Color-Tunable Amphiphilic Segmented π-Conjugated Polymer Nano-Assemblies and Their Bioimaging in Cancer Cells

Article abstract of DOI:10.1021/acs.macromol.6b00660

We report a unique color tunable amphiphilic segmented π-conjugated polymer design and their π-stack driven diverse self-assembled nanostructures and demonstrate their application as a new classes of aqueous luminescent nanoparticle probes for bioimaging in cervical and breast cancer cells. Oligo-phenylenevinylene (OPV) was employed as rigid luminescent π-core and oligo-ethyleneoxy chains were used as flexible spacers to construct new amphiphilic segmented π-conjugated polymers by Witting-Horner polymerization route. The rigidity of the π-core was varied using tricyclodecanemethyleneoxy, 2-ethylhexyloxy or methoxy pendants and appropriate π-core geometry was optimized to achieve maximum aromatic π-stacking interactions. Solvent-induced chain aggregation of the polymers exhibited a morphological transition from one-dimensional helical nanofibrous to three-dimensional spherical nanoassemblies in good/bad solvent combinations. This morphological transformation was accompanied by the fluorescence color change from blue-to-white-to-yellow. CIE color coordinates exhibited x = 0.25 and y = 0.32 for the white light followed by the collective emission from aggregated and isolated OPV chromophores. Electron and atomic microscopes, steady state photophysical studies, time-resolved fluorescent decay analysis, and dynamic light scattering method enabled us to establish the precise mechanism for the self-assembly of segmented OPV polymers. The polymers produced stable and luminescent aqueous nanoparticles of <200 nm diameter in water. Cytotoxicity studies in cervical and breast cancer cells revealed that these new aqueous luminescent polymer nanoparticles are highly biocompatible and nontoxic to cells up to 60 μg/mL. Cellular uptake studies by confocal microscope further exposed that these nanoparticles were internalized in the cancer cells and they were predominantly accumulated in the nucleus. The present investigation opens up new amphiphilic segmented π-conjugated polymer design for producing diverse supramolecular assemblies and also demonstrates their new application as biocompatible fluorescent nanoprobes for imaging in cancer cells.

Full text of DOI:10.1021/acs.macromol.6b00660

Article
pubs.acs.org/Macromolecules
Color-Tunable Amphiphilic Segmented π‑Conjugated Polymer Nano-
Assemblies and Their Bioimaging in Cancer Cells
Karnati Narasimha and Manickam Jayakannan*
Department of Chemistry, Indian Institute of Science Education and Research (IISER) Pune, Dr. Homi Bhabha Road, Pune 411008,
Maharashtra India
S
* Supporting Information
ABSTRACT: We report a unique color tunable amphiphilic
segmented π-conjugated polymer design and their π-stack driven
diverse self-assembled nanostructures and demonstrate their applica-
tion as a new classes of aqueous luminescent nanoparticle probes for
bioimaging in cervical and breast cancer cells. Oligo-phenylenevinylene
(OPV) was employed as rigid luminescent π-core and oligo-
ethyleneoxy chains were used as flexible spacers to construct new
amphiphilic segmented π-conjugated polymers by Witting−Horner
polymerization route. The rigidity of the π-core was varied using
tricyclodecanemethyleneoxy, 2-ethylhexyloxy or methoxy pendants and
appropriate π-core geometry was optimized to achieve maximum
aromatic π-stacking interactions. Solvent-induced chain aggregation of
the polymers exhibited a morphological transition from one-dimen-
sional helical nanofibrous to three-dimensional spherical nanoassemblies in good/bad solvent combinations. This morphological
transformation was accompanied by the fluorescence color change from blue-to-white-to-yellow. CIE color coordinates exhibited
x = 0.25 and y = 0.32 for the white light followed by the collective emission from aggregated and isolated OPV chromophores.
Electron and atomic microscopes, steady state photophysical studies, time-resolved fluorescent decay analysis, and dynamic light
scattering method enabled us to establish the precise mechanism for the self-assembly of segmented OPV polymers. The
polymers produced stable and luminescent aqueous nanoparticles of <200 nm diameter in water. Cytotoxicity studies in cervical
and breast cancer cells revealed that these new aqueous luminescent polymer nanoparticles are highly biocompatible and
nontoxic to cells up to 60 μg/mL. Cellular uptake studies by confocal microscope further exposed that these nanoparticles were
internalized in the cancer cells and they were predominantly accumulated in the nucleus. The present investigation opens up new
amphiphilic segmented π-conjugated polymer design for producing diverse supramolecular assemblies and also demonstrates
their new application as biocompatible fluorescent nanoprobes for imaging in cancer cells.
A−B diblocks of poly(3-triethyelene glycol thiophene)-block-
poly(3-hexylthiophene) was also reported.34 Thermo and pH-
responsive blocks of poly(3-hexylthiophene)-block-poly-
(triethyeleneglycolallene)s was reported as white luminescent
material.35 In most of these examples; the self-assembly was
primarily driven by the noncovalent interactions at the side
chains and the π-conjugated polymer backbone was almost
unaffected. Self-assembly of π-conjugated polymers that are
responsive to change in the backbone is particularly important
since it would provide new fundamental understanding of chain
folding phenomena and also enabling the color-tuning via
aromatic π−π stacking. Segmented π-conjugated polymers
having rigid aromatic π-core and flexible alkyl or oligo
ethyelene spacers in the backbone are unique classes of
materials for the above purpose. In this design, the aromatic
rigid core and flexible units can be selectively segregated in the
INTRODUCTION
■
Aqueous nanoassemblies of amphiphilic π-conjugated materials
are emerging as important luminescent molecular probes for
bioimaging1,2 and sensing toxic metals3−8 and as nanoscaffolds
for delivery of genes9 and drugs.10−12 Amphiphilicity in π-
conjugated aromatic polymers was typically introduced by
anchoring flexible oligo- (or poly-) ethylene glycols as side
chains in the π-conjugated backbones.13 PEG-lated oligomers
of thiophenes,14−17 fluorenes,18 phenylenes,19−23 phenylene-
ethylenes,24,25 phenylenevinylenes,26,27and perylenebisi-
mides28,29 were synthesized and self-assembled as vesicles,
nanoparticles, and toroids30,31 etc. Inspired by these self-
assemblies of small molecular π-conjugates; recently, amphi-
philic π-conjugated polymer based on rod−coil A−B−A
triblock copolymer of oligo-phenylenevinylene (OPV) core
and PEG corona were reported for nanofibrous and micellar
assemblies.32 Crown ether pendants were anchored on the
poly(phenylenevinylene) backbone and the role of K+ ion
binding to produce nanoribbon morphology was studied.33 The
effect of potassium ion on the helical nanofiber morphology of
Received: March 31, 2016
Revised: May 12, 2016
© XXXX American Chemical Society
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Figure 1. Segmented π-conjugated design for diverse polymer self-assembly and demonstrate their luminescent nanoassemblies as biomarkers in
cancer cell imaging.
polymer matrix by weak noncovalent forces such as aromatic π-
stacking and van der Waals forces. Karasz and co-workers
reported segmented polymers of oligo-(1, 4-
phenyelenevinylene)s cores and demonstrated their application
for blue light emitting diodes.36 Pang and co-workers developed
hybrid nanocomposites based on 1,3-meta-phenylene-bridged
conjugated polymer and single-walled carbon nanotubes.37,38
Segmented polymers with electron deficient and electron rich
aromatic cores were self-assembled into helical amyliod-
fibrils39,40 and charger-transfer complexes.41−43 Oligo-(1,4-
phenylenevinylene)s segmented polymer and PCBM blends
were prepared to study the photoinduced charge separation in
solid state.44 From our group, we reported segmented π-
conjugated polymers based on carboxylic substituted 1,3-oligo-
phenylenevinylenes as photosensitizer for Eu3+ ion45 and their
complexes were employed as triple detection probe for thermal,
toxic metal ion and amino acids.46 Further, oligo-1,4-phenyl-
enevinylenes (OPV) segmented polymers were studied for
helical donor−acceptor assemblies.47 Semicrystalline OPV
segmented polymers were designed and self-assembled as
organogels to demonstrate their photonic λ/4 wave plates for
optical switching applications.48 These studies emphasized the
importance of segmented π-conjugated polymers for photonic
and electronic applications. Unfortunately, no effort has been
taken until now to study the aqueous self-assembly of
segmented π-conjugated polymers for biomedical applications.
This is partially associated with the limited solubility of
segmented π-conjugated polymers in water. The present
investigation is aimed to address this problem by designing
new classes of water-soluble amphiphilic π-conjugated OPV
polymers and employ their aqueous stable nanoassemblies as
nontoxic fluorescent biomarkers for cellular imaging in cancer
cells. This new polymer design and imaging concept is shown
in Figure 1.
π-core and variable oligo-ethyleneoxy flexible spacer. For this
purpose, three OPV cores are chosen by varying the side chain
anchoring groups as 2-ethylhexyloxy (EH, branched unit),
tricyclodecanemethyleneoxy (TCD, rigid unit) and methoxy
(Me) units. The selection of these pendants was done based on
our recent efforts in resolving the crystal structures of OPVs to
trace the role of substitution on the planarity of the
molecules.49−51 It was found that the substitution of TCD,
EH, and Me in the vertical position and long tails in the
longitudinal positions of OPV aromatic core produced planar
geometry. Thus, these three planar OPV cores were chosen
here to make new classes of segmented polymers to study their
self-assembly. Thus, the combination of these OPV cores and
ethylene glycol spacers provide an excellent opportunity to fine-
tune the correct structural design to study the self-assembly
process of segmented π-conjugated polymers. The photo-
physical studies revealed that emissions characteristics of the
polymers were highly specific to their structure and the solvent
compositions. The polymers underwent strong aromatic π-
stacking and showed excellent color tunability from blue-to-
white-to-yellow. This color tunability was accomplished by the
morphological transition from nanofiber to spherical assem-
blies. The segmented polymers were found to produce stable
aqueous luminescent nanoparticles. Cytotoxicity studies in
cancer cells revealed that the polymer nanoparticles were
nontoxic to cells and confocal microscopic analysis exhibited
that the nanoparticles were readily taken by the cells and
accumulated in the perinuclear environment. The overall
investigation revealed that the new polymer design opens up
a new opportunity to fine-tune their diverse nanoassemblies of
segmented π-conjugated polymers and demonstrates their
applications as fluorescent biomarker for the first time in
cancer therapy.
