Fluorescent PEGulated Oligourethane Nanoparticles for Long-Term Cellular Tracing

Article abstract of DOI:10.1002/chem.201601718

We have introduced a new ABA-type amphiphilic block copolymer consisting of functional oligourethane hydrophobic blocks and two polyethylene glycol (PEG) hydrophilic blocks. The polymer was synthesized in a single step by step-growth polymerization betwee

Full text of DOI:10.1002/chem.201601718

DOI: 10.1002/chem.201601718
Full Paper
&
Biosensors
Fluorescent PEGulated Oligourethane Nanoparticles for Long-
Term Cellular Tracing**
[
a]
Tathagata Mondal, Jayita Sarkar, and Suhrit Ghosh*
Abstract: We have introduced a new ABA-type amphiphilic
block copolymer consisting of functional oligourethane hy-
drophobic blocks and two polyethylene glycol (PEG) hydro-
philic blocks. The polymer was synthesized in a single step
by step-growth polymerization between two monomers,
namely tetraphenylethylene (TPE)-diol and hexamehylene di-
isocyanate in the presence of a monofunctional impurity
PEG-2000. The polymer exhibits facile self-assembly in water
by synergistic effects of H-bonding and p–p interaction
among the oligourethane core, leading to the formation of
robust nanoparticles with remarkable aggregation-induced
emission (AIE). These nanoparticles show very low critical ag-
gregation concentration, stability over a large pH window,
and excellent biocompatibility as revealed by an MTT assay.
Cellular imaging with cancer cells showed facile cellular
uptake and, more importantly, retention of AIE in cellular
milieu for long times, which was successfully utilized for
long-term cancer cell tracking.
Introduction
they often exhibit cytotoxicity, photobleaching, and diffusion
so that the background fluorescence limits sensitivity. Further-
more, poor cellular uptake and insufficient retention time re-
sults in extrusion of dyes from the cells during cell division and
thus does not allow long-term tracking. Another major prob-
lem in small-molecule fluorescence dyes is their loss of flores-
cence due to external quenching by a large number of possi-
ble quenchers in the biological milieu or by self-quenching
Biocompatible fluorescent particles are of great importance for
vital biological applications such as biosensing and bioimag-
[1]
ing. The design of suitable fluorescent bio-probes remains
a challenging task because it requires optimization of several
parameters such as bright luminescence, toxicity, efficient cel-
lular uptake, specificity, and stability under physiological condi-
[2]
[3]
[7]
tions. Quantum dots (QDs), proteins, core–shell nanoparti-
due to aggregation in aqueous medium, which is not an easy
[
4]
[5]
[8]
cles, conjugated polymer-derived particles, small-molecule
problem address. In contrast, selected luminogens such as
[
6]
dyes, and others have been tested as fluorescent particles for
various biotechnological applications. However, heavy metal
components of QDs are known to release toxic components,
which limit their application in bioimaging. On the other hand,
green fluorescent protein (GFP) and its variants, which have
been routinely used as cell trackers, have some drawbacks
such as susceptibility to proteolytic enzymes, poor photostabil-
ity as well as disruption of normal cell function. Conjugated
polyelectrolytes or, more recently, polymer dots have attracted
great attention because of their bright fluorescence, low cyto-
toxicity and so on. Nevertheless, their synthesis is not trivial,
often involves metal catalysts, and the preparation of nanopar-
ticles by miniemulsion or other techniques requires substantial
effort for standardization. Small-molecule organic fluorophores
have also been used for cellular imaging because of their high
quantum yield and tunable optical properties. Nevertheless,
tetraphenylene ethylene (TPE) exhibit aggregation-induced
emission (AIE) because of the restriction of internal rotations
within the chromophore, which has inspired studies on wide-
ranging TPE-derivatives as emerging fluorescent organic mate-
[
9a–b]
[9c]
rials
and bioprobes. More recently, there has been up-
surge in attention to macromolecules with AIE chromo-
[
10,11]
phores,
which are attractive candidates for biological appli-
[
12]
cations when the polymer is water soluble. In the majority
of examples of TPE-based macromolecules, it is noted that the
dye is either attached as a pendant or at the terminal of a poly-
mer backbone made of CÀC bonds, which, upon immiscibility
[
13]
driven aggregation in a bad solvent, leads to AIE. These sys-
tems have a number of problems: (i) Loosely bound aggre-
gates may not allow maximum enhancement of the lumines-
cence; (ii) The polymer backbone (CÀC bond) is not biodegrad-
able and often not fully biocompatible; (iii) Significant dilution
after administration in the body may cause disassembly and
resultant turning off of the fluorescence. To circumvent these
issues, we searched for options for TPE-based water soluble
polymers in which the chromophore is part of the polymer
backbone itself. In this endeavor, we envisioned that the use
[
a] T. Mondal, J. Sarkar, Dr. S. Ghosh
Indian Association for the Cultivation of Science, Polymer Science Unit
2
A & 2B Raja S. C. Mullick Road, Kolkata 700032 (India)
Fax: ( +91)33-2473-2805
E-mail: psusg2@iacs.res.in
[
14]
