U.S. patent application number 14/826712 was filed with the patent office on 2016-03-10 for helical polycarbodiimide polymers and associated imaging, diagnostic, and therapeutic methods.
The applicant listed for this patent is Memorial Sloan Kettering Cancer Center. Invention is credited to Januka Budhathoki-Uprety, Daniel A. Heller.
Application Number | 20160067362 14/826712 |
Document ID | / |
Family ID | 55351147 |
Filed Date | 2016-03-10 |
United States Patent
Application |
20160067362 |
Kind Code |
A1 |
Heller; Daniel A. ; et
al. |
March 10, 2016 |
HELICAL POLYCARBODIIMIDE POLYMERS AND ASSOCIATED IMAGING,
DIAGNOSTIC, AND THERAPEUTIC METHODS
Abstract
Described herein are suspensions of helical polycarbodiimide
polymers that `cloak` nanotubes, thereby effecting control over
nanotube emission, providing a new mechanism of environmental
responsivity, and enabling precise control over sub-cellular
localization. The helical polycarbodiimide polymers described
herein are water soluble, easily modifiable, and have unique
architectures that facilitate their application in
radiopharmaceutical delivery and imaging methods, in therapeutics
and therapeutic delivery methods, and their use as sensors--both in
conjunction with carbon nanotubes, and without nanotubes.
Inventors: |
Heller; Daniel A.; (New
York, NY) ; Budhathoki-Uprety; Januka; (New York,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Memorial Sloan Kettering Cancer Center |
New York |
NY |
US |
|
|
Family ID: |
55351147 |
Appl. No.: |
14/826712 |
Filed: |
August 14, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62038235 |
Aug 16, 2014 |
|
|
|
Current U.S.
Class: |
424/1.37 ;
424/489; 435/34; 436/172 |
Current CPC
Class: |
A61K 51/065 20130101;
A61K 47/6927 20170801; A61K 47/59 20170801; G01N 33/587 20130101;
A61K 51/1251 20130101; G01N 33/542 20130101; C01B 32/168
20170801 |
International
Class: |
A61K 51/06 20060101
A61K051/06; G01N 33/58 20060101 G01N033/58; A61K 51/12 20060101
A61K051/12; A61K 51/10 20060101 A61K051/10; C01B 31/02 20060101
C01B031/02; A61K 47/48 20060101 A61K047/48 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This work was supported by National Institutes of Health
grant DP2-HD07569.
Claims
1. A suspension of helical-polymer-encapsulated carbon nanotubes,
wherein the helical polymer is a polycarbodiimide.
2. The suspension of claim 1, wherein the carbon nanotubes are
single-walled carbon nanotubes (SWCNTs).
3. The suspension of claim 1, wherein the helical polymer comprises
a clickable polymer scaffold.
4. The suspension of claim 1, wherein the polycarbodiimide
comprises one or more monomeric species selected from the group
consisting of ##STR00009## ##STR00010##
5. The suspension of claim 1, wherein the suspension is an aqueous
suspension.
6. The suspension of claim 1, wherein at least a plurality of the
helical-polymer-encapsulated carbon nanotubes in the suspension are
in van der Waals contact at a center-to-center distance between
adjacent nanotubes sufficient to exhibit inter-nanotube Forster
resonance energy transfer (INFRET).
7. The suspension of claim 6, wherein the center-to-center distance
is from 1 nm to 4 nm.
8. The suspension of claim 6, wherein the dispersed
helical-polymer-encapsulated carbon nanotubes in van der Waals
contact are not irreversibly bound.
9. The suspension of claim 6, comprising (i) a first set of
nanotubes each encapsulated by a helical polymer having at least a
first substituent functional group, and (ii) a second set of
nanotubes each encapsulated by a helical polymer having at least a
second substituent functional group, wherein the first substituent
functional group and the second substituent functional group imbue
the first and second sets of encapsulated nanotubes with
sufficiently strong coulombic attraction to each other to form
reversible fluorescent aggregates in the suspension.
10. The suspension of claim 1, wherein the helical polymer
comprises functional side chains.
11. The suspension of claim 10, wherein the functional side chains
comprise one or more members selected from the group consisting of
a primary amine, a carboxylic acid, a guanidine group, an
oligoethylene glycol, a methoxy-polyethylene glycol (PEG), a
hydroxyl-PEG, a folic acid, a trimethoprim, a peptide, an alkyne
peptide, an adenosine triphosphate (ATP) peptide, and an
opioid.
12. The suspension of claim 1, wherein the helical polymer
comprises one or more aromatic groups incorporated in its monomer
substituents.
13. The suspension of claim 12, wherein the one or more aromatic
groups promote multi-valent .pi.-.pi. interactions between the
polymer and the graphitic sidewall of the carbon nanotubes.
14. The suspension of claim 1, wherein the functional side chains
comprise a targeting group.
15. A biomolecular imaging probe and/or sensor comprising the
suspension of claim 1.
16. An imaging method comprising: administering the suspension of
claim 1 to a biological sample; exposing the biological sample
comprising the administered suspension to excitation light; and
detecting light emitted by suspension or fluorescent aggregates
formed by one or more components of the suspension.
17. The method of claim 16, further comprising disrupting the
fluorescent aggregates to reverse the emission of light.
18. The method of claim 17, further comprising alternating between
cycles of light emission and no light emission by re-aggregating
and disrupting, respectively, the fluorescent aggregates for high
resolution biomolecular imaging.
19. The method of claim 16, wherein the detecting step comprises
obtaining images of cellular nuclei of the biological sample.
20. A method of treating a disease or disorder, the method
comprising administering the suspension of claim 1 to a subject,
wherein the functional side chains of the helical polymer comprises
a therapeutic.
21. The method of claim 20, wherein the therapeutic comprises an
opiate.
22. A method for pretargeted radioimmunotherapy (PRIT), the method
comprising administering a polycarbodiimide functionalized with an
antibody, labeled with a radionuclide.
23. The method of claim 22, wherein the radionuclide comprises a
metallic lanthanide.
24. The method of claim 22, wherein the radionuclide is attached to
the polycarbodiimide via a chelator.
25. The method of claim 22, wherein the radionuclide comprises
.sup.89Zr.
26. The suspension of claim 1, wherein the suspension is a stable
suspension in aqueous solution or in serum.
27-71. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of, and
incorporates herein by reference in its entirety, U.S. Provisional
Patent Application No. 62/038,235.
FIELD OF THE INVENTION
[0003] This invention relates generally to compositions comprising
polycarbodiimide polymers, and related imaging, diagnostic, and
therapeutic methods.
BACKGROUND
[0004] Carbon nanotubes have several features which demonstrate
their potential for biomedical applications including cellular
sensing and imaging. For example, carbon nanotubes can be metallic
or semiconducting depending on their structure, which is due to the
symmetry and unique electronic structure of graphene. Thus, the
electronic structure and diameter of the carbon nanotube will
determine the spectral characteristics seen in absorption,
fluorescence, Raman scattering, etc. Moreover, the environmental
sensitivity and intrinsic photostability of single-walled carbon
nanotubes (SWCNTs) in the near-infrared wavelength range (ca. 900
nm-1600 nm) demonstrates the potential of biomedical applications.
However, such uses require the ability to simultaneously modulate
nanotube fluorescence and to biocompatibly derivatize the nanotube
surface using noncovalent methods.
[0005] Both covalent and non-covalent functionalization methods can
be used to solubilize carbon nanotubes for adaptation to biomedical
applications. Non-covalent functionalization of SWCNTs preserves
both the optical and structural properties of SWCNTs in solution.
Nanotubes can be encapsulated in various amphiphilic polymers.
Biopolymers such as single stranded DNA (ssDNA), peptides, or
proteins, and synthetic polymers, such as polyfluorenes,
polycarbazoles, aryleneethynylene polymers, polyethylene glycol
(PEG) derivatives, and dextran-based polymers have been
investigated for materials, and biological applications. However,
encapsulation with biopolymers can produce higher background signal
(e.g., DNA can produce high oxidation current and subsequently
higher background currents) or provide inadequate control of
coating the nanotube and therefore affect optical response (e.g.,
protein denaturation in unfavorable conditions). Moreover, the
above-mentioned synthetic polymers do not provide controllable
and/or tunable properties, which limit the ability to measure
(e.g., via imaging) the kinetics of dynamic self-assembly and
disassembly and translocation of photoluminescent nanotubes into
live cell nuclei.
[0006] Therefore, there is a need to better adapt carbon nanotubes
for biomedical applications, such as cellular imaging and sensing,
that provides control over nanotube emission, environmental
responsivity, precise control over sub-cellular localization,
ordered surface coverage, and systematic modulation of nanotube
optical properties.
SUMMARY OF INVENTION
[0007] Described herein are suspensions of helical polycarbodiimide
polymers that `cloak` nanotubes, thereby effecting control over
nanotube emission, providing a new mechanism of environmental
responsivity, and enabling precise control over sub-cellular
localization. The helical polycarbodiimide polymers described
herein are water soluble, easily modifiable, and have unique
architectures that facilitate their application in
radiopharmaceutical delivery and imaging methods, in therapeutics
and therapeutic delivery methods, and their use as sensors--both in
conjunction with carbon nanotubes, and without nanotubes.
[0008] For example, the helical polycarbodiimide polymers can be
modified with radionuclides or radionuclide-chelating agents.
Experiments performed with these polymers--for example,
DOTA-modified polymer with multiple chelation sites for
Lutetium-177--demonstrate rapid clearance and low organ update,
especially in the kidneys.
[0009] The helical polycarbodiimide polymers can also deliver
molecules and increase drug binding affinity via multivalency,
lending to their use as therapeutics and in therapeutic delivery,
for example, opiate-polymer conjugates that provide long-term
analgesic effects, as well as treatment of cancer, atherosclerosis,
skin disorders, infectious diseases, and other diseases. Due to the
semi-rigidity of the polymer, more binding sites are accessible,
compared with polymers having a globular form. Furthermore, the
helical polymer lengths are short and very controllable, allowing
for rapid clearance if desired.
[0010] Moreover, the helical polymers described herein are
demonstrated to encapsulate single-walled carbon nanotubes, which
are used as fluorescent sensors for in vitro, ex vivo, and in vivo
applications. The polymers provide both sensitivity to specific,
desired bioanalytes, and direct/target the sensors to specific
locations in the cell and body. Polymer-nanotube constructs are
shown that provide nuclear, cytosolic, and extracellular
localization. Moreover, a stable polymer-nanotube sensor is
presented for in vitro and in vivo redox potential
measurements.
[0011] In one aspect, the invention is directed to a suspension of
helical-polymer-encapsulated carbon nanotubes, wherein the helical
polymer is a polycarbodiimide. In certain embodiments, the carbon
nanotubes are single-walled carbon nanotubes (SWCNTs). In certain
embodiments, the helical polymer comprises a clickable polymer
scaffold. In certain embodiments, the polycarbodiimide comprises
one or more monomeric species selected from the group consisting
of
##STR00001## ##STR00002##
[0012] In certain embodiments, the suspension is an aqueous
suspension. In certain embodiments, at least a plurality of the
helical-polymer-encapsulated carbon nanotubes in the suspension are
in van der Waals contact at a center-to-center distance between
adjacent nanotubes sufficient to exhibit inter-nanotube Forster
resonance energy transfer (INFRET). In certain embodiments, the
center-to-center distance is from 1 nm to 4 nm. In certain
embodiments, the dispersed helical-polymer-encapsulated carbon
nanotubes in van der Waals contact are not irreversibly bound.
[0013] In certain embodiments, the suspension comprises (i) a first
set of nanotubes each encapsulated by a helical polymer having at
least a first substituent functional group (e.g., a primary amine),
and (ii) a second set of nanotubes each encapsulated by a helical
polymer having at least a second substituent functional group
(e.g., a carboxylic acid), wherein the first substituent functional
group and the second substituent functional group imbue the first
and second sets of encapsulated nanotubes with sufficiently strong
coulombic attraction to each other to form reversible fluorescent
aggregates in the suspension.
[0014] In certain embodiments, the helical polymer comprises
functional side chains. In certain embodiments, the functional side
chains comprise one or more members selected from the group
consisting of a primary amine, a carboxylic acid, a guanidine
group, an oligoethylene glycol, a methoxy-polyethylene glycol
(PEG), a hydroxyl-PEG, a folic acid, a trimethoprim, a peptide, an
alkyne peptide, an adenosine triphosphate (ATP) peptide, and an
opioid. In certain embodiments, the helical polymer comprises one
or more aromatic groups incorporated in its monomer substituents.