EXPERIMENTAL SECTION
The present investigation is emphasized to develop new
classes of amphiphilic segmented π-conjugated polymers based
on rigid and blue luminescent oligo-phenylenevinylene (OPV)
■
Materials. Hydroquinone, 4-hydroxybenzaldehyde, triethylene
glycol, hexaethylene glycol, p-toluenesulfonyl chloride, triethylamine,
B
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triethyl phosphite, dimethyl sulfate, and potassium tert-butoxide (1 M
in THF), were purchased from Aldrich Chemicals. HBr in glacial
acetic acid, paraformaldehyde, K2CO3, and KOH were purchased
locally. 1,8-Tricyclodecanemethanol was donated by Celanese
Chemicals and Co. and has used without further purification. Solvents
were purchased locally and purified by standard procedures. Tetraethyl
((2,5-dimethoxy-1,4-phenylene)bis(methylene))bis(phosphonate)
(3a), tetraethyl ((2,5-bis((2-ethylhexyl)oxy)-1,4-phenylene)bis-
(methylene))bis(phosphonate) (3b), and 1, 4-bis-
[(tricyclodecanemethyleneoxy)]-2,5-xylenediphosphonate (3c) were
synthesized according to our earlier procedures.48,49 Cervical cancer
(HeLa) cells, breast cancer (MCF-7) cells were maintained in DMEM
(phenol red containing, Gibco) containing 10% (v/v) fetal bovine
serum (FBS) and 1% (v/v) penicillin−streptomycin at 37 °C under a
5% CO2 atmosphere. For all the assays, cells were rinsed with 40%
DPBS (Gibco), trypsinized using 0.05% trypsin (Gibco) and seeded in
96 well or 6 well (as per experiment) flat bottomed plastic plates
(Costar). Fluoromount was obtained from Southern Biotech.
Na2SO4 and the solvent was removed to get product as white solid.
The product was further purified by passing through silica gel column
using ethyl acetate (30% v/v) in hexane as eluent. Yield = 13.7 g
1
(90%). H NMR (400 MHz, CDCl3), δ ppm: 7.79 (d, 4H, Ar−H),
7.35 (d, 4H, Ar−H), 4.11 (t, 4H, Ar−SO2OCH2CH2), 3.66 (t, 4H,
Ar−SO2OCH2CH2), 3.50 (s, 4H, Ar−SO2OCH2CH2−OCH2), and
2.45 (s, 6H, Ar−CH3). 13C NMR (100 MHz, CDCl3), δ ppm: 144.86,
132.87, 129.8, 127.9, 70.65, 69.18, 68.71, and 21.63. FT-IR (cm-1):
2869, 1744, 1448, 1344, 1293, 1170, 1124, 1014, 978, 906, 849, 807,
772, 704, and 660. MALDI-TOF: calculated MW = 458.55; found m/z
= 481.09 (M++Na+) and m/z = 497.43 (M++K+).
Synthesis of 3,6,9,12,15-Pentaoxaheptadecane-1,17-diyl
Bis(4-methylbenzenesulfonate) (1b). Hexaethylene glycol (2.0 g,
7.10 mmol) was reacted with p-toluenesulfonyl chloride (3.0 g, 15.60
mmol) in the presence of KOH (3.2 g, 56.80 mmol) in dry DCM (30
mL) as described for 1a. The product was further purified by column
chromatography using ethyl acetate (60% v/v) in hexane as eluent.
1
Yield = 3.14 g (75%). H NMR (400 MHz, CDCl3) δ ppm: 7.80 (d,
1
General Procedures. H and 13C NMR were recorded using 400
4H, Ar−H), 7.35 (d, 4H, Ar−H), 4.16 (t, 4H, Ar−SO2OCH2CH2),
3.69 (t, 4H, Ar−SO2OCH2CH2), 3.61−3.58 (m, 16H, Ar−
SO2OCH2CH2−OCH2−), and 2.45 (s, 6H, Ar−CH3). 13C NMR
(100 MHz, CDCl3) δ ppm: 144.79, 132.82, 129.78, 127.90, 70.63,
70.49, 70.43, 70.39, 69.22, 68.58, and 21.59. FT-IR (cm−1): 3394,
2878, 1719, 1646, 1600, 1453, 1351, 1294, 1215, 1171, 1097, 1031,
1005, 916, 813, 770, and 660. MALDI-TOF: calculated MW = 590.69;
found m/z = 613.69 (M+ + Na+) and m/z = 641.02 (M+ + K+).
Synthesis of 4,4′-(((Ethane-1,2-diylbis(oxy))bis(ethane-2,1-
diyl))bis(oxy))dibenzaldehyde (2a). 4-Hydroxybenzaldehyde (2.9
g, 24.0 mmol) and anhydrous K2CO3 (6.0 g, 44.0 mmol) were taken in
dry acetonitrile (40.0 mL) and it was refluxed under nitrogen
atmosphere. 1a (5.0 g, 11.0 mmol) was added and the reaction was
continued for 48 h at 80 °C under nitrogen atmosphere. The reaction
mixture was poured into the water and then extracted with ethyl
acetate. The organic layer was dried over anhydrous Na2SO4 and the
solvent was evaporated to get product as white solid. It was further
purified by column chromatography using 40% ethyl acetate/pet ether
MHz JEOL NMR spectrometer. Infrared spectra were recorded using
a Thermo-Scientific Nicolet 6700 FT-IR spectrometer in the solid
state in KBr in the range of 4000−600 cm−1. Mass analysis of
precursors was determined by the Applied Biosystems 4800 PLUS
MALDI TOF/TOF analyzer using TiO2 as a matrix. The molecular
weights of polymers were determined using gel permeation
chromatography (GPC) which was performed by Viscotek triple
detector setup and tetrahydrofuran as a solvent. TGA analysis was
done using PerkinElmer STA 6000 simultaneous thermal analyzer.
Differential scanning calorimetry (DSC) measurements had done by
using TA Q20 DSC. The data were recorded at heating and cooling
rate of 10 °C/min. The first heating cycle data was discarded since
they possessed prehistory of the sample. Absorption spectra were
recorded by using a PerkinElmer Lambda 45 UV spectrophotometer.
Steady state emission and excitation spectra were recorded using
Fluorolog-3 HORIBA JOBIN VYON fluorescence spectrophotometer.
The quantum yields of the polymer aggregates were determined using
quinine sulfate in 0.1 M H2SO4 (ϕ = 0.546) as standard. The solvent
induced aggregation studies were performed using methanol (MeOH)
and water as bad-solvents, respectively, for P-TCD-6EG by making
different good-solvent/bad-solvent combinations. The stock solution
was prepared by dissolving the polymer in THF, and chloroform and
various combinations of THF/chloroform and methanol/water
samples were prepared by maintaining the absorbance at ≈0.1. The
time-resolved fluorescence lifetime measurements (TCSPC) were
performed using a Fluorolog HORIBA JOBIN VYON fluorescence
spectrophotometer. Fluorescence intensity decays were collected by a
time-correlated single photon counting technique (TCSPC) setup
from Horiba Jobin Yvon. 371 nm LED used for sample excitation. FE-
SEM images were recorded using Zeiss Ultra Plus scanning electron
microscope and the samples were prepared by drop casting on silicon
wafers and coated with gold. Atomic force microscope images were
recorded for drop-cast samples using Agilent instruments. The sample
was drop casted on a freshly cleaved mica surface. The imaging was
carried out in tapping mode using TAP-190AL-G50 probe from
Budget sensors with a nominal spring constant of 48 N/m and the
resonance frequency of 190. Dynamic light scattering (DLS) was
performed using a Nano ZS-90 apparatus utilizing 633 nm red laser (at
90° angle) from Malvern Instruments. High Resolution-Transmission
Electron Microscopy (HR-TEM) images were recorded using
Technai-300 instrument by drop casting aqueous sample on
Formvar-coated copper grids. LSM confocal microscope was used
for cellular uptake images.
1
system. Yield = 2.7 g (69%). H NMR (400 MHz, CDCl3), δ ppm:
9.88 (s, 2H, Ar−CHO), 7.80 (d, 4H, Ar−H), 7.01 (d, 4H, Ar−H),
4.21 (t, 4H, Ar−OCH2CH2), 3.90 (t, 4H, Ar−OCH2CH2), and 3.77
(s, 4H, Ar−OCH2CH2−OCH2). 13C NMR (100 MHz, CDCl3), δ
ppm: 190.70, 163.78, 131.90, 130.11, 114.85, 71.93, 69.54, and 67.74.
FT-IR (cm-1): 2887, 1687, 1600, 1598, 1571, 1506, 1457, 1425, 1392,
1352, 1303, 1249, 1213, 1147, 1099, 951, 916, 826, 791, and 647.
MALDI-TOF: calculated MW = 358.39; found m/z = 381.10 (M+ +
Na+) and m/z = 397.07 (M+ + K+).
