Neutral N^C^N terdentate luminescent Pt(ii) complexes: Their synthesis, photophysical properties, and bio-imaging applications

Article abstract of DOI:10.1039/c4dt03165b

An emerging field regarding N^C^N terdentate Pt(ii) complexes is their application as luminescent labels for bio-imaging. In fact, phosphorescent Pt complexes possess many advantages such as a wide emission color tunability, a better stability towards pho

Full text of DOI:10.1039/c4dt03165b

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Neutral N^C^N terdentate luminescent Pt(II)
complexes: their synthesis, photophysical
Cite this: DOI: 10.1039/c4dt03165b
properties, and bio-imaging applications†
Alessia Colombo,‡a Federica Fiorini,‡b Dedy Septiadi,‡b Claudia Dragonetti,*a,c
Filippo Nisic,a Adriana Valore,a Dominique Roberto,a,c Matteo Mauro*b,d and
Luisa De Cola*b
An emerging field regarding N^C^N terdentate Pt(II) complexes is their application as luminescent labels
for bio-imaging. In fact, phosphorescent Pt complexes possess many advantages such as a wide emission
color tunability, a better stability towards photo- and chemical degradation, a very large Stokes shift, and
long-lived luminescent excited states with lifetimes typically two to three orders of magnitude longer
than those of classic organic fluorophores. Here, we describe the synthesis and photophysical character-
ization of three new neutral N^C^N terdentate cyclometallated Pt complexes as long-lived bio-imaging
probes. The novel molecular probes bear hydrophilic (oligo-)ethyleneglycol chains of various lengths to
increase their water solubility and bio-compatibility and to impart amphiphilic nature to the molecules.
The complexes are characterized by a high cell permeability and a low cytotoxicity, with an internalization
kinetics that depends on both the length of the ethyleneglycol chain and the ancillary ligand.
Received 14th October 2014,
Accepted 22nd December 2014
DOI: 10.1039/c4dt03165b
www.rsc.org/dalton
an adequate choice of the ligands; (ii) a better stability
towards photo- and chemical degradation; (iii) a very large
Introduction
The discovery of cisplatin {PtCl2(NH3)2} anticancer activity1 and Stokes shift that allows the detection of their emission at a
its subsequent significant clinical success have led to an much lower energy than the excitation energy; (iv) long-lived
increasing interest in the development of platinum-based luminescent excited states owing to their triplet-manifold
drugs.2–5 In addition, d8 platinum(II) complexes have attracted nature; (v) emission lifetimes typically two to three orders of
much attention due to their interesting luminescence magnitude longer than those of classic organic fluorophores.
properties.6–10 Cyclometallated derivatives are usefully employed Another important point is related to synthetic issues: the
for the preparation of triplet emitters for highly efficient OLED modification of many fluorescent organic structures can be
(organic light emitting diode) devices7,11–14 and for their appeal- difficult and laborious, whereas Pt complexes can be prepared
ing second-order nonlinear optical (NLO) properties.15–18
using well-defined and faster strategies, often involving
Another fast emerging field regarding luminescent Pt(II) stepwise introduction of different ligands. One argument
complexes is their application as luminescent labels for bio- sometimes raised against the use of platinum is its high
imaging.19–23 Although fluorescent organic labels are still the cost. However, for bio-imaging applications, the amount
leading choice for such applications,24–26 phosphorescent of material required is minimal and the cost of the metal is
Pt(II) complexes are slowly gaining attention and could out- often only a relatively minor fraction of the total synthetic
class organic molecules. In fact, Pt complexes display many efforts.
advantages such as: (i) a wide emission colour tunability by
Nonetheless, it is worth pointing out that UV light might
damage biological specimens and that tissues are relatively
transparent at wavelengths in the range of 600–1300 nm,
called the optical therapeutic window. Furthermore, red and
near infrared (NIR) light is highly desirable for both excitation
and detection due to the lack of overlap with auto-fluorescence
of biomolecules and the larger penetration of photons with
such a spectral energy. For these reasons, molecules displaying
a relatively high two-photon absorption cross-section in the
NIR are good candidates, and in this respect platinum com-
plexes could be key players.20,22
aDipartimento di Chimica dell’Università degli Studi di Milano, UdR-INSTM,
via Golgi 19, I-20133 Milano, Italy. E-mail: claudia.dragonetti@unimi.it
bISIS & icFRC, Université de Strasbourg & CNRS, 8 rue Gaspard Monge, 67000
Strasbourg, France. E-mail: mauro@unistra.fr, decola@unistra.fr
cISTM-CNR, via Golgi 19, I-20133 Milano, Italy
dUniversity of Strasbourg Institute for Advanced Study (USIAS), 5 allée du Général
Rouvillois, 67083 Strasbourg, France
†Electronic supplementary information (ESI) available: Additional photophysical
data, spectra and bio-imaging studies. See DOI: 10.1039/c4dt03165b
‡All these authors have contributed equally to this work.