In certain embodiments, the one or more aromatic groups promote
multi-valent .pi.-.pi. interactions between the polymer and the
graphitic sidewall of the carbon nanotubes.
[0015] In certain embodiments, the functional side chains comprise
a targeting group (e.g., an organelle targeting group, a protein
targeting group, a polysaccharide targeting group, or a targeting
group for another biological structure). In certain embodiments, a
biomolecular imaging probe and/or sensor comprises the
suspension.
[0016] In another aspect, the invention is directed to an imaging
method comprising: administering the suspension to a biological
sample (e.g., in vitro, ex vivo, or in vivo, e.g., wherein the
biological sample is a subject); exposing the biological sample
comprising the administered suspension to excitation light (e.g.,
near-infrared excitation light); and detecting light emitted by
suspension or fluorescent aggregates formed by one or more
components of the suspension (e.g., detecting light by
inter-nanotube Forster resonance energy transfer (INFRET)) in the
biological sample).
[0017] In certain embodiments, the imaging method comprises
disrupting the fluorescent aggregates (e.g., wherein disrupting the
fluorescent aggregates is performed by administering an agent) to
reverse the emission of light.
[0018] In certain embodiments, the imaging method comprises
alternating between cycles of light emission and no light emission
by re-aggregating and disrupting, respectively, the fluorescent
aggregates (e.g., for high resolution biomolecular imaging).
[0019] In certain embodiments, the detecting step comprises
obtaining images of cellular nuclei of the biological sample.
[0020] In another aspect, the invention is directed to a method of
treating a disease or disorder (e.g., cervical, pancreatic, or skin
cancer), the method comprising administering the suspension to a
subject, wherein the functional side chains of the helical polymer
comprises a therapeutic.
[0021] In certain embodiments, the therapeutic comprises an
opiate.
[0022] In another aspect, the invention is directed to a method for
pretargeted radioimmunotherapy (PRIT), the method comprising
administering a polycarbodiimide functionalized with an antibody,
labeled with a radionuclide (e.g., wherein administering the
labeled and functionalized polycarbodiimide delivers cytotoxic
radiation to a target cell of the subject).
[0023] In certain embodiments, the radionuclide comprises a
metallic lanthanide (e.g., yttrium or lutetium). In certain
embodiments, the radionuclide is attached to the polycarbodiimide
via a chelator (e.g.,
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or
desferoxamine (DFO)). In certain embodiments, the radionuclide
comprises .sup.89Zr.
[0024] In certain embodiments, the suspension is a stable
suspension (e.g., stable in aqueous solution or in serum).
[0025] In another aspect, the invention is directed to a
polycarbodiimide polymer having a helical conformation comprising
one or more functional groups (e.g., functional side chains).
[0026] In certain embodiments, the one or more functional groups
comprise at least one member selected from the group consisting of
a primary amine, a carboxylic acid, a guanidine group, an
oligoethylene glycol, a methoxy-PEG, a hydroxyl-PEG, a folic acid,
a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)
or another chelator or complexing agent, a trimethoprim, a
perphenazine, a peptide, an alkyne peptide, an ATP peptide, and an
opioid.
[0027] In certain embodiments, the polymer is labeled with a
radionuclide. In certain embodiments, the radionuclide comprises a
metallic lanthanide (e.g., yttrium or lutetium). In certain
embodiments, the radionuclide is attached to the polycarbodiimide
via a chelator (e.g.,
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or
deferoxamine (DFO)). In certain embodiments, the radionuclide
comprises .sup.89Zr.
[0028] In certain embodiments, the polymer is functionalized with
an antibody.
[0029] In certain embodiments, the polymer comprises a fluorophore.
In certain embodiments, fluorophore is an infrared (IR) dye.
[0030] In certain embodiments, the polymer comprises a therapeutic
(e.g., an opiate, e.g., octreotide).
[0031] In certain embodiments, the polymer comprises multimeric
targeting groups (e.g., for receptors in cancer cells).
[0032] In another aspect, the invention is directed to a method of
treating a disease or disorder, the method comprising administering
the polymer to a subject, wherein the polymer is functionalized
with a therapeutic.
[0033] In another aspect, the invention is directed to a method for
radiotherapy (e.g., PRIT), the method comprising administering the
polymer to a subject, wherein the polymer is functionalized with a
radionuclide.
[0034] In another aspect, the invention is directed to an imaging
method comprising: administering the polymer of any one of claims
27 to 37 to a biological sample (e.g., wherein the administering is
in vitro, ex vivo, or in vivo, e.g., wherein the biological sample
is a subject); exposing the biological sample comprising the
administered suspension to excitation light (e.g., near-infrared
excitation light); and detecting electromagnetic radiation emitted
by at least one of the one or more functional groups of the
polymer.
[0035] In certain embodiments, the method further comprises
exposing the biological sample to excitation light (e.g.,
near-infrared excitation light) prior to (and/or concurrent with)
the detecting step, wherein the detecting step comprises detecting
emitted fluorescent light.
[0036] In another aspect, the invention is directed to a
helical-polymer-encapsulated carbon nanotube, wherein the
helical-polymer is a polycarbodiimide.
[0037] In certain embodiments, the carbon nanotube is in a solid
form (e.g., powdered or adhered to a surface) and capable of
forming a stable suspension in solution (e.g., aqueous solution,
e.g., serum).
[0038] In another aspect, the invention is directed to a method of
utilizing a sensor to detect and/or monitor the presence of and/or
concentration of one or more analytes (e.g., pathogens or other
bioanalytes) in a sample, the method comprising: administering
and/or contacting the suspension and/or the polymer to/with the
sample; following the administering and/or contacting step,
allowing one or more components of the administered and/or
contacted suspension and/or polymer to accumulate in the sample,
wherein the one or more components exhibit a detectable sensitivity
to the one or more analytes to be assayed; and following the
accumulation, obtaining a measurement (e.g., a 1D, 2D, or 3D
measurement, map, or image (e.g., positron emission tomography
(PET) or single-photon emission computed tomography (SPECT) image))
indicative of the presence and/or concentration of the one or more
analytes in the sample.
[0039] In certain embodiments, the sample is a biological sample,
and the one or more analytes is/are bioanalyte(s).
[0040] In certain embodiments, the method comprises allowing the
one or more components of the administered and/or contacted
suspension and/or polymer to accumulate in an extracellular
location of the biological sample.
[0041] In certain embodiments, the method comprises allowing the
one or more components of the administered/contacted suspension
and/or polymer to accumulate in an intracellular location (e.g., in
cellular nuclei and/or in the cytosol) of the biological
sample.
[0042] In certain embodiments, the biological sample is an in
vitro, ex vivo, or in vivo sample (e.g., wherein the biological
sample is a subject).
[0043] In certain embodiments, the administering and/or contacting
step comprises administering and/or contacting a device (e.g., a
chip, microneedle delivery device, or transdermal patch) comprising
the suspension and/or the polymer to/with the sample (e.g., wherein
contacting comprises embedding, adhering, injecting, or placing the
device into or onto the sample).
[0044] In certain embodiments, the functional side chains of the
helical polymer comprise a radiolabel, and wherein the measurement
is a measurement of radiation emitted by the radiolabel. In certain
embodiments, functional side chains comprise a complex of
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)
with a metallic lanthanide (e.g., yttrium or lutetium).
[0045] In another aspect, the invention is directed to a method of
utilizing a sensor to detect and/or monitor a redox potential in a
sample, the method comprising: administering and/or contacting the
suspension of any one of claims 1 to 14 to/with the sample;
optionally, following the administering and/or contacting step,
allowing one or more components of the administered and/or
contacted suspension to accumulate in the sample (e.g., further
comprising, absorbing a quantity of the sample into a test strip or
isolating a quantity of the sample for redox measurement; e.g.,
further comprising, contacting the isolated sample with the
suspension, rather than the one or more components of the
suspension moving into the sample); and exposing the one or more
components of the administered and/or contacted suspension to an
applied voltage, and measuring the resulting redox potential (e.g.,
a 1D, 2D, or 3D measurement or map) of the sample.
[0046] In certain embodiments, the sample is a biological sample,
and the measured reduction potential is in the range from -150
millivolts to -400 millivolts.
[0047] In certain embodiments, the method further comprises
allowing the one or more components of the administered/contacted
suspension to accumulate in an extracellular location of the
biological sample.
[0048] In certain embodiments, the method further comprises
allowing the one or more components of the administered/contacted
suspension to accumulate in an intracellular location (e.g., a
cellular nuclei and/or a cytosol) of the biological sample.
[0049] In certain embodiments, the functional side chains of the
helical polymer comprise an organelle targeting group.
[0050] In certain embodiments, the biological sample is an in
vitro, ex vivo, or in vivo sample.
[0051] In certain embodiments, the administering and/or contacting
step comprises administering or contacting a device (e.g., a chip,
microneedle delivery device, or transdermal patch) comprising the
suspension to/with the sample (e.g., wherein contacting comprises
embedding, adhering, injecting, or placing the device into or onto
the sample).
[0052] In certain embodiments, the biological sample is skin and
wherein the one or more components of the administered and/or
contacted suspension are delivered to and embedded within an
epidermal layer of the skin (e.g., further comprising monitoring an
extracellular redox potential over time).
[0053] In another aspect, the invention is directed to a kit for
use in a radiopharmacy setting, the kit comprising: at least one
container, wherein the container has a type selected from an
ampule, a vial, a cartridge, a reservoir, a lyo-ject, or a
pre-filled syringe; the polymer, wherein the molecular weight
(e.g., weight average molecular weight or number average molecular
weight) is from 5 kDa to 75 kDa (e.g., from 10 kDa to 50 kD, e.g.,
from 15 kDa to 30 kDa); at least one disposable size exclusion
column; and at least one disposable filter, wherein the at least
one container holds (e.g., contains) the polymer.
DEFINITIONS
[0054] In order for the present disclosure to be more readily
understood, certain terms are first defined below. Additional
definitions for the following terms and other terms are set forth
throughout the specification.
[0055] In this application, the use of "or" means "and/or" unless
stated otherwise. As used in this application, the term "comprise"
and variations of the term, such as "comprising" and "comprises,"
are not intended to exclude other additives, components, integers
or steps. As used in this application, the terms "about" and
"approximately" are used as equivalents. Any numerals used in this
application with or without about/approximately are meant to cover
any normal fluctuations appreciated by one of ordinary skill in the
relevant art. In certain embodiments, the term "approximately" or
"about" refers to a range of values that fall within 25%, 20%, 19%,
18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%,
4%, 3%, 2%, 1%, or less in either direction (greater than or less
than) of the stated reference value unless otherwise stated or
otherwise evident from the context (except where such number would
exceed 100% of a possible value).
[0056] "Administration": The term "administration" refers to
introducing a substance into a subject. In general, any route of
administration may be utilized including, for example, parenteral
(e.g., intravenous), oral, topical, subcutaneous, peritoneal,
intraarterial, inhalation, vaginal, rectal, nasal, introduction
into the cerebrospinal fluid, or instillation into body
compartments. In some embodiments, administration is oral.
Additionally or alternatively, in some embodiments, administration
is parenteral. In some embodiments, administration is
intravenous.
[0057] "Analyte": As used herein, the term "analyte" broadly refers
to any substance to be analyzed, detected, measured, or quantified.
Examples of analytes include, but are not limited to, proteins,
peptides, hormones, haptens, antigens, antibodies, receptors,
enzymes, nucleic acids, polysaccharides, chemicals, polymers,
pathogens, toxins, organic drugs, inorganic drugs, cells, tissues,
microorganisms, viruses, bacteria, fungi, algae, parasites,
allergens, pollutants, and combinations thereof.
[0058] "Biocompatible": The term "biocompatible", as used herein is
intended to describe materials that do not elicit a substantial
detrimental response in vivo. In certain embodiments, the materials
are "biocompatible" if they are not toxic to cells. In certain
embodiments, materials are "biocompatible" if their addition to
cells in vitro results in less than or equal to 20% cell death,
and/or their administration in vivo does not induce inflammation or
other such adverse effects. In certain embodiments, materials are
biodegradable.