of a polyurethane scaffold would be attractive considering
its ease of synthesis, biocompatible, and biodegradable nature,
as well as the possibility of strong self-assembly by directional
[
**] PEG=polyethylene glycol.
Supporting information for this article can be found under http://
dx.doi.org/10.1002/chem.201601718.
Chem. Eur. J. 2016, 22, 10930 – 10936
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hydrogen-bonding interactions between the urethane func-
tional groups, which is not commonly seen in immiscibility
driven aggregation.
With this aim, we have synthesized a novel amphiphilic tri-
block copolymer (P1, Scheme 1) in which a short hydrophobic
block (only 28 wt%) consists of a TPE-derived oligourethane
Scheme 1. Synthesis of P1.
(
OU) that is capped at both ends by hydrophilic polyethylene
glycol (PEG) chains. In this article, we describe H-bonding-
driven robust self-assembly of P1 in water, leading to straight-
forward formation of highly fluorescent nanoparticles and its
[15]
utility in long-term cell imaging, which has been a difficult
task with fluorescent proteins or organic dyes because of their
1
Figure 1. a) H NMR spectrum of P1 in CDCl
CHCl
c) FTIR spectra of P1 in chloroform.
3
(* indicates peaks from residual
3
of the solvent and water); b) GPC traces of P1 and PEG-2000 in THF;
[16]
low photobleaching threshold.
À1
chloroform exhibits characteristic peaks at 3338 cm and
Results and Discussion
À1
1
729 cm for the NÀH and C=O stretching of the free ure-
Synthesis and characterization: Polymer P1 was synthesized
Scheme 1) by condensation polymerization between hexam-
thane group, respectively, further confirming the polymer
À1
(
structure. A sharp peak also appears at 1100 cm , which can
ethylene diisocyante (M1, BB) and a dihydroxy-TPE derivative
M2 (AA) in the presence of polyethylene glycol monomethyl
ether (PEG-2000) (A*) as a “monofunctional impurity” (MFI) and
be assigned to the C-O-C stretching frequency of the PEG
chains. Thermogravimetric analysis (TGA; Figure S1) shows an
initial 4% weight loss at 878C due to the dehydration of the
sample and then degradation at 2408C, suggesting excellent
[17]
1
,4-diazabicyclo[2.2.2]octane (DABCO) as the catalyst.
To ensure capping of the oligourethane chains at both ends
thermal stability. DSC studies (Figure S2) reveal a single melting
À1
by PEG, specific amounts of the two monomers and the mono-
transition at 518C (DH =77.17 Jg ) during heating and a re-
m
À1
functional impurity satisfying the equation 2N =2NAA + N
versible exothermic peak at 198C (DH =À73.39 Jg ) in the
BB
A*
18]
c
[
where N is the mole fraction of each component, was used.
cooling cycle due to crystallization of PEG.