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Fig. 1 Chemical structure of the investigated neutral platinum
complexes.
The investigation of platinum complexes for biological appli-
cations concerns mostly monomeric compounds, but aggre-
gates show some important features as well:19,20 (i) the emitter
is shielded from the environment and in particular from di-
oxygen that could induce quenching; (ii) the rigidity, due to the
packing of molecules in determined structures, decreases non-
radiative processes; (iii) the reactivity or toxicity of the com-
plexes is diminished by the difficult accessibility of the metal
centre; (iv) changes in the excited state nature and properties
lead to a bathochromic shift of excitation and emission towards
more biologically interesting spectral windows and sometimes
to an enhancement of the photophysical properties.
In this respect, amongst all luminescent platinum deriva-
tives, those bearing either an N^C^N22,23 or, more recently, an
N^N^N19,20,27 chromophoric ligand have shown very interest-
ing results. Despite the growing interest and the emerging
potential application as bio-imaging and/or theragnostic
agents that luminescent platinum complexes possess, the
Scheme 1 Synthetic pathway for the preparation of the reported plati-
num complexes.
chemical structure–internalization relationship remains
a
challenging and rather elusive goal.20,28,29
For this purpose, we decided to undertake a systematic study
of the bio-imaging and cytotoxicity features of a series of plati-
num complexes bearing a N^C^N cyclometallated 1,3-di(2-
pyridyl)-benzene by varying the molecular hydrophobic/hydro-
philic ratio (through the introduction of ethyleneglycol moieties
of different lengths) and the ancillary ligand, the incubating
medium and the staining concentration. The choice of oligo-
ethyleneglycol chains on the phosphorescent probe was based
on their known ability to increase the aqueous solubility and
bio-compatibility of luminescent iridium bio-labels.30
In the present work, we thus describe the synthesis and
photophysical characterization of three new neutral terdentate
N^C^N cyclometallated Pt complexes (Fig. 1) as long-lived bio-
imaging probes. As will be hereafter discussed, the novel com-
plexes bear hydrophilic moieties and are characterized by a
high cell permeability and low cytotoxicity, with an internaliz-
ation kinetics that depends on both the ethyleneglycol chain
length and the ancillary ligand.
toluene was distilled over Na/benzophenone. All reagents were
purchased from Sigma-Aldrich and were used without further
purification. Reactions requiring anhydrous conditions were
performed under argon. 1H NMR spectra were recorded at
400 MHz on a Bruker AVANCE-400 instrument. Chemical shifts
(δ) for 1H spectra are expressed in ppm relative to Me4Si as the
internal standard. Mass spectra were obtained with a FT-ICR
Mass Spectrometer APEX II & Xmass software (Bruker Dal-
tonics) – 4.7 Magnet and Autospec Fission Spectrometer (FAB
ionization). Thin layer chromatography (TLC) was carried out
with pre-coated Merck F254 silica gel plates. Flash chromato-
graphy (FC) was carried out with Macherey–Nagel silica gel 60
(230–400 mesh). The schematic synthetic route employed for
the preparation of the reported platinum complexes is dis-
played in Scheme 1.
Synthesis of compound 1
Under an argon atmosphere, 2-hydroxyethyl tosylate (1.02 g,
4.70 mmol) was added to a solution of 3,5-dibromophenol
(800 mg, 3.17 mmol) and K2CO3 (8.372 mg, 6.84 mmol) in
17.6 mL of dry DMF. The mixture was stirred at 90 °C over-
Experimental
General comments
Solvents were dried by standard procedures: N,N-dimethyl- night. The reaction mixture was diluted with ethyl acetate
formamide (DMF) was dried over activated molecular sieves; (100 mL) and washed with water (150 mL) and brine (100 mL),
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and then the organic layer was dried over Na2SO4 and the
1H NMR (400 MHz, CD2Cl2): δ 8.71 (d, J = 4.6 Hz, 2H), 8.22
solvent was removed under reduced pressure. The obtained (s, 1H), 7.78 (d, J = 8.1 Hz, 2H), 7.77 (t, J = 8.1, 2H), 7.68 (s,
crude product was purified by flash chromatography on silica 2H), 7.26 (t, J = 4.6 Hz, 2H), 4.34 (t, J = 4.0 Hz, 2H), 3.93 (t, J =
gel using hexane–ethyl acetate 4 : 1 as the eluent to give 4.0 Hz, 2H), 3.80–3.78 (m, 2H), 3.72–3.63 (m, 4H, 3.57–3.55 (m,
798 mg of the product (85% yield).
