9,105 1.3 H40-P(BLA-NCA)-PEG-OCH.sub.3/Mal 200,833 1.6
H40-P(Asp-AED)-PEG-OCH.sub.3/Mal 186,406 1.7
H40-P(Asp-AED-ICA)-PEG-OCH.sub.3/Mal 187,130 1.6
[0077] The cationic polymer H40-P(Asp-AED-ICA)-PEG, which had good
solubility in aqueous solutions, was able to form the unimolecular
NPs. Because of its covalent nature, the unimolecular NPs had
excellent stability in vitro and in vivo. siRNA (GFP-siRNA was used
as a model siRNA) was electrostatically complexed with the cationic
P(Asp-AED-ICA) polymer to form siRNA-complexed NPs. The
complexation was evaluated using agarose gel electrophoresis. As
shown in FIG. 4 (A), siRNA-complexed NPs with various N/P ratios
were tested and siRNA lost mobility in the electric field when the
N/P ratio reached 10, which was selected for the following tests.
The siRNA loading level, defined by the weight percentage of the
siRNA in the siRNA-complexed NP was 16.3% at N/P ratio of 10, and
the loading efficiency is 100%. The average hydrodynamic diameter
of the siRNA-complexed NPs was 68.3 nm (PDI=0.14) as measured by
dynamic light scattering (DLS). Transmission electron microscopy
(TEM) observation showed that the siRNA-complexed NPs were uniform,
with an average size of around 39 nm (FIG. 4 (B)).
Example 2: In Vitro Analyses
[0078] In Vitro siRNA Release. The release profiles of siRNA from
siRNA-complexed NPs were studied in a glass apparatus at 37.degree.
C. in a release medium at four conditions: (1) pH 7.4, (2) pH 5.3,
(3) pH 7.4+10 mM GSH, and (4) pH 5.3+10 mM GSH. siRNA-complexed NP
solutions (5 mL; 100 .mu.g/mL) were enclosed in a dialysis bag. The
dialysis bag was immersed in 50 ml of the release medium and kept
at 37.degree. C. under a horizontal laboratory shaker (Thermo
Scientific MaxQ Shaker, USA) at 100 rpm. At specific time points, 3
ml of release media were collected and replaced by the same volume
of fresh media. GFP-siRNA-Cy5.5 was used in this experiment. The
amount of released siRNA was analyzed based on the UV-vis intensity
of Cy5.5 at 649 nm.
[0079] Cellular Uptake. The cellular uptake behaviors of the NPs in
MDA-MB-468 TNBC cell lines were analyzed using a fluorescence
microscope based on the Cy5 dye conjugated on the NPs. Cells were
seeded (1.times.10.sup.5 cells/ml) onto 8-well high-optical-quality
plates and grown overnight. Cells were treated with either
non-targeted NPs, targeted NPs, or targeted NPs with free GE11
peptide (2 .mu.M; blocking assay) at an NP concentration of 100
.mu.g/ml. After 2 h incubation, cells were washed with PBS twice,
fixed with 4% PFA, and stained with DAPI for 4 h. Then the cells
were mounted with Prolong Gold anti-fade reagent. The cellular
uptake was observed using a fluorescence microscope (Nikon,
Melville, N.Y.). Digital monochromatic images were acquired using
NIS-Elements BR Software.
[0080] Endosomal/Lysosomal Escape. To assess the
endosomal/lysosomal escape behaviors of the NPs, MDA-MB-468 cells
were incubated with siRNA-complexed NPs for 2 h at 37.degree. C.
Cells treated with pure medium or free siRNA were used as negative
controls. The siRNA labeled with Cy5.5 was used for intracellular
tracking. The cells were washed three times with PBS, followed by
staining with LysoTracker Green DND-26 (100 nM) for
endosomes/lysosomes and Hoechst (5 ng/mL) for the nuclei, for 20
min at 37.degree. C. Cells were then washed three times with PBS.
The cellular localization of siRNA was visualized with a
fluorescence microscope (Nikon, Melville, N.Y.).
[0081] In Vitro siRNA Transfection. Cellular transfection was
performed on GFP-expressing MDA-MB-468717NBC cells using flow
cytometry and a fluorescence microscopy. GFP-expressing MDA-MB-468
cells were provided by Professor Wei Xu. For the flow cytometry
assay, cells were seeded at a density of 50,000 cells/well on a
24-well plate and incubated overnight. Cells were treated with pure
medium (control), siRNA-complexed non-targeted NPs (siRNA-NT),
siRNA-complexed targeted NPs iRNA-T), and siRNA complexed with
RNAiMAX (i.e., siRNA-RNAiMAX; positive control). The concentration
of siRNA was 40 nM. After 24 h incubation, cells were washed twice
with PBS and harvested with 0.25% trypsin. Cells were collected by
centrifugation at 200 g for 5 min, washed twice with PBS, fixed
with paraformaldehyde (PFA) for 15 min, and resuspended in 500
.mu.L PBS for analysis. The transfection efficiency was examined by
quantifying GFP expression levels in the cells using an
Accuri.upsilon. C6 flow cytometry system (BD Biosciences, USA). A
minimum of 10,000 cells was analyzed from each sample.
[0082] For fluorescence microscope imaging, cells were seeded
(50,000 cells/well) in an 8-well chamber slide system. Cells were
treated with the same five groups as described above. After 24 h
incubation, cells were washed twice with PBS, fixed with PFA for 15
min, stained with DAPI for 4 h, and mounted with ProLong Gold
Antifade Mountant. Images were acquired with a fluorescence
microscope (Nikon, Melville, N.Y.) to observe the GFP expression
levels in the cells. Digital monochromatic images were acquired
using NIS-Elements BR Software.
[0083] Cell Viability Assays. Cell viability tests were conducted
using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) assay. To test the cytotoxicity of the pure (empty)
NPs, MDA-MB-468 cells were seeded in quadruplicate on 96-well
plates and incubated overnight. Cells were treated with NPs at
different concentrations (i.e. ,10, 20, 50, 100, and 500 .mu.g/mL).
Cells treated with pure medium were used as the control group.
After 24 h of incubation, a standard MTT assay was performed by
aspirating the treatment media, adding 25 .mu.L of the medium
containing 0.5 mg/ml MTT agent, and incubating at 37.degree. C. for
4 h. Thereafter, the medium was aspirated and 75 .mu.L of DMSO was
added to each well. The plates were then measured at 570 nm using a
spectrophotometer (Quant, Bio-Tek Instruments, Winooski, Vt.), and
the average absorbance and percent of cell viability relative to
the control (pure medium) were calculated. The cytotoxicity of
siRNA-complexed NP systems was also studied. Similarly, cells were
treated with pure medium (control), siRNA-NT, siRNA-T,
siRNA-RNAiMAX, and RNAiMAX at the equivalent amount of siRNA (40
nM). After 24 h of incubation, the aforementioned MTT protocol was
performed and the cell viabilities relative to the control (pure
medium) were calculated.
Results and Discussion.
[0084] pH/redox Dual-Sensitive siRNA Release. NPs with pH/redox
dual-sensitive structures were designed to achieve decomplexation
of siRNA from cationic nanocarriers and controlled release of siRNA
to enhance gene silencing efficiency. To verify the pH/redox
dual-sensitive release behavior, in vitro release analyses were
conducted by monitoring Cy5.5-labeled siRNA. As shown in FIG. 4(C),
the release rate was very slow at neutral pH (7.4) without adding
GSH, with 7.1% of siRNA released after 48 h. In comparison, the
addition of GSH (10 mM) to the solution resulted in an increased
siRNA release rate (50.1% of siRNA released after 48 h). Meanwhile,
27.2% of siRNA was released at a pH of 5.3 after 48 h, which is
much faster compared to that at a neutral pH. Moreover, dual
stimuli (pH 5.3 and 10 mM GSH) led to the quickest siRNA release
(81.4% of siRNA released after 48 h). Taken together, these
observations suggest that siRNA can be decomplexed from
siRNA-complexed NPs inside of cells.