The triblock copolymer P1 was thus synthesized in a single
step and was isolated as a sticky solid in 60% yield. P1 was
characterized based on its H NMR spectra (Figure 1a), in
Self-assembly and AIE behavior of P1: Self-assembly of P1
in water was examined by dynamic light scattering (DLS) stud-
ies (Figure 2a), which revealed a sharp peak corresponding to
1
which all peaks could be assigned unambiguously. By compar-
hydrodynamic diameter (D ) of 25Æ5 nm. Transmission elec-
h
ing the intensity of the peak for H (terminal CH proton of the
tron microscopy studies of drop-cast aqueous P1 solution on
carbon-coated Cu grid indicated formation of spherical parti-
cles with a diameter in the range of 30–40 nm (Figure 2b, S3)
and thus corroborated the results obtained from DLS measure-
ments. The TEM images also showed the presence of a few
larger particles, which may be the consequence of multiple
e
3
PEG block) and H (backbone CH proton of the OU block), the
a
2
number average degree of polymerization (X ) was found to
n
be 6, which is consistent with the estimated value (X =7) de-
n
rived from Caruthers equation by feeding the relative mole
fractions of the two monomers and the MFI and assuming
[18]
[19]
a conversion of 99.0%. We intentionally opted for a low Xn
particle clustering during the drying process. To examine the
for the TPE-derived hydrophobic block to ensure facile water
effect of self-assembly on the emission properties of TPE-con-
taining P1, solutions of P1 in tetrahydrofuran (THF) (which is
a good solvent for the OU backbone) and water were exam-
ined under UV irradiation, which showed bright greenish blue
emission from the aqueous solution, whereas almost no light
was emitted from a vial filled with THF solution of P1, confirm-
dispersion and low cytotoxicity. The GPC trace of P1 (Fig-
À1
ure 1b) shows a unimodal peak with M =4500 gmol (PDI=
n
1
.2), which is consistent with the predicted number following
the NMR estimated X and molecular weight of the commer-
n
cially available PEG block. The FTIR spectra of P1 (Figure 1c) in
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Figure 2. a) Dynamic light scattering plot and b) TEM image of P1 in water;
c) Images of P1 in THF and water (1.0 mm) under UV light (lex =365 nm),
showing AIE in water; d) Fluorescence microscopy image of P1.
ing the AIE behavior of P1 (Figure 2c). This was further con-
firmed from fluorescence microscopy images, which showed
the presence of emitting particles (Figure 2d).
Figure 3. a) Emission spectra of P1 at different water content in THF and
b) variation of emission intensity at 470 nm as a function of % of water/THF;
To further elucidate the AIE behavior quantitatively, we re-
corded solvent-dependent emission spectra of P1 in THF/H O
2
c) Concentration-dependent emission spectra of P1 in H O and d) variation
2
mixtures (Figure 3a). In THF, negligible emission (380–390 nm)
was noted, which is characteristic of monomeric TPE. With
gradual increase in the water content, a new emission band
emerged at 470 nm and intensified (> 100 fold compared with
that in THF) in pure water. The emission intensity versus %
of emission intensity at 470 nm with concentration of P1; e) Emission spec-
tra of P1 in THF and H O at various pH, and f) normalized emission intensity
2
at 470 nm for different samples. [P1]=0.1 mm, lex =330 nm.
pared (Figure 4a). The stretching frequency for the C=O bond
H O plot (Figure 3b) suggests the onset of aggregation and re-
of the urethane group shifted to lower wavenumber by
2
À1
À1
sultant enhanced emission beyond 80% H O. The quantum
17 cm (1729 to 1712 cm ) in D O, indicating strong H-bond-
2
2
[
18]
yield of P1 was estimated to be very low (F=0.01) in THF, but
remarkably enhanced to 0.17 in aqueous medium, which ranks
among the top values reported for TPE-based polymers in
water. The critical aggregation concentration (CAC) of P1 was
ing interactions among the urethane groups. In addition, the
peaks characteristic for CÀH asymmetric and symmetric
stretching frequency appeared at significantly higher wave-
À1
numbers in D O (2928 and 2888 cm , respectively) than in
2
À1
estimated from the concentration-dependent emission spectra
chloroform (2910 and 2875 cm , respectively), indicating or-
À6
(
Figure 3c–d), which revealed a very low value of 7.6”10
m
dered packing of -CH - units within the nanoparticles. Further-
2
suggesting very strong aggregation, particularly considering
the hydrophobic TPE-derived OU block contributes only 28%
to the total mass of the polymer with the rest being made up
of hydrophilic PEG. The pH-dependent fluorescence spectra of
aqueous P1 solution (Figure 3e) revealed almost invariant
emission intensity in the window of pH 5.0–9.0, suggesting
very robust self-assembly and effective site isolation of the ure-
thane groups from bulk water. Even outside this window,
under highly acidic (pH 3.0) or basic (pH 11.0) conditions, 50–
more, in the presence of urea, which is used as a denaturing
agent for proteins because of its ability to disrupt H-bond-
[
21]
ing, the emission intensity significantly reduced (Figure 4b,
c) for P1, suggesting that the self-assembly was indeed primar-
ily governed by H-bonding.