2H), 3.38 (s, 3H). Elemental analysis: Calcd for C23H26N2O4:
1H NMR (400 MHz, CD2Cl2): δ 7.30 (s, 1H), 7.06 (s, 1H), C, 70.03; H, 6.64; N, 7.10. Found: C, 69.98; H, 6.69; N, 7.15.
7.04 (s, 1H), 4.1 (m, 2H), 3.95 (m, 2H). Elemental analysis: MS(FAB+): m/z 395 [PtL1].
Calcd for C8H8Br2O2: C, 32.47; H, 2.72. Found: C, 32.40;
Procedure for the synthesis of PtL1Cl and PtL2Cl
H, 2.80.
Under a nitrogen atmosphere, a solution of K2PtCl4 (1 equiv.)
Synthesis of compound 2
and HL1 or HL2 (1 equiv.) in an AcOH–H2O 9 : 1 mixture
A mixture of 3,5-dibromophenol (592 mg, 2.35 mmol), 2-(tri- (0.3 M) was placed in a microwave reactor at 160 °C (250 W) for
n-butylstannyl)pyridine (1.83 mL 5.65 mmol), PdCl2(PPh3)2 45 minutes while controlling the flow rate of cooling air. After
(100 mg, 0.14 mmol), and LiCl (1.8 g, 42.5 mmol) was sus- cooling to room temperature, the reaction mixture was filtered.
pended in 6 mL of degassed toluene, and heated under reflux The precipitate was washed successively with methanol, water,
for 48 hours. After cooling down to room temperature, ethanol, and diethyl ether.
aqueous NaOH (1M, 30 mL) was added. The phases were sep-
arated and the aqueous phase was extracted with ethyl acetate δ 9.30 (d, J = 4.7 Hz, 2H), 8.00 (q, J = 7.8 Hz, 2H), 7.74 (d, J =
(3 × 100 mL). 7.8 Hz, 2H), 7.36 (t, J = 4.7 Hz, 2H) 7.22 (s, 1H), 7.12 (s, 1H),
The combined organic layers were dried over Na2SO4 and 4.30 (m, 2H) 4.03 (m, 2H).
PtL1Cl. 150 mg, 80% yield 1H NMR (400 MHz, CD2Cl2):
concentrated in vacuo. The crude product was purified by flash
Elemental analysis: Calcd for C18H15ClN2O2Pt: C, 41.43;
chromatography on silica gel (ethyl acetate–hexane 7 : 3). The H, 2.90; N, 5.37. Found: C, 41.39; H, 2.88; N, 5.39.
product was isolated as a pale yellow oil in 60% yield.
MS(FAB+): m/z 486 [PtL1].
1H NMR (400 MHz, CDCl3): δ 8.71 (d, J = 4.64 Hz, 2H), 8.18
PtL2Cl. 100 mg, 70% yield 1H NMR (400 MHz, CD2Cl2):
(s, 1H), 7.82–7.75 (m, 2H), 7.63 (s, 2H), 7.44 (d, J = 4.64 Hz, δ 9.26 (d, J = 4.7 Hz, 2H), 8.00 (t, J = 7.8 Hz, 2H), 7.70 (d, J =
2H), 7.31 (m, 2H). Elemental analysis: Calcd for C16H12N2O: 7.8 Hz, 2H), 7.19 (s, 2H), 7.06 (s, 1H), 4.24 (t, J = 4.0 Hz, 2H),
C, 77.40; H, 4.87; N, 11.28. Found: C, 77.38; H, 4.85; N, 11.33. 3.90 (t, J = 4.0 Hz, 2H), 3.81–3.79 (m, 2H), 3.70–3.63 (m, 4H),
MS(FAB+): m/z 248.
3.57–3.55 (m, 2H), 3.38 (s, 3H).
Elemental analysis: Calcd for C23H25ClN2O4Pt: C, 44.27;
H, 4.04; N, 4.49. Found: C, 44.30; H, 4.07; N, 4.51.
MS(FAB+): m/z 623 [PtL2Cl], 588 [PtL2].