Synthesis of 4,4′-((3,6,9,12,15-Pentaoxaheptadecane-1,17-
diyl)bis(oxy))dibenzaldehyde (2b). 4-Hydroxybenzaldehyde (0.91
g, 7.4 mmol) was reacted with compound 1b (2.00 g, 3.4 mmol) using
anhydrous K2CO3 (1.87 g, 13.6 mmol) in dry acetonitrile (40 mL) as
described for 3a. It was further purified by column chromatography
using ethyl acetate and pet ether mixture (1:1 v/v). Yield = 1.0 g
(58%). 1H NMR (400 MHz, CDCl3), δ ppm: 9.88 (s, 2H, Ar−CHO),
7.82 (d, 4H, Ar−H), 7.02 (d, 4H, Ar−H), 4.21 (t, 4H, Ar−
OCH2CH2), 3.88 (t, 4H, Ar−OCH2CH2), and 3.65−3.73 (m, 16H,
Ar−OCH2CH2−OCH2). 13C NMR (100 MHz, CDCl3), δ ppm:
191.00, 163.78, 132.92, 130.00, 114.82, 70.85, 70.57, 70.53, 69.41, and
67.70. FT-IR (cm-1): 2870, 2740, 1690, 1600, 1590, 1510, 1450, 1430,
1390, 1350, 1310, 1260, 1220, 1160, 1100, 1050, 951, 835, and 615.
MALDI-TOF: calculated MW = 490.55; found m/z = 513.14 (M+ +
Na+) and m/z = 529.10 (M+ + K+).
Synthesis of Segmented π-Conjugated OPV Polymers.
Typical procedure is explained for P-TCD-3EG and other polymers
were synthesized following similar procedure. Compound 3c (0.5 g,
0.7 mmol) and compound 2a (0.25 g, 0.7 mmol) were dissolved in dry
THF (12 mL). The mixture was stirred under nitrogen atmosphere
about 15 min at 25 °C. Potassium tert-butoxide (5 mL in 1 M THF)
was added dropwise to the polymerization mixture under a nitrogen
atmosphere and the stirring was continued at 25 °C for 24 h. The
resultant yellow-green polymer solution was concentrated using
rotavapor and it was poured into a large amount of methanol (50
Synthesis of (Ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl)
Bis(4-methylbenzenesulfonate) (1a). Triethylene glycol (5.0 g,
33.33 mmol) and p-toluenesulfonyl chloride (13.9 g, 73.33 mmol)
were dissolved in dry DCM (50.0 mL). The solution was cooled (<5
°C), and powdered KOH (14.9 g, 266.00 mmol) was slowly added
under nitrogen atmosphere. The reaction mixture was stirred for 4 h at
0 °C. Water (60.0 mL) was added and the product was extracted into
DCM (100.0 mL). The organic layer was dried over anhydrous
C
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Scheme 1. Synthesis of the Main Chain Segmented Polymers Having Rigid OPV Core and Flexible Oligo-Ethyleneoxy Spacers
mL). The yellow-green polymer was filtered and washed with large
amount of methanol. The polymer was redissolved in tetrahydrofuran,
filtered and precipitated into methanol. The purification procedures
were followed at least twice. Yield = 0.55 g (91%). 1H NMR (CDCl3,
400 MHz), δ ppm: 7.45 (d, 4H, Ar−H), 7.32 (d, 2H, J = 16 Hz, CH
CH), 7.13 (d, 2H, J = 20 Hz, CHCH), 7.05 (s, 2H, Ar−H), 6.92 (d,
4H, Ar−H), 4.17 (t, 4H, OCH2), 3.89 (t, 4H, OCH2), 3.78 (d, 4H,
OCH2−TCD), and 2.5 to 1.50 (m, 30H, aliphatic-H). 13C NMR
(CDCl3, 100 MHz), δ ppm: 158.27, 151.11, 131.11, 128.30, 127.58,
126.77, 121.80, 114.80, 111.04, 73.61, 70.89, 69.81, 67.46, 45.67,
45.27, 43.96, 41.27, 40.30, 34.74, 29.07, 27.98, 27.00, and 26.51. FT-IR
(cm−1): 2940, 2867, 1602, 1508, 1458, 1419, 1385, 1345, 1294, 1249,
1178, 1125, 1055, 1019, 967, 922, 848, and 818.
14.12, and 11.31. FT-IR (cm−1): 2922, 2866, 1682, 1602, 1508, 1460,
1419, 1383, 1348, 1292, 1243, 1200, 1106, 1032, 959, 847, 818, 729,
and 643.
Aqueous Self-Assembly of P-TCD-6EG Polymer. In a typical
experiment, 2.0 mg of the polymer was dissolved in DMSO (1.5 mL)
and THF (0.5 mL). Deionized water (3.0 mL) was added dropwise
into the polymer solution. The resulting solution was stirred at 25 °C
under dark conditions for 4 h. The solution was transferred to the
semipermeable membrane having MWCO 2000 and then dialyzed
against a large amount of deionized water for 48 h. Fresh water was
replenished periodically to ensure the removal of THF and DMSO
from the dialysis membrane. The dialyzed solution was filtered,
lyophilized, and stored at 4 °C for further usage.
Synthesis of P-TCD-6EG. Compound 3c (0.5 g, 0.7 mmol) was
reacted with compound 2b (0.343 g, 0.7 mmol) using potassium tert-
butoxide (5 mL in 1 M THF) in dry THF (10 mL) described for P-
Cell-Viability Assay. The tetrazolium salt, 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed in
order to study the cytotoxic effect of the P-TCD-6EG polymer
nanoparticles in HeLa and MCF7 cells. For this experiment, a 96-well
plate (Corning, USA) was used and each of its wells were seeded with
1000 cells in 100 μL of DMEM with 10% FBS (fetal bovine serum)
and these were allowed to adhere for 16 h. The media from each well
was aspirated followed by addition of various concentrations of the
polymer to each well. These were added in triplicates corresponding to
individual experiments. Cells in DMEM with FBS in the absence of
polymer were also maintained in triplicates as a blank control. The 96
well plate was incubated for 72 h without changing the media. After 72
h medium from each well was aspirated and 100 μL of MTT solution
(a freshly prepared stock of MTT in sterile PBS (5 mg/mL) diluted to
50 μg/mL in DMEM) was added. These were allowed to incubate for
another 4 h at 37 °C. The formation of purple formazan crystals was
observed that developed due to the reduction of MTT by
mitochondrial dehydrogenase enzyme from viable cells, and the
media from each well was aspirated and 100 μL of DMSO was added
to the resultant crystals giving purple solutions in each well. The
absorbance from formazan crystals was immediately recorded using
microplate reader at 570 nm (Varioskan Flash), which gave a
representative of the number of viable cells per well. Values
corresponding to each triplicate (of control and polymer treated
cells) were determined and their mean was used for calculations. In the
triplicates, any value that deviated from rest two were discarded. The
mean of blank control set as 100% (corresponding to viable cells) and
relative percentage values for polymer nanoparticle were calculated
with respect to this.
1
TCD-3EG Yield = 0.61 g (87%). H NMR (CDCl3, 400 MHz) δ
ppm: 7.44 (d, 4H, Ar−H), 7.31 (d, 2H, J = 16 Hz, CHCH), 7.12 (d,
2H, J = 16 Hz, CHCH), 7.04 (s, 2H, Ar−H), 6.91 (d, 4H, Ar−H),
4.16 (t, 4H, OCH2), 3.87 (t, 4H, OCH2), 3.74 (m, 16H, OCH2), 3.72
(d, 4H, OCH2−TCD), and 2.5 to 1.50 (m, 30H, aliphatic-H). 13C
NMR (CDCl3, 100 MHz) δ ppm: 158.25, 151.08, 131.06, 128.30,
127.55, 126.63, 121.72, 114.78, 111.0, 73.60, 70.76, 70.55, 69.70,
67.43, 45.64, 45.24, 43.95, 41.26, 40.27, 34.73, 29.05, 28.0, 27.0, and
26.51. FT-IR (cm−1): 2936, 2864, 1647, 1600, 1506, 1457, 1417, 1382,
1349, 1292, 1241, 1178, 1102, 1019, 959, 844, and 814.
Synthesis of P-Me-6EG. Compound 3a (0.5 g, 1.1 mmol) was
reacted with compound 2b (0.343 g, 1.1 mmol) using potassium tert-
butoxide (6 mL in 1 M THF) in dry THF (10 mL) as described for P-
1
TCD-3EG. Yield = 0.48 g (77%). H NMR (CDCl3, 400 MHz), δ
ppm: 7.47 (d, 4H, Ar−H), 7.34 (d, 2H, J = 16 Hz, CHCH), 7.10 (s,
2H, Ar−H), 7.05 (d, 2H, J = 16 Hz, CHCH), 6.90 (d, 4H, Ar−H),
4.14 (t, 4H, OCH2), 3.90 (s, 6H, OCH3), 3.86 (t, 4H, OCH2), and
3.73−3.67 (m, 16H, OCH2). 13C NMR (CDCl3, 100 MHz), δ ppm:
158.34, 151.29, 130.82, 128.23, 127.70, 126.44, 121.14, 114.74, 108.84,
70.78, 70.58, 70.54, 69.69, 67.41, and 56.31. FT-IR (cm−1): 2969,
1688, 1598, 1503, 1456, 1404, 1348, 1293, 1239, 1209, 1098, 1033,
953, and 810.