[0085] Enhanced Endosomal/Lysosomal Escape Capability of NPs.
Another major obstacle in designing nanocarriers for siRNA delivery
is their poor endosomal/lysosomal escape capabilities. siRNA needs
to be released into the cytoplasm for efficient gene silencing.
Therefore, the siRNA nanocarriers were functionalized with
endosomal/lysosomal escape capabilities. As mentioned above, the
imidazole groups in the cationic segment promote
endosomal/lysosomal escape through the proton-sponge effect,
thereby facilitating the release of siRNA to the cytosol. To verify
endosomal/lysosomal escape, fluorescence microscopy was used to
assess the intracellular localizations of siRNA. The cells
(MDA-MB-468 TNBC cell line) were treated with pure medium (control)
or media containing free siRNA or siRNA-complexed nanoparticles.
siRNA was labeled with Cy5.5 for detection. After 2 h incubation,
the nucleus and endosomes/lysosomes of cells were stained with
Hoechst and Lysotracker, respectively. As shown in FIG. 5, siRNA
complexed with NPs were taken up efficiently as signified by the
strong Cy5.5 signal. The Cy5.5 signals barely overlapped with the
Lysotracker signals (endosomes/lysosomes), and they were
distributed relatively uniformly in the cytosol, demonstrating that
the majority of the siRNA escaped from the endosomes/lysosomes. The
z-stack images shown in FIG. 6 also confirmed the excellent
endososomal/lysosomal escape capabilities of the siRNA-complexed
NPs. NPs capable of effective endosomal/lysosomal escape should
lead to efficient gene silencing.
[0086] In Vitro Cellular Uptake. EGFR is overexpressed in many
common types of cancer. Here, an EGFR targeting peptide, GE11, was
used as an active-tumor-targeting ligand to enhance cellular
uptake. MDA-MB-468, a TNBC cell line that overexpresses EGFR, was
used as the model cell line. Cells were incubated with either
non-targeted (i.e., NPs without GE11 conjugation) or targeted
(i.e., GE11 conjugated) NPs for 2 h. Cells without any treatment
were used as a negative control. Cy5 was conjugated onto the NPs.
Fluorescence imaging analysis was performed to compare cellular
uptake. As shown in FIG. 7, the targeted NPs showed a markedly
higher Cy5 fluorescence intensity than non-targeted ones. In the
blocking experiment (co-incubated cells with free GE11 and targeted
NPs), after the EGFR was saturated with free GE11, the cellular
uptake of the targeted NPs returned to the level of the
non-targeted ones, thereby demonstrating the targeting ability of
GE11. Taken together, the targeted NPs increased the cellular
uptake of NPs through EGFR-mediated endocytosis.
[0087] In Vitro Gene Silencing Efficiency and Cell Viability
Analysis. To determine the potential of NPs to deliver siRNA, gene
silencing was assessed in vitro. The gene silencing capacity of
GFP-siRNA toward MDA-MB-468 cells stably expressing green
fluorescent protein was evaluated for siRNA-complexed non-targeted
and targeted NPs. Pure medium was used as the negative control.
RNAiMAX, a commercially available transfection agent, was used as
the positive control. As shown in FIG. 8(A) and (B), both targeted
and non-targeted groups induced GFP reduction compared to the
negative control. As expected based on the cellular uptake
analysis, the extent of knockdown was dependent on GE11
functionalization. Relative to the negative control group, the
siRNA-complexed non-targeted NPs produced a 47% GFP
down-regulation. In contrast, the targeted NPs induced a 79% GFR
reduction, which is comparable to that of RNAiMAX treatment (81%).
However, the assessment of cell viability on these treatments
revealed that siRNA-complexed RNAiMAX exhibited significant
cytotoxicity, inducing more than 25% cell death (FIG. 8(C)), which
is consistent with the previous report. See N. Segovia, M. Pont, N.
Oliva, V. Ramos, S. Borros, N. Artzi, Hydrogel doped with
nanoparticles for local sustained release of siRNA in breast
cancer, Advanced Healthcare Materials, 4 (2015) 271-280. However,
no apparent cytotoxicity associated with NPs was observed. In fact,
no significant cytotoxicity was observed for NPs alone up to 500
.mu.g/mL. Taken together, these findings reveal that
GE11-conjugated NPs are suitable nanocarriers for siRNA delivery
targeted at TNBC cells and, potentially, other EGFR-overexpressing
cells.
EQUIVALENTS
[0088] While certain embodiments have been illustrated and
described, a person with ordinary skill in the art, after reading
the foregoing specification, can effect changes, substitutions of
equivalents and other types of alterations to the conjugates and
nanoparticles of the present technology or derivatives, prodrugs,
or pharmaceutical compositions thereof as set forth herein. Each
aspect and embodiment described above can also have included or
incorporated therewith such variations or aspects as disclosed in
regard to any or all of the other aspects and embodiments.
[0089] The present technology is also not to be limited in terms of
the particular aspects described herein, which are intended as
single illustrations of individual aspects of the present
technology. Many modifications and variations of this present
technology can be made without departing from its spirit and scope,
as will be apparent to those skilled in the art. Functionally
equivalent methods within the scope of the present technology, in
addition to those enumerated herein, will be apparent to those
skilled in the art from the foregoing descriptions. Such
modifications and variations are intended to fall within the scope
of the appended claims. It is to be understood that this present
technology is not limited to particular methods, conjugates,
reagents, compounds, compositions, labeled compounds or biological
systems, which can, of course, vary. All methods described herein
can be performed in any suitable order unless otherwise indicated
herein or otherwise clearly contradicted by context. It is also to
be understood that the terminology used herein is for the purpose
of describing particular aspects only, and is not intended to be
limiting. Thus, it is intended that the specification be considered
as exemplary only with the breadth, scope and spirit of the present
technology indicated only by the appended claims, definitions
therein and any equivalents thereof. No language in the
specification should be construed as indicating any non-claimed
element as essential.
[0090] The embodiments, illustratively described herein may
suitably be practiced in the absence of any element or elements,
limitation or limitations, not specifically disclosed herein. Thus,
for example, the terms "comprising," "including," "containing,"
etc. shall be read expansively and without limitation.
Additionally, the terms and expressions employed herein have been
used as terms of description and not of limitation, and there is no
intention in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are
possible within the scope of the claimed technology. Additionally,
the phrase "consisting essentially of" will be understood to
include those elements specifically recited and those additional
elements that do not materially affect the basic and novel
characteristics of the claimed technology. The phrase "consisting
of" excludes any element not specified.
[0091] In addition, where features or aspects of the disclosure are
described in terms of Markush groups, those skilled in the art will
recognize that the disclosure is also thereby described in terms of
any individual member or subgroup of members of the Markush group.
Each of the narrower species and subgeneric groupings falling
within the generic disclosure also form part of the technology.
This includes the generic description of the technology with a
proviso or negative limitation removing any subject matter from the
genus, regardless of whether or not the excised material is
specifically recited herein.
[0092] As will be understood by one skilled in the art, for any and
all purposes, particularly in terms of providing a written
description, all ranges disclosed herein also encompass any and all
possible subranges and combinations of subranges thereof. Any
listed range can be easily recognized as sufficiently describing
and enabling the same range being broken down into at least equal
halves, thirds, quarters, fifths, tenths, etc. As a non-limiting
example, each range discussed herein can be readily broken down
into a lower third, middle third and upper third, etc. As will also
be understood by one skilled in the art all language such as "up
to," "at least," "greater than," "less than," and the like, include
the number recited and refer to ranges which can be subsequently
broken down into subranges as discussed above. Finally, as will be
understood by one skilled in the art, a range includes each
individual member, and each separate value is incorporated into the
specification as if it were individually recited herein.