Synthesis of the pro-ligand HL1
Under a nitrogen atmosphere, a mixture of the 5-substituted
m-dibromobenzene derivative 1 (150 mg, 0.51 mmol), 2-(tri-
n-butylstannyl)pyridine (495 μL 1.53 mmol), PdCl2(PPh3)2
Synthesis of PtL1NCS
(35.8 mg, 0.051 mmol), CuO (124 mg, 1.53 mmol), and DMF A solution of PtL1Cl (125 mg, 1 equiv.) in dichloromethane
(2 mL) was placed in a microwave reactor at 160 °C (250 W) for (300 mL) was treated with a solution of sodium thiocyanate
45 min while controlling the flow rate of cooling air. After (15.1 mg, 1.1 equiv.) in methanol (2 mL). After stirring at room
cooling to room temperature, the reaction mixture was poured temperature under nitrogen for 24 hours, the solution was fil-
into ethyl acetate (25 mL) and filtered. The filtrate was washed tered and the solvent was evaporated to dryness affording the
with water, the organic layer was dried over anhydrous Na2SO4 crude product that was washed first with methanol and then
and concentrated under reduced pressure. The crude product with ethanol.
was purified by flash chromatography using hexane–ethyl
1H NMR (400 MHz, CD2Cl2): δ 8.80 (d, J = 4.9 Hz, 2H); 8.06
(q, J = 7.8, 2H); 7.74 (d, J = 7.8 Hz, 2H); 7.39 (m, 2H); 7.17
acetate 1 : 9 as the eluent. Yield 62%.
1H NMR (400 MHz, CDCl3): δ 8.72 (d, J = 4.64 Hz, 2H), 8.20 (m, 2H); 4.49 (m, 2H); 4.30 (m, 2H).
(s, 1H), 7.83 (d, J = 7.20 Hz, 2H), 7.78 (d, J = 7.20 Hz, 2H), 7.67
(s, 2H), 7.27 (t, J = 4.64 Hz, 2H), 4.30 (m, 2H), 4.03 (m, 2H).
Elemental Analysis: Calcd for C19H15N3O2PtS: C, 41.91;
H, 2.78; N, 7.72. Found: C, 41.89; H, 2.79; N, 7.70.
MS(FAB+): m/z 486 [PtL1].
Synthesis of the pro-ligand HL2
Photophysical measurements
A mixture of the intermediate 2 (50 mg, 0.2 mmol), tosylate
derivative (96 mg, 0.3 mmol), and K2CO3 (55 mg, 0.4 mmol) in Absorption spectra were recorded on a Shimadzu UV-3600
dry DMF (1 mL) under argon was heated at 90 °C for 18 h. double-beam UV–vis–NIR spectrophotometer and baseline-
After cooling to room temperature the mixture was washed corrected. Steady-state emission spectra were recorded on a
with 2 mL of pentane in order to remove the unreacted tosylate Horiba Jobin-Yvon IBH FL-322 Fluorolog 3 spectrometer
and then 3 mL of diethyl ether were added, the precipitate was equipped with a 450 W xenon arc lamp, double-grating exci-
filtered, and the yellow solution containing the pure ligand tation, and emission monochromators (2.1 nm mm−1 of dis-
was dried under reduced pressure. The product was used persion; 1200 grooves mm−1) and a TBX-04 photomultiplier as
without further purification.
a detector. Emission and excitation spectra were corrected for
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source intensity (lamp and grating) and emission spectral PBS and fixed with a 4% paraformaldehyde (PFA) solution for
response (detector and grating) by standard correction curves.
Time-resolved measurements were performed using either
the time-correlated single-photon counting (TCSPC) elec-
10 min.