Based on the acquired evidence, we propose that, in aque-
ous medium, the TPE-OU segments assemble by lateral H-
bonding among the urethane groups, leading to restriction of
the conformational freedom of the TPE units, which contrib-
1
6
0% emission was retained (Figure 3 f), suggesting that self-as-
utes to robust self-assembly and AIE. When the H NMR spectra
sembly is not fully destroyed even under very harsh condi-
(Figure 4d) of P1 in CHCl and D O were compared, significant
peak broadening and reduction in intensity for all protons of
3
2
[
20]
tions.
Such remarkable stability may be attributed to multivalent
H-bonding interactions between the OU chains, in addition to
their hydrophobic collapse in water. To probe this, FTIR spectra
the TPE-OU segments in D O were observed, suggesting freez-
2
ing of the segmental motion in the aggregate state. The ob-
served upfield shift of the aromatic protons of the TPE chro-
of P1 in a good solvent (chloroform) and in D O were com-
mophore in D O is attributed to a shielding effect as a result of
2
2
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Figure 5. Microscopy images in bright field of (a, b) HeLa cells and (c,
À1
d) MCF-7 cells incubated without (a, c) or with (b, d) P1 (100 mgml ) for
24 h. Scale bars correspond to 100 mm; e) Cell viabilities of HeLa and MCF-7
cells treated with different concentrations of P1 for 24 h by MTT assay.
was examined with human breast cancer (MCF-7) cells as well
as human cervical cancer (HeLa) cells. Both MCF-7 and HeLa
cells showed no change in cell morphology (Figure 5, for
images obtained between these time points, see Figure S4)
after incubation with different concentrations of P1 for 24 h.
Cells kept their normal morphology even when the concentra-
3 2
Figure 4. a) FTIR spectra of P1 in CHCl and D O (c=2.0 mm); b) Emission
spectra of P1 in water (c=0.1 mm) before and after urea (2.0 mm) addition;
1
À1
c) Gradual decrease in emission intensity in the presence of urea; d) H NMR
tion of P1 was as high as 100 mgmL , which confirmed the
3 2
of P1 in CDCl and D O (* indicates solvent peaks); e) Proposed model for H-
bonded assembly of P1 leading to nanoparticle formation.
noncytotoxic nature of P1. To further confirm biocompatibility
of P1, cell viability of P1 to MCF-7 and HeLa cells was exam-
ined by conducting an MTT assay. As shown in Figure 5e,
almost 100% of cells were healthy after incubating with differ-
p-p interaction between them. Based on these data, we pro-
pose (Figure 4e) that P1 assembles through spontaneous inter-
chain H-bonding among the urethane groups and p-p stacking
among the TPE units, producing a rigid H-bonded hydrophobic
core containing conformationally locked TPE moieties, which is
responsible for the observed AIE. The hydrophobic core re-
mains encased by the flexible PEG chains, leading to formation
of stable fluorescence nanoparticles as observed under micros-
copy.
À1
ent concentrations of P1 (3–100 mgmL ) for 24 h. This result
was consistent with the optical microscopy observations.
Cellular uptake of P1 was evaluated by fluorescence micros-
copy. Strong signals in both HeLa and MCF-7 cells were ob-
À1
served after cells were incubated with 50 mgmL of P1 for
12 h (Figure 6). The areas with relatively weak fluorescence in-
tensity in the cells are possibly the location of the nuclei. This
observation demonstrated that P1 successfully escaped the en-
dosomal compartment with intact aggregation and selectively
stained the cytoplasmic region of both MCF-7 and HeLa cells.