Synthesis of P-EH-6EG. Compound 3b (0.5 g, 0.8 mmol) was
reacted with compound 2b (0.386 g, 0.7 mmol) using potassium tert-
butoxide (5 mL in 1 M THF) in dry THF (10 mL) as described for P-
1
TCD-3EG. Yield = 0.58 g (90%). H NMR (CDCl3, 400 MHz), δ
ppm: 7.45 (d, 4H, Ar−H), 7.35 (d, 2H, J = 16 Hz, CHCH), 7.08 (d,
2H, J = 16 Hz, CHCH), 7.10 (d, 2H, Ar−H), 6.91 (d, 4H, Ar−H),
4.15 (t, 4H, OCH2), 3.87 (t, 4H, OCH2), 3.67 (m, 16H, OCH2), 3.94
(d, 4H, OCH2−EH), 2.02 (m, 4H, OCH2-CH2−CH2), 1.82 (m, 2H,
OCH2−CH−CH2), 1.64−1.38 (m, 18H, aliphatic-H), 0.98 (t, 6H,
CH3), and 0.92 (t, 6H, CH3). 13C NMR (CDCl3, 100 MHz), δ ppm:
158.29, 151.00, 131.04, 128.00, 127.56, 126.69, 121.50, 114.79, 111.08,
71.76, 70.82, 70.61, 70.57, 69.72, 67.45, 39.76, 30.92, 29.25, 23.10,
Cellular Uptake Studies. Flame-dried coverslips were taken in 6
well plate in DMEM medium containing 10% FBS. On the surface of
these coverslips, cells were seeded at a density of 1 × 105 cells and
these were incubated at 37 °C for 16 h. The cells in each well, thus
grown, were treated with required concentrations of P-TCD-6EG
polymer nanoparticles. This was incubated at 37 °C for 4 h under CO2
environment and then the media was aspirated from each well, and the
cells were washed twice with PBS (2 × 1 mL) and fixed with 4%
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1
Figure 2. H NMR spectrum of P-TCD-3EG in CDCl3 (a). GPC chromatograms of the segmented polymers in THF (b). DSC thermograms of
polymers in heating cycles at 10 °C/min (c).
paraformaldehyde solution in PBS for 10 min at room temperature.
The cells were washed twice with PBS (2 × 1 mL) and stained with
phalloidin (red) conjugated to Alexa 610 (Invitrogen) diluted 1:100 in
5% BSA solution in PBS. These were incubated for 2 min, at room
temperature under dark conditions, and then the excess dye was
washed from the plate and the cells were gently rinsed with PBS. The
coverslips were mounted on slides using Fluoromount mounting
medium (Southern Biotech) and were left for drying overnight at
room temperature in the dark. The cells were imaged using a confocal
microscope using the λ 405 nm (blue channel) and λ 560 nm (red
channel) lasers. Images thus obtained were analyzed using ImageJ
analysis software, and the image for each channel was separated and
merged.
tricyclodecanemethyleneoxy units, respectively, and X stands
for a number of oligoether units present in the flexible alkyl
chains such as triethylene glycol (3EG) and hexaethylene glycol
(6EG), respectively. For example, the TCD polymer with 6-EG
unit is referred as P-TCD-6EG. The chemical structures of all
the polymers are shown in Scheme 1.
1
The structures of the polymers were characterized by H
1
NMR, 13C NMR, and FT-IR. H NMR spectra of the P-TCD-
3EG polymer are shown in Figure 2a, and different types of
protons in the chemical structure are assigned alphabetically
1
(see Figure S1 for NMR spectra of other polymers). The H
NMR spectrum of the P-TCD-3EG polymer showed peaks
from 7.46 to 6.90 ppm with an appropriate splitting pattern
corresponding to aromatic phenylenevinylene protons (see
Figure 2a). The two doublets which belong to trans-vinylene
protons (c and d protons) appeared at 7.36 and 7.09 ppm
(coupling constant J = 16 Hz). The peaks appeared at 7.43,
7.11, and 6.90 ppm were assigned to protons b, e, and a,
respectively. The broad triplets at 4.10 ppm, 3.86 ppm were
attributed to Ar−OCH2CH2 and 3.78 ppm is belonging to Ar−
OCH2−TCD protons, respectively. Among the two doublets in
the vinyl protons, one of them merged with solvent peak at
7.26 ppm (see Figure 2a). TCD protons appeared below 3.5
ppm. Similarly, 13C NMR spectra of the polymer P-TCD-3EG
showed the peaks for expected structure formation (see Figure
S2). The molecular weights of the polymers were determined
by gel permeation chromatography (GPC) in tetrahydrofuran
using polystyrene standards. The GPC chromatograms of the
polymers appeared as monomodal distribution except for P-
TCD-3EG which showed was partially soluble in THF (see
RESULTS AND DISCUSSION
■
Synthesis and Characterization of Segmented Poly-
mers. New classes of segmented polymers were synthesized
through multistep reactions as shown in Scheme 1. ω-Bis-
benzaldehydes (compounds 2a−2b) were synthesized by
reacting 4-hydroxybenzaldehyde with appropriate tosylates of
oligo-ethylene glycols (compounds 1a−1b). Three bisphosph-
onate esters having variable aliphatic and cycloaliphatic
substitutions such as methoxy (3a), 2-ethylhexyloxy (3b), and
tricyclodecanemethyleneoxy (3c), were synthesized following
our earlier procedure.48,49 The polymerization of monomers
3a−3c with equimolar amounts of bis-benzaldehydes (2a−2b)
under Wittig−Horner conditions produced respective seg-
mented polymers consisting of rigid oligo-phenylenevinylenes
and oligo-ethyleneoxy flexible spacers in the polymer backbone.
These polymers were referred as P-Y-X, where Y represents
Me, EH or TCD for the methoxy, ethylhexyloxy, or
E
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Figure 3. Emission spectra of P-TCD-3EG and P-TCD-6EG in CHCl3 (solid line) and thin film (dotted line) and the photographs are taken for the
powder sample on quartz plate upon photoexcitation (a). Absorbance spectra of P-TCD-3EG (b) and P-TCD-6EG (c) in methanol/chloroform
mixture. Emission spectra of P-TCD-3EG (d) and P-TCD-6EG (e) in methanol/chloroform mixture. Emission spectra of P-TCD-6EG (f) in water/
tetrahydrofuran solution. The photographs are taken for polymer solutions in vials upon photoexcitation by UV-light at 365 nm. The concentration
of the polymers were maintained as 2.5 × 10−5 M.
Figure 2b). The molecular weights of the polymers were
obtained as Mw = 16 800 to 28 000 g/mol with a polydispersity
of 2.0 to 3.5 (see Table ST1). The Witting−Horner
polymerization route is typically known to produce polymers
with 10−15 repeating units as observed by us51,47 and others.36
For biomedical applications, polymer molecular weights of <40
000 g/mol are preferred due to their easy clearance through the
kidney. Hence, the custom designed polymers have molecular
weight <28 000 g/mol which is less than the acceptable range
for biomedical imaging applications.
Aromatic π-Stack Aggregation and Emission Color-
Tuning. The segmented OPV polymers are designed with
different substitution in the central aromatic ring and flexible
oligoethyelneoxy chains. This arrangement facilitates the strong
aromatic π-stacking interaction among the OPV chromophores
to maximize the self-assembly process. To study this effect in
solution and solid state (in a thin film); the polymer samples
were subjected to steady state photophysical studies and time-
resolved fluorescent decay properties. The absorbance and
emission spectra of P-TCD-3EG and P-TCD-6EG polymers
were found to be almost identical in solution (see Figure 3a). In
the solid state, the emission spectra of the hexaethylene glycol
segmented P-TCD-6EG polymer showed 50 nm red-shift
compared to the P-TCD-3EG polymer (see absorbance spectra
in Figures S5 and S6). This suggests that the TCD-substituted
OPV chromophores became more facile to undergo self-
assembly via aromatic π-interaction when they were separated
by longer segmented spacers. The photographs of these
polymer samples in Figure 3a showed greenish and yellow
emission for P-TCD-3EG and P-TCD-6EG with respect to the
50 nm shift in their emission wavelength. This suggests that the
increasing oligoethyleneoxy segments between the adjacent
OPVs increased the π-stacking interaction among the
chromophores. The strong π−π stacking among the OPV
cores induced emission-shift. This trend is attributed to the
increasing the OPV self-aggregation with an increase in the
segmented spacer length. Unlike P-TCD-6EG, the other 6EG-
spacer polymers P-EH-6EG and P-ME-6EG did not show any
significant color change in their emission spectra (see Figure
S6). Thus, the aromatic π-stacking interaction in the segmented
polymer was mainly driven by the types of the OPV
chromophores and in the present case TCD-OPV was very
unique in exhibiting maximum packing among the various
OPV π-cores. The segmented polymer with hexaethylene glycol
(6EG) was found to be more flexible than triethylene glycol
spacer (3EG) which was evident from their Tg values (see
Figure 2c). The highly flexible chains possess high degree of
Thermogravimetric analysis (TGA) of the polymers showed
that the polymers were thermally stable up to 350 °C (see
Figure S3). Differential scanning calorimetry (DSC) analysis of
polymers was carried out at the heating/cooling cycle rate of
ο
10 °C/min. DSC thermograms of the polymers at 10 C/min
heating are shown in Figure 2c. All the polymers were found to
be completely amorphous and the DSC thermograms have
shown only glass transition temperature (Tg) in cooling and
heating cycles (see Figure S4 for their cooling cycles). The Tg’s
of the polymers gradually decreased from 108 to 70 °C upon
increasing the length of the oligoethylene glycol segment from
3EG to 6EG in P-TCD-3EG to P-TCD-6EG. This trend is
attributed to the increase in the flexibility of the polymer chains
with an increase in the number of oligo-ethyleneoxy units in the
segmented polymer chain. Further, the comparison among the
6EG spacer polymers revealed that the Tg values decreased with
a decrease in the rigidity of the polymers in the following order
TCD > Me > EH from 70 to 6 °C. This observation indicates
that the cycloaliphatic TCD units introduced much higher
rigidity in the OPV core compared to other two side chains.
The variation of Tg values from 108 to 6 °C in the segmented
polymers are clear indication that the rigidity of the π-
conjugated units and varying the EG-spacers in the backbone
are crucial factor for the rigidity of the structure. Among all
these polymers, TCD units appeared to be very unique in
producing highly rigid polymers with higher Tg values.
F
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Figure 4. CIE color coordinates of P-TCD-6EG polymer solution in THF + water (Δ) and CHCl3 + methanol (O) (a). TCSPC decay profiles of P-
TCD-6EG polymers in methanol+chloroform solvent mixtures (b). Plots of fluorescent lifetime τ1 and τ2 (c) and fractional contribution α1 and α2
(d) of the polymers in methanol + chloroform solvent mixtures. The concentrations of the polymers were maintained as 2.5 × 10−5 M.
freedom for both intrachain and interchain folding; as a result,
the OPV could undergo strong aromatic π-stacking inter-
actions. On the other hand, the shorter spacers (3EG) did not
have sufficient flexibility to bend the chains which restrict the
overlap between the OPV chromophores. These studies
suggested that the longer segmented OPV polymers (>6 EG
units) have high tendency to undergo self-organization via
aromatic π-stacking interaction. On the other hand, the shorter
segmented lengths (<3 EG units) did not provide enough
flexibility for TCD-OPV chromophores in the polymer
backbone for π-stacking induced self-assembly.
Typically polymer chains are expected to adopt either
expanded or coil-like conformation with respect to the solvents
in which they are present. For instance, a good solvent
maximizes the solvent−polymer interaction and solvates the
polymer chains in the expanded chain conformation whereas in
poor solvents the polymer chains predominately adopt coil-like
conformation and precipitate the polymers.47,50 Therefore, by
appropriately choosing the combination of good and poor
solvents; one can easily control the expanded or coil-like
conformation in polymer chains. Since the segmented polymers
showed very good spacer dependent self-assembly in the solid
state, their solvent induced aggregated self-assembly were
studied to trace their chain folding phenomena. For this
purpose, two solvent combinations are chosen: (i) methanol/
chloroform and tetrahydrofuran/water. Absorbance spectra of
polymers (see Figure 3, parts b and c) showed a decrease in
absorbance with an increase in methanol content in the solvent
mixture. This trend is attributed to the variation in the molar
extinction coefficient of OPV chromophores in the solvent
combination.51 Interestingly P-TCD-6EG polymer showed an
additional peak at 470 nm (shown by the arrow) with respect
to the formation of π-induced OPV aggregates. This trend
clearly indicates that P-TCD-6EG polymers have a high
tendency for π-induced aggregation upon increasing the
amount of methanol in the polymer solution. The emission
spectra of P-TCD-3EG and P-TCD-6EG polymers in
methanol/chloroform solvent combinations are shown in
Figure 3, parts d and e, respectively (see Figure S7 for P-EH-
6EG and P-Me-6EG). The drastic decrease in the emission
intensity of the spectra was attributed to the reduction in their
molar extinction coefficient in their absorbance spectra.
Interestingly, the long segmented P-PCD-6EG polymer showed
excellent color tuning ability with the formation of new
emission peak at higher wavelength region (which was absent
in their lower counterpart P-TCD-3EG, see Figure 3d). The
emission peak at λem = 450 nm was assigned to isolated OPV
chromophores and the new peak at λem = 535 nm was assigned
to OPV chromophore in the aggregated state (shown by the
arrow). The comparison of the solid state emission maxima of
P-TCD-6EG at 535 nm (see Figure 3a) and solvent induced
aggregated emission peak at 537 nm (see Figure 3e) revealed
that the OPV chromophores had attained maximum aromatic
π-staking in methanol + chloroform mixture as equivalent to
that of their interaction in the solid state. Further, to quantify
the extent of aggregation in P-TCD-6EG (also P-TCD-3EG),
the ratio of the peaks λem = 535/450 nm were plotted against
the solvent composition and shown as an inset in Figures 3d
and 3e. These plots showed distinct break points at 40−50%
methanol in chloroform for long segmented polymer for P-
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TCD-6EG (see Figure 3e) whereas the change is insignificant
in short segmented polymer P-TCD-3EG (see Figure 3d).
Additionally, the polymer solution of P-TCD-6EG showed
excellent color tuning from blue-to-white-to-yellowish with an
increase in the methanol composition in the solvent mixture
(see the photographs in Figure 3e). Similar solvent induced
aggregation experiments were carried out for P-EH-6EG and P-
Me-6EG and these studies indicated that these polymers did
not show π-stacking interaction unlike P-TCD-6EG (see Figure
S7). Based on these studies, it can be concluded that the TCD-
OPV chromophores exhibited excellent π-induced aggregation
compared to ethylhexyloxy and methoxy units in the
segmented polymers. Furthermore, it is rather clear that the
placing of the π-conjugated chromophores apart in the
segmented backbone maximize the π-stack induced aggregation
in segmented π-conjugated polymers.
In order to study the aqueous self-assembly of π-induced
aggregation, the P-TCD-x polymers were dialyzed against large
amount of water using cellulose semipermeable membrane of
MWCO of 2000 g/mol for 48 h. The reservoir was
continuously replenished with fresh water to ensure the
removal of DMSO and THF from the polymer solution.
Among the two segmented polymers, only P-TCD-6EG was
found to produce a stable aqueous solution whereas P-TCD-
3EG (also P-Me-6EG) precipitated from water and was not
stable during the dialysis in water. The optical density of P-
TCD-6EG was chosen as 0.1 and the composition of the
solutions were varied from 10 to 90 v/v of THF + water. The
emission spectra of the P-TCD-6EG (see Figure 3f) showed
the decrease in the emission intensity of the isolated OPV
chromophores (at λem = 450 nm) and new emission peak
appeared for aggregated OPV chromophores (at λem = 535
nm). The comparison of emission characteristics in water +
THF combinations (see Figure 3f) with its solid state emission
spectra of P-TCD-6EG (see Figure 3a) [or its aggregation in
CHCl3 + methanol in Figure 3e] revealed that TCD-OPV
chromophores attained maximum aromatic π-stacking in >50%
water in THF or in 100% water. Further, to quantify the extent
tuning unlike the P-TCD-6EG polymers (both polymer have
same spacer length; however, their substitution in the OPV
cores varied). The difference for these behaviors can be
correlated based on their structural arrangements from the
single crystal data of methoxy substituted OPVs.49 The three
aryl rings in the methoxy substituted OPVs do not occupy the
same plane and they were tilted at either side by more than
34.02°. As a result, the OPVs became nonplanar and expected
to have less probability for the aromatic π-stacking for color
tune-ability. On the other hand, the π-core consisting of the aryl
rings in TCD substituted OPVs showed planar geometry in the
single crystal structure.50 Thus, strong aromatic π-stacking
interactions were feasible among the TCD-OPVs for their
unique color tuning-ability in the present segmented polymer
system. This observation clearly supports that the P-TCD-6EG
segmented polymer is very unique in structural design to
undergo aromatic π-stacking to produce diverse luminescent
colors. It is important to note that the present segmented OPV
polymer design is one of the first examples to report color
tuning in single π-conjugated polymer system. The fluorescence
quantum yields for the polymer solutions were determined
using quinine sulfate as standard.47,51 The quantum yields are
plotted for both chloroform+methanol and THF+water solvent
combinations (see Figure S9). The quantum yields were found
to decrease with the increase in the poor solvent amount
(methanol or water) in the polymer solutions. The decrease in
the quantum yield is attributed to the aggregated induced
fluorescence quenching of the π-conjugated OPV chromo-
phores.52
Time correlated single photon counting (TCSPC) was
employed to study the excited state luminescent life times of P-
TCD-6EG polymer in methanol/chloroform solvent combina-
tions. The OPV chromophore was excited with 371 nm Nano
LED source and the TCSPC decay profiles of the polymer are
shown in Figure 4b. The decay profiles of the polymer did not
show many changes; however, a small decrease in the decay
with an increase in the methanol compositions could be
observed (see Figure 4b). These luminescence decay profiles
were fitted with biexponential decay and their lifetime values
are given in Table ST-2 in the Supporting Information. The τ1
and τ2 values and their fractional contributions are plotted and
shown in Figure 4, parts c and d. τ1 and τ2 represent the decay
rate constant for isolated and aggregated OPV chromophores,
respectively. On increasing the methanol concentration, the
value of τ2 increased up to 60% MeOH/CHCl3 and then
attained a plateau. The higher τ2 values indicate that the
aggregated species indeed can fluorescence for a longer period.
The fractional contributions were also showed break-points at
40−50% methanol in the solvent mixtures. Hence, it may be
concluded that the TCSPC decay studies supported the color
tuning ability of the P-6EG polymer with a high luminescent
lifetime in all three colors blue, white and yellow. On the basis
of the photophysical studies, it may be concluded that the
segmented OPV polymer possessed unique polymer geometry
to undergo chain folding by the strong aromatic π-stacking on
the backbone of the polymer chains.
of aggregation in P-TCD-6EG, the ratio of the peaks λem
=
535/450 nm were plotted against the water/THF composition
and shown as an inset in Figure 3f. This plot showed a distinct
break point at 40−50% of THF in water for P-TCD-6EG. The
polymer P-TCD-6EG showed excellent color tuning from blue
to white to yellowish with an increase in the water content in
the solution (see Figure 3f) or in water. The emission spectra of
P-TCD-6EG in the solvent combinations were fed into CIE
color coordinate diagram and the details are shown in Figure
4a. It is very interesting to notice that the P-TCD-6EG chains
could be transforming its emission color from blue-to-white-to-
yellow. At the intermediate solvent combination (50:50%),
both expanded and coil-like conformation coexisted in the
polymer solution to produce white emission. This was further
supported by the break points seen in the inset in Figure 3,
parts e and f. White light emission in the present system is
observed due to the merging of blue and greenish yellow
emission of isolated and aggregated OPV chromophores,
respectively. The CIE color coordinates for the white emission
was obtained as x = 0.25 and y = 0.32 which is in accordance for
white emission as reported by us and others.53,54,35 On the
other hand, other polymers P-TCD-3EG, P-Me-6EG, and P-
EH-6EG did not show such variation in the emission colors in
the CIE color coordinates (see Figure S8). The segmented
polymers with P-Me-6EG polymers did not show the color
Nanofibers, Hollow Spheres, and Nanoparticles. It is
very clear from the photophysical studies that the P-TCD-6EG
chains are capable of undergoing π-induced aggregation for
color tuning from blue-to-white-to-yellow. To visualize the size
and shape of the polymer aggregates accompanied by the above
transition, the P-TCD-6EG samples in CHCl3 + methanol
solvent combinations were subjected to FE-SEM and AFM
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Figure 5. FE-SEM images of P-TCD-6EG polymer in chloroform (a) and 20% (b), 40% (c), and 60% (d) of methanol in chloroform. AFM images
of P-TCD-6EG polymer in chloroform (e) and 20% (f), 40% (g), and 60% (h) of methanol in chloroform/methanol. The concentrations of the
polymers were maintained as 1 × 10−5 M.
analysis. These morphological features are given in Figure 5.
