U.S. patent application number 16/799375 was filed with the patent office on 2020-06-18 for modulated guanidine-containing polymers or nanoparticles.
This patent application is currently assigned to The Florida International University Board of Trustees. The applicant listed for this patent is Joong Ho BARRIOS MOON. Invention is credited to Alfonso BARRIOS, Joong Ho MOON.
Application Number | 20200188520 16/799375 |
Document ID | / |
Family ID | 71072199 |
Filed Date | 2020-06-18 |
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United States Patent
Application |
20200188520 |
Kind Code |
A1 |
MOON; Joong Ho ; et
al. |
June 18, 2020 |
MODULATED GUANIDINE-CONTAINING POLYMERS OR NANOPARTICLES
Abstract
A modulated guanidine substituted polymer or nanoparticle has a
guanidine moiety or on a plurality of repeating units of a polymer
or on the surface of a nanoparticle where the guanidine moiety is
modulated as a substituted amidinourea or amidinocarbamate or salt
thereof. The modulated guanidine substituted polymer or
nanoparticle can be prepared by direct amination of a N-Boc
protected guanidine substituted conjugated polymer or N-Boc
protected guanidine substituted nanoparticle, where an amine or
alcohol is combined in solution or suspension with the protected
conjugated polymer or nanoparticle and the resulting mixture is
heated. The modulated guanidine substituted polymer or nanoparticle
can be used in a cancer treatment formulation.
Inventors: |
MOON; Joong Ho; (Weston,
FL) ; BARRIOS; Alfonso; (Miami, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MOON; Joong Ho
BARRIOS; Alfonso |
Weston
Miami |
FL
FL |
US
US |
|
|
Assignee: |
The Florida International
University Board of Trustees
Miami
FL
|
Family ID: |
71072199 |
Appl. No.: |
16/799375 |
Filed: |
February 24, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16220801 |
Dec 14, 2018 |
10568902 |
|
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16799375 |
|
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62598578 |
Dec 14, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C08F 34/02 20130101;
A61K 47/32 20130101; A61K 9/5146 20130101; C07D 491/18 20130101;
A61K 47/34 20130101 |
International
Class: |
A61K 47/32 20060101
A61K047/32; C07D 491/18 20060101 C07D491/18; C08F 34/02 20060101
C08F034/02 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under
DMR1352317 awarded by the National Science Foundation. The
government has certain rights in the invention.
Claims
1. A modulated guanidine substituted polymer, comprising a
guanidine moiety on a plurality of repeating units of a polymer,
the repeating unit comprises the following structure: ##STR00033##
wherein L is a linker and can be null; and R is selected from
hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heteroalkyl, substituted
heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl,
substituted heterocycloalkyl, cycloalkenyl, and substituted
cycloalkenyl, alkenyl and substituted alkenyl, alkynyl, haloalkyl,
acyl, amino, alkylamino, arylamino and hydroxylalkyl.
2. The modulated guanidine substituted polymer according to claim
1, R being an N-alkylamino; N-arylamino; N-(alkylaryl)amino;
N-(aryalkyl)amino; N, N-dialkylamino; N, N-diarylamino; N,
N-di(alkylaryl)amino; N, N-di(aryalkylamino); N-alkyl,N-arylamino;
N-alkyl,N-(alkylaryl)amino; N-alkyl,N-(arylalkyl)amino;
N-aryl,N-(alkylaryl)amino; or N-aryl,N-(arylalkyl)amino group.
3. The modulated guanidine substituted polymer according to claim
1, R being an unsubstituted or substituted morpholine, pyrolidine,
pyrrole, piperidine, ethyleneimine, indole, isoindole, carbazole,
imidazole, purine, aminoethanol, amino terminal polyethylene oxide,
substituted or unsubstituted alky carbamate, substituted or
unsubstituted aryl carbamate, substituted or unsubstituted
alkylaryl carbamate, or substituted or unsubstituted aryalkyl
carbamante.
4. The modulated guanidine substituted polymer according to claim
1, R being selected from hexylamine (HA), benzylamine (BA), and
aminoethoxyethanol (AEE).
5. The modulated guanidine substituted polymer according to claim
1, the polymer being a homopolymer having a structure of
##STR00034## wherein m'.gtoreq.2; and R is selected from hexylamine
(HA), benzylamine (BA), and aminoethoxyethanol (AEE).
6. The modulated guanidine substituted polymer according to claim
1, the polymer being a copolymer, the copolymer further comprising
one or more types of monomer species selected from ##STR00035##
7. The modulated guanidine substituted polymer according to claim
6, the copolymer being a random copolymer selected from
##STR00036## wherein ##STR00037## means that the two monomer
species are randomly distributed; both m and n.gtoreq.1; R is
selected from hexylamine (HA), benzylamine (BA), and
aminoethoxyethanol (AEE).
8. The modulated guanidine substituted polymer according to claim
6, the copolymer being a block copolymer comprising a structure of
##STR00038## wherein m and n can be the same or different, and both
m and n.gtoreq.1; and R is selected from hydrogen, alkyl,
substituted alkyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heteroalkyl, substituted heteroalkyl, cycloalkyl,
substituted cycloalkyl, heterocycloalkyl, substituted
heterocycloalkyl, amino, alkylamino, arylamino and
hydroxylalkyl.
9. The modulated guanidine substituted polymer according to claim
8, the block copolymer being selected from ##STR00039## wherein m
and n.gtoreq.1, and m and n can be the same or different.
10. The modulated guanidine substituted polymer according to claim
6, the copolymer being a block copolymer comprising a structure of
##STR00040## wherein ##STR00041## means that the two monomer
species are randomly distributed; m, n and o can be the same or
different, and m, n and o.gtoreq.1; and R is selected from
hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heteroalkyl, substituted
heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl,
substituted heterocycloalkyl, amino, alkylamino, arylamino and
hydroxylalkyl.
11. The modulated guanidine substituted polymer according to claim
10, the block copolymer being selected from ##STR00042## wherein m,
n and o.gtoreq.1, and m, n and o can be the same or different;
##STR00043## means that the two monomer species are randomly
distributed.
12. The modulated guanidine substituted polymer according to claim
6, the copolymer being a block copolymer comprising a boronic acid
moiety on the modulated guanidine moiety.
13. The modulated guanidine substituted polymer according to claim
12, the block copolymer having a structure of ##STR00044## wherein
m and n.gtoreq.1, and m and n can be the same or different; and L
is a linker and can be null.
14. The modulated guanidine substituted polymer according to claim
13, the linker being a C1-C10 alkyl or heteroalkyl.
15. The modulated guanidine substituted polymer according to claim
1, which is conjugated to a nanoparticle, the nanoparticle
comprising silica, alumina, titania, zinc oxide, tin oxide, silver
oxide, cuprous oxide, cupric oxide, ceria, vanadium oxide zirconia,
molybdenum, tungsten oxide, barium oxide, calcium oxide, iron
oxide, or nickel oxide.
16. A therapeutic formulation comprising the modulated guanidine
substituted polymer of claim 1, a therapeutic agent and a
pharmaceutically acceptable carrier.
17. A method for treating cancer comprising administering, to a
subject in need of such treatment, an effective amount of the
therapeutic formulation of claim 16.
18. A method for delivering a therapeutic agent into a cancer cell,
comprising contacting the cancer cell with the modulated guanidine
substituted polymer of claim 1, and the therapeutic agent.
19. A method for transporting a therapeutic agent across a
biological membrane, comprising contacting the biological membrane
with the modulated guanidine substituted polymer of claim 1, and
the therapeutic agent.
20. A method according to claim 19, the biological membrane being
selected from cell membranes, organelle membranes, mucous
membranes, basement membranes, and serous membranes.
Description
CROSS-REFERENCE TO A RELATED APPLICATION
[0001] This application is a continuation-in-part (CIP) application
of U.S. application Ser. No. 16/220,801, filed Dec. 14, 2018, which
claims the benefit of U.S. Provisional Application Ser. No.
62/598,578, filed Dec. 14, 2017, the disclosure of which is hereby
incorporated by reference in its entirety, including all figures,
tables and drawings.
BACKGROUND OF INVENTION
[0003] Ovarian cancer (OVCA) is the most lethal gynecologic
malignancy. The majority of patients are diagnosed with advanced
disease, which ultimately recurs, and they die from the disease.
OVCA is becoming resistant to current chemotherapies, including the
two most commonly used first-line drugs taxol and cisplatin, and
patients are exhausting their treatment options.
[0004] Multidrug resistance (MDR) is closely related to
overexpression of membrane efflux proteins (e.g., P-glycoprotein)
and anti-apoptotic proteins [e.g., survivin and myeloid cell
leukemia 1 (Mc1-1)]. Because small RNA molecules, including small
interfering RNA (siRNA), have an extraordinary ability to knock
down gene expression, RNA interference (RNAi) induced by small RNA
molecules can be an excellent solution for overcoming MDR.
[0005] RNA molecules are highly susceptible to enzymatic
degradation and too big to penetrate the cellular membranes.
Although various types of delivery materials have been developed
and used at the in vitro tissue culture level, gene regulation at
the ex vivo or in vivo level has been largely unsuccessful due to
poor intracellular siRNA availability.
[0006] Lack of targeting and inefficient intracellular entry of
drugs requires over dosing, which is also responsible for poor
therapeutic outcomes. Efficient delivery of negatively charged RNA
molecules to target cells is pivotal for a successful application
of RNAi technology. Innovative therapeutic delivery techniques are
urgently needed to address the drug resistance and poor
intracellular entry efficiency.
[0007] In human airway epithelium possessing additional
extracellular barriers, such as mucus layers, transfection using
conventional lipid-based or positively charged carriers is
extremely limited. As the critical physical barrier interfacing
environmental stimuli, the mucosal surfaces of epithelium tightly
regulate various physiological and immunological processes. In the
mucus layer, dense mucin fibers and negatively charged
proteoglycans provide the adhesive and viscous protective layer
that often trap and remove positively charged carriers, resulting
in poor delivery of payloads to the underlying epithelial cells.
Very few options are currently available for delivering nucleic
acids to the airway epithelium. Mucus-altering or mucolytic agents
can be used as adjuvants of gene carriers, although high millimolar
concentrations are often needed to disrupt or disturb the mucus
layers.
[0008] Alternatively, block copolymers of polyethyleneimine (PEI)
and polyethyleneglycol (PEG) have been developed to deliver plasmid
DNA (pDNA) to the lung airways. Positively charged PEI and
negatively charged pDNA form ionic complexes, while the PEG block
shields the positively charged block from the negatively charged
mucus layers and provides diffusion through nanometer sized mucus
meshes. However, the PEG block often causes poor gene complexation
and reduced cellular entry; and pDNA can form smaller ionic
complexes with PEI-PEG copolymers due to the molecular topography
of pDNA, enabling compaction to nanoparticles. Although
optimization provides the opportunity to balance the ratios between
charged and PEG segments, block copolymer architectures in
biological fluids containing ions and proteins complicate surface
properties and influence biological functions.
[0009] Short peptides or their synthetic mimics of the protein's
translocation domains are excellent materials to introduce
therapeutic agents to intracellular compartments rapidly. The fast
entry of those materials is associated with a combination of
membrane pore formation and non-receptor-mediated endocytosis.
Combinations of ionic bonding, hydrogen bonding, and hydrophobic
interactions influence the entry pathways. However, coupling cell
penetrating peptides to therapeutic proteins or nucleic acids often
alters the entry pathways, resulting in decreased intracellular
availability. Fluorescent labels needed to study the entry
mechanism and the localization of synthetic materials influence the
material's physical properties and cellular behaviors toward the
materials. The development of nontoxic biomaterials exhibiting
superior cellular entry and therapeutic delivery is needed to
substantially increase therapeutic efficacy of these systems.
[0010] In another approach, nanometer sized particles accumulate in
relatively loosely organized tumor tissues as opposed to tight
normal tissue. When the particulates are modified with ligands
specific to the receptors overexpressed on cancer cell surfaces,
targeting at the tissue level can be further improved.
Unfortunately, overall therapeutic efficacy remains unsatisfactory
due to poor intracellular entry and a lack of organelle targeting.
Endocytosis mediated by cell surface receptors is the primary entry
pathway, but it is often slow and inefficient. Endocytosed
therapeutic agents undergo degradation in endosomes and lysosomes
or in recycling processes, such as exocytosis, which lower the
intracellular concentration of therapeutic agents. By not involving
an endosome escaping process, direct membrane translocation offers
high intracellular concentrations of therapeutics. Nanometer sized
particles with modulated surface properties are pivotal for
efficient intracellular delivery and labeling because the surface
properties are closely related to their initial interaction
following entry.
[0011] Aromatic it-electron conjugated polymers (CPs) are
innovative fluorescent materials that have a high potential as
therapeutic carriers. Because of excellent photophysical
properties, such as high brightness and sensing ability, and
excellent biophysical properties, such as biocompatibility,
nontoxicity, high cellular interaction, and ease of entry, CPs have
been used for live cell and tissue imaging, biochemical sensing,
and gene and drug delivery. In addition to intrinsic fluorescent
properties that are highly advantageous for labeling and tracking,
the charged CPs are structurally similar in charge density and
backbone rigidity to materials known for exhibiting efficient
cellular entry, such as tyrosine aminotransferase (TAT) as shown in
FIG. 1. Because of a rigid hydrophobic backbone and a flexible
hydrophilic charged side chains, CPs can bind to and enter through
cellular membranes.
[0012] Moon et al. U.S. Pat. Nos. 9,676,886 and 9,757,410 disclose
biodegradable CPs that are made by introducing flexible degradable
functional groups along the backbone of the CP that can be used for
quantitative labeling of mitochondria. Cellular interaction and
internalization of CPs are dependent on the chemical structures of
both the backbone and side chains of the CPs. CPs with guanidine
units (G-CPs) having molecular weights of .about.14,000 g/mol enter
live cells quickly, within 10 minutes upon incubation, through the
cancer cell membrane.