Moreover, we monitored strong signals from P1 inside cells
Biocompatibility and long-term cell imaging: Robust and
highly emissive nanoparticles of P1 were tested in cellular
tracking. For successful bioimaging application, it is essential
to evaluate cytotoxicity of the materials. This crucial aspect
À1
even after incubation for 48 h with 50 mgmL of P1, which in-
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Figure 7. Fluorescence microscopy images of HeLa cells stained with P1
À1
(
50 mgmL ) at different passages. The scale bar is 100 mm.
linkage is cleaved under biological conditions and thus no sig-
nificant fluorescence was observed.
Figure 6. Cell imaging using P1: a) DIC image of HeLa cell without polymer
treatment; b) fluorescence microscopy image obtained with 405 nm laser
shows no fluorescence; c) DIC image of HeLa cell after treatment with
À1
Conclusion
50 mgmL polymer shows nice morphology of cell; d) fluorescence micros-
copy image obtained with 405 nm laser shows blue color fluorescence from
We have shown a much simplified way of making biocompat-
cytoplasm of HeLa cell; e) DIC image of MCF-7 cell after treatment with
À1
ible, robust, and highly emissive (F=0.17) organic nanoparti-
5
0 mgmL polymer shows nice morphology of cell; f) fluorescence micro-
[
22]
scopic image obtained with 405 nm laser shows blue color fluorescence
cles
by H-bonding-driven self-assembly of PEGulated oli-
from cytoplasm of MCF-7 cell. Scale bars correspond to 20 mm.
gourethane containing a TPE chromophore. Retaining high
fluorescence of the majority of organic chromophores in water
remains a challenge because of their aggregation-induced
quenching of fluorescence. In contrast, the presence of TPE
chromophores makes the present system highly emissive in
the assembled state. The polymeric aggregates showed very
efficient cellular uptake and, more interestingly, could be sus-
tained in a cellular environment for a long time, as evident by
the strong fluorescence of P1-treated cells. This property
helped the facile tracking of HeLa cells for as long as four pas-
sages, which can be considered a much superior performance
compared with commercial cellular trackers, which lose effi-
ciency after one or two passages. A suitable noncytotoxic dye
for long-term cell tracking either in vitro or in vivo is of increas-
ing demand for biomedical research because it has enormous
potential for monitoring various cellular events such as cell di-
vision, migration, fusion, lysis as well as transplantation. The
excellent biocompatibility and long-term cell tracking ability of
P1, we believe, makes it a promising supramolecular biomate-
dicated that aggregation of P1 within the cellular environment
remained intact for a long time. This observation inspired us to
further evaluate the potential use of P1 as a long-term cell
tracker.
To investigate the potential use of P1 as long-term cell track-
À1
er, HeLa cells treated with 50 mgmL P1 were repeatedly pas-
saged every 48 h, and the intracellular fluorescence of P1 in
every passage was checked by fluorescence microscopy. It was
found that P1 could track HeLa cells after up to as many as
four passages (Figure 7) and could retain inside cells for up
to168 h. It is well known that during passaging of confluent
cells stained with conventional fluorophores, the internalized
fluorophore may be extruded to the fresh culture media due
to reverse gradient in the fluorophore concentrations and the
compounds are rarely retained in the live cells after single pas-
sage, which limits their application in long-term cellular track-
[16]
[23]
ing.
Figure S5) by using ImageJ software. Pleasingly, more than
0% of the initial fluorescence remained for as long as four
Fluorescence intensity inside the cells was quantified
rial with great prospect for use not only as an in vitro cell
(
tracker but also for in vivo imaging in biomedical research.
2
passages, which can be ranked among the very few recent ex-
amples of polymeric materials that are suitable for long-term
cellular tracking. It is possible that beyond the fourth passage,
dilution below CAC causes disassembly or the oligourethane
Experimental Section
Materials and methods: Solvents and reagents were purchased
from commercials sources and purified by prescribed protocols.