[0093] All publications, patent applications, issued patents, and
other documents (for example, journals, articles and/or textbooks)
referred to in this specification are herein incorporated by
reference as if each individual publication, patent application,
issued patent, or other document was specifically and individually
indicated to be incorporated by reference in its entirety.
Definitions that are contained in text incorporated by reference
are excluded to the extent that they contradict definitions in this
disclosure.
[0094] Other embodiments are set forth in the following claims,
along with the full scope of equivalents to which such claims are
entitled.
Sequence CWU 1
1
42112PRTHuman immunodeficiency virus 1Gly Arg Lys Lys Arg Arg Gln
Arg Arg Arg Pro Gln1 5 10218PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 2Lys Leu Ala Leu Lys Leu Ala
Leu Lys Ala Leu Lys Ala Ala Leu Lys1 5 10 15Leu Ala315PRTDrosophila
sp. 3Arg Gln Ile Lys Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys1 5
10 15416PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Arg Gln Ile Arg Ile Trp Phe Gln Asn Arg Arg Met
Arg Trp Arg Arg1 5 10 15517PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 5Cys Ser Ile Pro Pro Glu Val
Lys Phe Asn Lys Pro Phe Val Tyr Leu1 5 10 15Ile613PRTRana
temporaria 6Phe Val Gln Trp Phe Ser Lys Phe Leu Gly Arg Ile Leu1 5
10710PRTMaurus palmatus 7Gly Asp Cys Leu Pro His Leu Lys Leu Cys1 5
10818PRTMus sp. 8Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln
Ala His Ala His1 5 10 15Ser Lys924PRTHomo sapiens 9Leu Gly Thr Tyr
Thr Gln Asp Phe Asn Lys Phe His Thr Phe Pro Gln1 5 10 15Thr Ala Ile
Gly Val Gly Ala Pro 201030PRTUnknownDescription of Unknown
Neurturin sequence 10Gly Ala Ala Glu Ala Ala Ala Arg Val Tyr Asp
Leu Gly Leu Arg Arg1 5 10 15Leu Arg Gln Arg Arg Arg Leu Arg Arg Glu
Arg Val Arg Ala 20 25 301118PRTDrosophila sp. 11Arg Gln Ile Lys Ile
Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10 15Gly
Gly1230PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 12Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly Gly
Asp Ile Met Gly Glu1 5 10 15Trp Gly Asn Glu Ile Phe Gly Ala Ile Ala
Gly Phe Leu Gly 20 25 301311PRTHuman immunodeficiency virus 13Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5
101418PRTUnknownDescription of Unknown SynB1 sequence 14Arg Gly Gly
Arg Leu Ser Tyr Ser Arg Arg Arg Phe Ser Thr Ser Thr1 5 10 15Gly
Arg1510PRTUnknownDescription of Unknown SynB3 sequence 15Arg Arg
Leu Ser Tyr Ser Arg Arg Arg Phe1 5 101611PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Pro
Ile Arg Arg Arg Lys Lys Leu Arg Arg Leu1 5 101712PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 17Arg
Arg Gln Arg Arg Thr Ser Lys Leu Met Lys Arg1 5 101815PRTFlock house
virus 18Arg Arg Arg Arg Asn Arg Thr Arg Arg Asn Arg Arg Arg Val
Arg1 5 10 151919PRTBrome mosaic virus 19Lys Met Thr Arg Ala Gln Arg
Arg Ala Ala Ala Arg Arg Asn Arg Trp1 5 10 15Thr Ala Arg2013PRTHuman
T-cell leukemia virus II 20Thr Arg Arg Gln Arg Thr Arg Arg Ala Arg
Arg Asn Arg1 5 102113PRTHuman immunodeficiency virus 21Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln1 5 102213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 22Gly
Arg Arg Arg Arg Arg Arg Arg Arg Arg Pro Pro Gln1 5
102327PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 23Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly
Lys Ile Asn Leu1 5 10 15Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu
20 252417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 24Lys Leu Ala Leu Lys Leu Ala Leu Lys Leu Ala Leu
Ala Leu Lys Leu1 5 10 15Ala2527PRTHomo sapiens 25Met Gly Leu Gly
Leu His Leu Leu Val Leu Ala Ala Ala Leu Gln Gly1 5 10 15Ala Trp Ser
Gln Pro Lys Lys Lys Arg Lys Val 20 252627PRTUnknownDescription of
Unknown FBP sequence 26Gly Ala Leu Phe Leu Gly Trp Leu Gly Ala Ala
Gly Ser Thr Met Gly1 5 10 15Ala Trp Ser Gln Pro Lys Lys Lys Arg Lys
Val 20 252727PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 27Gly Ala Leu Phe Leu Gly Phe Leu Gly
Ala Ala Gly Ser Thr Met Gly1 5 10 15Ala Trp Ser Gln Pro Lys Lys Lys
Arg Lys Val 20 252827PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 28Gly Ala Leu Phe Leu Gly Phe
Leu Gly Ala Ala Gly Ser Thr Met Gly1 5 10 15Ala Trp Ser Gln Pro Lys
Ser Lys Arg Lys Val 20 252921PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 29Lys Glu Thr Trp Trp Glu Thr
Trp Trp Thr Glu Trp Ser Gln Pro Lys1 5 10 15Lys Lys Arg Lys Val
203021PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Lys Glu Thr Trp Phe Glu Thr Trp Phe Thr Glu Trp
Ser Gln Pro Lys1 5 10 15Lys Lys Arg Lys Val 203117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMISC_FEATURE(1)..(17)This sequence may encompass 4-17
residues 31Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg1 5 10 15Arg3217PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMISC_FEATURE(1)..(17)This
sequence may encompass 4-17 residues 32Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys1 5 10 15Lys3313PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(2)..(2)Aminocaproic acidMOD_RES(4)..(4)Aminocaproic
acidMOD_RES(6)..(6)Aminocaproic acidMOD_RES(8)..(8)Aminocaproic
acidMOD_RES(10)..(10)Aminocaproic acidMOD_RES(12)..(12)Aminocaproic
acid 33Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg1 5
103413PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(2)..(2)Aminobutyric
acidMOD_RES(4)..(4)Aminobutyric acidMOD_RES(6)..(6)Aminobutyric
acidMOD_RES(8)..(8)Aminobutyric acidMOD_RES(10)..(10)Aminobutyric
acidMOD_RES(12)..(12)Aminobutyric acid 34Arg Xaa Arg Xaa Arg Xaa
Arg Xaa Arg Xaa Arg Xaa Arg1 5 103513PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 35Arg
Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg1 5
103613PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 36Arg Met Arg Met Arg Met Arg Met Arg Met Arg Met
Arg1 5 103713PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 37Arg Thr Arg Thr Arg Thr Arg Thr Arg
Thr Arg Thr Arg1 5 103813PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 38Arg Ser Arg Ser Arg Ser Arg
Ser Arg Ser Arg Ser Arg1 5 103910PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 39Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg1 5 104013PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 40Arg Ala Arg Ala Arg Ala Arg
Ala Arg Ala Arg Ala Arg1 5 10417PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 41Arg Arg Arg Arg Arg Arg
Arg1 54217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42Tyr His Trp Tyr Gly Tyr Thr Pro Gln Asn Val Ile
Gly Gly Gly Gly1 5 10 15Cys
* * * * *