The polymer showed nanofibrous morphology in chloroform
alone (see Figure 5a) and the length of helical nanofibers was
estimated to be 10 to 20 μm and their widths were determined
to be 20−50 nm in diameter. The closer observation revealed
that thin and highly helical nanofibers were produced by the
longer segmented P-TCD-6EG. HR-TEM image was also
further confirmed the existence of nanofibrous morphology in
P-TCD-6EG (see Figure S10). The P-TCD-6EG chains
showed an excellent morphological transition from nanofibers
to spherical nanoassemblies upon increasing the methanol
amount in the solvent mixture (see Figure 5b−d). The closer
observation of spherical assemblies in Figure 5c revealed that
they are hollow. The observation was attributed to the folding
or collapsing of the nanofibers into hollow spheres. At higher
methanol content, the morphology was turned completely into
Figure 6. P-TCD-6EG polymer FE-SEM image in THF (a); AFM
image in THF (b), in 20% (c), 40% (d), and 60% (e) water/THF
composition, and in water (f). The concentrations of the polymers
a spherical particle in nature (see Figure 5d).
Atomic force microscope (AFM) images of P-TCD-6EG in
chloroform showed highly twisted helical nanofibrous (see
were maintained as 1 × 10−5 M.
Figure 5e) as seen in their FE-SEM images. AFM analysis
showed the length of the fibers 5 to 10 μm with a thickness of
60 nm. AFM images of the polymer samples in solvent mixtures
in Figure 6f. FE-SEM and high resolution TEM (HR-TEM)
were recorded and shown in Figure 5f−h. At 20% methanol
compositions, the polymer exhibited both helical nanofibers
and hollow spheres (see Figure 5f). Upon increasing the
methanol amount, the formation of hollow spheres became
predominant and the nanofibrous morphology vanished
completely (see Figure 5, parts f and h). At 40−60% methanol,
the polymer chains produced exclusively hollow spheres of 400
nm in size. The depth profile of these objects clearly confirms
their hollow structures (see inset in Figure 5h). The P-TCD-
6EG polymer was also showed an excellent morphological
transition from nanofibers to spherical nanoassemblies in THF
+ water solvent mixture (see Figure 6a−f). FE-SEM and AFM
images showed that the polymer chains exhibited the helical
nanofibrous morphology in THF (see Figure 6, parts a and b).
Upon increasing the water amount in the solvent mixture, the
polymer transformed into hollow spherical assemblies as
evident from their AFM images in Figure 6c−f. At 60 and
80% THF in water, the polymer produced exclusively hollow-
spheres (see Figure 6, parts c and d). At 40% THF in water
(see Figure 6e) and the dialyzed aqueous solution (see Figure
6f, in water alone), the polymers were self-assembled into <200
nm tiny nanoparticles, which was confirmed by the AFM image
images of the dialyzed nanoparticles were recorded and showed
in Figure S11. These electron microscopic images exhibited the
formation of spherical nanoparticles.
In order to prove that the spherical nanoassemblies were pre-
existed in the polymer solution and visualized as individual
objects during imaging rather than they were produced during
the solvent evaporation process; the polymer solutions were
subjected to dynamic light scattering analysis. DLS histograms
of THF + water and chloroform + methanol mixtures are
shown in Figure 7. The dialyzed aqueous solution exhibited
monomodal distribution with respect to particles in the ranges
of 200 20 nm (see Figure 7a). DLS histograms retained its
monomodal; however the size of the spherical objects became
larger with a decrease in the amount of water in THF+water
combinations. The size of the particles increase from 200 nm to
1.0 μm for 60% and 40% of water in THF (see Figure 7, parts b
and c). This trend clearly suggests that upon increasing the
THF concentration, the tightly packed nanoparticles slowly
uncoiled into large size aggregates. The DLS histograms of
chloroform+methanol solvent combination is also exhibited
similar trend (see Figures 7d to 7f). For example, the polymers
were self-assembled as monomodal aggregated species in 60%
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Figure 7. DLS histograms of P-TCD-6EG in water (a) and in 60% (b), and 40% (c) water/THF composition. The concentration of the polymers
were maintained as 2.5 × 10−5 M (d). DLS histograms of P-TCD-6EG in 60% (d), 40%(e), and 20% (f) methanol/chloroform composition. The
concentrations of the polymers were maintained as 2.5 × 10−4 M.
and 40% methanol/chloroform (see Figures 7, parts d and e),
and it became multimodal with larger aggregates in 20%
methanol/chloroform (see Figure 7f). These trends indicate
that the polymer self-assembled as tightly packed spherical
objects in bad solvents (in methanol or water) and expanded to
larger sizes in good solvents (THF or chloroform) (for the DLS
histograms of other good and bad solvent combination, see
Figures S12 and S13). Thus, the DLS data direct evidence that
the morphological transitions from spherical to nanofibrous and
vice versa are indeed occurred by the ability of the polymer
chains to preassemble in the solvent combinations and not
occurred by the solvent evaporation process while sample
preparation for imaging. The segmented polymer chains were
solvated in good solvents (like chloroform and THF) in
expanded chain conformation. These solvated chains undergo
interchain aggregation to produce long fibril structure in the
drop castes films (see Figures 5a and 6a). On the other hand, in
poor solvents (like methanol and water), the polymer chains
are collapsed to adapt coil-like conformation and these coiled
chains would prefer to aggregate together to produce
nanoparticles (see Figures 5d and 6f). Thus, polymer topology
played a crucial role on the self-assembly of these custom
designed segmented OPV polymers.
Based on the photophysical studies, color-tuning ability,
morphological evidence, and DLS studies; the self-assembly of
the segmented polymers are schematically drawn and shown in
Figure 1. The following conclusion may be drawn for the
unique self-assembly of custom-designed segmented TCD-
OPV polymer. The polymer existed as solvated chains in good
solvents (THF or chloroform) and exhibits blue-luminescence.
The drop casted film from the good solvent produces helical
nanofibrous morphology. Upon increasing the bad solvent
composition (water or methanol) in the polymer solution
(THF + water or chloroform + methanol); the polymer chains
experienced strong aromatic π−π stacking and produced
layered intermediates via hydrophobic and hydrophilic
interaction by the amphiphilic backbones. At this stage, the
OPV π-core exists both in the aggregated (greenish yellow) and
isolated form (blue) and their combination produced white
color emission. The increase in the bad solvent amount
(methanol or water) increase the packing further and the
layered assembly folded as spherical hollow particles and emit
greenish yellow emission. The dialyzed polymer solution (in
water alone); self-assembled structure appeared as tiny
nanoparticles (no hollow structures are observed in the
microscopic analysis) and luminescence as bright yellow
color. Hence, the present segmented polymer design is very
unique in producing diverse nanoassemblies such as nanofibers
and hollow spheres and further capable of exhibiting
luminescence color-tuning from blue-to-white-to-yellow in a
single π-conjugated system.
Cytotoxicity and Bioimaging. Water-soluble π-conju-
gated polymers are emerging as important classes of
luminescent probes for cellular imaging in biological
system.1,2,55 The custom designed segmented TCD-OPV
polymer produced very stable luminescent aqueous nano-
particles. Thus, these nanoparticles were employed as a
bioimaging probe for cancer cells. The cytotoxicity of the
polymer nanoparticles were investigated in cervical (HeLa) and
breast cancer (MCF 7) cell lines. The cytotoxicity of the
polymer was tested in these cell lines by varying their
concentration up to 60 μg/mL and the data are summarized
in Figure 8. The nascent polymer nanoparticles were found to
be nontoxic to cells and more than 90% cell viability was
Figure 8. Cytotoxicity of P-TCD-6EG polymer nanoparticles (a) in
HeLa cells and (b) in MCF-7 cells.
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observed in both cell lines. This data confirmed that the
amphiphilic nanoparticles made by the segmented TCD-OPV
polymers are very good biocompatible probes for cancer cells.
For cellular imaging, the polymer nanoparticle was adminis-
trated to HeLa cells and their confocal images were recorded
and shown in Figure 9. Since the polymer nanoparticles are
carries along with intrinsic fluorescence as dual function
delivery-cum-biomarkers in cancer therapy. Currently, research
work is focused on these directions to explore these segmented
conjugated polymers in cancer therapy.
CONCLUSION
■
In summary, a new series of amphiphilic segmented π-
conjugated polymers were designed and developed for studying
their solvent-mediated diverse supramolecular assemblies and
employed their aqueous nanoassemblies as a fluorescent probe
for bio- imaging in cancer cells. The π-conjugated core was
constituted by oligo-phenylenevinylene (OPV) units with
variable anchoring units at the periphery to tune their rigidity
and solubility for self-organization. The flexible segments were
chosen from oligoethyleneoxy units and they were placed in the
backbone of the chains to yield amphiphilic segment OPV
polymers. Solvent induced aggregation ability of the segmented
polymers were investigated in chloroform + methanol, THF +
water, and water alone. The π-conjugated OPV core was found
to undergo strong aromatic π−π stacking upon increasing the
composition of the bad solvent (water or methanol) in the
polymer solution. Both types of the OPV cores as well as the
oligoethyleneoxy chain lengths were found to play an important
role in directing the segmented polymers for diverse self-
assembly. The polymer P-TCD-6EG was found to produce
helical nanofibrous morphology in good solvents (chloroform
or THF) and it underwent a morphological transition from
fibers to hollow spheres upon varying the solvent composition.
This morphological transition was also accompanied by the
fluorescence color tuning by the OPV core. It was found that
the chromophores exhibited blue-to-white-to-yellow color
change along with the morphological transitions. Dynamic
light scattering studies confirmed that the segmented polymers
indeed produced stable spherical nanoaggregates of 200 nm in
the solution. Steady state fluorescence studies and time-
resolved fluorescent decay dynamic analysis confirmed the
color tunability in the segmented π-conjugated polymers. The
polymers produced stable nanoparticles assemblies in water
with luminescent properties. Cytotoxicity in cervical and breast
cancer cells revealed that these segmented polymer nano-
particles are nontoxic to cells. Confocal microscopy imaging
revealed that the polymer nanoparticles selectively accumulated
in the nucleus. The present design explored new segmented π-
conjugated polymers for producing stable aqueous nano-
particles which are successfully demonstrated as potential
candidates for bioimaging applications. The segmented
polymer design reported here not only restricted to OPV π-
core, and in general it can be expanded to wide ranges of other
π-conjugated systems to explore this new concept for material
and biomedical applications.
Figure 9. Confocal images of HeLa cells incubated with P-TCD-6EG
polymer at 4, 8, and 12 h. The cells were observed through the blue
channel to locate the polymer (OPV chromophore) fluorescence. The
actin cytoskeleton network in cells was stained with phalloidin (red).
blue fluorescent, the cytoplasm of the cell was stained with
Phalloidin (red fluorescent dye) and merged images distinctly
showed the nucleus and cytoplasm. To study the influence of
the incubation period on the polymer nanoparticles uptake; the
cells were treated at various time intervals of 4, 8, and 12 h with
the polymer nanoparticles. The comparison of these images
revealed that the cellular uptake of the polymer nanoparticles at
4 h is almost similar to that at 8 and 12 h.
Most of the polymer nanoparticles are typically up taken by
the cells through endocytosis process. Our recent studies on the
polymer nanoparticles in the treatment of the breast and colon
cancer cell lines supported the similar observation.56 Typically
polyaromatic drugs or nuclear staining agents bind to the DNA
in the nucleus by intercalation and aromatic π-stacking
interaction.56 Thus, the newly designed OPV aromatic π-core
may be underwent similar binding mechanism at the nucleus.
Very recently we observed a similar trend for the binding of
OPV based biodegradable block copolymer nanoassemblies.55
These nanoparticles were found to be selectively accumulated
in the nucleus of the cells. The merged image did not show any
trace of the polymer particles in the cytoplasm. This
observation makes these segmented OPV nanoparticles as
ideal markers for the nucleus. These experiments confirmed
that the blue luminescent nascent polymer nanoparticles are
highly cell penetrable and very useful for cellular imaging
applications in cancer diagnostics. Since OPV cores are very
new entries for cellular imaging and the present manuscript
explored their reproducibility in both cervical (HeLa) and
breast (MCF 7) cancer cell lines; the nuclear staining of OPVs
would be expected to be similar for other cells as well. Further
detail studies of DNA-OPV chromophores interactions are
required to confirm this hypothesis. Owing to their
biocompatibility, the structure of the segmented polymer can
be further improvised to them as efficient anticancer drug
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.macro-
mol.6b00660.
Synthesis of polymers, structural and molecular weights
characterization, TGA and DSC plot, absorption and
emission data, FE-SEM and AFM, and HR-TEM images,
photophysical data, and NMR spectra of the monomers
and polymers (PDF)
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(17) Schenning, A. P. H. J.; Kilbinger, A. F. M.; Biscarini, F.;
Cavallini, M.; Cooper, H. J.; Derrick, P. J.; Feast, W. J.; Lazzaroni, R.;
Leclere, P.; McDonell, L. A.; Meijer, E. W.; Meskers, S. C. J.
Supramolecular Organization of α,α‘-Disubstituted Sexithiophenes. J.
Am. Chem. Soc. 2002, 124, 1269.
(18) Abbel, R.; van der Weegen, R.; Meijer, E. W.; Schenning, A. P.
H. J. Multicolour self-assembled particles of fluorene-based bolaam-
phiphiles. Chem. Commun. 2009, 1697.
(19) Shin, S.; Lim, S.; Kim, Y.; Kim, T.; Choi, T.; Lee, M.
Supramolecular Switching between Flat Sheets and Helical Tubules
Triggered by Coordination Interaction. J. Am. Chem. Soc. 2013, 135,
2156−2159.
(20) Lee, E.; Jeong, Y.; Kim, J.; Lee, M. Controlled Self-Assembly of
Asymmetric Dumbbell-Shaped Rod Amphiphiles: Transition from
Toroids to Planar Nets. Macromolecules 2007, 40, 8355−8360.
(21) Ryu, J.; Lee, M. Transformation of Isotropic Fluid to Nematic
Gel Triggered by Dynamic Bridging of Supramolecular Nanocylinders.
J. Am. Chem. Soc. 2005, 127, 14170−14171.
(22) Moon, K.; Kim, H.; Lee, E.; Lee, M. Self-Assembly of T-Shaped
Aromatic Amphiphiles into Stimulus-Responsive Nanofibers. Angew.
Chem., Int. Ed. 2007, 46, 6807.
AUTHOR INFORMATION
Corresponding Author
*(M.J.) E-mail: jayakannan@iiserpune.ac.in.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors express thanks for a research grant from the
Department of science and Technology (DST), New Delhi,
India, under SERB Project SB/OC/OM-10/2014. The authors
thank Bapurao Surnar and Sonashree Saxane from our group
for cytotoxicity and cellular imaging data, respectively. We
thank MHRD, New Delhi, India, for funding under FAST
Scheme. K.N. thanks the UGC, New Delhi, India, for a research
fellowship.
REFERENCES
■
(1) Li, K.; Liu, B. Polymer-encapsulated organic nanoparticles for
fluorescence and photoacoustic imaging. Chem. Soc. Rev. 2014, 43,
6570−6597.
(23) Moon, K.; Lee, E.; Lee, M. Synthesis and self-assembly of
propeller-shaped amphiphilic molecules. Chem. Commun. 2008, 3061−
3063.
(24) Rest, C.; Mayoral, M. J.; Fucke, K.; Schellheimer, J.; Stepanenko,
̀
V.; Fernandez, G. Self-Assembly and (Hydro)gelation Triggered by
(2) Zhu, C.; Liu, L.; Yang, Q.; Lv, F.; Wang, S. Water-Soluble
Conjugated Polymers for Imaging, Diagnosis, and Therapy. Chem. Rev.
2012, 112, 4687−4735.
(3) Bunz, U. H. F.; Seehafer, K.; Bender, M.; Porz, M.
Poly(aryleneethynylene)s (PAE) as paradigmatic sensor cores. Chem.
Soc. Rev. 2015, 44, 4322−4336.
Cooperative π−π and Unconventional C[BOND]H···X Hydrogen
Bonding Interactions. Angew. Chem., Int. Ed. 2014, 53, 700−705.
(25) Ajayaghosh, A.; Varghese, R.; Mahesh, S.; Praveen, V. K. From
Vesicles to Helical Nanotubes: A Sergeant-and-Soldiers Effect in the
Self-Assembly of Oligo(p-phenyleneethynylene)s. Angew. Chem., Int.
Ed. 2006, 45, 7729.
(26) Shin, S.; Gihm, S. H.; Park, C. R.; Kim, S.; Park, S. Y. Water-
Soluble Fluorinated and PEGylated Cyanostilbene Derivative: An
Amphiphilic Building Block Forming Self-Assembled Organic Nano-
rods with Enhanced Fluorescence Emission. Chem. Mater. 2013, 25,
3288−3295.
(27) Hulvat, J. F.; Sofos, M.; Tajima, K.; Stupp, S. I. Self-Assembly
and Luminescence of Oligo(p-phenylene vinylene) Amphiphiles. J.
Am. Chem. Soc. 2005, 127, 366−372.
(28) Zhang, X.; Gorl, D.; Wurthner, F. White-light emitting dye
micelles in aqueous solution. Chem. Commun. 2013, 49, 8178−8180.
(29) Zhang, X.; Chen, Z.; Wurthner, F. Morphology control of
fluorescent nanoaggregates by co-self-assembly of wedge- and
dumbbell-shaped amphiphilic perylene bisimides. J. Am. Chem. Soc.
2007, 129, 4886−4887.
(30) Kim, Y.; Li, W.; Shin, S.; Lee, M. Development of Toroidal
Nanostructures by Self-Assembly: Rational Designs and Applications.
Acc. Chem. Res. 2013, 46, 2888−2897.
(31) Molla, M. R.; Ghosh, S. Aqueous self-assembly of chromophore-
conjugated amphiphiles. Phys. Chem. Chem. Phys. 2014, 16, 26672−
26683.
(32) Wang, H.; Wang, H. H.; Urban, V. S.; Littrell, K. C.;
Thiyagarajan, P.; Yu, L. Syntheses of Amphiphilic Diblock Copolymers
Containing a Conjugated Block and Their Self-Assembling Properties.
J. Am. Chem. Soc. 2000, 122, 6855−6861.
(33) Luo, Y.; Liu, H.; Xi, F.; Li, L.; Jin, X.; Han, C. C.; Chan, C.
Supramolecular Assembly of Poly(phenylene vinylene) with Crown
Ether Substituents To Form Nanoribbons. J. Am. Chem. Soc. 2003,
125, 6447−6451.
(34) Lee, E.; Hammer, B.; Kim, J. K.; Page, Z.; Emrick, T.; Hayward,
R. C. Hierarchical Helical Assembly of Conjugated Poly(3-
hexylthiophene)-block-poly(3-triethylene glycol thiophene) Diblock
Copolymers. J. Am. Chem. Soc. 2011, 133, 10390−10393.
(35) Hu, Y.; Su, M.; Ma, C.; Yu, Z.; Liu, N.; Yin, J.; Ding, Y.; Wu, Z.
Multiple Stimuli-Responsive and White-Light Emission of One-Pot
Synthesized Block Copolymers Containing Poly(3-hexylthiophene)
and Poly(triethyl glycol allene) Segments. Macromolecules 2015, 48,
5204−5212.
(4) Thomas, S. W., III; Joly, G. D.; Swager, T. M. Chemical Sensor
Based on Amplifying Fluorescent Conjugated Polymers. Chem. Rev.
2007, 107, 1339−1386.
(5) Kim, H. N.; Ren, W. X.; Kim, J. S.; Yoon, J. Fluorescent and
colorimetric sensors for detection of lead, cadmium, and mercury ions.
Chem. Soc. Rev. 2012, 41, 3210−3244.
(6) Wu, Y.; Tan, Y.; Wu, J.; Chen, S.; Chen, Y. Z.; Zhou, X.; Jiang, Y.;
Tan, C. Fluorescence Array-Based Sensing of Metal Ions Using
Conjugated Polyelectrolytes. ACS Appl. Mater. Interfaces 2015, 7,
6882−6888.
(7) McQuade, D. T.; Pullen, A. E.; Swager, T. M. Conjugated
Polymer-Based Chemical Sensors. Chem. Rev. 2000, 100, 2537−2574.
(8) Bunz, U. H. F. Poly(aryleneethynylene)s: Syntheses, Properties,
Structures, and Applications. Chem. Rev. 2000, 100, 1605−1644.
(9) Xu, X.; Liu, R.; Li, L. Nanoparticles made of π-conjugated
compounds targeted for chemical and biological applications. Chem.
Commun. 2015, 51, 16733−16749.
(10) Ding, D.; Li, K.; Zhu, Z.; Pu, K.; Hu, Y.; Jiang, X.; Liu, B.
Conjugated polyelectrolyte−cisplatin complex nanoparticles for
simultaneous in vivo imaging and drug tracking. Nanoscale 2011, 3,
1997−2002.
(11) Feng, L.; Liu, L.; Lv, F.; Bazan, G. C.; Wang, S. Preparation and
Biofunctionalization of Multicolor Conjugated Polymer Nanoparticles
for Imaging and Detection of Tumor Cells. Adv. Mater. 2014, 26,
3926−3930.
(12) Feng, X.; Tang, Y.; Duan, X.; Liu, L.; Wang, S. Lipid-modified
conjugated polymer nanoparticles for cell imaging and transfection. J.
Mater. Chem. 2010, 20, 1312−1316.
(13) Akcelrud, L. Electroluminescent polymers. Prog. Polym. Sci.
2003, 28, 875.
(14) Kilbinger, A. F. M.; Schenning, A. P. H. J.; Goldoni, F.; Feast,
W. J.; Meijer, E. W. Chiral Aggregates of α,ω-Disubstituted
Sexithiophenes in Protic and Aqueous Media. J. Am. Chem. Soc.
2000, 122, 1820−1821.
(15) Jiang, L.; Hughes, R. C.; Sasaki, D. Y. Supramolecular assembly
of a quaterthiophene surfactant. Chem. Commun. 2004, 1028−1029.
(16) Henze, O.; Feast, W. J.; Gardebien, F.; Jonkheijm, P.; Lazzaroni,
R.; Leclere, P.; Meijer, E. W.; Schenning, A. P. H. J. Chiral Amphiphilic
Self-Assembled α,α‘-Linked Quinque-, Sexi-, and Septithiophenes:
Synthesis, Stability and Odd−Even Effects. J. Am. Chem. Soc. 2006,
128, 5923.
L
DOI: 10.1021/acs.macromol.6b00660
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
(36) Yang, Z.; Sokolik, I.; Karasz, F. E. A Soluble Blue-Light-Emitting
Polymer. Macromolecules 1993, 26, 1188−1190.
(37) Chen, Y.; Xu, Y.; Perry, K.; Sokolov, A. P.; More, K.; Pang, Y.
Achieving Diameter-Selective Separation of Single-Walled Carbon
Nanotubes by Using Polymer Conformation-Confined Helical Cavity.
ACS Macro Lett. 2012, 1, 701−705.
(56) Pramod, P. S.; Shah, R.; Chaphekar, S.; Balasubramanian, N.;
Jayakannan, M. Polysaccharide Nano-vesicular Multidrug Carrier for
Synergistic Killing of Cancer Cells. Nanoscale 2014, 6, 11841−11855.
(38) Chen, Y.; Malkovskiy, A.; Wang, X.-Q.; Lebron-Colon, M.;
Sokolov, A. P.; Perry, K.; More, K.; Pang, Y. Selection of Single-Walled
Carbon Nanotube with Narrow Diameter Distribution by Using a
PPE−PPV Copolymer. ACS Macro Lett. 2012, 1, 246−251.
(39) Lokey, R. S.; Iverson, B. L. Synthetic molecules that fold into a
pleated secondary structure in solution. Nature 1995, 375, 303−305.
(40) Gabriel, G. J.; Sorey, S.; Iverson, B. L. Altering the Folding
Patterns of Naphthyl Trimers. J. Am. Chem. Soc. 2005, 127, 2637−
2640.
(41) Ghosh, S.; Ramakrishnan, S. Structural Fine-Tuning of
(Donor−spacer−acceptor−spacer)n Type Foldamers. Effect of Spacer
Segment Length, Temperature, and Metal-Ion Complexation on the
Folding Process. Macromolecules 2005, 38, 676−686.
(42) Ghosh, S.; Ramakrishnan, S. Aromatic donor-acceptor charge-
transfer and metal-ion-complexation-assisted folding of a synthetic
polymer. Angew. Chem., Int. Ed. 2004, 43, 3264−3268.
(43) Ghosh, S.; Ramakrishnan, S. Small molecule induced folding of
a synthetic polymer. Angew. Chem., Int. Ed. 2005, 44, 5441−5447.
(44) Tan, T. A. T.; Clarke, T. M.; James, D.; Durrant, J. R.; White, J.
M.; Ghiggino, K. P. Synthesis and photo-induced charge separation of
confined conjugation length phenylene vinylene-based polymers.
Polym. Chem. 2013, 4, 5305−5309.
(45) Balamurugan, A.; Reddy, M. L. P.; Jayakannan, M. π-conjugated
Polymer-Eu3+ Complexes: A Versatile Luminescent Molecular probe
for Temperature Sensing. J. Mater. Chem. A 2013, 1, 2256−2266.
(46) Balamurugan, A.; Kumar, V.; Jayakannan, M. Triple action
Polymer Probe: Carboxylic Distilbene Fluorescent Polymer Chemo-
sensor for Temperature, Metal-ion and Biomolecules. Chem. Commun.
2014, 50, 842−845.
(47) Goel, M.; Narasimha, K.; Jayakannan, M. Helical Self-assemblies
of Segmented Poly(phenylenevinylene)s and their Hierarchical
Donor-Acceptor Complexes. Macromolecules 2014, 47, 2592−2603.
(48) Narasimha, K.; Jayakannan, M. π-Conjugated Polymer
Anisotroipic Organogel Nano-fibrous Assemblies for Thermo-
responsive Photonic Switches. ACS Appl. Mater. Interfaces 2014, 6,
19385−19396.
(49) Goel, M.; Narasimha, K.; Jayakannan, M. Direct Evidence for
Secondary Interactions in Planar and Nonplanar Aromatic π-
Conjugates and Their Photophysical Characteristics in Solid-State
Assemblies. J. Phys. Chem. B 2015, 119, 5102−5112.
(50) Goel, M.; Jayakannan, M. Herringbone and Helical Self-
assembly of π -conjugated Molecules in Solid State through CH/π
Hydrogen Bond. Chem. - Eur. J. 2012, 18, 11987−11993.
(51) Amrutha, S. R.; Jayakannan, M. Probing the π -stack Induced
Molecular Aggregation in π -Conjugated Polymers, Oligomers and
Their Blends of Poly(phenylenevinylene)s. J. Phys. Chem. B 2008, 112,
1119−1129.
́
(52) Resta, C.; Di Pietro, S. D.; Majeric Elenkov, M.; Hamersak, Z.;
Pescitelli, G.; Di Bari, L. D. Consequences of Chirality on the
Aggregation Behavior of Poly[2-methoxy-5-(2′-ethylhexyloxy)-p-phe-
nylenevinylene] (MEH-PPV). Macromolecules 2014, 47, 4847−4850.
(53) Sonawane, S. L.; Asha, S. K. Blue, Green, and Orange-Red
Emission from Polystyrene Microbeads for Solid-State White-Light
and Multicolor Emission. J. Phys. Chem. B 2014, 118, 9467−9475.
(54) Balamurugan, A.; Reddy, M. L. P.; Jayakannan, M. Single
polymer Photosensitizer for Tb3+ and Eu3+ ions: A Novel Approach
for White Light Emission based on Carboxylic Functionalized poly(m-
phenylenevinylene)s. J. Phys. Chem. B 2009, 113, 14128−14138.
(55) Kulkarni, B.; Surnar, B.; Jayakannan, M. Dual Functional
Nanocarrier for Cellular Imaging and Drug Delivery in Cancer Cells
Based on π -Conjugated Core and Biodegradable Polymer Arms.
Biomacromolecules 2016, 17, 1004−1016.
M
DOI: 10.1021/acs.macromol.6b00660
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