[
24]
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Hexamethylene-diisocyanate, 4-hydroxy benzophenone, Zn-dust,
TiCl4 suspension, and 1,4-diazabicyclo[2.2.2]octane (DABCO) were
purchased from Sigma Aldrich chemicals and TCI chemicals. Hex-
amethylene-diisocyante was purified by distillation under reduced
pressure using Kugelrohr equipment. DABCO was recrystallized
from hexane; other reagents were used as received. Spectroscopy
grade solvents were used for all physical and spectroscopic studies.
dent PL experiments, an aqueous solution of P1 (c=0.1 mm) was
adjusted with either aqueous NaOH or HCl solution and spectra
were recorded after incubating the sample for 1 h. For DLS meas-
urements, P1 solution was prepared by using the method de-
scribed above and the experiment was performed at 0.1 mm con-
centration. The same solution was drop-cast on a carbon-coated
copper grid for TEM experiments and images were captured after
drying the sample for 12 h in air. For fluorescence microscopy
images, an aqueous solution of P1 (1.0 mm) was drop-cast on
a glass slide and the florescence microscopy images were captured
after drying the sample in air. FTIR spectra were recorded with a so-
lution of P1 (2.0 mm) either in CHCl or H O, which was placed be-
1
H NMR spectra were recorded with a Bruker DPX-500 MHz spec-
trometer and the peak positions were calibrated by using TMS as
an internal standard. UV/Vis experiments were performed with
a PerkinElmer Lambda 25 spectrometer. Emission spectral studies
were carried out with a FluoroMax-3 spectrophotometer from
HORIBA Jobin Yvon. FTIR spectra were recorded with a PerkinElmer
Spectrum 100FTIR spectrometer. Dynamic light scattering (DLS)
measurements were carried out with a Malvern instrument. Trans-
mission electron microscopy (TEM) images were captured with
a JEOL-2010EX machine operating at an accelerating voltage of
3
2
tween two CaF2 windows (path length=0.2 mm), and spectral
measurements were carried out with scan range of 4000–
À1
À1
1000 cm , resolution 1.0 cm , number of scans=32, and T=
258C.
Quantum yield (F) determination: For the determination of fluo-
2
00 kV. Fluorescence microscopy images were taken with an Olym-
[25]
rescence quantum yield, series of solutions of P1 and standard
pus (1”2-KSP, 6m 24413) machine, Japan. The molecular weight of
(
Fluorescein in 0.1 N NaOH, Q =0.95) were prepared in water and
D
À1
the polymer was estimated in THF (1.0 mgmL ) at 308C with re-
THF with varying concentrations and their absorption and emission
spectra were recorded under identical conditions. Then F was esti-
mated by using Equitation (1)
spect to poly(methyl methacrylate) (PMMA) standards with
a Water’s GPC machine equipped with a 515 HPLC pump, Waters
2
414 RI detector, and HSPgel HT 4.0/HSPgel HT 2.5 columns con-
nected in series. The flow rate of the eluent was maintained at
ꢀ
ꢁꢀ
ꢁ
2
x
2
ST
À1
0
.6 mLmin .
Grad
GradST
x
h
ꢀx ¼ ꢀST
ð1Þ
h
Synthesis of P1: TPE-diol M2 (50 mg, 0.137 mmol) and polyethy-
À1
lene glycol mono-methyl ether (M =2000 gmol
; 205 mg,
w
0
.102 mmol) were placed in a glass ampoule and the mixture was
where ST and x are standard and sample, f is the fluorescence
quantum yield. Grad denotes the gradient from the plot of inte-
grated fluorescence intensity vs. absorbance, and h is the refractive
index of the solvent.
dissolved in anhydrous THF (0.5 mL). Dry argon gas was passed
through the reaction mixture for 10 min, then a solution of hexam-
ethylene di-isocyanate (32 mg, 0.188 mmol) and DABCO (1 mg,
0
.005 mmol) in anhydrous THF (0.2 mL) was added to the reaction
Cell culture conditions: Human breast cancer cell line MCF-7 and
human cervical cancer cell line HeLa were cultured in high glucose
Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal
mixture and subsequently argon was purged to that reaction mix-
ture for an additional 15 min. The reaction mixture was stirred at
6
lution became significantly viscous, indicating polymerization. The
viscous material was then dissolved in THF (0.5 mL) and precipitat-
ed from excess diethyl ether. The crude product was then dis-
solved in water (2 mL) and subjected to dialysis for 48 h (MWCO
58C under an inert atmosphere for 8 h during which the clear so-
bovine serum and 1% l-Glutamine-penicillin-streptomycin and
À1
1
0 mgmL Insulin (in case of MCF-7) at 378C in a humidified envi-
ronment containing 5% CO . Cells were maintained by passaging
2
them regularly at ca. 80% confluency.