[0013] Conventional methods of synthesizing CPs with diverse
functional group are tedious and problematic. In addition to
intrinsic synthetic challenges of optimizing polymerization
conditions for each monomer, the resulting CP with different
functional groups will have different molecular weight and
polydispersity, which will influence their physical and biophysical
behaviors. It is therefore desirable to form a nanoparticle or a CP
that has attached modified guanidine moieties. These may provide
rapid and tailored cellular delivery of anti-MDR siRNA for
dramatically enhanced chemotherapy efficiencies that can impact
cancer treatment.
[0014] Cell membranes are impermeable to most macromolecules. Many
drug candidates fail to advance clinically because they do not have
the properties needed to cross biological membranes and reach their
intracellular target. Additionally, poor pharmacokinetics,
stability, and off-target effects lead to undesirable biological
responses. Thus, there is a need to develop novel delivery
materials that overcome the biological barrier.
BRIEF SUMMARY
[0015] The subject invention provides materials and methods for
disrupting the mucus layer and intracellularly delivering
therapeutic agents such as drugs, nucleic acids and proteins. The
subject invention also provides methods for design and synthesis of
nanomaterials that enhance or assist the passage of therapeutic
agents across biological membranes.
[0016] In one embodiment, the subject invention provides
nanomaterials as molecule transporters for targeted delivery of
therapeutic agents into cells, preferably, cancer cells for
inhibiting the growth of cancer cells and altering gene expression
in these cells.
[0017] In one embodiment, the nanomaterial of the subject invention
comprises a modulated guanidine substituted polymer or
nanoparticle, the modulated guanidine substituted polymer or
nanoparticle comprising a guanidine moiety on a plurality of
repeating units of a polymer, or on the surface of a nanoparticle,
the modulation comprising a substituted amidinourea or
amidinocarbamate or salt thereof. Preferably, the modulation
comprises the substituted amidinourea or salt thereof.
[0018] In one embodiment, the substituted amidinourea or salt
thereof comprises a hydrophobic modulation, wherein the hydrophobic
modulation can be, for example, an N-alkylamino; N-arylamino;
N-(alkylaryl)amino; N-(aryalkyl)amino; N, N-dialkylamino; N,
N-diarylamino; N, N-di(alkylayrl)amino; N, N-di(aryalkylamino);
N-alkyl, N-arylamino; N-alkyl, N-(alkylaryl)amino; N-alkyl,
N-(arylalkyl)amino; N-aryl, N-(alkylaryl)amino; or N-aryl,
N-(arylalkyl)amino group.
[0019] In a further embodiment, the alkyl group is a C2 to C22
straight, branched, cycloalkyl or alkyl substituted cycloalkyl
group, and the aryl group is a C6 to C22 mono- or polycyclic
aromatic group.
[0020] In one embodiment, the hydrophobic modulation comprises a
heterocyclic modulation, wherein the heterocyclic modulation
comprises an unsubstituted or substituted morpholine, pyrolidine,
pyrrole, piperidine, ethyleneimine, indole, isoindole, or
carbazole.
[0021] In one embodiment, the substituted amidinourea or salt
thereof comprises a hydrophilic modulation, wherein the hydrophilic
modulation can be, for example, imidazole, purine, aminoethanol, or
amino terminal polyethylene oxide.
[0022] In one embodiment, the modulated guanidine substituted
polymer or nanoparticle comprises a guanidine moiety on a plurality
of repeating units of a polymer, or on the surface of a
nanoparticle, the modulation comprises a substituted or
unsubstituted amidinocarbamate or salt thereof. Preferably, the
amidinocarbamate or salt thereof comprises a substituted or
unsubstituted alky carbamate, aryl carbamate, alkylaryl carbamate
or aryalkyl carbamante.
[0023] In one embodiment, the modulated guanidine substituted
polymer comprises a conjugated polymer. The conjugated polymer
comprises, for example, poly(phenyleneethynylene),
poly(phenylenevinylene), poly(phenylene), poly(fluoreine),
polythiophene, or any p-electron conjugated polymer. Preferably,
the conjugated polymer comprises a polymer chain/structure
according to the subject invention.
[0024] The polymer of the modulated guanidine substituted polymer
can be a natural or synthetic polymer.
[0025] In one embodiment, the nanoparticle comprises silica,
alumina, titania, zinc oxide, tin oxide, silver oxide, cuprous
oxide, cupric oxide, ceria, vanadium oxide zirconia, molybdenum,
tungsten oxide, barium oxide, calcium oxide, iron oxide, or nickel
oxide.
[0026] In one embodiment, the subject invention provides a method
of preparing a modulated guanidine substituted polymer or
nanoparticle, the method comprising:
[0027] providing a N-Boc protected guanidine substituted polymer or
N-Boc protected guanidine substituted nanoparticle and a solvent to
form a solution or suspension; adding an amine or an alcohol to the
solution or suspension to make a reaction solution or
suspension;
[0028] heating the solution or suspension to a temperature of at
least 80.degree. C.; and isolating the modulated guanidine
substituted conjugated polymer or nanoparticle or a suspension or
solution thereof.
[0029] In one embodiment, the subject invention provides methods
for treating cancer using the modulated guanidine substituted
polymer or nanoparticle according to the subject invention. The
cancer treatment, comprising:
[0030] providing a modulated guanidine substituted polymer or
nanoparticle of the subject invention;
[0031] combining the modulated guanidine substituted polymer or
nanoparticle with a vehicle and, optionally, adjuvants to deliver
the modulated guanidine substituted conjugated polymer or
nanoparticle to form a therapeutic formulation;
[0032] delivering the therapeutic formulation to a cancer
patient.
BRIEF DESCRIPTION OF DRAWINGS
[0033] FIG. 1 shows a schematic comparison of flexible tyrosine
aminotransferase (TAT) synthetic mimics vs the backbone rigidity of
the guanidine comprising conjugated polymer, (G-CP).
[0034] FIG. 2A shows a scanning ion-conductance microscopy (SICM)
image of HeLa cells treated with G-CP.
[0035] FIG. 2B shows a magnified image where the pore formation on
the surface of the HeLa cells has been induced by the G-CP.
[0036] FIG. 2C shows a SICM topographic image of the HeLa cells
treated with G-CP and labeled with m-Cherry protein.
[0037] FIG. 2D shows a SICM potential map of the same area as FIG.
2C showing the presence of numerous pores.
[0038] FIG. 2E shows a confocal microscopic image of HeLa cells
incubated with fluorescing G-CP for 10 min supporting fast cellular
entry of G-CP.
[0039] FIG. 3 shows a scheme for the modulation of the chemical
environment at the guanidine group by amidinourea formation,
according to an embodiment of the invention.
[0040] FIG. 4A show a fluorescent microscopic images of HeLa cells
incubated with modulated guanidine complex for 1 h where the
modulating amine was aminoethoxyethanol to form a hydrophilic G-CP,
Poly-2, according to an embodiment of the invention, with CP shown
in the left panel and siGLO on the right panel.
[0041] FIG. 4B show a fluorescent microscopic images of HeLa cells
incubated with guanidine complex for 1 h to form a G-CP, Poly-2,
with CP shown in the left panel and siGLO on the right panel.
[0042] FIG. 4C show a fluorescent microscopic images of HeLa cells
incubated with modulated guanidine complex for 1 h where the
modulating amine was morpholine to form a slightly hydrophobic
G-CP, according to an embodiment of the invention, with CP shown in
the left panel and siGLO on the right panel.
[0043] FIG. 4D show a fluorescent microscopic images of HeLa cells
incubated with modulated guanidine complex for 1 h where the
modulating amine was diisopropylamine to form a hydrophobic G-CP,
according to an embodiment of the invention, with CP shown in the
left panel and siGLO on the right panel.
[0044] FIG. 5 is a reaction scheme for the preparation of Poly-1
and Poly-2 of a G-CP, according to an embodiment of the
invention.
[0045] FIG. 6A shows UV absorbance (left) and emission (right)
spectra of Poly-1 in DMSO and 95% water/5% DMSO.
[0046] FIG. 6B shows UV absorbance (left) and emission (right)
spectra of Poly-2 in DMSO and 95% water/5% DMSO.
[0047] FIG. 7A shows a nanoparticle tracking analysis (NTA) of
Poly-1.
[0048] FIG. 7B shows a NTA of Poly-2.
[0049] FIG. 8A shows a plot of zeta potential for Poly-1 with
siRNA.
[0050] FIG. 8B shows a plot of zeta potential for Poly-2 with
siRNA.
[0051] FIG. 9 shows a bar chart for Cell viability inhibition by
Poly-1 and Poly-2.
[0052] FIG. 10 shows a polyacrylamide gel electrophoresis
retardation assay, top, where Lane 1: siRNA (400 nM); Lane 2: siRNA
(400 nM)+Poly1 (5 .mu.M); Lane 3: siRNA (400 nM)+Poly 1 (10 .mu.M);
Lane 4: siRNA (400 nM)+Poly 1 (20 .mu.M); Lane 5: siRNA (400
nM)+Poly 1 (40 .mu.M); Lane 6: siRNA (400 nM)+Poly 1 (5 .mu.M);
Lane 7: siRNA (400 nM)+Poly 1 (10 .mu.M); Lane 8: siRNA (400
nM)+Poly 2 (20 .mu.M); and Lane 9: siRNA (400 nM)+Poly 2 (40
.mu.M), where significance at p<0.05.
[0053] FIG. 11A shows a confocal microscopic image of BEAS-2B cells
incubated with CP/siGLO polyplexes for 1 h show CP-mediated siRNA
delivery for Poly-1 located in the cytosol having a green signal
from the CP.
[0054] FIG. 11B shows a confocal microscopic image of BEAS-2B cells
incubated with CP/siGLO polyplexes of Poly-1 for 1 h where a red
signals from siGLO is observed.
[0055] FIG. 11C shows a combined signal from the CP and siGLO in
from cells treated with Poly-1 in a confocal microscopic image of
BEAS-2B cells incubated with CP/siGLO polyplexes of Poly-1 for 1
h.
[0056] FIG. 11D shows a confocal microscopic image of BEAS-2B cells
incubated with CP/siGLO polyplexes for 1 h show CP-mediated siRNA
delivery for Poly-2 located in the cytosol having a green signal
from the CP.
[0057] FIG. 11E shows a confocal microscopic image of BEAS-2B cells
incubated with CP/siGLO polyplexes of Poly-1 for 1 h where a red
signals from siGLO is observed.
[0058] FIG. 11F shows a combined signal from the CP and siGLO in
from cells treated with Poly-2 in a confocal microscopic image of
BEAS-2B cells incubated with CP/siGLO polyplexes of Poly-1 for 1 h
where a portion of siGLO were released from Poly 2 and localized in
the nucleus.
[0059] FIG. 12 shows a plot of polarization upon addition of a CFTR
activator (albuterol to block the epithelial sodium channel)
followed by an inhibitor (CFTR.sub.inh172) to NHBE cells mounted in
a Ussing chamber where a large apical current change with the
cultured NHBE cells exhibiting the characteristic membrane
polarization of epithelium.
[0060] FIG. 13 is a bar chart of the HDAC mRNA knockdown efficiency
in BEAS-2B cells for Lipofectamine, Poly-1 and Poly-2.
[0061] FIG. 14 is a bar chart of the relative knockdown efficiency
of BEAS-2B cell treated with Poly-2/siHDAC, where Poly-A is
Poly-2.
[0062] FIG. 15 is a bar chart of the relative HDAC mRNA expression
level at primary NHBE cell treated with Poly2/siHDAC, where Poly-A
is Poly-2.
[0063] FIG. 16 is a bar chart of the relative HDAC mRNA expression
levels of NHBE cells treated with Lipofectamine 2000 (a
lipid-based), Poly 1 (cationic), and Poly 2 (with a modulated
chemical environment at its positive charge), where *p<0.0003
and **p<0.007.
[0064] FIG. 17 shows a scheme for the various synthetic methods of
formation of modulated guanidine functionalized nanoparticles or
polymers, according to embodiments of the invention.
[0065] FIG. 18 shows a reaction scheme for the preparation of a
G-CP.
[0066] FIG. 19 shows a reaction scheme for the preparation of a
modulated G-CP, according to an embodiment of the invention.
[0067] FIG. 20 shows the flow cytometric analysis of Hela cells
treated with polymers (10 .mu.M)/Enhanced Green Fluorescent Protein
(EGFP) (60 nM) complexes overnight at 37.degree. C. in serum
containing medium. Values represent the mean relative MFI as
compared to untreated cells.+-.CV for two independent
experiments.
DETAILED DISCLOSURE
[0068] The subject invention provides materials and methods for
disrupting the mucus layer and intracellularly delivering
therapeutic agents such as drugs, nucleic acids, peptides, and
proteins. The subject invention also provides methods for the
design and synthesis of polymeric systems and nanomaterials that
enhance or assist the passage of therapeutic agents across
biological membranes.
[0069] In one embodiment, the subject invention provides polymeric
systems comprising cell-penetrating peptide (CPP)-like moieties for
transporting therapeutic agents and/or biological molecules across
biological membranes. The polymeric systems can be used as
molecular transporters that facilitate the internalization of
therapeutic agents and/or biological molecules by cells.
Advantageously, the properties of the polymeric systems can be
tuned by modulating the chemistry and architecture of the
materials. Specifically, by retaining only the key features of CPPs
necessary for sufficient internalization and delivery of the cargo,
CPP synthetic mimics (CPPMs) have improved properties compared to
naturally-occurring CPPs.