[0059] "Biodegradable": As used herein, "biodegradable" materials
are those that, when introduced into cells, are broken down by
cellular machinery (e.g., enzymatic degradation) or by hydrolysis
into components that cells can either reuse or dispose of without
significant toxic effects on the cells. In certain embodiments,
components generated by breakdown of a biodegradable material do
not induce inflammation and/or other adverse effects in vivo. In
some embodiments, biodegradable materials are enzymatically broken
down. Alternatively or additionally, in some embodiments,
biodegradable materials are broken down by hydrolysis. In some
embodiments, biodegradable polymeric materials break down into
their component polymers. In some embodiments, breakdown of
biodegradable materials (including, for example, biodegradable
polymeric materials) includes hydrolysis of ester bonds. In some
embodiments, breakdown of materials (including, for example,
biodegradable polymeric materials) includes cleavage of urethane
linkages.
[0060] "Carrier": As used herein, "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the compound is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water or aqueous solution
saline solutions and aqueous dextrose and glycerol solutions are
preferably employed as carriers, particularly for injectable
solutions. Suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin.
[0061] "Detector": As used herein, the term "detector" includes any
detector of electromagnetic radiation including, but not limited
to, CCD cameras, photodiodes, optical sensors, and infrared
detectors.
[0062] "Functionalization": As used herein, the term
"functionalization" refers to any process of modifying a material
by bringing physical, chemical or biological characteristics
different from the ones originally found on the material.
Typically, functionalization involves introducing functional groups
to the material. As used herein, functional groups are specific
groups of atoms within molecules that are responsible for the
characteristic chemical reactions of those molecules. As used
herein, functional groups include both chemical (e.g., ester,
carboxylate, alkyl) and biological groups (e.g., adapter, or linker
sequences).
[0063] In some embodiments, click reactive groups are used (for
`click chemistry`). Examples of click reactive groups include the
following: alkyne, azide, thiol (sulfydryl), alkene, acrylate,
oxime, maliemide, NHS (N-hydroxysuccinimide), amine (primary amine,
secondary amine, tertiary amine, and/or quarternary ammonium),
phenyl, benzyl, hydroxyl, carbonyl, aldehyde, carbonate,
carboxylate, carboxyl, ester, methoxy, hydroperoxy, peroxy, ether,
hemiacetal, hemiketal, acetal, ketal, orthoester, orthocarbonate
ester, amide, carboxyamide, imine (primary ketimine, secondary
ketamine, primary aldimine, secondary aldimine), imide, azo
(diimide), cyanate (cyanate or isocyanate), nitrate, nitrile,
isonitrile, nitrite (nitrosooxy group), nitro, nitroso, pyridyl,
sulfide, disulfide, sulfinyl, sulfonyl, sulfino, sulfo,
thiocyanate, isothiocyanate, caronothioyl, thione, thial,
phosphine, phosphono, phosphate, phosphodiester, borono, boronate,
bornino, borinate, halo, fluoro, chloro, bromo, and/or iodo
moieties.
[0064] "Image": The term "image", as used herein, is understood to
mean a visual display or any data representation that may be
interpreted for visual display. For example, a three-dimensional
image may include a dataset of values of a given quantity that
varies in three spatial dimensions. A three-dimensional image
(e.g., a three-dimensional data representation) may be displayed in
two-dimensions (e.g., on a two-dimensional screen, or on a
two-dimensional printout). The term "image" may refer, for example,
to an optical image, an x-ray image, an image generated by:
positron emission tomography (PET), magnetic resonance, (MR) single
photon emission computed tomography (SPECT), and/or ultrasound, and
any combination of these.
[0065] "Radiolabel": As used herein, "radiolabel" refers to a
moiety comprising a radioactive isotope of at least one element.
Exemplary suitable radiolabels include but are not limited to those
described herein. In some embodiments, a radiolabel is one used in
positron emission tomography (PET). In some embodiments, a
radiolabel is one used in single-photon emission computed
tomography (SPECT). In some embodiments, radioisotopes comprise
.sup.99mTc, .sup.111In, .sup.64Cu, .sup.67Ga, .sup.186Re,
.sup.188Re, .sup.153Sm, .sup.177Lu, .sup.67Cu, .sup.123I,
.sup.124I, .sup.125I, .sup.11C, .sup.13N, .sup.15O, .sup.18F,
.sup.186Re, .sup.188Re, .sup.153Sm, .sup.166Ho, .sup.177Lu,
.sup.149Pm, .sup.90Y, .sup.213Bi, .sup.103Pd, .sup.109Pd,
.sup.159Gd, .sup.140La, .sup.198Au, .sup.199Au, .sup.169Yb,
.sup.175Yb, .sup.165Dy, .sup.166Dy, .sup.67Cu, .sup.105Rh,
.sup.111Ag, .sup.89Zr, .sup.225Ac, and .sup.192Ir.
[0066] "Sample": The term "sample" refers to a volume or mass
obtained, provided, and/or subjected to analysis. In some
embodiments, a sample is or comprises a tissue sample, cell sample,
a fluid sample, and the like. In some embodiments, a sample is
taken from (or is) a subject (e.g., a human or animal subject). In
some embodiments, a tissue sample is or comprises brain, hair
(including roots), buccal swabs, blood, saliva, semen, muscle, or
from any internal organs, or cancer, precancerous, or tumor cells
associated with any one of these. A fluid may be, but is not
limited to, urine, blood, ascites, pleural fluid, spinal fluid, and
the like. A body tissue can include, but is not limited to, brain,
skin, muscle, endometrial, uterine, and cervical tissue or cancer,
precancerous, or tumor cells associated with any one of these. In
an embodiment, a body tissue is brain tissue or a brain tumor or
cancer. Those of ordinary skill in the art will appreciate that, in
some embodiments, a "sample" is a "primary sample" in that it is
obtained from a source (e.g., a subject); in some embodiments, a
"sample" is the result of processing of a primary sample, for
example to remove certain potentially contaminating components
and/or to isolate or purify certain components of interest.
[0067] "Subject": As used herein, the term "subject" includes
humans and mammals (e.g., mice, rats, pigs, cats, dogs, and
horses). In many embodiments, subjects are be mammals, particularly
primates, especially humans. In some embodiments, subjects are
livestock such as cattle, sheep, goats, cows, swine, and the like;
poultry such as chickens, ducks, geese, turkeys, and the like; and
domesticated animals particularly pets such as dogs and cats. In
some embodiments (e.g., particularly in research contexts) subject
mammals will be, for example, rodents (e.g., mice, rats, hamsters),
rabbits, primates, or swine such as inbred pigs and the like.
[0068] "Therapeutic agent": As used herein, the phrase "therapeutic
agent" refers to any agent that has a therapeutic effect and/or
elicits a desired biological and/or pharmacological effect, when
administered to a subject.
[0069] "Treatment": As used herein, the term "treatment" (also
"treat" or "treating") refers to any administration of a substance
that partially or completely alleviates, ameliorates, relives,
inhibits, delays onset of, reduces severity of, and/or reduces
incidence of one or more symptoms, features, and/or causes of a
particular disease, disorder, and/or condition. Such treatment may
be of a subject who does not exhibit signs of the relevant disease,
disorder and/or condition and/or of a subject who exhibits only
early signs of the disease, disorder, and/or condition.
Alternatively or additionally, such treatment may be of a subject
who exhibits one or more established signs of the relevant disease,
disorder and/or condition. In some embodiments, treatment may be of
a subject who has been diagnosed as suffering from the relevant
disease, disorder, and/or condition. In some embodiments, treatment
may be of a subject known to have one or more susceptibility
factors that are statistically correlated with increased risk of
development of the relevant disease, disorder, and/or
condition.
[0070] Drawings are presented herein for illustration purposes, not
for limitation.
BRIEF DESCRIPTION OF DRAWINGS
[0071] The foregoing and other objects, aspects, features, and
advantages of the present disclosure will become more apparent and
better understood by referring to the following description taken
in conjunction with the accompanying drawings, in which:
[0072] FIGS. 1A-1B depict preparation of polycarbodiimide-SWCNT
complexes.
[0073] FIG. 1A shows synthesis of polycarbodiimide polymers
(Poly-1-8).
[0074] FIG. 1B depicts an exemplary scheme showing preparation of
the polycarbodiimide-SWCNT aqueous suspension.
[0075] FIGS. 2A-2F show optical and morphological properties of
polycarbodiimide-SWCNTs.
[0076] FIG. 2A shows vis-nIR absorption spectra.
[0077] FIG. 2B shows nIR emission spectra of
polycarbodiimide-SWCNTs (16 mg/L nanotubes) excited at 659 nm.
[0078] FIG. 2C shows center wavelengths of nanotube emission peaks
collected from photoluminescence excitation/emission profiles of
polycarbodiimide-SWCNTs and surfactant suspended SWCNTs.
[0079] FIG. 2D shows atomic force micrograph of Amine-Poly-8-SWCNT
complexes showing periodic banding along the nanotube surface.
[0080] FIG. 2E shows a magnified AFM image of a single
Amine-Poly-8-SWCNT complex.
[0081] FIG. 2F shows a height profile of a single complex denoted
by the white arrow in FIG. 2E.
[0082] FIGS. 3A-3B show two-dimensional near-infrared
photoluminescence excitation/emission (PLE) plots showing
normalized emission intensity from polycarbodiimide-SWCNTs and
surfactant suspended SWCNTs (SDS and SDC) as a function of
excitation wavelength.
[0083] FIGS. 4A-C show atomic force microscopy and transmission
electron microscopy images of polycarbodiimide-SWCNTs.
[0084] FIG. 4A shows atomic force microscopy height images of
Amine-Poly-6-SWCNT.
[0085] FIG. 4B shows atomic force microscopy height images of
Amine-Poly-8-SWCNTs.
[0086] FIG. 4C shows atomic force microscopy height images of
transmission electron microscopy images of
polycarbodiimide-SWCNTs.
[0087] FIGS. 5A-5G show reversible inter-nanotube Forster resonance
energy transfer (INFRET) in polycarbodiimide-SWCNTs.
[0088] FIG. 5A shows a schematic representation of the INFRET
process and its reversal upon addition of amine-functionalized
polycarbodiimide.
[0089] FIG. 5B shows photoluminescence excitation-emission (PLE)
map of Amine-Poly-6-SWCNTs, Carboxy-Poly-7-SWCNTs, mixture of
Amine-Poly-6-SWCNTs and Carboxy-Poly-7-SWCNTs, and the mixture
after subsequent addition of amine-polymer.
[0090] FIG. 5C shows a nanotube (n, m) species-dependent PL
intensity change upon initiating INFRET.
[0091] FIG. 5D shows a nanotube (n, m) species-dependent PL
intensity change upon INFRET reversal.
[0092] FIG. 5E shows individual spectra acquired during a time
course acquisition of INFRET kinetic data. Intensity was normalized
to the area under the curve.
[0093] FIG. 5F shows INFRET dynamics show a monotonic relative PL
intensity increase in small bandgap nanotubes (Peaks 4 and 5) and
simultaneous relative PL intensity decrease in large bandgap
nanotubes (Peaks 1-3).
[0094] FIG. 5G shows INFRET ratio, plotted using Peak 5 as the
acceptor and Peak 1 as the donor. The final data point was acquired
after initiating INFRET reversal using amine-polymer.
[0095] FIG. 6A shows near-infrared images of
polycarbodiimide-SWCNTs immobilized on glass surfaces showing
discrete fluorescent nanotubes (left and middle panels). Dilute
solutions were placed on 35 mm glass bottom petri dishes for 10
seconds and excess solution was removed prior to imaging the
nanotubes on the surface. The right panel shows nIR fluorescent
aggregates of polycarbodiimide-SWCNTs after mixing the two nanotube
complexes in solution. Carboxy-Poly-7-SWCNT was added to
Amine-Poly-6-SWCNT (1:1 ratio) and left to stand for 10 sec; excess
solution was removed from the surface prior to imaging.
[0096] FIG. 6B shows height projection near-infrared image of
polycarbodiimide-SWCNTs aggregates, generated by 3D deconvolution
of a stack of images acquired in 10 .mu.m Z-steps.
[0097] FIG. 6C shows PL Intensity change upon initiating and
reversing self-assembly of Amine-Poly-7-SWCNTs and
Carboxy-Poly-8-SWCNTs.
[0098] FIGS. 7A and 7A-1 show that the kinetic data from FIG. 5F
was fit with a logistic function (of the form
y = A 1 - A 2 1 + ( x x 0 ) p + A 2 ##EQU00001##
to obtain the parameters in the accompanying table.