Cytotoxicity assay using MTT: To assess the metabolic activity of
MCF-7 and HeLa cells using a methylthiazolyldiphenyltetrazolium
bromide (MTT) assay, cells were seeded in 96-well plates with
ꢀ
3000) (to remove any excess PEG 2000) and freeze-dried to
obtain the desired polymer. (60% yield) as a white sticky solid.
1
H NMR (500 MHz, CDCl , TMS): d=6.85–7.27 (br., 18H; TPE pro-
3
À1
a seeding density of 10000 cells well . After 24 h incubation, the
tons), 4.18 (br., 4H), 3.49–3.76 (PEG protons), 3.36 (s, 6H; PEG ter-
minal -OCH protons), 3.2 (br., 4H), 1.54 (br., 4H), 1.33 ppm (br.,
4
medium was replaced by the P1 suspension at concentrations of
3
À1
1
00, 50, 25, 12.5, 6.25, and 3.12 mgmL made by serial dilution.
H); FTIR: n˜ =3338 (N-H stretching urethane), 2910 and 2875
The cells were then incubated for 24 h and 50 mL of freshly pre-
(
-CH - asymmetric and symmetric stretching), 1729 (C=O urethane),
2
À1
pared MTT (5 mgmL in 1” PBS) solution was added to each well.
1
610 (aromatic C=C stretching), 1499 (N-H urethane secondary),
The medium with MTT solution was carefully removed after 4 h in-
cubation in the 378C incubator. DMSO (200 mL) was then added
into each well and the plate was gently shaken for 10 min at RT to
dissolve all precipitates formed. The absorbance of MTT at 570 nm
was monitored with a microplate reader (VARIOSKAN, Thermo
Fisher). Cell viability was expressed by the ratio of absolute absorb-
ance of the cells incubated with polymer suspension to that of the
cells incubated with culture medium only.
À1
1100 (C-O-C stretching PEG) cm .
Preparation of nanoparticles: A known amount of P1 was dis-
solved in THF to make a stock solution. From this an aliquot was
transferred to a vial, THF was evaporated and subsequently water
was added and then water was added to the film and sonicated
for 10 min to obtain a stable dispersion of the organic nanoparti-
cles. Concentration of P1 in water was adjusted to 1.0 mm.
Physical studies: Solvent-dependent emission spectra (l =
Fluorescence microscopy imaging of cells using P1: MCF-7 cells
and HeLa cells were cultured at 378C in a coverglass-bottom con-
focal dish (Genetix Biotech Asia, India) with a seeding density of
20000 cells per dish. After 24 h, the medium was removed and the
adherent cells were washed once with 1”PBS, pH 7.4. The P1 in
ex
3
30 nm) of P1 were recorded in different ratios of THF and water,
keeping the concentration of P1 fixed at 0.1 mm. For concentra-
tion-dependent PL experiments, a 0.1 mm aqueous solution of P1
was gradually diluted with a measured amount of P1 and spectra
À1
were recorded after each dilution until the concentration reached
DMEM medium at 50 mgmL of P1 was then added to the cham-
À7
1
.8”10 m. For urea addition experiments, 20.0 mole equivalent
bers. After incubation for 12 h, the cells were washed again with
1”PBS, pH 7.4 and then fixed by using 4% formaldehyde for
30 min, and further washed with 1”PBS, pH 7.4. Cell images were
of urea was added in a 0.1 mm aqueous solution of P1, and PL
spectra were recorded after different time intervals. For pH-depen-
Chem. Eur. J. 2016, 22, 10930 – 10936
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Full Paper
obtained by using an inverted fluorescence microscope (Olympus
IX83, Tokyo, Japan) with an excitation wavelength of 405 nm.
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Keywords: biosensors · block copolymers · fluorescence ·
nanoparticles · self-assembly
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Published online on June 30, 2016
Chem. Eur. J. 2016, 22, 10930 – 10936
www.chemeurj.org
10936
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim