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Fluorescence imaging of a new monofunctional

platinum(II) complex containing a thioflavin-T
Cite this: New J. Chem., 2015,
(ThT)-based fluorophore†
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39, 1592

Received (in Montpellier, France)

14th January 2015, Zhanfen Chen,*a Shuping Zhang,a Lei Shen,a Zhenzhu Zhub and Jian Zhanga
Accepted 20th January 2015

DOI: 10.1039/c5nj00107b

www.rsc.org/njc

A new fluorescent monofunctional Pt(II) complex, [PtLCl]Cl (L =
4 0 -bis(pyridine-2-ylmethyl)amino-2-phenylbenzothiazole), has been
synthesized and characterized. The complex is suitable for fluores-
cence imaging in living cells, which is valuable for studying the
cellular processing of anticancer monofunctional platinum(II) com-
pounds and for bringing new insights into their cellular mechanism.
Platinum-based drugs play an important role in cancer chemo-
therapy, but the side effects, toxicity and drug resistance have
limited their wide range of clinical applications.1 Therefore,
numerous novel platinum complexes have been designed and
synthesized to develop new antitumor drugs in the past decades.2
Among them, monofunctional platinum complexes, whose struc-
tural features and DNA-binding modes are different from those
of current platinum drugs but still display an impressive anti-
tumour activity, have the potential to overcome the drawbacks of
traditional platinum-based drugs and have emerged as a new
class of antitumor drugs.2a,3 For example, monofunctional
platinum–acridine agents do not form DNA crosslinks but still
demonstrate significant cytotoxicity both in vitro and in vivo.4
cis-Diammine(pyridine)chloridoplatinum(II), pyriplatin, exhibits
remarkable anticancer properties with a different spectrum of
activity, forming monofunctional DNA adducts that can inhibit
transcription and better elude DNA repair.5 Guo et al. have also
reported a series of monofunctional anticancer platinum(II)
complexes with pyridine–quinoline derivatives and many of
them display anticancer spectra different from that of cisplatin.6
Nevertheless, the cellular distribution and processing of such
monofunctional platinum(II) complexes in tumour cells are
largely unknown and the mechanism of their action at the
cellular level is still poorly understood.
Recently, fluorescence imaging has emerged as one of the
most powerful techniques to monitor the cellular distributions
and travelling pathways of platinum drugs in living cells with high
temporal and spatial resolution.7 It is valuable for studying the
cellular processing of anticancer platinum compounds and bring-
ing new insights into their cellular mechanism. In a relatively
noninvasive method, visualization of platinum complexes within
the cell is commonly realized by tethering fluorophores to their
structures.8 For example, fluorescent-labelled cisplatin derivatives,
with carboxyfluorescein,7b fluorescein,9 and Alexa-Flour 546,10 have
been reported to explore the intracellular distribution of cisplatin
and to provide valuable imformation. Several fluorophore-tagged
monofunctional platinum(II) complexes suitable for biological
imaging applications in living systems have been developed.11
Herein, a new fluorescent monofunctional platinum(II) complex,
[PtLCl]Cl (shown in Fig. 1), was obtained by covalently tethering
a fluorophore thioflavin-T (ThT) derivative to a tridentate chelating
PtII center, Pt(BPA) motif (BPA = N,N-bis(pyridin-2-ylmethyl)amine).
The fluorescence properties, DNA binding ability, cytotoxic activity,
and cellular processing in human cervical cancer HeLa cells
were studied.
The ligand 4 0 -bis(pyridine-2-ylmethyl)amino-2-phenylbenzo-
thiazole (L) was synthesized according to reported procedures.12
The platinum complex [PtLCl]Cl was synthesized and characterized

a
Hubei Collaborative Innovation Center for Rare Metal Chemistry,
Hubei Key Laboratory of Pollutant Analysis & Reuse Technology,
College of Chemisry & Chemical Engineering, Hubei Normal University,
Huangshi 435002, China. E-mail: chenzf1979@163.com; Fax: +86 714 6515602
b
State Key Laboratory of Coordination Chemistry, Coordination Chemistry
Institute, School of Chemistry and Chemical Engineering,
Nanjing University, Nanjing, 210093, China
† Electronic supplementary information (ESI) available: Characterization of L and Fig. 1 Schematic drawing of ligand L and its monofunctional platinum(II)
[PtLCl]Cl, and other experimental data and figures. See DOI: 10.1039/c5nj00107b complex [PtLCl]Cl.