[0070] In one embodiment, the subject invention provides molecular
transporters for intracellularly delivering therapeutic agents such
as drugs, nucleic acids, peptides and proteins.
[0071] In one embodiment, the molecular transporter comprises a
modulated guanidine substituted polymer or nanoparticle, the
modulated guanidine substituted polymer or nanoparticle comprising
a guanidine moiety on a plurality of repeating units of a polymer,
or on the surface of a nanoparticle, the modulation comprising a
substituted amidinourea or amidinocarbamate or salt thereof.
Preferably, the modulation comprises the substituted amidinourea or
salt thereof.
[0072] In one embodiment, the molecular transporters of the subject
invention can be synthesized using ring-opening metathesis
polymerization (ROMP). ROMP has great benefits over other
polymerization techniques, which include controlled polymer length,
low polydispersity index (PDI) and easy copolymer design.
[0073] In one embodiment, the polymer is a conjugated polymer (CP).
Biodegradable CPs can be formed by introducing flexible degradable
functional groups along the backbone of the CP that can be used for
quantitative labeling of mitochondria. Cellular interaction and
internalization of CPs are dependent on the chemical structures of
both the backbone and side chains of the CPs. CPs with guanidine
units (G-CPs), as disclosed in Moon et al. U.S. Pat. Nos. 9,676,886
and 9,757,410, and incorporated herein by reference, that have
molecular weights of .about.14,000 g/mol, enter live cells quickly
through the cancer cell membrane. After generating positive charges
on guanidine, the resulting polymers are soluble in DMSO, and form
nanoparticles in PBS buffer with a hydrodynamic diameter of about
56 nm. Live cells treated with CPs containing various functional
groups display surface morphologies at the submicron level that are
dependent on the chemical functionalities of the CPs.
[0074] As can be seen in FIGS. 2A and 2B, the scanning ion
conductance microscopy images indicate pores on the cells surface.
The potential at the pores are lower than for the rest of the area,
suggesting formation of pore-like features on the membrane (FIGS.
2C and 2D). Based on the different potentials at the topographic
pores, different stages of cellular entry of CP are suggested (FIG.
2E). Confocal microscopic imaging also supports fast and efficient
cellular entry of G-CP. Within 10 min, a significant amount of G-CP
was found in the intracellular compartments.
[0075] Ideal small interfering RNA (siRNA) delivery requires RNA
protection, excellent pharmacokinetics, targeting, cellular entry,
and release of siRNA in the cytosol of target cells. The
biophysical properties of positively charged carriers complexed
with negatively charged siRNA can be tailored by introducing
functional groups at the positive charge. Positively charged
carriers often exhibit toxic effects and promote high blood
clearance through opsonization, limiting clinical applications.
[0076] According to one embodiment of the invention, on-ionic
group, including, but not limited to, hydrophobic lipids and
hydrophilic PEGs, at the positive charge improve carriers'
biophysical properties, provided that the modification does not
diminish the ionic complexation and cellular entry. Unlike
modulated guanidine on TAT peptides, which decreases entry
efficiency, G-CPs modulated with various functional groups exhibit
enhanced biophysical properties.
[0077] Modulated G-CPs, according to one embodiment of the
invention, as shown in FIG. 3, have an aminoethoxyethanol groups at
the positive charge of the guanidines to increase the efficiency of
cellular entry and siRNA delivery, as illustrated in FIG. 4A vs
FIG. 4B for guanidines, while exhibiting no viability inhibition.
Addition of a morpholine group at the charged guanidine changed the
cellular entry behaviors dramatically, as shown in FIG. 4C, as
indicated by the diffused uniform staining of the cytosol. Addition
of a bulky diisopropylamine group at the charged guanidine appears
to diminish the cellular entry behavior, as shown in FIG. 4D.
[0078] Modulating the charged group with specialized functional
groups, such as, but not limited to, tumor cell surface targeting
ligands, PEG, or drugs, can offer tailored cellular targeting and
entry to achieve optimized therapeutic efficacy. Because the drug
and gene knockdown efficacy is highly unique on each tumor due to
the unique tumor microenvironment, no generalized carrier will work
equally on each tumor. Modulation, as indicated above, allows
facile optimization for a tumor because of the straightforward
chemical modulation chemistry that can be carried out. Any
commercially available amine or alcohol, which can be readily
transformed to an amine, or alcohol can be directly coupled to
guanidine via the Mitsnobu reaction, can be added to the positively
charged guanidine group. Using this synthetic approach to modulate
G-CPs, a library of potential agents for screening and ultimate use
against specific tumor types can be tailored for cellular entry and
efficacy. Because the conventional and frequently used tumor cell
lines have significantly different cellular features from patient
tumors, drug delivery systems developed and optimized using the
conventional cell lines can have large discrepancies in therapeutic
efficacy when drugs are administered to patients.
[0079] Modulating the chemical environments at the positively
charged guanidine functional group of the CP is used to optimize
siRNA delivery. Embodiments of the invention are directed to a
method to form CPs where there is introduced various functional
groups of hydrophilic or hydrophobic molecules at the guanidine
group, as can be seen in FIG. 3 of such CPs. The modulated CPs
exhibit enhanced cellular entry, modulated intracellular
localization, and better siRNA delivery. The modulated CPs promote:
enhanced siRNA delivery by G-CP modulated with functional groups
including short ethylene glycol (EG); efficient knockdown of MDR
associated genes; and increased drug potency over cancer cells. The
fine-tuning allows better protection, cellular entry, and release
of siRNA. Less siRNA can be used for controlling the gene
expression levels.
[0080] The conjugated polymers (CPs), according to embodiments of
the invention, are macromolecules with highly delocalized
.pi.-conjugated backbones and amphiphilic side chains. CPs display
large absorption extinction coefficients, amplified quenching, high
quantum yields, and tunable absorption and emission maxima.
Guanidine is a part of the side chain of arginine and remains
charged over a wide pH range, which is reflected in the high pKa
value (12.48) of its protonated counterpart. There are a great
number of guanidine moieties peptides available due to their ease
of modification and straight-forward synthetic strategy. Guanidinum
groups provide the CPs with cationic properties and act as mimics
of cell-penetrating peptides (CPPs), molecular recognition, and
antimicrobial agent.
[0081] Chemical modulation at the positive charge of a
guanidinium-containing modulated CP is shown to efficiently
knockdown a target gene of well-differentiated primary human
bronchial epithelium cells, which closely mimic many in vivo
phenotypes of airway epithelium including: regulation of ion
transport; mucous secretion; and mucociliary clearance. Not to be
bound by a mechanism, the positive charges needed for ionic
complexation of siRNA appear to increase adsorption to a mucus
layer, resulting in decreased transfection efficiency and higher
blood clearance by absorbing various serum proteins. In embodiments
of the invention, amidinourea formation introduces hydrophilic
PEG-like functional groups at the positive charge by reacting
Boc-protected guanylurea to address these shortcomings.
[0082] Hydrophilic PEG-like functional groups are introduced at the
positive charge by reacting Boc-protected guanidine with
aminoethoxyethanol followed by Boc deprotection as shown in FIG. 5.
According to an embodiment of the invention, Poly-2 of FIG. 5 is
successfully synthesized in high yields, with formation of a
guanylurea group in every repeating unit, as characterized by an
amide proton signal in .sup.1H-NMR spectra at about 12.3 ppm.
Poly-2 exhibits good solubility in common organic solvents,
characteristic absorption/emission profiles of CPs, as shown in
FIGS. 6A and 6B, and has about a three-fold greater fluorescent
quantum yield than Poly-1. Poly-1 and Poly-2 exhibit very weak
dynamic light scattering signals even at concentrations in excess
of the mM levels, implying that the non-aqueous soluble CPs are
relatively well-solvated due to the highly charged guanidine and
guanylurea, preventing hydrophobic backbone aggregation. Upon
complexation of the CPs with the negatively charged siRNA,
nanometer-sized polymer/siRNA polyplexes are formed. The
hydrodynamic diameters (HDs) of Poly-1 and 2 are 137.+-.40 and
152.+-.44 nm, respectively, as shown in FIGS. 7A and 7B. Zeta
potentials of both polyplexes are slightly positively charged,
about+13 mV, as shown in FIGS. 8A and 8B.
[0083] No cell viability inhibition was exhibited at up to 40 .mu.M
concentration of either Poly-1 or Poly-2, demonstrating that
supporting the modulation at guanidine does not raise viability
inhibition. As indicated in FIG. 9, the siRNA against histone
deacethylase (HDAC) (siHDAC) was delivered by both Poly-1 and
Poly-2 to BEAS-2B cell lines, where mRNA expression levels are
quantified by RT-qPCR using glyceraldehye 3-phosphate dehydrogenase
(GAPDH) as a control gene. By Gel retardation assay, as shown in
FIG. 10, the guanylurea-functionalized Poly-2 exhibits much better
siRNA complexation than the guanidine-containing Poly-1. The entire
siRNA is complexed by Poly-2 at an N (nitrogen)/P (phosphate) ratio
of about 5, whereas Poly-1 showed only complexes about 20% at that
ratio.
[0084] Using a fluorescently labeled control siRNA (i.e., siGLO
Red), CP-mediated siRNA delivery for Poly-1 and Poly-2 is confirmed
by confocal microscopy, as shown in FIGS. 11A, 11B, and 11C and
FIGS. 11D, 11E, and 11F, respectively. After an hour of incubation
at the N/P ratio of about 9, the CPs and siGLO reside inside
BEAS-2B cells. The relatively high amount of siGLO observed in
cells incubated with Poly-2, indicates that guanylurea modification
increases siRNA delivery efficiency. The strong siGLO complexation
by Poly-2, results in higher amounts of intracellular siGLO due to
enhanced cellular entry of the complex resulting from a balance of
hydrophobicity and charge density. When the positive charge of
guanidine is balanced with hydrophobic moieties, polymers with many
guanidine-containing carriers exhibit efficient membrane
interaction followed by high intracellular entry. A portion of
siGLO is found in the nuclei of cells treated with Poly-2/siGLO, as
indicated in FIGS. 11E and 11F;
[0085] indicating siGLO is released from Poly-2. From this result,
it appears that hydrophilic modification at the positive charge of
CPs allows better siRNA complexation, efficient cellular entry, and
subsequent intracellular release of siRNA.
[0086] The guanylurea-functionalized CP for delivery of siRNA,
according to an embodiment of the invention, was evaluated in a
physiological setting, where ex vivo primary bronchial epithelial
cells were incubated with Poly-2/siGLO polyplex. Primary bronchial
epithelial cells obtained from nasal turbinates or cadaver lungs
were grown in plastic dishes or on porous supports at the
air-liquid interface. While the cells grown on plastic dishes
present a poorly differentiated squamous phenotype, the cells grown
on porous supports at the air-liquid interface closely recapitulate
their normal in vivo morphology, including: the cell-matrix and
cell-cell interactions; differentiation of mucus, goblet, and
ciliary cells; polarized epithelial ion transport;
[0087] and regenerating the native bronchial epithelium ex vivo.
Therefore, ex vivo primary human bronchial epithelial cells are an
excellent model of the constituted airway epithelial and are used
for ex vivo drug delivery studies before extrapolating to large
animal models or human clinical studies.
[0088] Conformation of the polarity and integrity of the epithelium
carried out by trans-epithelial electrical resistance (TEER)
measured after 21 days of differentiation at the air-liquid
interface, where primary NHBE cells exhibit a mean TEER value of
731 ohms/cm.sup.2, indicating efficient barrier formation. The
apical chloride ion flux, monitored by treatment with albuterol
activates the cystic fibrosis transmembrane conductance regulator
(CFTR) protein to stimulate chloride ion flux. As shown in FIG. 12,
a sharp current increase occurs immediately upon addition of
albuterol. The specificity of CFTR-mediated efflux is indicated by
the decreased current after addition of a CFTR inhibitor (i.e.,
CFIR.sub.inh172).
[0089] Confocal microscopic images clearly indicate that Poly-2
delivers siGLO to NHBE cells, while cells treated with Poly-1/siGLO
and Lipofectamine/siGLO, respectively, exhibited only background
signals. The added hydrophilic groups near the positive charges
promote diffusion of the ionic complex through the mucus layer
followed by efficient intracellular entry.
[0090] The gene knockdown efficiency of Poly-2 was initially
evaluated at the in vitro level using siRNA against histone
deacethylase (HDAC) (siHDAC) in BEAS-2B cells. The mRNA expression
levels were quantified by real time quantitative polymerase chain
reaction (RT-qPCR) using glyceraldehye 3-phosphate dehydrogenase
(GAPDH) as a control gene. Inhibition of HDACs has been shown to
suppress proliferation of non-small-cell lung cancer (NSCLC) and
restore the drug sensitivity to NSCLC. The guanidine-modified
Poly-1 exhibits relatively poor knockdown efficiency even in
immortalized cell lines. Poly-2 and Lipofectamine exhibit a
dose-dependent target gene knockdown, supporting that the
guanylurea modification enhances RNAi efficiency, as indicated in
FIG. 13. As FIG. 14 indicates, Poly-2 exhibit sufficient knockdown
of the target gene at the mRNA level and is comparable to
Lipofectamine. The HDAC mRNA expression level at primary NHBE cell
treated with Poly-2/siHDAC indicates knockdown, as shown in FIG.
15, whereas Lipofectamine does not knockdown the target gene.
[0091] Both lipid-based and purely positively charged carriers,
such as Poly-1, exhibit no or poor HDAC knockdown efficiency in
well-differentiated NHBE cells, as shown in FIG. 16, due to poor
cellular siRNA delivery to the epithelium cells. Meanwhile, Poly-2
consistently exhibits the average of about 30% knockdown efficiency
over six independent lung samples. Due to the negatively charged
hydrophobic mucus layers, positively charged Poly-1 and lipid-based
carriers experience difficulty in diffusing through the mucus
layer. The hydrophilic environment introduced at the positive
charge of guanidine allows efficient ionic complexation and
diffusion through the mucus layer.
[0092] Chemical modification of the guanidine group often destroys
the function available for guanidine, where nitrogen atoms of
guanidines, bearing electron-withdrawing substituents, act as a
reactive nucleophile. Few methods for guanidine modification are
known and they include the reaction between guanidine and alcohols
under Mitsunobu reaction condition and alkylation of guanidine with
electrophiles, such as alkyl halides, under basic conditions.
Recently, Kessler et al. Angew Chem Int Ed Engl., 2016,
55(4):1540-3 taught the modification of the guanidine group of
Cilengitide ligand by N-methylation, N-alkylation, or N-acylation
and successfully demonstrated an increasing selectivity of
Cilengitide ligands. Takemoto et al. J. Org. Chem., 2009, 74 (1),
pp 305-11 taught the use of palladium- or iridium-catalysts and
displayed a direct modification of guanidines. The direct
modification of guanidine head group received much less attention
and there are no reports of modification of guanidine moiety in
polymer.
[0093] According to an embodiment of the invention, a catalyst free
post polymerization reaction incorporates a variety of hydrophilic
and hydrophobic functional groups onto conjugated polymers (CPs).
By this method, structurally diverse polymers are synthesized
without tedious polymerization steps. The modified polymers are
easily analyzed using NMR spectroscopy and are prepared with high
yields overall. The guanidine head group reacts with
diisopropylamine (DIPA), which yields a CP with solubility and
physical properties that differ from the CP with the guanidine head
group. Incorporation of hydrophobic groups, like piperidine, and
hydrophilic group, like morpholine and aminoethoxyethanol, provide
other CPS with varied properties.
[0094] In embodiments of the invention, the polymer need not be a
conjugated polymer, which is generally ridged, but can be a
non-conjugated polymer that has a flexible backbone. In embodiments
of the invention, the polymer can have flexible side chains that
enhance the water solubility of the polymer. In embodiments of the
invention, the guanidine or protected guanidine is sufficient to
impart water solubility. Synthetic and natural polymers that can be
employed can be, but are not limited to, amine functionalized
polymethacrylates and polyacrylates, branched and linear
polyehtyleneimines, polyamidoamine, amine functionalized
dendrimers, poly-L-lysine, chitosan, amine functionalized dextran,
amine functionalized alginates, amine functionalized heparin, and
amine functionalized oligo or polysaccharide.
[0095] In embodiments of the invention, nanoparticles are used for
efficient intracellular delivery and labeling after modulating
surface properties to enhance their initial interaction following
entry. As shown in FIG. 17, a nanoparticle bearing a surface
functional group or a polymer with a functional group can be
converted by reaction at the functional group to a guanidine,
Boc-protected guanidine, or any other protected guanidine. The
nanoparticles can be those that are metal oxides, metal carbides,
metal nitrides, metals, diamond, or any other type of nanoparticle.
The polymer can be in the form of a soluble polymer or can be a
nanoparticle where the functional groups are of sufficient
concentration at the nanoparticle surface to yield a nanoparticle
that is decorated with the guanidine, Boc-protected guanidine, or
any other protected guanidine. The nanoparticles can be of a single
structure or a core-shell particle. The particles can inherently
have surface functionality that react with guanidine or protected
guanidine. The particle surface can be functionalized by reaction
with an agent, for example, a silane coupling agent, such as, but
not limited to, 3-aminopropyltrimethoxy silane, or thiol or
disulfide containing alkyls with hydroxyl or amine groups for
functionalization of metal nanoparticles.
[0096] In one embodiment, the polymeric system according to the
subject invention comprises a modulated guanidine substituted
polymer, the modulated guanidine substituted polymer comprising a
guanidine moiety on a plurality of repeating units of a polymer.
The modulation comprises a substituted amidinourea or
amidinocarbamate or salt thereof.
[0097] In one embodiment, the polymeric system is a guanylurea
functionalized molecular transporter, the guanylurea functionalized
molecular transporter comprises a modulated guanidine substituted
polymer, the modulated guanidine substituted polymer comprising a
guanidine moiety on a plurality of repeating units of a polymer.
The modulation comprises a substituted amidinourea or
amidinocarbamate or salt thereof.
[0098] Advantageously, amidinoureas, also called guanylureas, are
moieties with extended hydrogen bonding capacity as compared to
guanidine. The interaction between the polymeric transporter system
and cell membrane is enhanced by a polyvalent effect. The
replacement of guanidine with guanylurea moieties in the polymeric
system improves the binding of molecular transporters to the cell
membrane and the delivery of cargo.
[0099] In one embodiment, the amidinocarbamate or salt thereof
comprises a substituted or unsubstituted alky carbamate, aryl
carbamate, alkylaryl carbamate or aryalkyl carbamante
[0100] In one embodiment, the modulated guanidine substituted
polymer comprises a conjugated polymer. In specific embodiments,
the conjugated polymer comprises poly(phenyleneethynylene),
poly(phenylenevinylene), poly(phenylene), poly(fluoreine),
polythiophene, or any p-electron conjugated polymers.
[0101] In one embodiment, the guanylurea functionalized molecular
transporter comprises a modulated guanidine substituted polymer,
the modulated guanidine substituted polymer comprising a polymer
chain that comprises one or more types of constituent units or
repeating units.
[0102] Preferably, the repeating unit or monomer comprises or has
the following structure:
##STR00001##
[0103] wherein L is a linker and can be null; and R is selected
from, for example, hydrogen, alkyl, substituted alkyl, aryl,
substituted aryl, heteroalkyl, substituted heteroalkyl, heteroaryl,
substituted heteroaryl, cycloalkyl, substituted cycloalkyl,
heterocycloalkyl, substituted heterocycloalkyl, cycloalkenyl,
substituted cycloalkenyl, alkenyl substituted alkenyl, alkynyl,
haloalkyl, acyl, amino, alkylamino, arylamino and
hydroxylalkyl.
[0104] Preferably, R is selected from N-alkylamino; N-arylamino;
N-(alkylaryl)amino; N-(aryalkyl)amino; N, N-dialkylamin; N,
N-diarylamino; N, N-di(alkylaryl)amino; N, N-di(aryalkylamino);
N-alkyl, N-arylamino; N-alkyl, N-(alkylaryl)amino; N-alkyl,
N-(arylalkyl)amino; N-aryl, N-(alkylaryl)amino; N-aryl,
N-(arylalkyl)amino; unsubstituted or substituted morpholine;
unsubstituted or substituted pyrolidine; unsubstituted or
substituted pyrrole; unsubstituted or substituted piperidine;
unsubstituted or substituted ethyleneimine; unsubstituted or
substituted indole; unsubstituted or substituted isoindole;
unsubstituted or substituted carbazole; imidazole or substituted
imidazole; purine or substituted purine; aminoethanol; amino
terminal polyethylene oxide, substituted or unsubstituted alky
carbamate, substituted or unsubstituted aryl carbamate, substituted
or unsubstituted alkylaryl carbamate and substituted or
unsubstituted aryalkyl carbamante. More preferably, R is selected
from hexylamine (HA), benzylamine (BA), and aminoethoxyethanol
(AEE).
[0105] In some embodiments, L is selected from alkyl, alkenyl,
alkynyl, aromatics, heteroalkyl, heteroaryl, cycloalkyl, and
heterocyclyl. Preferably, L is selected from C1-C10 alkyls, C2-C10
alkenyls, C2-C10 alkynyls, C3-C10 cycloalkyls, and C3-C10
heterocyclyls.
[0106] In one embodiment, the modulated guanidine substituted
polymer comprises a homopolymer, the homopolymer comprising the
following structure:
##STR00002##
[0107] wherein L is a linker and can be null; m'.gtoreq.2; and R is
selected from hydrogen, alkyl, substituted alkyl, aryl, substituted
aryl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted
heteroaryl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl,
substituted heterocycloalkyl, cycloalkenyl, and substituted
cycloalkenyl, alkenyl and substituted alkenyl, alkynyl, haloalkyl,
acyl, amino, alkylamino, arylamino and hydroxylalkyl. Preferably, R
is selected from N-alkylamino; N-arylamin; N-(alkylaryl)amino;
N-(aryalkyl)amino; N, N-dialkylamino; N, N-diarylamino; N,
N-di(alkylaryl)amino; N, N-di(aryalkylamino); N-alkyl, N-arylamino;
N-alkyl, N-(alkylaryl)amino; N-alkyl, N-(arylalkyl)amino; N-aryl,
N-(alkylaryl)amino; N-aryl, N-(arylalkyl)amino; unsubstituted or
substituted morpholine; unsubstituted or substituted pyrolidine;
unsubstituted or substituted pyrrole; unsubstituted or substituted
piperidine; unsubstituted or substituted ethyleneimine;
unsubstituted or substituted indole; unsubstituted or substituted
isoindole; unsubstituted or substituted carbazole; imidazole or
substituted imidazole; purine or substituted purine; aminoethanol;
amino terminal polyethylene oxide, substituted or unsubstituted
alky carbamate, substituted or unsubstituted aryl carbamate,
substituted or unsubstituted alkylaryl carbamate and substituted or
unsubstituted aryalkyl carbamante. More preferably, R is selected
from HA, BA and AEE.
[0108] In one embodiment, the modulated guanidine substituted
polymer comprises a homopolymer having a structure of
##STR00003##
wherein m'.gtoreq.2; and R is selected from HA, BA and AEE.
[0109] In specific embodiments, the modulated guanidine substituted
polymer comprises a homopolymer selected from
##STR00004##
wherein m'.gtoreq.2.
[0110] In one embodiment, the modulated guanidine substituted
polymer is a copolymer comprises a polymer chain that comprises two
or more types of constituent units or repeating units. In a further
embodiment, the copolymer may be a bipolymer that is obtained by
copolymerization of two monomer species, terpolymer that is
obtained by copolymerization of three monomer species, or
quaterpolymer that is obtained by copolymerization of four monomer
species.
[0111] In one embodiment, the modulated guanidine substituted
polymer of the subject invention further comprises one or more
repeating units or monomer species selected from
##STR00005##
[0112] In one embodiment, the copolymer is an alternating
copolymer, periodic copolymer, random copolymer or block copolymer.
An alternating copolymer is a copolymer comprising two species of
monomeric units distributed in alternating sequence, for example,
--ABABABAB-- or --(AB)n--. A random copolymer is a copolymer
comprising two or more types of monomer species with each monomer
residue located randomly in the polymer molecule, for example,
--AAAABBBABBA--, or --AAAABCBBCBACCBC--. A periodic copolymer a
copolymer comprising two or more types of monomer species and has
units arranged in a repeating sequence, for example,
--(ABABBAAAABBB)n--, or --(AABCBAABBBCCAB)n--. A block copolymer is
a copolymer comprising two or more blocks of different homopolymers
chemically attached to each other, e.g., by covalent bonds. For
example, a block copolymer having repeating units A and B may be
arranged as --AAAAABBBBB-- or --AAAAABBBBBAAAAA--.
[0113] In one embodiment, the modulated guanidine substituted
polymer is a random copolymer comprising a polymer chain selected
from
##STR00006## ##STR00007##
[0114] wherein
##STR00008##
means that the two monomer species are randomly distributed; both m
and n.gtoreq.1; R and R' are independently selected from hydrogen,
alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, heteroalkyl, substituted heteroalkyl,
cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted
heterocycloalkyl, cycloalkenyl, and substituted cycloalkenyl,
alkenyl and substituted alkenyl, alkynyl, haloalkyl, acyl, amino,
alkylamino, arylamino and hydroxylalkyl; wherein m and n may be the
same or different, and R and R' are different.
[0115] Preferably, R and R' are independently selected from
N-alkylamino; N-arylamino; N-(alkylaryl)amino; N-(aryalkyl)amino;
N, N-dialkylamino; N, N-diarylamino; N, N-di(alkylaryl)amino; N,
N-di(aryalkylamino); N-alkyl,N-arylamino; N-alkyl,
N-(alkylaryl)amino; N-alkyl, N-(arylalkyl)amino; N-aryl,
N-(alkylaryl)amino; N-aryl, N-(arylalkyl)amino group; unsubstituted
or substituted morpholine; unsubstituted or substituted pyrolidine;
unsubstituted or substituted pyrrole; unsubstituted or substituted
piperidine; unsubstituted or substituted ethyleneimine;
unsubstituted or substituted indole; unsubstituted or substituted
isoindole; and unsubstituted or substituted carbazole; and more
preferably, R and R' are independently selected from HA, BA and
AEE.
[0116] In one embodiment, the modulated guanidine substituted
polymer is a block copolymer that induces nanostructure formation.
By introducing a hydrophobic block, as in the block copolymers
according to the subject invention, the hydrophobic segment may
"collapse" in aqueous environment and create a micelle type
nanostructure. The block copolymers of the subject invention were
synthesized with various guanylurea modifications on one or more of
the blocks.
[0117] Advantageously, by varying the local environment around the
positive charge in the polymers using both hydrophobic and
hydrophilic moieties, the polymers may be used to encapsulate
drugs/small molecules. Additionally, these copolymers may be used
to complex macromolecules such as proteins where the block
structure may enhance the nanoparticle formation.
[0118] In one embodiment, the modulated guanidine substituted
polymer is a block copolymer comprising a polymer chain that
comprises one or more blocks of homopolymer,
##STR00009##
wherein m'.gtoreq.2; L is a linker and can be null; and the polymer
chain further comprises one or more blocks of homopolymer
comprising monomer
[0119] species selected from
##STR00010##
[0120] In one embodiment, the modulated guanidine substituted
polymer is a block copolymer comprising a polymer chain that
comprises one or more blocks of polymer comprising a plurality
[0121] of monomers
##STR00011##
and the polymer chain further comprises one or more blocks of
polymer comprising a plurality of monomer species selected from
##STR00012##
[0122] In one embodiment, the modulated guanidine substituted
polymer is a block copolymer comprising a polymer chain of
##STR00013##
[0123] wherein m and n can be the same or different, and both m and
n.gtoreq.1; and R is selected from hydrogen, alkyl, substituted
alkyl, aryl, substituted aryl, heteroalkyl, substituted
heteroalkyl, heteroaryl, substituted heteroaryl, cycloalkyl,
substituted cycloalkyl, heterocycloalkyl, substituted
heterocycloalkyl, cycloalkenyl, and substituted cycloalkenyl,
alkenyl and substituted alkenyl, alkynyl, haloalkyl, acyl, amino,
alkylamino, arylamino and hydroxylalkyl. Preferably, R is selected
from N-alkylamino; N-arylamino; N-(alkylaryl)amino;
N-(aryalkyl)amino; N, N-dialkylamino; N, N-diarylamino; N,
N-di(alkylaryl)amino; N, N-di(aryalkylamino); N-alkyl, N-arylamino;
N-alkyl, N-(alkylaryl)amino; N-alkyl, N-(arylalkyl)amino; N-aryl,
N-(alkylaryl)amino; N-aryl, N-(arylalkyl)amino; unsubstituted or
substituted morpholine; unsubstituted or substituted pyrolidine;
unsubstituted or substituted pyrrole; unsubstituted or substituted
piperidine; unsubstituted or substituted ethyleneimine;
unsubstituted or substituted indole; unsubstituted or substituted
isoindole; unsubstituted or substituted carbazole; imidazole or
substituted imidazole; purine or substituted purine; aminoethanol;
amino terminal polyethylene oxide, substituted or unsubstituted
alky carbamate, substituted or unsubstituted aryl carbamate,
substituted or unsubstituted alkylaryl carbamate and substituted or
unsubstituted aryalkyl carbamante. More preferably, R is selected
from HA, BA and AEE.
[0124] In a specific embodiment, the modulated guanidine
substituted polymer is a block copolymer comprising a polymer chain
selected from
##STR00014##
wherein m and n.gtoreq.1, and m and n can be the same or
different.
[0125] In one embodiment, the homopolymer and/or copolymer of the
subject invention comprises one or more boronic acid moieties.
Boronic acids have strong binding affinity to biomolecules
containing vicinal diols, such as sialic acid. Specifically,
boronic acids can target low abundance biomolecules in glucose rich
environments. Increased levels of sialic acid have been observed in
many cancer cell lines. Thus, such copolymer containing boronic
acid moieties may be used as a targeted therapy for cancer
treatments.
[0126] In one embodiment, the modulated guanidine substituted
polymer is a block copolymer comprising a polymer chain of
##STR00015##
wherein
##STR00016##
means that the two monomer species may be randomly distributed; m,
n and o can be the same or different, and m, n and o.gtoreq.1; and
R is selected from hydrogen, alkyl, substituted alkyl, aryl,
substituted aryl, heteroalkyl, substituted heteroalkyl, heteroaryl,
substituted heteroaryl, cycloalkyl, substituted cycloalkyl,
heterocycloalkyl, substituted heterocycloalkyl, cycloalkenyl, and
substituted cycloalkenyl, alkenyl and substituted alkenyl, alkynyl,
haloalkyl, acyl, amino, alkylamino, arylamino and hydroxylalkyl.
Preferably, R is selected from N-alkylamino; N-arylamino;
N-(alkylaryl)amino; N-(aryalkyl)amino; N N-dialkylamino; N,
N-diarylamino; N, N-di(alkylaryl)amino; N, N-di(aryalkylamino);
N-alkyl, N-arylamino; N-alkyl, N-(alkylaryl)amino N-alkyl,
N-(arylalkyl)amino; N-aryl, N-(alkylaryl)amino; N-aryl,
N-(arylalkyl)amino; unsubstituted or substituted morpholine;
unsubstituted or substituted pyrolidine; unsubstituted or
substituted pyrrole; unsubstituted or substituted piperidine;
unsubstituted or substituted ethyleneimine; unsubstituted or
substituted indole; unsubstituted or substituted isoindole;
unsubstituted or substituted carbazole; imidazole or substituted
imidazole; purine or substituted purine; aminoethanol; amino
terminal polyethylene oxide, substituted or unsubstituted alky
carbamate, substituted or unsubstituted aryl carbamate, substituted
or unsubstituted alkylaryl carbamate and substituted or
unsubstituted aryalkyl carbamante. More preferably, R is selected
from HA, BA and AEE. In a specific embodiment, the modulated
guanidine substituted polymer is a block copolymer comprising a
polymer chain selected from
##STR00017## ##STR00018##
[0127] wherein m, n and o.gtoreq.1, and m, n and o can be the same
or different;
##STR00019##
means that the two monomer species may be randomly distributed.
[0128] Specifically, ligand affinity and specificity can be tuned
by functionalizing boronic acid probes. Functionalization of
phenylboronic acid derivatives changes the pKa of the boronic acid
probes and the stability of tumor tissue in the acidic
microenvironment.
[0129] In one embodiment, the modulated guanidine substituted
polymer is a guanylurea boronic acid derivative that comprises a
boronic acid moiety on the modulated guanidine moiety of the
polymer according to the subject invention.
[0130] In a specific embodiment, the guanylurea boronic acid
derivative comprises a polymer chain of
##STR00020##
wherein m and n.gtoreq.1, and m and n can be the same or different;
and L is a linker and can be null. The linker may be alkyl or
heteroalkyl, preferably, a short (e.g., C1-C10) alkyl or
heteroalkyl.
[0131] In a specific embodiment, the guanylurea boronic acid
derivative comprises a polymer chain of
##STR00021##
wherein m and n.gtoreq.1, and m and n can be the same or
different.
[0132] In one embodiment, the subject invention provides a
nanomaterial that comprises a medulated guanidine substituted
polymer of the subject invention conjugated to the surface of a
nanoparticle. The nanoparticle comprises one or more selected from
silica, alumina, titania, zinc oxide, tin oxide, silver oxide,
cuprous oxide, cupric oxide, ceria, vanadium oxide zirconia,
molybdenum, tungsten oxide, barium oxide, calcium oxide, iron
oxide, and nickel oxide.
[0133] In one embodiment, the subject invention also provides a
therapeutic formulation comprising the modulated guanidine
substituted polymer or nanoparticle of the subject invention and a
pharmaceutically acceptable carrier, wherein the therapeutic
formulation further comprises one or more therapeutic agents,
wherein one or more therapeutic agents are encapsulated by the
modulated guanidine substituted polymer.
[0134] In one embodiment, the therapeutic formulation of the
subject invention comprises a mixture/complex of the modulated
guanidine substituted polymer or nanoparticle of the subject
invention and one or more therapeutic agents, wherein the modulated
guanidine substituted polymer or nanoparticle is mixed with the
therapeutic agent at a concentration ratio ranging, for example,
from about 1:1 to about 1000: 1, from about 1:1 to about 900: 1,
from about 1:1 to about 800: 1, from about 1:1 to about 700: 1,
from about 1:1 to about 600: 1, from about 1:1 to about 500: 1,
from about 1:1 to about 400: 1, from about 1:1 to about 300: 1,
from about 1:1 to about 200: 1, from about 1:1 to about 100: 1,
from about 1:1 to about 90: 1, from about 1:1 to about 80: 1, from
about 1:1 to about 70: 1, from about 1:1 to about 60: 1, from about
1:1 to about 50: 1, from about 1:1 to about 40: 1, from about 1:1
to about 30: 1, from about 1:1 to about 20: 1, or from about 1:1 to
about 10: 1.
[0135] "Pharmaceutically acceptable carrier" refers to a diluent,
adjuvant or excipient with which the one or more active agents
disclosed herein can be formulated. Typically, a "pharmaceutically
acceptable carrier" is a substance that is non-toxic, biologically
tolerable, and otherwise biologically suitable for administration
to a subject, such as an inert substance, added to a
pharmacological composition or otherwise used as a diluent,
adjuvant or excipient to facilitate administration of the
composition disclosed herein and that is compatible therewith.
[0136] Examples of carriers suitable for use in the pharmaceutical
compositions are known in the art and such embodiments are within
the purview of the invention. The pharmaceutically acceptable
carriers and excipients, including, but not limited to, aqueous
vehicles, water-miscible vehicles, non-aqueous vehicles,
stabilizers, solubility enhancers, isotonic agents, buffering
agents, suspending and dispersing agents, wetting or emulsifying
agents, complexing agents, sequestering or chelating agents,
cryoprotectants, lyoprotectants, thickening agents, pH adjusting
agents, and inert gases. Other suitable excipients or carriers
include, but are not limited to, dextran, glucose, maltose,
sorbitol, xylitol, fructose, sucrose, and trehalose.
[0137] In one embodiment, the subject invention further provides
methods for treating a cancer, the method comprising administering,
to a subject in need of such treatment, an effective amount of the
therapeutic formulation of the subject invention.
[0138] The term "subject" or "patient," as used herein, describes
an organism, including mammals such as primates. Mammalian species
that can benefit from the disclosed methods of treatment include,
but are not limited to, apes, chimpanzees, orangutans, humans, and
monkeys; domesticated animals such as dogs, cats; live stocks such
as horses, cattle, pigs, sheep, goats, and chickens; and other
animals such as mice, rats, guinea pigs, and hamsters.
[0139] The terms "treatment" or any grammatical variation thereof
(e.g., treat, treating, etc.), as used herein, includes but is not
limited to, the application or administration to a subject (or
application or administration to a cell or tissue from a subject)
with the purpose of delaying, slowing, stabilizing, curing,
healing, alleviating, relieving, altering, remedying, less
worsening, ameliorating, improving, or affecting the disease or
condition, the symptom of the disease or condition, or the risk of
(or susceptibility to) the disease or condition. The term
"treating" refers to any indication of success in the treatment or
amelioration of a pathology or condition, including any objective
or subjective parameter such as abatement; remission; lessening of
the rate of worsening; lessening severity of the disease;
stabilization, diminishing of symptoms or making the pathology or
condition more tolerable to the subject; or improving a subject's
physical or mental well-being. Treating can also include preventing
a condition or disorder, which, as used herein, means delaying the
onset of, or progression of, a particular sign or symptom of the
condition or disorder.
[0140] In one embodiment, the subject invention provides methods
for targeted delivery of a compound or molecule, including
therapeutic agents (e.g., drugs, antibodies, DNAs, RNAs such as
siRNAs and miRNAs, peptides and proteins), into cells, preferably,
cancer cells and epithelium cells, the method comprising contacting
the cells with the polymeric system or therapeutic formulation of
the subject invention.
[0141] In one embodiment, the subject invention provides methods
for targeted delivery of a therapeutic agent into cells,
preferably, cancer cells and epithelium cells, the method
comprising contacting the cells with the modulated guanidine
substituted polymer of the subject invention and the therapeutic
agent.
[0142] In one embodiment, the subject invention provides methods
for targeted delivery of a compound or molecule, including
therapeutic agents (e.g., drugs, antibodies, DNAs, RNAs such as
siRNAs and miRNAs, peptides and proteins), into the nuclei of
cells, preferably, cancer cells and epithelium cells, the method
comprising contacting the cells with the polymeric system or
therapeutic formulation of the subject invention.
[0143] In one embodiment, the subject invention provides methods
for targeted delivery of a therapeutic agent into the nuclei of
cells, preferably, cancer cells and epithelium cells, the method
comprising contacting the cells with the modulated guanidine
substituted polymer of the subject invention and the therapeutic
agent.
[0144] In one embodiment, the subject invention provides methods
for transporting a compound or molecule, including therapeutic
agents (e.g., drugs, antibodies, DNAs, RNAs such as siRNAs and
miRNAs, peptides and proteins), across a biological membrane, the
method comprising contacting the biological membrane with the
polymeric system or formulation of the subject invention. The
biological membrane may be, for example, cell membranes, organelle
membranes, mucous membranes, basement membranes, and serous
membranes.
[0145] In one embodiment, the subject invention provides methods
for transporting a therapeutic agent across a biological membrane,
the method comprising contacting the biological membrane with the
modulated guanidine substituted polymer of the subject invention
and the therapeutic agent.
[0146] In one embodiment, the subject invention further provides
methods for altering/modulating gene expression in a cell,
preferably, cancer cell and epithelium cell, the method comprising
contacting the cell with the polymeric system or therapeutic
formulation of the subject invention. Altering/modulating gene
expression in a cell includes inhibiting or promoting gene
expression in said cell.
[0147] In one embodiment, the subject invention further provides
methods for altering/modulating gene expression in a cell,
preferably, cancer cell and epithelium cell, the method comprising
contacting the cell with the modulated guanidine substituted
polymer of the subject invention and the therapeutic agent.
[0148] In one embodiment, the subject invention further provides
methods for inhibiting gene expression in a cell, preferably,
cancer cell and epithelium cell, the method comprising contacting
the cell with the polymeric system or therapeutic formulation of
the subject invention, wherein the polymeric system or therapeutic
formulation comprises one siRNA targeting the gene of interest.
[0149] As used herein, the singular forms "a," "an," and "the" are
intended to include the plural forms as well, unless the context
clearly indicates otherwise. Further, to the extent that the terms
"including," "includes," "having," "has," "with," or variants
thereof are used in either the detailed description and/or the
claims, such terms are intended to be inclusive in a manner similar
to the term "comprising." The transitional terms/phrases (and any
grammatical variations thereof), such as "comprising," "comprises,"
and "comprise," can be used interchangeably.
[0150] The transitional term "comprising," "comprises," or
"comprise" is inclusive or open-ended and does not exclude
additional, unrecited elements or method steps. By contrast, the
transitional phrase "consisting of" excludes any element, step, or
ingredient not specified in the claim. The phrases "consisting" or
"consists essentially of" indicate that the claim encompasses
embodiments containing the specified materials or steps and those
that do not materially affect the basic and novel characteristic(s)
of the claim. Use of the term "comprising" contemplates other
embodiments that "consist" or "consisting essentially of" the
recited component(s).0
[0151] The term "about" or "approximately" means within an
acceptable error range for the particular value as determined by
one of ordinary skill in the art, which will depend in part on how
the value is measured or determined, i.e., the limitations of the
measurement system. For example, "about" can mean within 1 or more
than 1 standard deviation, per the practice in the art.
Alternatively, "about" can mean a range of up to 0-20%, 0 to 10%, 0
to 5%, or up to 1% of a given value. Alternatively, particularly
with respect to biological systems or processes, the term can mean
within an order of magnitude, preferably within 5-fold, and more
preferably within 2-fold, of a value. Where particular values are
described in the application and claims, unless otherwise stated
the term "about" meaning within an acceptable error range for the
particular value should be assumed. In the context of compositions
containing amounts of concentrations of ingredients where the term
"about" is used, these values include a variation (error range) of
0-10% around the value (X.+-.10%).
[0152] As used herein, each m', m, n, and o is intended to include
.gtoreq.2, .gtoreq.3, .gtoreq.4, .gtoreq.5, .gtoreq.6, .gtoreq.7,
.gtoreq.8, .gtoreq.9, .gtoreq.10,
.gtoreq.11,.gtoreq.12,.gtoreq.13,.gtoreq.14,.gtoreq.15,.gtoreq.16,.gtoreq-
.17,.gtoreq.18,.gtoreq.19, .gtoreq.20, .gtoreq.21, .gtoreq.22,
.gtoreq.23, .gtoreq.24, .gtoreq.25, .gtoreq.26, .gtoreq.27,
.gtoreq.28,.gtoreq.29, .gtoreq.30, .gtoreq.31, .gtoreq.32,
.gtoreq.33, .gtoreq.34, .gtoreq.35, .gtoreq.36, .gtoreq.37,
.gtoreq.38, .gtoreq.39, .gtoreq.40, .gtoreq.41, .gtoreq.42,
.gtoreq.43, .gtoreq.44, .gtoreq.45, .gtoreq.46, .gtoreq.47,
.gtoreq.48, .gtoreq.49, .gtoreq.50, .gtoreq.51, .gtoreq.52,
.gtoreq.53, .gtoreq.54, .gtoreq.55, .gtoreq.56, .gtoreq.57,
.gtoreq.58, .gtoreq.59, .gtoreq.60, .gtoreq.61, .gtoreq.62,
.gtoreq.63, .gtoreq.64, .gtoreq.65, .gtoreq.66, .gtoreq.67,
.gtoreq.68, .gtoreq.69, .gtoreq.70, .gtoreq.71, .gtoreq.72,
.gtoreq.73, .gtoreq.74, .gtoreq.75, .gtoreq.76, .gtoreq.77,
.gtoreq.78, .gtoreq.79, .gtoreq.80, .gtoreq.81, .gtoreq.82,
.gtoreq.83, .gtoreq.84, .gtoreq.85, .gtoreq.86, .gtoreq.87,
.gtoreq.88, .gtoreq.89, .gtoreq.90, .gtoreq.91, .gtoreq.92,
.gtoreq.93, .gtoreq.94, .gtoreq.95, .gtoreq.96, .gtoreq.97,
.gtoreq.98, .gtoreq.99, and .gtoreq.100.
[0153] The recitation of a listing of chemical groups in any
definition of a variable herein includes definitions of that
variable as any single group or combination of listed groups. The
recitation of an embodiment for a variable or aspect herein
includes that embodiment as any single embodiment or in combination
with any other embodiments or portions thereof.
EXAMPLES
Methods and Materials
Materials
[0154] Reagents and solvents were purchased from Fisher Scientific
and used without further purification. Deuterated solvents were
purchased from Cambridge Isotope Laboratories (Cambridge, Mass.).
All solutions were prepared using deionized (DI) water
(.about.18M.OMEGA.) from water purification system (Ultra Purelab
system, ELGA/Siemens). The number average molecular weight
(M.sub.n), weight average molecular weight (M.sub.w), and
polydispersity index (PDI=Mw/Mn) of CPs were determined by gel
permeation chromatography (GPC) against polystyrene standards using
a Shimadzu high performance liquid chromatography (HPLC) system
fitted with PLgel 5 .mu.m MIXED-D columns and SPD-20A
ultraviolet-visible (UV-vis) detector at a flow rate of 1.0 mL/min.
Samples for GPC, small amount (.about.100 .mu.L) of polymer in
dimethylformamide (DMF) or dichloromethane (DCM) was diluted with 1
mL of HPLC grade THF and then filtered through a 0.45 .mu.M
polytetrafluoroethylene (PTFE) syringe filter prior injection.
UV-vis spectra were recorded using a Varian Cary 50 Bio
spectrophotometer. Fluorescence spectra were obtained using a
FluoroLog-3 Spectrofluorometer (Jobin Yvon/Horiba).
9,10-diphenylanthracene (QY=0.9) in cyclohexane was used as a
fluorescence standard. Fourier transform infrared (FTIR) spectra
were recorded on a PerkinElmer Spectrum 100 FTIR Spectrometer. Fine
polymer powders were directly mounted on an attenuated total
reflection (ATR) cell of the spectrometer. Nuclear magnetic
resonance (NMR) spectra were recorded on a 400 MHz Avance Bruker
NMR spectrometer. Chemical shifts were reported in parts per
million (ppm) for 1H NMR on the .delta. scale based on the middle
peak (.delta.=2.50 ppm) of the dimethylsulfoxide (DMSO-d6) solvent
as an internal standard.
Monomer Synthesis
[0155] Synthesis of monomer A. Guanidinium-containing aryldiiodide
monomer A was synthesized as described in Ahmed et al.,
Bioconjugate Chem. 2018, 29 (4), 1006-9. [0156] Synthesis of
monomer B. A round-bottomed flask was charged with compound A (2.00
g, 1.96 mmol), trimethylsilylacetylene (0.77 g, 7.84 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (137.6 mg, 0.20 mmol), and CuI (18.6
mg, 0.10 mmol). The round-bottomed flask was evacuated and filled
with N.sub.2 three times. A solution of tetrahydrofuran (THF) and
diisopropylamine (DIPA) was mixed in a 4:1 ratio (v/v) and degassed
with N.sub.2 for 10 minutes, and then 50 mL was transferred to the
ROUND-BOTTOMED FLASK via a cannula. The reaction mixture was
stirred at room temp for 3 h equipped with a N.sub.2 balloon. The
yellow reaction mixture was filtered to remove insoluble particles
and THF was distilled from the mixture in vacuo. The reaction
mixture was dissolved in DCM and washed with 1M NH.sub.4Cl two
times followed by brine. Column chromatography using 30% ethyl
acetate in hexane yielded a trimethylsilyl (TMS)-protected compound
as a white solid (1.22 g, 65% yield). .sup.1H NMR (400 MHz),
CDCl.sub.3, .delta.: 11.46 (s, 0.95H), 8.67 (s, 0.96H), 7.21 (s,
0.98H), 4.1 (t, J=4.4 Hz, 2H), 3.88 (t, J=4.8 Hz, 2.H), 3.78 (t,
J=4.8 Hz, 2H), 3.66 (q, J=5.2 Hz, 2H), 1.50 (s, 9.1H), 1.45 (s,
9.21H), 0.10 (s, 18.2H).
[0157] In a round-bottomed flask, the trimethylsilyl
(TMS)-protected compound (1.00 g, 1.04 mmol), and potassium
carbonate (0.36 g, 2.60 mmol) were mixed in methanol (40 mL). The
round-bottomed flask was then stirred at r.t. for 20 min. Upon
confirmation of TMS deprotection by TLC, the solvent was dried in
vacuo. The reaction mixture was then purified by short-path column
chromatography using 35% ethyl acetate in hexane, yielding a
yellowish solid (0.51 g, 60% yield). .sup.1H NMR (400 MHz),
CDCl.sub.3, .delta.: 11.45 (s, 1H), 8.67 (s, 1H), 6.98 (s, 1H),
4.15 (t, J=4.4 Hz, 2H), 3.86 (t, J=4.8 Hz, 2H), 3.75 (t, J=4.8 Hz,
2H), 3.65 (q, J=5.2 Hz, 2H), 3.35 (s, 1H), 1.50 (s, 9H), 1.45 (s,
9H). .sup.13C NMR (400 MHz), CDCl.sub.3, .delta.: 156.4, 154.2,
153.1, 118.3, 113.8, 83.2, 83.1, 79.6, 79.4, 70.0, 69.8, 69.4,
40.9, 28.4, 28.2. FT-IR (neat): 3330.9, 3281.4, 2975.3, 2930.5,
1720.2, 1636.1, 1613.1, 1568.2, 1495.8, 1410.3, 1319.7, 1222.7,
1129.0, 1049.9 cm.sup.-1. HRMS [M+H].sup.+=817.4342 (theoretical)
and 817.4365 (observed). [0158] Synthesis of monomer 5. The
Diels-Alder adduct 1 was synthesized according to previously
published procedures. In short, furan and maleic anhydride were
stirred in toluene at 80.degree. C. The product precipitated at
room temperature and was used without further purification with a
yield of 80%.
##STR00022##
[0159] Ethylenediamine was single side N-Boc protected by addition
of 0.1 eq of di-tert-buyl decarbonate dropwise over 24 h. Product
was purified by extraction with water (3.times.) and brine
(1.times.). Compound was obtained as a yellow oil at 83% yield.
##STR00023##
[0160] Compounds 3-4 were synthesized using modified literature
procedures. 1 was dissolved in methanol and 1.5 eq of each amine
and 1 eq of triethylamine were added. The reaction was complete
after 24 h. Compound 3 precipitated at room temperature and 4 was
purified by extraction and collected in DCM. Yields for 3 and 4
were 50% and 68% respectively.
##STR00024##
[0161] Compound 3 was deprotected in 1:1 mixture of dichloromethane
and TFA and precipitated into diethyl ether. The precipitate was
dried, dissolved in a 9:1 mixture of acetonitrile and water along
with 3 eq of TEA, 1.5 eq of N,N'-Di-Boc-1H-pyrazole-1-carboxamidine
and allowed to react overnight at room temperature. The reaction
mixture was diluted with DCM and extracted with water (3.times.).
Monomer 5 was purified by recrystallization using dichloromethane
and methanol system affording pure compound at 68% yield.
##STR00025##
Homo Polymer Synthesis
[0162] PN-G-R
[0163] Boc-protected guanidine polymers were synthesized by
dissolving monomer 5 in dry DCM and adding varying molar
equivalents of Grubbs' 3.sup.rd gen catalyst. The solutions were
stirred for 45 minutes before the addition of 1 mL of ethyl vinyl
ether to terminate the polymerization. The polymer solutions were
precipitated (3.times.) into diethyl ether and precipitates were
collected and dried.
##STR00026##
[0164] Boc-protected homopolymer was either deprotected (PN-G) or
modified to PN-G-R (R=BA, HA, AEE or other primary/secondary
amines) by reaction in THF at 75.degree. C. 1.5 eq of the
corresponding amine was added to the polymer solution and left
stirring for 24 hours. The resulting polymer was purified by
precipitation (3.times.) into diethyl ether. The resulting
precipitates were deprotected in a 1:1 DCM:TFA mixture.
##STR00027##
Guanidine Homo Polymer (Boc-protected Poly-1)
[0165] As indicated in the reaction scheme of FIG. 18, a Schlenk
flask was charged with guanidine substituted p-diiodiaromatic
monomer A 50 mg, 0.04 mmol, guanidine substituted
p-diacetylenodiaromatic comonomer B (40.0 mg, 0.04 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (3.43 mg, 0.005 mmol), and CuI (0.47
mg, 0.0024 mmol). The Schlenk flask was evacuated and filled with
N.sub.2. A solution of tetrahydrofuran (THF) and diisopropylamine
(DIPA) was mixed in 4:1 volume ratio and degassed with N.sub.2, and
2 mL was transferred to the Schlenk flask via a cannula. The
reaction mixture was stirred at RT for 16 h. The solution was
filtered through a glass wool filter and added dropwise to methanol
to precipitate the GCP. The supernatant was decanted; the
precipitate was re-dissolved in dichloromethane (0.5 mL) and
purification method was repeated using methanol. The resulting Boc
protected polymer in DCM was characterized by gel permeation
chromatography (GPC) and their absorption/emission profile was
measured. The final polymer was allowed to dry under high vacuum
for 16 h before .sup.1H NMR characterization. .sup.1H NMR (400
MHz), CDCl.sub.3, .delta.: 11.46 (s, 1H), 8.62 (s, 1H), 7.05 (s,
1H), 4.24 (s, 2H), 3.91 (s, 2H), 3.74 (s, 2H), 3.62 (s, 2H), 1.48
(s, 9H), 1.45 (s, 9H). FT-IR (neat): 3329.4, 3131.7, 2975.3,
2931.3, 1720.1, 1635.2, 1614.0, 1567.7, 1503.9, 1411.8, 1364.5,
1319.8, 1280.5, 1249.7, 1131.0, 1048.8 cm.sup.-1. GPC: Mn=13,500
g/mol, Mw =18,000 g/mol, PDI=1.30, UV-Vis (THF) .lamda..sub.max=442
nm, Fluo .lamda..sub.max=469 nm.
Guanidine-DIPA (PG-D)
[0166] As illustrated in FIG. 19, using the general polymerization
procedure for Boc-protected Poly-1, above, through the addition of
the 4:1 THF/DIPA mixture a latent reaction mixture was formed with
the Boc-protected Poly-1. The reaction mixture was heated at
80.degree. C. for 16 h. Upon precipitation, as above, an overall
yield of 63% (26.9 mg) was achieved. The resulting polymer in DCM
was characterized by gel permeation chromatography (GPC) and its
absorption/emission profiles were measured. The final polymer was
allowed to dry under high vacuum for 16 h before .sup.1H NMR
characterization.
[0167] .sup.1H NMR (400 MHz), CDCl.sub.3, .delta.: 12.42 (s,
0.92H), 8.21 (s, 0.88H), 7.05 (s, 0.8811), 4.23 (s, 2H), 3.90 (s,
2H), 3.75 (s, 3.46H), 3.60 (s, 0.61H), 3.54 (s, 1.92H), 1.42 (s,
9.23H), 1.23 (s, 12.2511) GPC: Mn=13155 g/mol, Mw=22363 g/mol,
PDI=1.70, UV-Vis (THF) .lamda..sub.max=434 nm, Fluo
.lamda..sub.max=472 nm.
Boc-Deprotection to Poly-1
[0168] A solution of Boc-protected Poly-1 in DCM (1.00 mL) was
treated with trifluoroacetic acid (TFA) at room temperature for 48
hours. The solvent was removed under reduced pressure and the crude
material was dissolved in minimum amount of dimethylformamide (DMF)
to have a clear homogeneous solution. The polymer solution in DMF
was transferred to diethyl ether, resulting in yellowish fiber like
precipitates that were collected by decantation. The polymer was
dissolved in DMF and then re-precipitated in ethyl acetate (EA).
This process was repeated twice and the final Boc-deprotected
polymer was collected by decantation followed by vacuum dry. After
drying in a high vacuum, the final deprotected polymer was a yellow
gel (74.6% yield) with complete Boc-deprotection confirmed by
.sup.1H NMR. .sup.1H NMR (400 MHz, DMSO-d.sub.6, .delta.): 8.13 (s,
1H), 7.92 (s, 2H), 7.27 (s, 2H), 6.87 (s, 1H), 5.77 (s, 1H), 5.03
(s, 1H), 4.30 (s, 2H), 3.98 (s, 2H), 3.59 (s, 4H), 3.50 (m, 6H),
2.98 (m, 10H), 2.87 (s, 1H), 2.01 (m, 6H). FT-IR (neat): 3360.36,
2160.37, 1736.79, 1681.18 cm.sup.-1. UV-Vis (DMSO)
.lamda..sub.max=434 nm, Fluo .lamda..sub.max=490 nm, QY=0.08.
Guanidine-Morpholine (PG-M)
[0169] A Schlenk flask was charged with GCP (10 mg, 0.012 mmol) and
Morpholine (2.57 mg, 0.03 mmol). The Schlenk flask was evacuated
and filled with N.sub.2. A degassed tetrahydrofuran (THF) (1.5 mL)
was transferred to the Schlenk flask via a cannula. The reaction
was stirred at 80.degree. C. for 16 h. The viscous polymer solution
was filtered through glass wool followed by precipitation in
diethyl ether and re-precipitating in methanol. The final polymer
was yellow gel (7.72 mg with 76% yield). .sup.1H NMR (400 MHz),
CDCl.sub.3, .delta.: 12.2 (s, 1H), 8.3 (s, 1.05H), 7.04 (s, 0.94H),
4.22 (s, 211), 3.90 (s, 2.30), 3.74 (s, 4.47H), 3.60 (s, 4.06H),
3.52 (s, 4.21H), 1.42 (s, 9.9011). GPC: Mn=12364 g/mol, Mw=18598
g/mol, PDI=1.50. UV-Vis (THF) .lamda..sub.max=428 nm, Fluo
.lamda..sub.max=469 nm.
Guanidine-Piperidine (PG-P)
[0170] A Schlenk flask was charged with GCP (10 mg, 0.012 mmol) and
Piperidine (2.51 mg, 0.03 mmol). The Schlenk flask was evacuated
and filled with N.sub.2. Degassed tetrahydrofuran (THF) (1.5 mL)
was transferred to the Schlenk flask via a cannula. The reaction
was stirred at 80.degree. C. for 16 h. The viscous polymer solution
was filtered through glass wool followed by precipitating in
diethyl ether and re-precipitating in methanol. The final polymer
was yellow gel (5.36 mg with 53% yield). .sup.1H NMR (400 MHz),
CDCl.sub.3, .delta.: 12.30 (s, 0.98H), 8.22 (s, 0.91H), 7.05 (s,
0.86H), 4.22 (s, 1.92H), 3.90 (s, 1.99H), 3.74 (s, 2.15H), 3.65 (s,
2.42H), 3.59 (s, 0.83H), 3.53 (s, 1.78H), 3.47 (s, 2.71H) 1.42 (s,
9H). GPC: Mn=14606 g/mol, Mw=27751 g/mol, PDI=1.90. UV-Vis (THF)
.lamda..sub.max=436 nm, Fluo .lamda..sub.max=461 nm.
Guanidine-Aminoethoxyethanol (Poly-1)
[0171] A Schlenk flask was charged with Guanidine Homo Polymer (10
mg, 0.012 mmol) and Aminoethoxyethanol (3.1 mg, 0.03 mmol). The
Schlenk flask was evacuated and filled with N.sub.2. A degassed
tetrahydrofuran (THF) (1.5 mL) was transferred to the Schlenk flask
via a cannula. The reaction was stirred at 80.degree. C. for 16 h.
The viscous polymer solution was filtered through glass wool
followed by re-precipitation in diethyl ether and re-precipitating
in methanol. The final polymer was yellow gel (8.5 mg with 82.5%
yield). .sup.1H NMR (400 MHz), CDCl.sub.3, .delta.: 12.05 (s,
0.73H), 8.27 (s, 0.63H), 7.04 (s, 0.87H), 6.03 (s, 0.55H), 4.22 (s,
2H), 3.90 (s, 2.38H), 3.72 (s, 5.42H), 3.53 (s, 8.26H), 3.36 (s,
02.31H), 1.42 (s, 7.22H); .sup.1H NMR (400 MHz, DMSO-d.sub.6,
.delta.): 10.03 (s, 1H), 9.25 (s, 1H), 8.60 (s, 2H), 7.53 (s, 2H),
4.38 (s, 1H), 4.03 (s, 1H), 3.81 (s, 2H). GPC: Mn=13767 g/mol,
Mw=19529 g/mol, PDI=1.41. UV-Vis (THF) .lamda..sub.max=435 nm, Fluo
.lamda..sub.max=465 nm; UV-Vis (DMSO) .lamda..sub.max=434 nm, Fluo
.lamda..sub.max=494 nm, QY=0.20.
Random Copolymer Synthesis
##STR00028##
[0173] Random copolymer PN-co-G-BA was synthesized by mixing both
respective monomers in DCM and adding varying molar equivalents of
Grubbs 3.sup.rd gen catalyst. Polymerizations were terminated by
the addition of 1 mL of ethyl vinyl ether and precipitation into
diethyl ether (3.times.), followed by deprotection in TFA.
Block Copolymer Synthesis
##STR00029##
[0175] Block copolymer Boc-Block-PN-co-G-BA was synthesized by
stirring 4 with varying molar ratios of Grubbs catalyst for 10
minutes prior to the addition of monomer 5. Polymerization was
terminated with ethyl vinyl ether and precipitated (3.times.) in
diethyl ether.
##STR00030##
[0176] Boc-Block-PN-co-G-BA was either deprotected to yield
Block-PN-co-G-BA or reacted with varying amines in THF followed by
deprotection to yield Block-PN-co-R-BA where R=AEE, HA, or BA.
##STR00031##
[0177] Boc-Block-PN-co-R above was synthesized in similar manner as
previous polymers using varying molar ratios of Grubbs catalyst.
This polymer was deprotected or modified using guanylurea
modification to yield Block-PN-co-R where R=G, AEE, HA or BA. These
polymers were modified with various phenyl boronic acid derivatives
to yield the final Block-PN-co-R-BoronicAcid
##STR00032##
where the boronic acid position varies on the phenyl group.
Physical and Photophysical Properties of CPs
[0178] For the data tabulated in Table 1, below, the UV absorbance
and emission spectra of Poly-1 and Poly-2 were determined in DMSO
and 95% water+5% DMSO. In a good solvent, such as DMSO, polymer
absorbance and emission did not change. But in a poor solvent, such
as water, their emission spectra were significantly changed. FIGS.
6A and 6B show the UV absorbance and emission spectra. The HD and
zeta potential of Poly-1 and Poly-2 were determined using
Nanoparticle Tracking Analysis (NTA) and Dynamic Light Scattering
(DLS), respectively. For NTA and DLS, all cuvette, pipette, and
pipette tips were autoclaved. The working area was cleaned with 70%
ethanol to avoid cross contamination. Stock polymer samples were
prepared at a concentration of 100 .mu.M in DMSO solvent. 10 .mu.L
of a stock polymer solution in DMSO was added to 90 .mu.L of RNAse
water. The polymer solution in DMSO-water added to 11.11 nM and 900
.mu.L siRNA containing RNAse water. The mixture of polymer and
siRNA solution was gently pipetted, and 1 mL sample solution was
then injected to the NTA chamber, and images of scattering
particles in the sample were collected for 90 seconds. Software
identified each individual particle and tracked its motion
throughout the duration of the recorded video. The measured
particle displacement is a function of Brownian motion, which is
related to the particle size through the Stokes-Einstein equation.
The final data was collected under the detection threshold at 4, to
obtain the acceptable data meeting the concentration requirements.
All measurements were performed in triplicate at 25.degree. C.
using a temperature controller. The values in Table 2, below, are
averaged from three independent measurements. Selected
representative NTA graphs are presented in FIGS. 7A and 7B.
TABLE-US-00001 TABLE 1 Physical and Photophysical Properties of CPs
Zeta .LAMBDA..sub.max, abs .LAMBDA..sub.max, em Potental, HD, Poly
Mn.sup.a PDI.sup.b n.sup.c (nm).sup.d (nm).sup.d,e mV.sup.f
nm.sup.g QY.sup.h 1 13,500 1.30 16 434 490 8.0 .+-. 2.0 99 .+-. 29
8.0 2 13,800 1.40 15 434 494 4.0 .+-. 1.0 139 .+-. 43 20
.sup.aDetermined by gel permeation chromatography in THF,
.sup.bPolydispersity index (Mw/Mn), .sup.cDegree of polymerization,
.sup.dMeasured in DMSO, .sup.eExcitation wavelength 430 nm,
.sup.fZeta potential in water, .sup.gDetermined by nanoparticle
tracking analysis, .sup.hQuantum yield in DMSO measured relative to
diphenylanthracene standard
TABLE-US-00002 TABLE 2 Size in nm of Poly-1 and Poly-2 with siRNA
Poly Polymer, nm.sup.a,b Polymer-siRNA, nm.sup.a,b 1 99 .+-. 29 137
.+-. 40 2 139 .+-. 43 152 .+-. 44 .sup.aDMSO (1%) and water (99%),
.sup.bConc. of final polymer and siRNA were 1 .mu.M and 10 nM,
respectively
[0179] Zeta potentials of Poly-1 and Poly-2 in complexation with
siRNA were measured using Zetasizer Nano-ZS (Zen 3600, Malvern
Instruments Ltd.). The viscosity and refractive index of water were
used for estimation of relative zeta potential difference among the
samples. Stock polymer samples were prepared at a concentration of
1000 .mu.M in DMSO. 10 .mu.L of stock polymer solution was
dissolved in 90 .mu.L RNAse water. Then the polymer solution in
RNAse water was transferred to 900 .mu.L of siRNA containing RNAse
water (siRNA concentration 11.11 nM) and the solution was mixed by
pipetting. The final polymer and siRNA concentration in the
solution were 10 .mu.M and 100 nM, respectively. All measurements
were performed in triplicates at 25.degree. C. and the average
values were reported in Table 3, below.
TABLE-US-00003 TABLE 3 Zeta potential of Poly-1 and Poly-2 with
siRNA Poly Polymer, mV.sup.a Polymer-siRNA, mV.sup.b 1 8.0 .+-. 2
11 .+-. 1.3 2 4.0 .+-. 1 12 .+-. 0.8 .sup.aDMSO (1%) and water
(99%), .sup.bConc. of final polymer and siRNA were 1 .mu.M and 10
nM
Cell Culture
[0180] Primary human bronchial epithelial cells were isolated and
re-differentiated at the air-liquid interface cultures as per
Unwalla et al. Am. J. Respir. Cell. Mol. Biol. 2012, 46(4), 551-8
and Unwalla et al. Am. J. Respir. Cell. Mol. Biol. 2015, 52 (1),
65-74. Cells were obtained from properly consented donors whose
lungs were not suitable for transplantation for the causes
unrelated to airway complications and supplied by University of
Miami Life Alliance Organ Recovery Agency. Since the material was
obtained from deceased individuals with minor, de-identified
information, its use does not constitute human subjects research as
defined by CFR 46.102. A signed and well documented consent of each
individual or legal healthcare proxy for the donation of lungs for
research purpose is on file with the Life Alliance Organ Recovery
Organization allows research purpose of this material. Unless
otherwise indicated, experiments used cells from non-smokers to not
confound the findings in unknown ways. These primary cultures
undergo mucociliary differentiation at the air-liquid interface
reproducing in vivo morphology and key physiologic processes to
recapitulate the native bronchial epithelium ex vivo. Primary NHBE
cells isolated from human lungs were provided by University of
Miami Life Alliance Organ Recovery Agency and re-differentiated on
porous supports at the air-liquid interface. Re-differentiated NHBE
cells were tested for ciliation by staining acetylated tubulin.
[0181] The immortalized normal human bronchial epithelial cell line
BEAS-2B (ATCC CRL-9609) was purchased from the American Type
Culture Collection (Manassas, Va., USA). BEAS-2B cells were
cultured in BioLite 75 cm.sup.2 flasks (Thermo Scientific)
containing Bronchial Epithelial Cell Growth Medium (BEGM). BEGM
media was supplemented with 0.1% (v/v) human recombinant epidermal
growth factor, 0.1% (v/v) insulin, 0.1% (v/v) hydrocortisone, 0.1%
(v/v) ethanolamine, 0.1% phosphoryl ethanolamine, 0.1% (v/v)
retinoic acid, 0.1% (v/v) epinephrine, 0.24% (v/v) transferrin, 1%
(v/v) penicillin/streptomycin and 0.1% (v/v) bovine pituitary
extract. The cells were cultured in 95% air and 5% CO2 at
37.degree. C. and maintained free of mycoplasma contamination.
Cell Viability Assay
[0182] BEAS-2B cells (.about.15,000 cells per well) in 200 .mu.L of
a complete medium, were seeded into a 96-well plate and allowed to
attach for one day at 37.degree. C. under humidified atmosphere of
5% CO2/95% air. Final concentrations of 40 .mu.M, 20 .mu.M, 10
.mu.M, 5 .mu.M, and 1 .mu.M of CPs were added into the complete
media by dilution with CPs stock solutions. After addition of CPs,
cells were incubated for another 18 h. The cells were treated with
10 .mu.L of methylthiazole tetrazolium (MTT) (5 mg/mL in PBS,
CALBIOCHEM, Germany) and incubated for 4 h at 37.degree. C.
Subsequently, 200 .mu.L of medium was removed by using a pipette
and then 100 .mu.L of biological grade DMSO (Sigma Aldrich, St.
Louis, Mo., USA) was added to solubilize the purple formazan
crystals formed by proliferating cells. Absorbance was measured by
a microplate well reader (infinite M1000 PRO, TECAN, Switzerland)
at 570 nm. Relative cell viability (%), FIG. 9, as a function of
CPs concentration was expressed as the percentage relative to the
untreated control cells. All measurements were performed in
triplicate and standard deviation was included in the error
bar.
Gel Retardation Assay
[0183] The siRNA binding capabilities of Poly-1 and Poly-2,
respectively, were examined by a gel retardation assay as indicated
in FIG. 10. 10 .mu.L of siHDAC (400 nM) (Santa Cruz Biotechnology)
was mixed with 10 .mu.L of the polymers with different
concentrations. Samples were gently vortexed and kept for 30 m at
room temperature. Then, polyplexes solutions (20 .mu.L) were mixed
with 20 .mu.L of 2xRNA loading buffer (Thermo Fisher Scientific).
The polyplexes solutions (40 .mu.L) were loaded in to 40% poly
(acrylamide) gel (cross-linking of 2.67) and run in 1X TBE buffer
at 90V for 80 m. Free siHDAC bands were visualized using 0.5
.mu.g/mL ethidium bromide solution. The bands were visualized by
using the Quantity One software (Bio-Rad Laboratories, USA) and the
density values are normalized to free siRNA.
Immunocytochemistry for Cilia to Determine Differentiation.
[0184] NHBE cells were allowed to re-differentiate for 21 days at
the air-liquid interface on transwell filters. Re-differentiation
was determined by staining for ciliation as described in
Chinnapaiyan et al. PLoS One 2017. 12(1): p. e0169161. Cells were
fixed in 4% paraformaldehyde in PBS, pH 7.4 for 15 min and
permeabilized with 1% Triton X-100 in PBS for 20 min at room
temperature (RT). After permeabilization, cells were washed with
PBS and then blocked with 3% BSA in PBS for 1 h at room
temperature. Cells were treated with the primary antibody [mouse
anti human acetylated .alpha.-tubulin (Sigma Cat. #T6793; 1:1000)]
in blocking solution and incubated overnight at 4.degree. C. Cells
were washed three times and then incubated with Alexa 647 anti
mouse IgG for 45 min. Cells were washed three times with blocking
solution and counterstained with 4,6-diamidine-2-phenylindole
(DAPI, KPL) to label nuclei for 10 min. Transwell filters were
excised and placed directly on the slide and images were acquired
on visualized using a Zeiss fluorescence microscope with high
resolution Axiocam 506 mono microscope camera (Zeiss, Germany).
Cilia appear green at the apical side of the NHBE cultures with
nuclei stained in blue.
Confocal Microscopic Images of BEAS-2B Cells.
[0185] BEAS-2B cells (.about.0.5.times.10.sup.6/well) were seeded
into a 12-well plate with glass coverslip one day prior to CP
treatment, and then cultured in a complete media for 24 h under 5%
CO.sub.2 at 37.degree. C. Cells were washed three times with
1.times.PBS after removing the media. The polyplex formed by mixing
10 .mu.M CPs and siGLO (100 nM) was added to cells and then
incubated for 48 h. After 48 h incubation, cells were washed three
times with 1.times.PBS and fixed with 4% PFA for 10 m. Cells were
then washed three times with 1.times.PBS and coverslips were
mounted on microscope slides using a 1:1 glycerol/PBS mounting
medium. Fluorescent images of the cells were obtained using an
Olympus Fluorview FV1200 confocal microscope (Melville, N.Y. USA)
equipped with a bandpass filter for green (513-556 nm) and a 60X
oil immersion len (NA 1.35, n=1.519 immersion oil). Image J
software (Version 1.50b, U.S. National Institute of Health,
Bethesda, Md., USA) was used to process the image.
Confocal Fluorescence Microscopic Images of Primary NHBE Cells.
[0186] NHBE cells were treated with polyplex containing siGLO as
described, above. After 48 h incubation, cells were washed three
times with 1.times.PBS and fixed with 4% PFA for 10 m. Cells were
then washed three times with 1.times.PBS. The cells grown on
semipermeable membrane were separated from the chamber and then
mounted on a microscope slide using a 1:1 glycerol/PBS mounting
medium followed by sealing with nail polish. Fluorescent images of
the cells were obtained using an Olympus Fluorview FV1200 confocal
microscope (Melville, N.Y. USA) equipped with a bandpass filter
(513-556 nm) and a 60X oil immersion lens (NA 1.35). Image J
software (Version 1.50b, U.S. National Institute of Health,
Bethesda, Md., USA) was used to process the image.
Gene Knockdown Experiment in BEAS-2B Cells
[0187] Lipofectamine RNAiMAX-Mediated Transfection of siHDAC in
BEAS-2B Cells.
[0188] High-capacity cDNA reverse transcription kit was purchased
from Applied Biosystems. Taqman Fast Advanced Master Mix was
purchased from Life Technologies. Lipofectamine.RTM. RNAiMAX
Transfection Reagent and Opti-MEM.TM. Reduced-Serum Medium were
purchased from Thermo Fisher Scientific. BEAS-2B cells were plated
on collagen coated tissue culture plates at a density of
0.6.times.10.sup.6. Twenty-four hours following plating, cells were
transfected with siHDAC complexed with Lipofectamine RNAiMax in
Opti-MEM medium according to manufacturer's instructions using
different concentrations of the siRNA (i.e., 12.5, 25, 50, 75, and
100 nM). BEAS-2B cells treated with equivalent amounts of
lipofectamine RNAiMAX in Opti-MEM was used as transfection control,
as shown in FIG. 13. The mixture was vortexed and incubated at room
temperature for 30 m before adding to the cells. After eight-hour
post-transfection, experiments were terminated, and total RNA was
isolated and analyzed by quantitative RT-PCR.
Polymer-Mediated Transfection of siHDAC in BEAS-2B Cells.
[0189] The polyplex solutions were freshly prepared prior to
transfection experiments. One day after plating BEAS-2B cells
(0.6.times.10.sup.6) in a 12-wells plate, cells were transfected by
adding polyplex solutions. 5 mM polymer stock solution was diluted
to 5, 10, 20, and 40 .mu.M, respectively, in 50 .mu.l of RNase and
DNase free water, and then mixed with various amounts of siHDAC
(i.e., 12.5, 25, 50, 75, and 100 nM). Polyplex solution was
vortexed for 30 m. Cells were incubated with polyplex for 48 h. The
total RNA was analyzed by quantitative RT-PCR, as indicated in FIG.
13.
Gene Knockdown Experiment in NHBE Cells
[0190] The polyplex solutions were freshly prepared prior to
transfection experiments. NHBE cultures re-differentiated at the
air-liquid interface (ALI) were transfected by adding polyplex
solutions using a protocol identical to that for BEAS2B cells
above. Separately, another set of NHBE air-liquid interface
cultures were treated with siRNA complexed with Lipofectamine
RNAimax for comparison. Experiments proceeded for 48 hours and
total RNA was isolated and analyzed by quantitative RT-PCR, as
indicated in FIG. 13.
[0191] Total RNA was extracted from cells treated with transfection
agents after 48 h incubation using an RNeasy mini kit (Qiagen Inc.
Valencia, Calif.). The concentration and integrity of the extracted
RNA were analyzed by measurement of the OD260/280 (Synergy.TM. HTX
Multi-Mode Microplate Reader, Winooski, Vt., USA). Complementary
DNA (cDNA) was reversely transcribed by using the Applied
Biosystems High performance kit (Applied Biosystem, Carlsbad,
Calif.). Reverse transcription of 2 .mu.g of total cellular RNA was
performed in a final volume of 20 .mu.l containing 10 .mu.l of RNA,
2 .mu.l of 10X RT buffer, 0.8 .mu.l of dNTP Mix (100 mM), 2.0 .mu.l
of 10X RT random hexamer primers, 1.0 .mu.l of MultiScribe.TM.
reverse transcriptase, 1 .mu.l of RNase inhibitor, and 3.2 .mu.l of
nuclease-free water. The reverse transcription reaction was allowed
to proceed using cycling parameters recommended by the
manufacturer: an initial incubation at 25.degree. C. for 10 m
followed by incubation at 37.degree. C. for 120 m. The reverse
transcription was terminated by incubating at 85.degree. C. for 5
sec. cDNA samples were stored at -20.degree. C. until further use
for quantitative PCR. Quantitative PCR was performed on the Bio-Rad
CFX96 real-time system (BioRad, Hercules, Calif., USA) using
validated TaqMan probes (GAPDH, HDAC2) according to manufacturer
recommended cycling parameters (an initial denaturation cycle of
95.degree. C. for 20 s followed by 40 cycles of 95.degree. C./3 s
and 60.degree. C./30 s. qRT-PCR results are represented as relative
quantification normalized to internal control (GAPDH).
Ussing Chamber Method to Determine Apical CFTR Activity
[0192] Ussing chamber electrophysiology was used to confirm
re-differentiation and polarization as per: Unwalla et al., Am. J.
Respir. Cell. Mol. Biol. 2015, 52 (1), 65-74; and Chinnapaiyan et
al., Sci Rep. 2018, 8(1), 7984. CFTR is located at the apical side
(mucosal) of the airway epithelium. Briefly, NHBE cultures were
re-differentiated at the air-liquid interface. Following
re-differentiation for 21 days, the snap wells were removed from
supports and mounted in Ussing chambers. The short circuit current
was measured and allowed to stabilize followed by addition of
amiloride (10 .mu.M) added apically to eliminate epithelial sodium
channel (ENaC) influences. CFTR activation was affected by addition
of albuterol (10 .mu.M) and change in short circuit current
(.DELTA.I.sub.SC) was determined. CFTR specificity was confirmed by
addition of CFTR inhibitor CFTR.sub.inh172 (20 .mu.M) and the
decrease in .DELTA.I.sub.SC was recorded.
Protein Delivery
[0193] Various homopolymers, random copolymers and block copolymers
of the subject invention have been synthesized for the
intracellular delivery of proteins. The role of the guanylurea
functional group was explored by comparing the PN-G as a
guanidine-rich control, PN-G-BA that contains a hydrophobic phenyl
moiety directly attached on the positive charged side chain through
guanylurea modification, and a copolymer variant, PN-co-G-BA, which
isolates the phenyl groups from the positive charge by having the
two on isolated repeating units. The chain length of polymers
PN-G-BA and PN-co-G-BA was controlled to have the same number of
positive charges and same relative hydrophobicity since these may
be factors that influence the protein complexation and cellular
entry.
[0194] To understand the ability of the synthesized polymers on
protein delivery and the role of the guanylurea functionality, EGFP
delivery into Hela cells was chosen as a model system. In short,
the polymers were mixed with EGFP at ratios of 10 uM and 60 nM
respectively. The complexes were allowed to form for 30 minutes
prior to treating Hela cells in serum containing medium. Hela cells
were treated overnight with complexes, and rinsed thoroughly with
PBS prior to flow cytometric analysis.
[0195] Results show .about.3 fold improvement in delivery
efficiency using the guanylurea modified polymer, PN-G-BA, as
compared to the other polymers (FIG. 20). PN-co-G-BA containing the
same degree of hydrophobicity as PN-G-BA shows similar delivery
efficiency as the guanidine rich polymer PN-G (FIG. 20). This
result indicates that the guanylurea modification that maintains
the hydrophobic segment on the positive charge exhibits the best
EGFP delivery efficiency.
[0196] Various homopolymers, random copolymers and block copolymers
containing boronic acid moieties may be used as a targeted therapy
for cancer treatments. Improvements in cell entry efficiency, cargo
complexation and nanoparticle stability are explored with these
polymeric systems.
[0197] All patents and publications referred to or cited herein are
incorporated by reference in their entirety, including all figures
and tables, to the extent they are not inconsistent with the
explicit teachings of this specification.
[0198] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application.
* * * * *