[0099] FIG. 7B shows the curves of peaks P1 and P5 fit the
classical solutions for the reactant and product, respectively, in
a consecutive series of first order chemical reactions.
[0100] FIGS. 8A-8C show near infrared (nIR) fluorescence images of
living HeLa cells incubated with polycarbodiimide-SWCNTs.
[0101] FIG. 8A, comprising panels A1-A5, shows cells incubated with
specified polymernanotube complexes at 37.degree. C. for 18 h.
Panel A5, for example, is a micrograph of cells incubated at
4.degree. C. for 4 h. A 730 nm laser was used for excitation and
light was collected over 900 nm-1400 nm.
[0102] FIG. 8B, comprising panels B1-B5, shows combined nIR
fluorescence and brightfield images of living HeLa cells.
[0103] FIG. 8C, comprising panels C1-C2, shows brightfield, nIR
fluorescence and Hoechst nuclear stain of live HeLa cells incubated
in the presence of Amine-Poly-8-SWCNTs and Guanidine-Poly-4-SWCNTs,
respectively.
[0104] FIGS. 9A-9B show near-infrared fluorescence images of live
HeLa cells incubated with Guanidine-Poly-4-SWCNTs.
Near-infrared--bright-field overlays illustrate nuclear
localization.
[0105] FIG. 10 shows viability assays on HeLa cells incubated with
polycarbodiimide-SWCNT complexes. HeLa cells were incubated in the
presence of specified polymer-nanotube complexes in a 35 mm petri
dish for 24 hours before imaging. Tali.TM. Image-Based Cytometer
was used to measure cell viability test performed using Tali.TM.
viability kit-Dead cell red (Invitrogen) following manufacturer's
protocol.
[0106] FIG. 11 shows near-infrared and brightfield overlay
micrographs of human skin, showing the lack of penetration of
polycarbodiimide-SWCNTs, regardless of functionalization.
Polycarbodiimide-SWCNTs deposits mainly in the stratum corneum
layer of the skin. Skin was acquired from human patients after
Moh's surgery and immediately incubated with
polycarbodiimide-SWCNTs on the surface of the skin specimen. Skin
sections without nanotube treatment were used as control.
[0107] FIGS. 12A-12C show a synthesis scheme and molecular
structures for helical polycarbodiimide polymers as described
herein.
[0108] FIGS. 13A-13B depict schematics showing the manufacturing of
a nano-sensor dispersion, according to an illustrative
embodiment.
[0109] FIG. 13A shows representative scheme of sensor fabrication
into final sensor dispersion (top) and examples of applications for
the nano-sensor (bottom).
[0110] FIG. 13B shows example of sensor modification allowing
specific targeting to sub-cellular locations.
[0111] FIGS. 14A-14B show illustrative data from a nano-sensor
dispersion.
[0112] FIG. 14A shows data showing the linear response of a
nano-sensor to decreasing reduction potential mediated in a
buffered aqueous biological solution of cysteine and ascorbate as
major redox couples. Each marked line represents a unqiuely
responding chiral nanotube within the same sensor population.
[0113] FIG. 14B shows data showing different chiralities of the
sensor can act together to form a ratiometric fluorescence
response, thereby allowing quantitative measurement.
[0114] FIG. 15 shows the activities in liver, spleen, and kidney
ranged from 1-2% ID/g suggesting that clearance from blood
primarily occurred from the kidney, as typically hepatic/RES
clearance is slow in comparison and shows high and prolonged
uptake/retention in the liver and spleen (e.g. liposomes).
[0115] FIG. 16 shows representative images of polycarbodiimide
polymers with opiate substituent groups.
[0116] FIG. 17 shows synthesis of polymers: DFO-conjugated polymer
DFO-JBP1 and polymer without DFO, mPEG-JBP2. DFO-JBP1 was
synthesized to measure radiolabeling efficiency, in vitro
stability, and in vivo performance of polycarbodiimide polymers
without targeting ligands. mPEG-JBP2 serves as a negative control
to ensure ligand specific binding of radiometal, 89Zr. Polymer
probe design integrates targeting ligands, radiometal chelators,
and solubilizing groups in a single polymer chain.
[0117] FIGS. 18A-18C show radiolabeling of Polymers and Serum
Stability Tests.
[0118] FIG. 18A shows .sup.89Zr labeling of DFO-JBP1 showed greater
than 99% radiolabeling.
[0119] FIG. 18B depicts that a control polymer mPEG-JBP2 showed
negligible labeling (less than 3%). Radiolabeling was performed at
room temperature for 30 minutes.
[0120] FIG. 18C depicts that .sup.89Zr-labeled polymer, or
.sup.89Zr-DFO-JBP1, showed 99% stability in human serum at
37.degree. C. over seven days.
[0121] FIGS. 19A-19B show PET imaging and biodistribution of
.sup.89Zr-Radiolabeled Polymer, .sup.89Zr-DFO-JBP1.
[0122] FIG. 19A shows coronal PET images of .sup.89Zr-DFO-JBP1.
Healthy BALB/c mice (n=2) were intravenously administered
.sup.89Zr-DFO-JBP1 (.about.10 mg, .about.100 .mu.Ci, 250 mL saline)
and imaged at 2 h, 24 h, and 72 h post injection. Initial time
points showed high activities in the blood, whereas at 72 h p.i
most of the activities were detected in the liver.
[0123] FIG. 19B shows ex vivo biodistribution of .sup.89Zr-DFO-JBP1
in select major organs. At 96 h p.i, the mice (n=2) were
sacrificed, organs were extracted, and radioactivities and organ
weights were measured and expressed as the % injected dose per gram
of tissue.
[0124] FIG. 20 shows synthesis of urea derivatives, monomers, and
corresponding polymers as described herein.
[0125] FIGS. 21A-21B show the FTIR spectra of polymers, Poly-1 and
Poly-2, respectively.
[0126] FIG. 22 shows azide compounds 5-7 that were synthesized and
characterized in certain embodiments as described herein.
[0127] The features and advantages of the present disclosure will
become more apparent from the detailed description set forth below
when taken in conjunction with the drawings, in which like
reference characters identify corresponding elements throughout. In
the drawings, like reference numbers generally indicate identical,
functionally similar, and/or structurally similar elements.
DETAILED DESCRIPTION
[0128] Throughout the description, where compositions are described
as having, including, or comprising specific components, or where
methods are described as having, including, or comprising specific
steps, it is contemplated that, additionally, there are
compositions of the present invention that consist essentially of,
or consist of, the recited components, and that there are methods
according to the present invention that consist essentially of, or
consist of, the recited processing steps.
[0129] It should be understood that the order of steps or order for
performing certain action is immaterial so long as the invention
remains operable. Moreover, two or more steps or actions may be
conducted simultaneously.
[0130] The mention herein of any publication, for example, in the
Background section, is not an admission that the publication serves
as prior art with respect to any of the claims presented herein.
The Background section is presented for purposes of clarity and is
not meant as a description of prior art with respect to any
claim.
[0131] Described herein are suspensions of helical polycarbodiimide
polymers that `cloak` nanotubes, thereby effecting control over
nanotube emission, providing a new mechanism of environmental
responsivity, and enabling precise control over sub-cellular
localization. The helical polycarbodiimide polymers described
herein are water soluble, easily modifiable, and have unique
architectures that facilitate their application in
radiopharmaceutical delivery and imaging methods, in therapeutics
and therapeutic delivery methods, and their use as sensors--both in
conjunction with carbon nanotubes, and without nanotubes.
[0132] For example, the helical polycarbodiimide polymers can be
modified with radionuclides or radionuclide-chelating agents.
Experiments performed with these polymers--for example,
DOTA-modified polymer with multiple chelation sites for
Lutetium-177--demonstrate rapid clearance and low organ update,
especially in the kidneys.
[0133] The helical polycarbodiimide polymers can also deliver
molecules and increase drug binding affinity via multivalency,
lending to their use as therapeutics and in therapeutic delivery,
for example, opiate-polymer conjugates that provide long-term
analgesic effects, as well as treatment of cancer, atherosclerosis,
skin disorders, infectious diseases, and other diseases. Due to the
semi-rigidity of the polymer, more binding sites are accessible,
compared with polymers having a globular form. Furthermore, the
helical polymer lengths are short and very controllable, allowing
for rapid clearance if desired.
[0134] Moreover, the helical polymers described herein are
demonstrated to encapsulate single-walled carbon nanotubes, which
are used as fluorescent sensors for in vitro, ex vivo, and in vivo
applications. The polymers provide both sensitivity to specific,
desired bioanalytes, and direct/target the sensors to specific
locations in the cell and body. Polymer-nanotube constructs are
shown that provide nuclear, cytosolic, and extracellular
localization. Moreover, a stable polymer-nanotube sensor is
presented for in vitro and in vivo redox potential
measurements.
[0135] In addition, the helical polymers described herein are
demonstrated to be radiolabeled and serve as multimodal targeted
molecular imaging probes for early cancer, such as pancreatic
cancer, detection. The polymers integrate multimeric targeting
ligands for receptors in cancer cells to achieve high tumor
specific uptake and retention, contain multiple chelators to
chelate multiple radiometals for enhanced specific activity and
quantitative PET imaging, and allow tunable hydrophilicity through
minimal structural changes to increase plasma stability, prolong
probe circulation in vivo, improve pharmacokinetics, and reduce
immunogenicity.
Example 1
Helical Polycarbodiimide Cloaking of Carbon Nanotubes
[0136] In the examples described herein, a platform of helical
polycarbodiimide polymers was synthesized to `cloak` the nanotubes
which affected control over nanotube emission, provided a new
mechanism of environmental responsivity, and enabled precise
control over sub-cellular localization. The helical polymers
exhibited ordered surface coverage on the nanotubes, allowed
systematic modulation of nanotube optical properties, and produced
up to 12-fold differences in photoluminescence efficiency. The
polymers facilitated controllable and reversible inter-nanotube
Forster resonance energy transfer, allowing kinetic measurements of
dynamic self-assembly and disassembly. Tailored polycarbodiimide
substituent groups also enabled sub-cellular targeting for imaging,
including stable translocation of photoluminescent nanotubes within
live cell nuclei.
[0137] Synthetic helical polymers mimic the basic structural motifs
of vital biomolecules such as DNA and peptides. The functions of
helical polymers depend on conformation, chain flexibility, and on
the array of functional moieties along the polymer backbone.
Polycarbodiimides are synthetic helical polymers with tunable
properties. Polycarbodiimide regioregularity is shown through
.sup.15N-isotope labeling studies demonstrating precise control of
the polymer microstructure and post-modification in a regioregular
polycarbodiimide, resulting in a polymer chain with a regular array
of functional side chains.
[0138] A modular polycarbodiimide polymer system is presented
herein that cloaks nanotubes in repeating chemical functional units
and suspends pristine nanotubes in aqueous solutions. Alkyne
polycarbodiimides (Poly-1 and Poly-2) were synthesized and organic
azides were subsequently coupled to terminal alkyne groups in these
polymers via Cu(I) catalyzed alkyne-azide cycloaddition, as
depicted in FIG. 1A. Side chains in these polymers such as primary
amines, carboxylic acids, guanidine groups, and oligoethylene
glycols were incorporated to mimic side chains in polylysines,
polyglutamic acids, and polyarginines, and to increase water
solubility. Additionally, aromatic groups were incorporated in each
monomer substituent 3 and 4 (FIG. 1A) to promote multi-valent
.pi.-.pi. interactions between the polymer and the graphitic
sidewall of SWCNTs. Raw SWCNTs (Unidym, HiPCO) were sonicated in
the presence of a polycarbodiimide from the library (Poly 3-8) to
render them soluble in an aqueous solution. The insoluble materials
were pelleted via ultracentrifugation and removed, yielding a dark
aqueous supernatant (FIG. 1B). Excess free polymer was then removed
from the suspensions by centrifugal filtration. The aqueous
suspensions were stable under ambient conditions for several
months, with no visible aggregation.
[0139] Polycarbodiimide-SWCNT complexes were characterized by
absorption spectroscopy in the vis-nIR region. Absorption spectra
of all polycarbodiimide-SWCNT complexes in FIG. 2A shows
characteristic E.sub.22 and E.sub.11 transition features of
semi-conducting SWCNTs. Sharp, discrete peaks in the absorption
spectra are indicative of well-dispersed nanotubes. The
photoluminescence efficiencies of the polymer-nanotube complexes
varied with the encapsulating polymer. FIG. 2B shows the
photoluminescence intensities from polycarbodiimide-SWCNTs
differing up to 12-fold, depending on the polymer substituent
functional group as well as the polymer microstructure. Such trends
are similar to findings reported for DNA-encapsulated SWCNTs.
[0140] Two-dimensional photoluminescence excitation/emission (PLE)
spectroscopy was conducted on polycarbodiimide-SWCNTs by recording
emission spectra upon varying the excitation wavelength, as
described below. Fourteen distinct nanotube species detected in 2D
PLE plots on polycarbodimide-SWCNT complexes (FIGS. 3A and 3B) were
assigned (n, m) chirality indices. Excitation and emission
wavelength maxima, collected from the PLE plots fell within a
narrow range which was red-shifted relative to surfactant-suspended
SWCNT emission (FIG. 2C).
[0141] Atomic force microscopy (AFM) and transmission electron
microscopy (TEM) were conducted to characterize
polycarbodiimide-SWCNT morphology (FIGS. 2D-2F, FIGS. 4A-4C).
Images of Amine-Poly-8-SWCNTs, deposited and dried on freshly
cleaved mica surface, show a distinct, periodic banding pattern
along the nanotube surface. The patterns exhibit a spacing of
.about.20 nm along the nanotube axis and band heights up to
.about.0.8 nm-0.5 nm above the surface of the nanotubes. Without
having to be bound by theory, these observations, coupled with the
long-term stability of the polymer-nanotube suspension, suggest a
uniform conformation of these aromatic polymers along the SWCNTs.
These AFM micrographs are comparable to those from DNA encapsulated
SWCNTs, where a regular banding pattern of DNA strands with a pitch
of 14-20 nm along the nanotubes have been reported. Without having
to be bound by theory, based on this regular pattern and the
similarity to the pattern in DNA-SWCNTs which are predicted (by all
atom molecular dynamics (MD) simulations) to helically wrap
nanotubes via the .pi.-.pi. interactions, the polymer also likely
helically-encapsulates the nanotubes.
[0142] Forster resonance energy transfer (FRET), also described as
exciton energy transfer (EET) in SWCNTs, has been observed between
adjacent semiconducting nanotubes in van der Waals contact wherein
large band gap donors transfer energy to smaller band gap
acceptors. In small bundles, a center to center distance of 1-4 nm
between nanotubes was shown to optimize energy transfer in SWCNTs.
With a functionally-diverse set of polymer-SWCNTs in hand, the
possibility of inter-nanotube Forster resonance energy transfer
(INFRET) events between individually-encapsulated nanotubes in
aqueous solutions was investigated. FIG. 5A is a schematic
representation of the process. FIG. 5B shows 2D PLE plots of two
oppositely-charged polymer-nanotube complexes (zeta potential
values 67.93.+-.2.73 mV for Amine-Poly-6-SWCNTs and -62.93.+-.1.28
mV for Carboxy-Poly-7-SWCNTs) and the resulting mixture. Complexes
were chosen to take advantage of strong coulombic attraction
between basic primary amine groups and acidic carboxylic acid
groups to bring nanotubes encapsulated in corresponding polymers
into a favorable distance for INFRET, without creating irreversibly
formed van der Waals bundles. Mixing the two polymer-SWCNT
complexes resulted in fluorescent aggregates (FIGS. 6A-6B). Overall
emission in PLE measurement decreased likely due to quenching
induced by metallic SWCNTs in aggregates. However, the emission
from smaller band gap SWCNTs increased with respect to that of
large band gap SWCNTs (FIG. 5B). Extra peaks appeared in the short
wavelength excitation/long wavelength emission range, a signature
of energy transfer. The relative intensity increase was found to
exhibit an (n, m) dependence which was virtually monotonic with
emission wavelength (FIG. 5C). Excess amine-functionalized
polycarbodiimide was later introduced to disrupt aggregation.
Addition of the free polymer resulted in a recovery of the original
relative PL intensities concomitant with the disappearance of large
aggregates (FIG. 5B, FIG. 6C). Plotting the net recovery of (n, m)
intensities showed a semi-monotonic trend with emission wavelength
and apparent mod-dependent behavior (FIG. 5D).
[0143] Real-time measurements of INFRET dynamics illustrate that
the process is spontaneous, controllable, and reversible. Upon
mixing the aforementioned oppositely-charged nanotubes, the
fluorescence exhibited a monotonic decrease in PL intensities from
large bandgap nanotubes (Peaks 1-3, FIG. 5E) and simultaneous
relative increase from small bandgap nanotubes (Peaks 4 and 5, FIG.
5E). The relative fluorescence intensities of each peak, plotted
over time, illustrate the INFRET dynamics between large and small
band gap nanotubes (FIG. 5F). Each intensity-time curve was fit
with the logistic function to obtain the time for half-maximal
intensity change (FIGS. 7A and 7A-1), due to this function's use in
the approximation of protein aggregation kinetics. However, the
kinetics of Peak 1 and Peak 5 fit well as the reactant and the
final product in a series of first order forward reactions,
respectively (FIG. 7B). The first order behavior suggests that the
larger bandgap nanotubes within Peak 1 act almost purely as energy
donors and the smaller bandgap nanotubes within Peak 5 as energy
acceptors. After 110 minutes, amine-functionalized polycarbodiimide
polymer (0.5 mg/mL) was added and gently mixed, resulting in a
near-instantaneous reversal of INFRET back to the initial ratio.
Using the above information, The INFRET ratio, plotted as
I.sub.a/(I.sub.a+I.sub.d) where I.sub.a is the acceptor intensity
and I.sub.d is the donor intensity, was obtained using Peak 5 as
the acceptor and Peak 1 as the donor (FIG. 5G).
[0144] The finding demonstrates FRET produced between nanotubes not
contained within an irreversible bundle, but rather employing
coulombic attraction between polymers permitted spontaneous forward
and directed reversibility. Therefore, the described compositions
are useful, for example, for the measurement of dynamic
processes.
[0145] The biological fate of the polycarbodiimide-cloaked carbon
nanotubes was found to depend almost completely on the
encapsulating polymer substituent groups. Cellular interactions of
polycarbodiimide-SWCNTs in human cervical cancer cells (HeLa cells)
were investigated. Polycarbodiimide-nanotube complexes exhibited
substituent-dependent uptake and localization into specific
sub-cellular spaces (FIGS. 8A-8C). The cellular uptake was highly
diminished in the case of polyethylene glycol polymer pendant
groups (FIG. 8A, panel A1), comparable to lack of cellular uptake
of PEGylated gold nanoparticles.
[0146] Upon internalization, sub-cellular distribution of nanotubes
was dictated by the nature of the encapsulating polymer substituent
groups. The anionic Carboxy-Poly-7-SWCNTs accumulated in
perinuclear areas (FIG. 8A, panel A2), resembling the cellular
distribution of DNA-encapsulated nanotubes. The two
amine-functionalized polymer-nanotube constructs exhibited
sub-cellular distribution profiles where a small fraction localized
within the nucleus and the majority situated elsewhere within the
cell (FIGS. 8A, panel A3 and 8C, panel C1). Nanotubes encapsulated
in polymers with guanidine side chains localized almost completely
within the nuclear region (FIGS. 8A, panel A4 and 8C, panel C2,
FIGS. 9A and 9B) as confirmed by co-localization of nIR fluorescent
nanotubes with Hoechst nuclear dye (Molecular Probes). The nuclear
translocation of cargoes by polyarginines and their derivatives has
been attributed to the presence of guanidine moieties. Multiple
copies of adjacent guanidine side chains in Guanidine-Poly-4
presumably mimicked polyarginine side chains, delivering their
encapsulated nanotube cargos into the nuclei. The energy-dependence
of complex internalization was confirmed by the lack of noticeable
cellular uptake upon incubation at 4.degree. C. (FIG. 8A, panel
A5). Under experimental conditions, the polymer-SWCNTs posed no
obvious toxicity to cells (viability greater than 90%, FIG.
10).
[0147] Micrographs were obtained to determine whether the variable
surface chemistries of polycarbodiimide-SWCNTs allow penetration of
intact human skin tissue topical exposure. The micrographs show
accumulation of all tested polymer-nanotube complexes on the
stratum corneum, the outermost layer of the skin, without evident
penetration (FIG. 11).
[0148] Thus, the experiments show that non-covalent
functionalization of SWCNTs through encapsulation in designed
helical polycarbodiimides forms water soluble, well-dispersed, and
nIR fluorescent nanotubes that are stable under ambient conditions.
The polymers, used in certain embodiments as described herein,
demonstrated controllable, reversible inter-nanotube FRET, enabling
a mechanism for switchable biomolecular probes and sensors. The
polymers, as used in certain embodiments as described herein, also
demonstrate a system substituent-dependent sub-cellular
localization of nanotubes, including stable localization in cell
nuclei.
Example 2
Synthesis of Helical Polycarbodiimide Polymers, for Use in
Therapeutic and Diagnostic Applications
[0149] A synthesis scheme and molecular structures for helical
polycarbodiimide polymers described herein are presented in FIGS.
12A-12C.
[0150] For synthesis of urea derivatives, a primary amine compound
(RNH.sub.2) (1.0 equiv) was diluted in anhydrous dichloromethane
and added to an isocyanate compound (RNCO) (1.2 equiv) in
dichloromethane, stirred at low temperature, and kept cold in an
ice bath. The reaction mixture was stirred at room temperature or
refluxed overnight until the completion of the reaction. The
solvent was removed in a rotary evaporator and crude white solid
was purified by recrystallization in ethanol at 4.degree. C. and
dried to obtain white crystalline solid.
[0151] For synthesis of carbodiimide monomers, triethyl amine (2.5
equiv) was added to a suspension of dibromotriphenylphosphorane
(1.2 mol equiv) in dichloromethane at low temperature and the
reaction mixture was stirred at low temperature under inert
atmosphere for 5 minutes. A urea derivative (1.0 equiv) was added
to the reaction mixture and stirred until completion. The
dehydration of the urea derivative into carbodiimide monomer was
monitored by the formation of a very strong FTIR signal at
.about.2120-2140 cm.sup.-1. Upon completion of the reaction, hexane
was added to precipitate side products. The monomer compound was
then extracted from the solid by hexanes. Crude monomer was further
purified by column chromatography on silica gel using ethyl
acetate:hexanes (1:2) and dried under reduced pressure to obtain a
carbodiimide monomer as a colorless oil.
[0152] The catalyst was synthesized and characterized as described
in Tang, H.; Boyle, P.; Novak, B., Chiroptical switching
polyguanidine synthesized by helix-sense-selective polymerization
using
[(R)-3,3'-dibromo-2,2'-binaphthoxy](di-tert-butoxy)titanium(IV)
catalyst. Journal of the American Chemical Society 2005,
2136-2142.
[0153] The polymers were synthesized following the procedure
described in Budhathoki-Uprety, J.; Novak, B., Synthesis of
Alkyne-Functionalized Helical Polycarbodiimides and their Ligation
to Small Molecules using `Click` and Sonogashira Reactions.
Macromolecules 2011, 44 (15), 5947-5954. Briefly, the catalyst,
either neat or dissolved in chloroform (0.2 mL per 500 mg monomer)
was added to the monomer at room temperature and under inert
atmosphere. The reaction mixture turned to dark red and solidified
to an orange red solid. The polymerization process was monitored in
FTIR by disappearance of IR signals from carbodiimide
(.about.2140-2120 cm.sup.-1) and formation of new IR absorption at
.about.1620-1640 cm.sup.-1 of the polymer backbone. Upon completion
of the polymerization (ca. 24 h), the solid was dissolved in
chloroform, precipitated in methanol, separated, and dried to
obtain light yellow solid.
[0154] Organic azides were coupled to the polymers via `click`
chemistry. To the stirring polymer solution in tetrahydrofuran
under inert atmosphere, azide compound (1.0 mol equiv per alkyne
unit), triethyl amine or DBU (6.0 mol equiv per alkyne unit) and
CuI (10 mol %) were added. The reaction mixture was stirred
overnight under an argon atmosphere. Coupling of small molecules
azides to alkyne side chains in polymers was monitored by FTIR
analysis. Upon completion of the reaction, the resulting polymer
was washed with THF and/or diethyl ether, separated by filtration
and dried under reduced pressure. Basic polymers were acidified
with a few drops of dilute HCl and carboxylic acid functionalized
polymer was treated with a few drops of saturated solution of
NaHCO.sub.3 to increase water solubility. Acidic and basic polymer
solutions were then filtered through centrifugal filters (Amicon
Ultracel.RTM., MWCO 3K Da, Merck Millipore Ltd) to remove residual
small molecules and washed with water until free from free acid or
base as tested with litmus paper. The polymers were then used to
suspend SWCNTs.
Example 3
Nanoscale Sensors for Quantitative Redox Potential Measurement
[0155] Reduction potential (or Redox) is a physical concept used to
measure the tendency of chemical compounds (couples) to transfer
electrons during a reaction, and by extension the chemical
potential energy in a system or couple. The direction, regulation,
and capacity for cellular activity depends upon the state of these
redox reactions, quantifiable with an electric potential voltage,
for phenomena as diverse as energy production, biosynthesis, gene
expression, signaling and detoxification. Redox Biology currently
remains largely qualitative. Recent linkage between perturbations
in redox state and cancerous transformation, cell growth and
division, cell viability, drug efficacy, and numerous pathologies
have increased interest in quantitative Redox Biology.
[0156] In certain embodiments, the compositions described herein
allow for a Single-Walled Carbon Nanotube (SWCNT) based optical
sensor for this purpose. Current art is not capable of measuring
this parameter in living samples or using materials that have
commercialization capability for wide spread use across diverse
markets.
[0157] In certain embodiments, this sensor utilizes the optical
fluorescence properties of SWCNTs dispersed with a unique
Polycarbodiimide (JB-2-18 or JB-2-104) which enables the aqueous
dispersed SWCNT to assume an electronic structure responsive to
voltage change in the physiologically relevant redox potential
range of approximately -150 millivolts to -400 millivolts. The
ability of SWCNTs to sensitively respond to applied voltage has
been tested and modeled in non-aqueous systems. The sensor directly
measures this parameter within aqueous systems using non-invasive
near infrared fluorescence emission.
[0158] The link between redox state and disease makes accurate
measurement of redox increasingly important, both as a direct
mechanism of pathology or as an indirect biomarker for screening.
Human pathologies linked to aberrant redox state at either the
mechanistic or biomarker level include, but are not limited to,
sepsis, renal disease, cardiovascular disease, cancer
carcinogenesis and therapy, inflammation, Alzheimer's disease,
Parkinson's disease, Traumatic Brain Injury, Autism,
atherosclerosis, Schizophrenia and Bipolar Disorder, Metabolic
disorders, wounding and tissue regeneration, skin and cellular
aging, skin damage and carcinogenesis, and gastrointestinal
inflammation and disease. All basic research applications on
various diseases can benefit greatly from a commercial tool for the
measurement of redox potential for discovery of mechanisms,
biomarkers, and screening therapies.
[0159] Current and future biomarkers relating disease to aberrant
redox potentials or abnormal redox couples must be measured and
detected in clinical chemistry laboratories for patient
diagnostics. In certain embodiments, this sensor is useful as a
measurement tool for diagnosing patients in clinical settings. The
sensors are non-degradable and require no special storage, reducing
the need for upkeep of traditional machinery and biochemical tools
like antibodies and enzymes.
[0160] Given increasing evidence for the role of oxidants and redox
couples in skin aging, skin cancer, and skin damage, in certain
embodiments, an available microneedle delivery process delivers
nano-sensors to, and embeds within, the epidermal layers of the
skin for constant monitoring of extracellular redox. For example,
current delivery platforms are commercially available from 3M
Company. Measurement of redox via fluorescence emission is obtained
with light directed at the skin at wavelengths innocuous to tissue.
This gives consumers and physicians the ability to track skin
exposure and damage from oxidants/chemicals and radiation, tracking
of possible pre-cancerous abnormalities, and/or determination of
post-cancer treatment efficacy and progression. Furthermore,
because epidermal skin is constantly shed in 2-3 week cycles, this
sensor is temporary, therefore affording no personal risk.
[0161] In other embodiments, these nano-sized sensors are
fabricated on chip platforms and integrated with technology thereby
giving consumers the ability to measure the redox potential of
consumer products or solutions in daily life in a mobile fashion
where, for example, this sensor is integrated into smart phone
platforms as an additional plug-in application and attachment.
Furthermore, physicians can similarly use this technology as a
quick tool to analyze fluids. The redox potential of fluids, for
example, changes markedly if pathogens are present and
proliferating. Given that many consumer products on the market are
formulated with strict chemical constituents or various solutions
(skin care products, foods) the redox potential measured can
indicate the quality or harm of a product and its stability over
time.
[0162] Industrial and process engineering sectors require
measurement of redox potential to monitor solution quality of dyes,
foods, chemicals, microorganism growth media, and cosmetics. For
example, fermentation of yeast for industrial scale alcoholic
beverage production requires quality control including redox
measurement of samples at various stages of development. Similar
process control exists for other industries. Most of these
measurements currently require large expensive probes and
machinery. Furthermore, the volume of sample taken from production
to measure can be significant. In certain embodiments, nano-sensors
described herein, developed in the redox range of interest, can be
used to continuously monitor this measurement in real time and
decrease significantly the volume needed for measurement.
[0163] In an experimental example, a nano-sensor was fabricated by
mixing Single-Walled Carbon Nanotubes (SWCNTs), available from
various distributors, in a 1:10 ratio by weight with
polycarbodiimide, solubilized in water. The mixture is then probe
tip sonicated at 30% amplitude and approximately 4-5 Watts for 20
minutes. This resulting dispersion is then worked up:
ultracentrifugation for 30 minutes, cut-off filtration using a
benchtop centrifuge
[0164] 2-3 times for 6 minutes each, re-dilution in water, and a
final benchtop centrifugation at maximum force for 20 minutes. The
resulting dispersion is ready for use, but may be subjected to an
additional optional step.
[0165] Using the sensor merely requires addition of the final
dispersion into the medium to be measured, or into the cell culture
media for incubation and uptake via cellular processes. The
concentration of the sensor dispersion can be gathered by taking
the absorption of the solution at 630 nm, and dividing the valley
by a known coefficient, for a result in mg/L.
[0166] In certain embodiments, detection of fluorescence emission
requires an excitation source, preferentially a laser, at a
wavelength near the resonant absorption of the proper nano-sensor
chirality. Unlike organic fluorophores, nanotubes absorb
off-resonant light; therefore, many lasers commonly used today are
compatible. As with other optical tools, an appropriate filter set
and infrared camera are used to detect the emission signal.
[0167] Analysis of data is similar to analysis of other
fluorescence data currently in use. Information with nanotubes is
usually gathered as spectra where differences, intensity, and
chromatic shifting in peaks are analyzed, or via tracking of
individual sensors in microscopy, whereby spectral and spatial
information is collected from samples (i.e. cells).
[0168] FIGS. 13A-13B depict schematics showing the manufacturing of
a nano-sensor dispersion, according to an illustrative embodiment.
FIGS. 14A-14B show illustrative data from a nano-sensor dispersion.
As shown in FIG. 14A, the data show the linear response of a
nano-sensor to decreasing reduction potential, mediated in a
buffered aqueous biological solution of cysteine and ascorbate as
major redox couples. Each marked line represents a uniquely
responding chiral nanotube within the same sensor population. As
shown in FIG. 14B, the data shows that different chiralities of the
sensor act together to form a ratiometric fluorescence response,
thereby allowing quantitative measurement.
Example 4
Helical Polymers for Pretargeted Radioimmunotherapy
[0169] Recently Orcutt et al. reported a novel scFv antibody
("C825") with pM affinity for low molecular weight (MW)
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)
complexes with various metallic lanthanides including yttrium (Y)
and lutetium (Lu) (Orcutt K D, Slusarczyk A L, Cieslewicz M,
Ruiz-Yi B, Bhushan K R, Frangioni J V, et al. Engineering an
antibody with picomolar affinity to DOTA chelates of multiple
radionuclides for pretargeted radioimmunotherapy and imaging.
Nuclear medicine and biology. 2011; 38:223-33.). Specifically
intended for pretargeted radioimmunotherapy (PRIT), Orcutt and
colleagues also prepared bi-specific antibodies having the format
IgG-scFv which incorporated the sequences for C825, as well as
those for IgG antibodies with high affinity and specificity for
cancer cell-surface targets (e.g. carcinoembryonic (CEA) antigen).
During PRIT in vivo with the IgG-C825 constructs, the IgG-C825 was
initially administered and ample time was allowed for accumulation
at the tumor, followed with a clearing agent to remove freely
circulating IgG-C825. In the last step, a low-MW DOTA-hapten would
be administered, which would be recognized by prelocalized
IgG-C825. In order to obtain optimum therapeutic index, the
DOTA-hapten would show rapid blood clearance via the renal route,
as well as low non-specific uptake and retention in normal tissues,
including those associated with the reticuloendothelial system
(RES). With rapid clearance and minimum retention in tissues, the
residence time of the radioactivity is minimized, thus reducing the
absorbed dose to those tissues (and consequently limiting the
maximum tolerated dose). The biodistribution and clearance
properties of various DOTA-haptens have been described by Orcutt
and colleagues.
[0170] Experiments described herein show that the DOTA-Bn-polymers
can be radiolabeled with Lu-177 with radiochemical purities
sufficient for in vivo biodistribution studies. Radioactivity in
blood was 0.131.+-.0.125% ID/g at 2 hr post-injection, indicating
rapid clearance from circulation. As shown in FIG. 15, the
activities in liver, spleen, and kidney ranged from 1-2% ID/g
suggesting that clearance from blood primarily occurred from the
kidney, as typically hepatic/RES clearance is slow in comparison
and shows high and prolonged uptake/retention in the liver and
spleen (e.g. liposomes). In all other organs assayed (including
s.c. human tumor xenograft, heart, lungs, stomach, small and large
intestines, muscle, and bone), the radioactivity concentrations
were consistently less than 1% ID/g at 2 hr p.i. suggesting low
uptake and retention in those tissues.
[0171] These DOTA-Bn-polymers are useful not only because of their
favorable clearance and biodistribution properties, but also
because of their multivalent design (i.e. greater than 1
DOTA/polymer). It has been reported that there is improvement in
overall tumor uptake during pretargeted radioimmunotherapy with
radioactive bivalent "janus" haptens in comparison with monovalent
haptens. The DOTA/polymer stoichiometry allows for addition of a
radioactive DOTA-metal complex or for which C825 does not show pM
affinity (e.g. copper or actinium), followed by cold lutetium or
yttrium metal. This allows for the non-radioactive Y/Lu-DOTA
present on the polymer to serve as an affinity handle for antibody
recognition and capture. Pretargeting GD2-positive solid tumors in
mice with antibody-streptavidin fusions has been shown. However,
instead of using radioactive biotin as the targeting hapten, two
radiolabeled biotinylated peptides and radiolabeled and
biotinylated bovine serum albumin can also be effectively used.
Thus, small peptides and proteins can be targeted via biotinylation
and the pretargeting strategy.
[0172] As described herein, the DOTA-Bn-polymer was supplied as a
light yellow dry powder. A stock solution was prepared by adding
200 .mu.L of 0.5 M ammonium acetate pH 5.3 to 5 mg of
DOTA-Bn-polymer (25 mg/mL). The resulting solution appeared as a
suspension, and stored at -20.degree. C. To radiolabel with Lu-177,
20 .mu.L of the stock was added to an acid-washed plastic Eppendorf
tube, followed with 10 .mu.L of DMSO and an additional 100 .mu.L
0.5 M ammonium acetate pH 5.3 (e.g., to solubilize the
DOTA-Bn-polymer). To this solution, 11.55 mCi (427.4 MBq) of Lu-177
was added (as .sup.177LuCl.sub.3 in 0.05 N HCl, specific activity:
170 MBq/nmol; Perkin Elmer), the reaction was vortexed to mix, and
the reaction was incubated at 80.degree. C. for 90 min. To chelate
any remaining free metal, 15 .mu.L of 50 mM DTPA pH 7 was added,
and the reaction was allowed to incubate for an additional 10 min
at room temperature. To separate the .sup.177Lu-DOTA-Bn-polymer
from .sup.177Lu-DTPA, the crude reaction was applied to a PD-10
desalting column (Sephadex G-25; greater than 5000 M.sub.r; GE
Healthcare) that was pre-equilibrated with saline for injection,
and eluted with additional saline. According to the manufacturer,
the void volume is .about.2.5 mL, and the total column volume is
8.3 mL. The radioactivity concentrations in each elution fraction
as well as the column itself were determined by assay in a Capintec
CRC-25R dose calibrator using the manufacturer's recommended
settings for the isotope.