1592 | New J. Chem., 2015, 39, 1592--1596 This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015

Letter
Fig. 2 ESI-MS spectrum (positive mode) for [PtLCl]Cl in DMSO/methanol
(v/v, 1/5). Assignment (m/z): 639.73, [PtLCl]+ (PtC25H20N4SCl, calcd 639.07).
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The isotopic distribution pattern of the major peak matching perfectly with
the simulated one.
(see the Experimental section for details). The purity and
composition of the complex were examined by 1H NMR, ESI-MS,
molar conductivities, and elemental analysis. In the 1H NMR
spectrum of the complex in DMSO-d6, the identical proton signal
of the two methylene groups at d 4.84 ppm visible in free BPA units
was split into two doublets at d 5.95 and 5.52 ppm due to their
inequivalency after the coordination of Pt(II) with L (Fig. S1 and S2,
ESI†). Additionally, almost all of phenyl and pyridyl signals shifted
significantly to the lower field compared with those of free L, also
suggesting the coordination. The ESI-MS spectrum of the complex
in DMSO/methanol (v/v, 1/5) solution gave major peaks at an m/z
value of 639.73, which could be attributed to the positively
charged species [PtLCl]+ (PtC25H20N4SCl) (Fig. 2). The isotopic
distribution patterns of the peak match perfectly with the
simulated one. The signal of [PtLCl]+ still exists after about
4 days, indicating that the platinum complex is stable in DMSO/
methanol. The molar conductivity of the complex (LM) in DMF
at room temperature is 80 S cm2 mol1, which falls within the
range of 1 : 1 electrolytes and suggests that only one chloride
ion is present in the outer sphere of the complex.13 The
fluorescence spectrum of L in Tris-HCl–NaCl buffer exhibits
intense fluorescence with emission and excitation maxima at
438 nm and 360 nm, respectively (Fig. 3). The coordination of
platinum(II) with L leads to a 2.5-fold decrease in emission
without any distinct shift in emission. Even so, the mono-
functional platinum(II) complex exhibits considerable and
stable fluorescence which should be visible by fluorescence
microscopy, and is suitable for the confocal microscopy experi-
ments.14 The cytotoxicities of [PtLCl]Cl and L on tumour cells
were tested using the MTT assay on the human cervical
carcinoma (HeLa), human pulmonary carcinoma (A549), and
human breast carcinoma (MCF-7) cell lines. The IC50 values are
presented in Table S1 (ESI†). The platinum complex [PtLCl]Cl
shows lower cytotoxicity towards these cell lines compared to
cisplatin. For example, its IC50 value against the HeLa cell line is
87.26 mM, approximately 5.0-fold higher than that of cisplatin.
However, the free ligand L is not active for the three tested cell
lines, which indicates that the cytotoxicity of [PtLCl]Cl results
from the presence of PtII. The moderate activity of [PtLCl]Cl
makes it a good model compound to study the cellular behavior
of monofuncional platinum(II) complexes.
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Fig. 3 Excitation (  ) and emission spectra (—) of [PtLCl]Cl (0.01 mM) and
L (0.01 mM) in a Tris-HCl–NaCl buffer containing 10% DMSO (pH 7.4).
Since DNA is the primary target of Pt(II)-based antitumor
complexes, the potential DNA binding ability of [PtLCl]Cl was
evaluated by UV, CD and fluorescence spectroscopy. The time-
dependent UV experiment with calf thymus DNA (CT-DNA)
showed a 53% hypochromism of the metal to ligand charge
transfer (MLCT) absorption band at 353 nm of [PtLCl]Cl after
4 h at a molar ratio r([DNA]/[complex] = 0.5) (Fig. S3, ESI†), which
is much more evident than those observed for DNA intercalators,
suggesting that [PtLCl]Cl could strongly bind to DNA.15 The CD
spectra of CT-DNA upon the addition of [PtLCl]Cl displayed a
slight decrease in ellipticity for both positive and negative bands
of DNA (Fig. S4, ESI†), which indicated that the platinum(II)
complex could indeed interact with DNA and induce a confor-
mational change in DNA. The fluorescence titration experiments
showed that the emission intensity of [PtLCl]Cl at 438 nm
gradually decreased with an increase in the amount of DNA
(Fig. S5, ESI†), implying that the cellular DNA binding would
quench the native fluorescence of [PtLCl]Cl and possibly inter-
fere with its imaging properties in cells. However, under the
same conditions the fluorescence intensity of ligand L gradually
increased with an increase in the amount of DNA (Fig. S6, ESI†),
which suggests that the DNA binding mode of L is different
from that of [PtLCl]Cl and should be due to intercalation
between the adjacent DNA base pairs similar to ethidium
bromide (EB).16 Guanine-N7 is the preferred binding site in
DNA for platinum-based drugs,17 therefore, the reactivity of
[PtLCl]Cl with a model compound 5 0 -GMP at a 1 : 1 molar ratio
was monitored by 1H NMR spectroscopy. As shown in Fig. 4,
small amounts of adducts appeared after the platinum complex
and 5 0 -GMP were incubated together for 24 h. A typical downfield
shift from 8.18 to 8.83 ppm with an apparent line broadening is
clearly observed for the guanine H8 signal in 5 0 -GMP, which
indicated that the N7-GMP has coordinated with the Pt(II)
center.18 In the meantime, the signal of sugar H10 at 5.91 ppm
shifted to 4.94 ppm, which was similar to the changes induced by
cisplatin.19 The results suggest that [PtLCl]+ has the potential to
form monofunctional Pt-DNA adducts in vivo. But the DNA bind-
ing mode of [PtLCl]Cl could not preclude the possibility of inter-
calation from the ligand itself of the complex.
The cellular response of the platinum(II) complex [PtLCl]Cl
in HeLa cells was investigated by confocal fluorescence micro-
scopy in HEPES buffer (pH = 7.40, 0.5% DMSO), which is
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Fig. 4 1H NMR spectra of 5 0 -GMP (2.5 mM), [PtLCl]Cl (2.5 mM), the
reaction between 5 0 -GMP and [PtLCl]Cl (1 : 1) for 24 h in DMSO-d6 at
room temperature. The labeled peaks (*) correspond to the unspecified
reaction products.
compared to that of ligand L. Since the excitation maximum of
L and [PtLCl]Cl is 360 nm, the laser beam of 405 nm was
adopted as excitation light for the confocal imaging (Fig. 5). As
shown in Fig. 5b and e, there appears to be readily detectable
green fluorescence localized in the area close to the dim nucleus
no matter whether HeLa cells were incubated with L or [PtLCl]Cl
over 1 h. And in this period of time, the intracellular fluorescence
of the platinum complex [PtLCl]Cl is observed certainly later than
that of ligand L (images not shown). When the incubation time is
longer than 1 h, the fluorescence distribution remains constant.
However, there is no fluorescence in the cell nucleus over 1 h
and even overtime up to 20 h after incubation. The results
suggest that both L and [PtLCl]Cl are permeable through the
cell membrane and could enter into cells and could be visua-
lized in living cells. The absence of fluorescence because of
the platinum(II) complex in the nucleus does not preclude the
possibility that the complex could reach the nucleus, as the
complex could interact with DNA, resulting in quenching of its
Fig. 5 Confoncal microscopic images of HeLa cells (lex, 405 nm; band path,
410490 nm) in HEPES buffer after treatment with ligand L (20 mM, a, b, c) or
[PtLCl]Cl (20 mM, d, e, f) for 1 h. Images (a)/(d): bright-field transmission images
of the stained cells; (b)/(e): the green fluorescence images; (c)/(f): overlay
between bright-field and fluorescence images.
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fluorescence. However free ligand L could not enter into
the nucleus because of its very strong fluorescence intensity
whether on its own or after binding to DNA, which may explain
why the free ligand exhibits no cytotoxicity.11b And the speed of
the cellular entry of [PtLCl]Cl is slightly slower than that of the
electrically neutral L, which should be attributed to the different
mechanisms of the cellular uptake. As reported for pyriplatin, the
cellular uptake of [PtLCl]Cl may be mediated by an organic cation
transporter.3a,5 In comparison to the platinum(II) complex, the
intracellular fluorescence area of ligand L displays brighter and
additional scattering bright dots surrounding the dim nucleus,
which is identical with the fluorescence properties of ligand L
and its platinum(II) complex in vitro.
Besides the nucleus, the mitochondria and lysosomes are the
potential subcellular compartments in which platinum(II) com-
plexes may accumulate.20 Thus here localization studies of [PtLCl]Cl
in HeLa cells were carried out by costaining with mitochondria
marker Red CMXRos (a rhodamine derivative) and lysosome maker
Red DND-99, respectively (Fig. 6). As shown in Fig. 6a–c, the
convincing yellow stains in the superimposed image are observed,
which are indicative of fine co-localisation. The results suggest
that [PtLCl]Cl is preferentially accumulated in mitochondria. The
phenomenon has been observed in the system of other mono-
functional platinum(II) complexes, which is different from that of
conventional fluorescent-tagged cisplatin analogues.11a,7b Moreover,
overlapping the fluorescent images of [PtLCl]Cl and Red DND-99
showed extensive orange scattered spots, revealing the partial
colocalization (Fig. 6d–f). This suggests that [PtLCl]Cl could be
partially accumulated in the lysosome apparatus, which is different
from that reported for fluorescent monofunctional platinum(II)
complexes.11 The latter could not be sequestered in the lysosomal
vesicles and have high affinity for vacuolations or mitochondria.
However accumulation of neutral fluorescent platinum complexes
in acidic lysosomes has been observed as well.21 So sub-cellular
sequestration of anticancer platinum(II) complexes should have no
direct connection with their charge in solution. The mitochondrial/
lysosomal trapping and sequestration of [PtLCl]Cl will contribute to
Fig. 6 Confocal fluorescence images of HeLa cells co-stained with 50 nM
MitoTracker Red CMXRos (or 1 mM LysoTracker Red DND-99) and 20 mM
[PtLCl]Cl for 1.5 h. Images (a)/(d): fluorescence image of cells visualized by
green emission of [PtLCl]Cl; (b): fluorescence image visualized by red emis-
sion of red CMXRos; (c): overlay between (a) and (b); (e): fluorescence image
visualised by red emission of red DND-99; (f): overlay between (d) and (e).

Letter
the reduced efficacy of the compound to cause DNA damage and
resultant apoptosis, and consequently may limit its effectiveness as
an anticancer drug.20,22 But the mitochondrial or lysosomal accu-
mulation, most probably leading to swelling or disruption, may
trigger cell death, which has also been identified as a paraptosis/
necrosis-inducing pathway triggered for some other platinum com-
plexes.9,11a,21,23 So the cytotoxicity of [PtLCl]Cl may result from such
cell paraptosis or necrosis. Nevertheless, it has been found that
lysosomal disruption is also a putative case of the nephrotoxicity of
cisplatin.22b Therefore, [PtLCl]Cl may also be similar to cisplatin,
and the mechanism of its cytotoxicity is still likely to involve DNA
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damage, since [PtLCl]Cl could interact with DNA as described in the
former analysis in vitro. Different from cisplatin [PtLCl]+ could form
monofunctional Pt-DNA adducts. The precise cellular distribution
and the process by [PtLCl]Cl still need further research.
In summary, a new cytotoxic fluorescent monofunctional Pt(II)
complex, [PtLCl]Cl (L = 40 -bis(pyridine-2-ylmethyl)amino-2-phenyl-
benzothiazole), has been successfully constructed by covalently
tethering a fluorophore thioflavin-T (ThT) derivative to a tridentate
chelating PtII center, which is suitable for cellular imaging in living
cells. The current fluorescence imaging shows that [PtLCl]Cl could
enter the cells slowly, and can be sequestrated in mitochondria and
acidic lysosomes. The finding of mitochondria and lysosomal seques-
tration of the positive charge of the complex ion provides new
insights into the cellular antitumor mechanism of monofunctional
platinum(II) complexes. Such information is of great value for the
rational design of novel monofunctional platinum anticancer drugs.
Experimental
[PtLCl]Cl was synthesized as follows: to a stirred solution of L
(81 mg, 0.2 mmol) in 5 mL of methanol, a methanol solution of
cis-Pt(DMSO)2Cl2 (84 mg, 0.2 mmol) was added. The reaction mixture
was then refluxed at 60 1C for 4 h. A yellow precipitate was formed
when the mixture was cooled to room temperature, which was then
collected by filtration and washed with methanol (yield: 62%).
Elemental anal. found (calcd) for [PtLCl]Cl (PtC25H20N4SCl2) (%): C
44.50 (44.51); H 2.89 (2.97); N 16.65 (16.62). ESI-MS m/z: 639.73
[PtLCl]+. 1H NMR for [PtLCl]Cl (400 MHz, DMSO-d6, 25 1C): d = 8.542
(d, 2H, J 3.6 Hz), 8.26 (d, 2H, J 7.2 Hz), 8.24 (d, 2H, J 7.6 Hz), 8.14
(t, 2H, J 8.2 Hz), 8.06 (t, 2H, J 7.8 Hz), 7.77 (d, 2H, J 7.4 Hz), 7.69
(t, 2H, J 6.9 Hz), 7.53 (q, 2H, J 7.6 Hz), 5.95 (d, 2H, J 6.8 Hz), 5.57
(d, 2H, J 7.5 Hz) ppm. LM = 80 S cm2 mol1 (in DMF).
Acknowledgements
We are grateful for the financial support from the National
Natural Science Foundation of China (no. 21301056) and the
China Postdoctoral Science Foundation (no. 2012M521419).
Notes and references
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