TABLE-US-00001 TABLE 1 Fraction Volume Lu-177 activity 1 reaction
(~160 .mu.L), 0 2 1 mL 0 3 1 mL 66.8 4 1 mL 436 5 0.5 mL 470 6 0.5
mL 674 7 0.5 mL 910 8 0.5 mL 1025
[0173] After collection of the 8.sup.th fraction (total
load+elution=.about.5.2 mL), the column was assayed in the dose
calibrator (7.68 mCi, 66% of applied radioactivity). The
radiochemical purity (RCP) of fractions 4 and 5 were assayed by
thin-layer chromatography (Baker-flex Silica Gel IB-F; elution
solvent 1/1 methanol/10% sodium acetate (aq);
.sup.177Lu-DOTA-Bn-polymer R.sub.f=0.125-0.15, .sup.177Lu-DTPA
R.sub.f.about.1). The plate radioactivity was assayed using a
Bioscan radioTLC scanner. Fraction 4 showed a single peak with an
R.sub.f=0.125, while fraction 5 showed 2 peaks (major peak: 86.1%
of total radioactivity on plate R.sub.f=0.125; minor peak: 13.9% of
plate radioactivity R.sub.f=0.15). Fraction 3 was assumed to have
the same radiochemical purity as fraction 4. Fractions 3, 4, and 5
were combined for injection (overall RCP .about.90% of radioactive
species with R.sub.f=0.125). For injection, doses comprising of
82.4-90.4 .mu.Ci of .sup.177Lu-activity (presumably as
.sup.177Lu-DOTA-Bn-polymer) were formulated in 200 .mu.L final
volume of saline.
[0174] Two groups (n=5/group) of athymic nu/nu female nude mice
(6-8 weeks old; Harlan Sprague Dawley) bearing IMR32-Luc
subcutaneous xenografts in the lower flank (average size 1.47 g or
1.39 cm.sup.3 assuming a density of 1.05 g/mL) were injected
intravenously with .sup.177Lu-DOTA-Bn-polymer using the tail vein.
One of the groups was sacrificed 2 hr post-injection (p.i.) and the
other at 24 hr p.i. for ex vivo assay of radioactive
biodistribution. Mice were euthanized, and tumor and selected
organs were harvested, weighed, and radioassayed by gamma
scintillation counting (Perkin Elmer Wallac Wizard 3"). Count rates
were converted to activities using a system calibration factor,
decay corrected and normalized to the administered activity, and
expressed as percent injected dose per gram (% ID/g).
Example 5
Opiate Polycarbodiimide Conjugates for Drug Delivery and Peripheral
Analgesia
[0175] Two polycarbodiimide polymers containing opiate substituent
groups were synthesized.
[0176] The first (P32) was found to translocate rapidly into the
nuclei of certain cells. The polymer is able to translocate fully
into the nucleus within three hours after administration in vitro.
The construct is able to transport large materials, including
carbon nanotubes, into the nucleus. Representative images are shown
in FIG. 16.
[0177] The P33 construct was constructed to function as a
peripherally acting opiate analgesic with a preferable side-effect
profile over morphine (low euphoria, respiratory depression,
physical dependence, addiction), and long-lasting analgesic
efficacy above morphine. In vitro and in vivo data of both P32 and
P33 polymers are presented in Table 2 below.
TABLE-US-00002 TABLE 2 Binding in Opioid transfected CHO cell lines
Analgesia in CD1 mice given subcutaneously Ki (nM) MOR DOR KOR
6TM/E11 ED50 (mg/kg) P32 1.25 .+-. 0.34 13.11 .+-. 2.88 0.56 .+-.
0.01 12.67 .+-. 2.1 not analgesic P33 3.45 .+-. 0.55 5.88 .+-. 0.79
0.85 .+-. 0.18 10.75 .+-. 0.9 10 Terminology: MOR = Mu opiate
receptor DOR = Delta opiate receptor KOR = Kappa opiate receptor
6TM/E11 = 6 transmembrane domain E11 splice variant of MOR
Example 6
Radiolabeled Polymers as Multimodal Targeted Molecular Imaging
Probes for Early Pancreatic Cancer Detection
[0178] The present disclosure describes dual-modal positron
emission tomography (PET) and fluorescent imaging agents with
multimeric targeting ligands for enhanced receptor binding and
multiple radiometal chelators for improved signal and
high-resolution imaging. Molecular imaging probes based on the
disclosed polymer-conjugates are well suited for various
applications (e.g., cancer detection and therapeutics) because the
polymers described herein i) integrate multimeric targeting ligands
for receptors in cancer cells to achieve high tumor specific uptake
and retention, ii) contain multiple chelators to chelate multiple
radiometals for enhanced specific activity and quantitative PET
imaging, and iii) allow tunable hydrophilicity through minimal
structural changes to increase plasma stability, prolong probe
circulation in vivo, improve pharmacokinetics, and reduce
immunogenicity. The modular aspect of these `clickable` polymer
scaffolds allows for a library of derivatives to be quickly
synthesized to tune their in vivo properties, as described above.
In certain embodiments, a major advantage to these polymer
scaffolds is the ability to easily change the peptide to change the
molecular target and to change the chelator (e.g. DOTA instead of
DFO) to change the radiometal. Changing the targeting peptide
allows for these systems to target a theoretically limitless number
of molecular targets, and changing the radiometal allows for PET or
SPECT imaging with a variety of radiometals with different emission
properties and half-lives. Moreover, the disclosed polymer
scaffolds also provide opportunities for therapy using isotopes
such as .sup.177Lu and .sup.90Y.
[0179] In certain embodiments, one advantage of this modular
polymer scaffold is facile purification. For example, small peptide
conjugates typically require HPLC purification and subsequent
heating and solvent evaporation prior to formulation for injection.
However, these polymer systems reach molecular weights of 15-30
kDa, allowing for efficient purification using disposable size
exclusion columns (PD-10) and disposable spin-filters (Amicon).
This type of system is amenable for making kit formulations. As a
result, these systems can be deployed in a hospital radiopharmacy
setting, unlike conventional small peptide conjugates which require
HPLC purification by an expert radiochemist.
Synthesis of a DFO-Conjugated Polymer, Radiolabeling, and In Vitro
Stability Tests
[0180] Polycarbodiimide polymers conjugated with the radiometal
chelator, desferrioxamine B (DFO), a hexadentate ligand that
chelates .sup.89Zr under mild conditions, fluorophore (IR650 dye),
and PEG side chains (DFO-JBP1) were synthesized as shown in FIG.
17. .sup.89Zr PET tracer emits low energy positrons and exhibits a
relatively long half-life (78.41 h) that facilitates high imaging
resolution and allows imaging at multiple time points, for example
as described by Deri et al J. Med. Chem. 2014. The synthesized
DFO-conjugated polymer (DFO-JBP1) was efficiently (greater than
99%) radiolabeled with .sup.89Zr within 60 minutes at room
temperature. The radiochemical purity was greater than 99.9% as
determined from radio-iTLC as is shown in FIG. 18A. Without having
to be bound by theory, negligible radiolabeling in control
experiments with a similar polymer without DFO-conjugation
(mPEG-JBP2, FIG. 18B) suggests lack of non-specific labeling from
polymer backbone. Table 3 shows that the concentration dependent
.sup.89Zr labeling of DFO-JBP1 showed near quantitative
radiolabeling with 100 .mu.Ci activities in 10 .mu.g polymer.
TABLE-US-00003 TABLE 3 Concentration Dependent Radiolabeling
Polymer Initial 89Zr-Radiolabeling (%) weight (.mu.g) activity
(.mu.Ci) of DFO-JBP1 1 100 16.6 10 100 97.6 30 100 >99 100 100
>99 200 100 >99 400 100 >99
[0181] As shown in Table 3, the highest specific activities
detected were 13 mCi/mg polymers. Serum stability test on the
.sup.89Zr-radiolabeled polymer (.sup.89Zr-DFO-JBP1) in the presence
of human blood serum showed 98-99% stability over seven days (FIG.
18C). The .sup.89Zr radiolabeling of the polymer achieved high
specific activities (e.g., 13 mCi/mg) enabling a low dose injection
in mice.
PET Imaging and Biodistribution of Radiolabeled Polymer in Healthy
Mice
[0182] .sup.89Zr-labeled polymer without targeting ligands was i.v.
injected (.about.100 .mu.Ci, .about.10 .mu.g) into healthy mice
(BALB/c) and imaged at three time points (2 h, 24 h, and 72 h)
using PET (FIG. 19A). At 2 h post injection, the majority of
radioactivity was detected in the blood, and at 24 h significant
radioactivity was still detected in blood indicating a long
circulation time. Polymer derivatives with conjugated targeting
peptides can benefit from this long circulation time for higher
tumor uptake. Overtime, most radioactivity was detected in the
liver. The ex vivo biodistribution performed at 96 h post injection
matched the trend with PET images with the highest activity
detected in the liver (FIG. 19B).
Materials and Methods
Chemicals
[0183] Reagents were purchased from Sigma-Aldrich, Milwaukee, Wis.,
Acros Organics, and Fisher Scientific, Fair Lawn, N.J., and used as
received. Neutral silica gel (Ultrapure 60-200 .mu.m, 60 .ANG.,
Acros Organics) was used in column chromatography purification of
monomers. Anhydrous and inhibitor-free tetrahydrofuran (THF) was
used for click chemistry.
Material Characterization
[0184] NMR data were recorded on a Bruker Advance III Ultrashield
Plus 500 MHz spectrometer at room temperature. The chemical shift
values were reported relative to TMS (.delta.=0.00 ppm) as an
internal standard. Fourier transform infrared (FTIR) spectra were
acquired using a Bruker Optics Tensor 27 FTIR spectrometer using
ATR cell (Pike technologies). Wavenumbers in cm.sup.-1 are reported
for characteristic peaks. All manipulations for polymerization were
done at room temperature inside an MBraun UNIlab drybox under inert
atmosphere. High resolution mass spectra (HRMS) were obtained on a
Waters LCTPremier XE mass spectrometer by electrospray ionization.
Size exclusion chromatography (SEC) was performed on a Viscotek
GPCmax system (Malvern Instruments) equipped with ViscoGEL columns
(IMBMMW-3078 and I-MBLMW-3078 in series) connected to a Viscotek
TDA 305 triple detector array at 30.degree. C. using THF as an
eluent to determine relative molecular weights of the polymers.
Polystyrene standards were used for the calibration of the
instrument. Polymer samples were dissolved in the solvent system
containing 0.12 M diethanolamine in THF, and the solutions were
filtered through 0.45 .mu.m PTFE filters prior to injection. The
flow rate was 1.0 mL/min, and injector volume was 100 .mu.L.
OmniSEC software was used to calculate the molecular weight. The
polymer-SWCNTs zeta potential measurements were carried out in a
Zetasizer Nanoseries (Malvern Instruments).
Synthesis and Characterization of Compounds
[0185] Urea derivatives, monomers, and corresponding polymers (FIG.
20) were prepared as described below (Budhathoki-Uprety, J.; Novak,
B., Synthesis of Alkyne-Functionalized Helical Polycarbodiimides
and their Ligation to Small Molecules using `Click` and Sonogashira
Reactions Macromolecules 2011, 44 (15), 5947-5954). Molar ratio of
monomer to catalyst was limited to 25:1 (Poly-1) or 32:1 (Poly-2)
to obtain low molecular weight polymers to improve aqueous
solubility.
1-(3-ethynylphenyl)-3-propylurea, Compound 1
##STR00003##
[0187] 3-Amino phenylacetylene (1.0 g, 8.53 mmol, 1.0 equiv) was
diluted in anhydrous dichloromethane (25 mL) and added to
n-propylisocyanate (0.87 g, 10.24 mmol, 1.2 equiv) in
dichloromethane (10 mL), stirred at low temperature, and kept cold
in an ice bath. The reaction mixture was allowed to warm to room
temperature followed by reflux overnight. The solvent was removed
in a rotary evaporator and crude white solid was purified by
recrystallization in ethanol at 4.degree. C. and dried to obtain
white crystalline solid 1. 1H NMR (500 MHz, CDCl3, .delta. ppm):
reference TMS=0 ppm, .delta.=7.99 (s, 1H), 7.39 (s, 1H), 7.26 (d,
1H), 7.15-7.08 (m, 2H), 6.02 (s, br, 1H), 3.11-3.07 (m, 2H), 2.99
(s, 1H), 1.45-1.38 (m, 2H), 0.83 (t, J=7.5 Hz, 3H). 13C NMR (125
MHz, CDCl3, .delta. ppm): reference CDCl3=77.23 ppm, .delta.=156.9,
139.4, 129.1, 126.6, 123.4, 122.8, 120.7, 83.5, 77.3, 42.0, 23.4,
11.4. HRMS (ESI) [M+H]+m/z calcd for C12H15N2O, 203.1184. found,
203.1187.
1-phenyl-3-(prop-2-yn-1-yl)urea, Compound 2
##STR00004##
[0189] Propargyl amine (0.60 g, 10.89 mmol, 1.1 equiv) was diluted
in anhydrous dichloromethane (20 mL) and added to phenylisocyanate
(1.18 g, 9.90 mmol, 1.0 mol equiv) in dichloromethane (20 mL),
stirred at low temperature, and kept cold in an ice bath. The
reaction mixture was then allowed to warm to room temperature. A
white precipitate resulted shortly after mixing with
phenylisocyanate. The reaction mixture was allowed to stir for 3
hours. The white solid was then separated and purified by
recrystallization in dichloromethane at 4.degree. C. to obtain
white crystalline solid 2. 1H NMR (500 MHz, DMSO-d6, .delta. ppm):
reference DMSO-d6=2.50 ppm, .delta.=8.56 (s, 1H), 7.40 (d, J=7.65
Hz, 2H), 7.23 (t, J=7.60 Hz, 2H), 6.91 (t, J=7.35 Hz, 1H), 6.45 (t,
J=5.60 Hz, 1H), 3.90 (dd, J=5.70 Hz, 2.45 Hz, 2H), 3.09 (t, J=2.45
Hz, 1H). 13C NMR (125 MHz, DMSO-d6, .delta. ppm): reference
DMSO-d6=39.51 ppm, .delta.=154.7, 140.1, 128.6, 121.4, 117.8, 82.1,
72.9, 28.7. HRMS (ESI) [M+H]+m/z calcd for C10H11N2O, 175.0871.
found, 175.0863.
3-ethynyl-N-((propylimino)methylene)aniline, Compound 3
##STR00005##
[0191] Triethyl amine (2.07 g, 20.51 mmol, 2.5 equiv) was added to
a suspension of dibromotriphenylphosphorane (4.15 g, 9.84 mmol, 1.2
mol equiv) in dichloromethane (2 mL) and stirred at low temperature
under inert atmosphere. After stirring the mixture for 5 minutes,
compound 1 (1.66 g, 8.20 mmol, 1.0 equiv) was added and the
reaction mixture and stirred until completion. The dehydration of
the urea derivative into carbodiimide monomer was monitored by the
formation of a very strong FTIR signal at .about.2120-2140 cm-1.
Upon completion of the reaction, hexane was added to precipitate
side products. The monomer compound was then extracted from solid
by hexanes. Crude monomer was further purified by column
chromatography on silica gel using ethyl acetate:hexanes (1:2) and
dried under reduced pressure to obtain 3 as a colorless oil. 1H NMR
(500 MHz, CDCl3, .delta. ppm): reference TMS=0 ppm, .delta.=7.20
(m, 3H), 7.07-7.04 (m, 1H), 3.39 (t, J=6.8 Hz, 2H), 3.07 (s, 1H),
1.73-1.1.69 (m, 2H), 1.01 (t, J=7.4 Hz, 3H). 13C NMR (125 MHz,
CDCl3, .delta. ppm): reference CDCl3=77.23 ppm, .delta.=141.3,
129.5, 128.4, 127.1, 124.3, 123.3, 120.9, 83.2, 77.7, 48.7, 24.9,
11.6. FTIR (thin film, cm-1): characteristic absorption from
terminal alkyne group and monomer; 3290 (terminal alkyne), 2123
(vs, carbodiimide).
N-((prop-2-yn-1-ylimino)methylene)aniline, Compound 4
##STR00006##
[0193] The same procedure as described in the synthesis of compound
3 was employed. 1H NMR (500 MHz, CDCl3, .delta. ppm): reference
TMS=0 ppm, .delta.=7.31-7.28 (m, 2H), 7.16-7.14 (m, 3H), 4.08 (d,
J=2.45 Hz, 2H), 2.44 (t, J=2.50 Hz, 1H). 13C NMR (125 MHz, CDCl3,
.delta. ppm): reference TMS=0 ppm, .delta.=139.5, 139.0, 129.4,
125.5, 124.0, 79.0, 73.5, 36.0. FTIR (thin film, cm-1):
characteristic absorption from terminal alkyne group and monomer;
3302 (terminal alkyne), 2119 (vs, carbodiimide). HRMS (ESI) [M+H]+
m/z calcd for C10H9N2, 157.0766. found, 157.0761.
Synthesis of Catalyst
[0194] The catalyst was synthesized and characterized following
previously described procedure (Tang, H.; Boyle, P.; Novak, B.,
Chiroptical switching polyguanidine synthesized by helix-sense
selective polymerization using
[(R)-3,3'-dibromo-2,2'-binaphthoxy](di-tert-butoxy)titanium(IV)
catalyst. J. Am. Chem. Soc. 2005, 2136-2142).
Synthesis of Polymers
[0195] Polymers were synthesized following the reported procedure
1. Briefly, the catalyst, either neat or dissolved in chloroform
(0.2 mL per 500 mg monomer) was added to the monomer at room
temperature and under inert atmosphere. The reaction mixture turned
to dark red and solidified to an orange red solid. The
polymerization process was monitored in FTIR by disappearance of IR
signals from carbodiimide (.about.2140-2120 cm.sup.-1) and
formation of new IR absorption at .about.1620-1640 cm.sup.-1 of the
polymer backbone. Upon completion of the polymerization (ca. 24 h),
the solid was dissolved in chloroform, precipitated in methanol,
separated, and dried to obtain light yellow solid.
##STR00007##
[0196] FTIR (thin film, cm-1): characteristic absorption from
terminal alkyne group and polymer backbone; 3304 (terminal alkyne
C--H), 2123 (alkyne triple bond, C.ident.C), 1631 (imine in polymer
backbone, C.dbd.N). 1H NMR (500 MHz, CDCl3, .delta. ppm): reference
TMS=0 ppm, .delta.=7.28-6.84 (br), 5.35-5.29 (br), 4.37-4.20 (br),
3.14 (br), 2.07-0.75 (br).
##STR00008##
[0197] FTIR (thin film, cm-1): characteristic absorption from
terminal alkyne group and polymer backbone, 3300 (terminal alkyne
C--H), 2123 (w, alkyne triple bond, C.ident.C), 1624 (imine in
polymer backbone, C.dbd.N). Mn=13, 346, PDI=1.29. 1H NMR (500 MHz,
CDCl3, .delta. ppm): reference TMS=0 ppm, .delta.=7.15-6.49 (br),
3.45 (br), 3.19 (br), 3.03 (br), 2.53 (br) 1.01-0.70 (br).
[0198] FIGS. 21A-21B show the FTIR spectra of polymers, Poly-1 and
Poly-2, respectively.
Synthesis of Azides
[0199] Azide compounds, as shown in FIG. 22, were synthesized and
characterized following literature procedures (Inverarity, I. A.;
Hulme, A. N., Marked small molecule libraries: a truncated approach
to molecular probe design. Organic & Biomolecular Chemistry
2007, 5 (4), 636-643; Srinivasan, R.; Tan, L. P.; Wu, H.; Yang,
P.-Y.; Kalesh, K. A.; Yao, S. Q., High-throughput synthesis of
azide libraries suitable for direct "click" chemistry and in situ
screening. Organic & Biomolecular Chemistry 2009, 7 (9),
1821-1828; Budhathoki-Uprety, J.; Peng, L.; Melander, C.; Novak, B.
M., Synthesis of Guanidinium Functionalized Polycarbodiimides and
Their Antibacterial Activities. ACS Macro Lett. 2012, (1),
370-374).
Coupling of Azides 5-7 to Poly-1 and Poly-2 Via `Click` Chemistry
to Prepare Poly-3-8.
[0200] To the stirring polymer solution in tetrahydrofuran under
inert atmosphere, azide compound (2.0 mol equiv per alkyne unit),
triethyl amine or DBU (6.0 mol equiv per alkyne unit) and CuI (10
mol %) were added. The reaction mixture was stirred overnight under
an argon atmosphere. Coupling of small molecules azides to alkyne
side chains in polymers was monitored by FTIR analysis. Upon
completion of the reaction, the resulting polymer was washed with
THF and/or diethyl ether, separated by filtration and dried under
reduced pressure. FTIR analysis of final polymers showed full
conversion of all alkyne repeat units in click reaction. Limited
solubility of final polymers posed difficulty in GPC measurements.
Amine-Poly-6, Amine-Poly-8, and Guanidine-Poly-4 were acidified
with a few drops of dilute HCl to increase water solubility.
Carboxy-Poly-7 was treated with a few drops of saturated solution
of NaHCO3. Acidic and basic polymer solutions were then filtered
through centrifugal filters (Amicon Ultracel.RTM., MWCO 3K Da,
Merck Millipore Ltd) to remove residual small molecules and washed
with water until free from free acid or base as tested with litmus
paper. The polymers were then used to suspend SWCNTs.
Photoluminescence Excitation/Emission Contour Plots
[0201] Photoluminescence (PL) plots were constructed using a
home-built apparatus comprising of a tunable white light laser
source, inverted microscope, and InGaAs nIR detector. The laser was
a SuperK EXTREME supercontinuum white light laser source (NKT
Photonics) with a VARIA variable bandpass filter accessory capable
of tuning the output 500-825 nm with a bandwidth of 20 nm. A
longpass dichroic mirror (900 nm) was used to filter the excitation
beam. The light path was shaped and fed into the back of an
inverted IX-71 microscope (Olympus) where it passed through a
20.times.nIR objective (Olympus) and illuminated a 200 .mu.L
nanotube sample in a 96-well plate (Greiner). Emission from the
nanotube sample was collected again by the 20.times. objective and
diverted, via a long-pass dichroic mirror (875 nm), matched to the
f/# of the spectrometer using several lenses, injected into an
Isoplane nIR spectrograph (Princeton Instruments) with a slit width
of 410 .mu.m, and dispersed by a grating of 86 g/mm and 950 nm
blaze wavelength. The light was collected by a PIoNIR InGaAs
640.times.512 pixel array (Princeton Instruments).
[0202] Excitation, emission, and wavelength corrections and
calibrations were performed as follows. The power at each
excitation wavelength was measured at the objective with a PM100D
power meter (Thorlabs) from which a power spectrum was constructed
and used to correct the emission intensities for nonuniform
excitation. A HL-3-CAL-EXT halogen calibration light source (Ocean
Optics) was used to correct for non-uniformities in the emission
path arising from grating, detector, and lens inefficiencies. A
Hg/Ne pencil style calibration lamp (Newport) was used to calibrate
emission wavelengths ranging from 950-1350 nm.
[0203] Acquisition was conducted in semi-automated fashion
controlled by Labview code which iteratively increased the
excitation laser source from 491-824 nm in steps of 3 nm and saved
the data in ASCII format. Using a center wavelength of 1135 nm, the
emission spectra range was 915-1354 nm with has a resolution of 0.7
nm. Background subtraction was conducted using a well filled with
DI H.sub.2O. Following acquisition, the data was processed with a
Matlab code which applied the corrections for non-uniform
excitation and emission (as mentioned previously), created the
contours with a Gaussian smoothing function, and output the figures
to be used for nanotube peak picking.
Topical Application of Polycarbodiimide-SWCNT Complexes on Human
Skin.
[0204] Polycarbodiimide-SWCNT complexes (nanotube concentration in
aqueous suspension: 70-90 mg/L) were deposited onto normal human
skin after harvesting from patients during Moh's surgery. Normal
skin at the periphery of the tumor was used; the tumor tissue was
discarded. After a two-hour exposure to nanotubes, the skin surface
was wiped off to remove unabsorbed nanotubes. The skin samples were
then microtomed into 5 mm thick slices and imaged under 730 nm
excitation.
* * * * *