Organelle stainings
tronics PicoHarp300 or the Multi Channel Scaling (MCS) elec- The cell layer was washed twice with PBS and rinsed in 0.1%
tronics NanoHarp 250 of the PicoQuant FluoTime 300 Triton X-100 in PBS for 5 minutes and afterwards in 1% bovine
(PicoQuant GmbH, Germany), equipped with a PDL 820 laser serum albumin (BSA; Sigma Aldrich) in PBS for another
pulse driver. A pulsed laser diode LDH-P-C-405 (λ = 406 nm, 20 min. The cell layer on glass cover slips was stained with
pulse FWHM < 70 ps, repetition rate 200 kHz–40 MHz) was Phalloidin Alexa Fluor® 647 (Invitrogen), for F-actin/
used to excite the sample and mounted directly on the sample membrane staining for 20 min in the dark at room tempera-
chamber at 90°. The photons were collected using
a
ture, and washed twice with PBS. For nucleoli staining
PMA-C-192 photomultiplier (PMT) single photon counting purpose, a 500 nM SYTO® RNASelect™ Green Fluorescent Cell
detector. The data were acquired using the commercially avail- Stain (Invitrogen) solution was added to cells for 20 minutes
able software EasyTau (PicoQuant GmbH, Germany), while followed by washing with PBS. For visualizing the nuclear
data analysis was performed using the commercially available region, cell nuclei were stained with 4′,6-diamidino-2-phenyl-
software FluoFit (PicoQuant GmbH, Germany). For multi-expo- indole carboxamidine (DAPI) and washed twice with PBS. The
nential decays, the intensity, namely I(t), has been asumed to cover slips were mounted onto glass slides for microscopy
decay as the sum of individual single exponential decays:
measurements.
ꢀ
ꢁ
N
X
t
τi
Fluorescence confocal microscopy
IðtÞ ¼
αi exp ꢀ
i¼1
Reported fluorescence images were taken using a Zeiss LSM
710 confocal microscope setup with 63× magnification and
numerical aperture (NA) 1.3 of Zeiss LCI Plan-NEOFLUAR
water immersion objective lens (Zeiss GmbH, Germany). The
samples were excited by a continuous wave (cw) laser at
405 nm. The emission of the complexes was collected in the
range of 500–720 nm. For co-localization experiments, the
samples already co-stained with different dyes, namely DAPI
(excitation/emission wavelength: 358 nm/461 nm), SYTO®
RNASelect™ (excitation/emission wavelength: 490 nm/
530 nm), and Alexa Fluor® 647 Phalloidin (excitation/emission
wavelength: 650 nm/668 nm), were excited independently at
405, 488 and 633 nm, respectively. All image processings were
performed using the ZEN software (Zeiss GmbH, Germany)
and the FigureJ plugin (IBMP, UniStra) within ImageJ (NIH).
False colour images were adjusted to better distinguish
different complexes and complexes from cellular organelles.
Excited state lifetime values are given with 10% relative
uncertainty.
All the photoluminescence quantum yield measurements
(PLQY) in solution were recorded at a fixed excitation wave-
length (λexc = 400 nm) using a Hamamatsu Photonics absolute
PLQY measurement Quantaurus system equipped with a con-
tinuous wave xenon light source (150 W), a monochromator,
an integrating sphere, and a photonics multi-channel analyzer
and employing the commercially available PLQY measurement
software (Hamamatsu Photonics Ltd, Shizuoka, Japan). PLQY
values are given with 0.01 uncertainty.
Cell culture
All materials for cell culture were purchased from Gibco.
Human cervical carcinoma, HeLa, cells were cultured inside
growth medium containing 88% Dulbecco’s Modified Eagle’s
Medium (DMEM), 10% Fetal Bovine Serum (FBS), 1% penicil-
lin–streptomycin, and 1% L-glutamine (200 mM) under the
conditions of 37 °C and 5% of CO2 for 48 hours until reaching
80–85% cell confluency. Subsequently, the cells were washed
twice with Phosphate Buffer Solution (PBS), trypsinated and
approximately 50 000 cells were re-seeded on monolayer glass
cover slips placed inside a six-well plate culture dish and glass
bottom dishes (MatTek). Fresh culture medium (2 mL) was
added gently and cells were grown overnight.
Kinetics of internalization of the complex
The culture medium of live cells grown on glass bottom dishes
was removed and 1 mL of complex staining solution (5 μM in
less than 1% DMSO containing PBS) was added. The cells
were subsequently imaged using
setup for one minute acquisition time for a total duration of
30 minutes.
a confocal microscopy
Cell viability studies
Cellular viability was measured using an automatic cell
counter CASY® (Roche Innovatis AG). Approximately 50 000
cells were grown in 2 mL of culture medium in six-well plates
Platinum complex incubation
The culture medium was removed and 1 mL of the staining at 37 °C in a 5% CO2 environment for 48 hours. Culture
solution containing the corresponding platinum complexes medium was removed and replaced by 1 mL staining solution
(50 μM in less than 1% DMSO containing PBS) was gently of complexes PtL1Cl, PtL1NCS, and PtL2Cl (50 μM in less than
added to the cells. After incubation at 37 °C and 5% of CO2 for 1% DMSO in culture medium). After 30 minutes of incubation,
10 minutes, the staining medium was removed and the cell the staining solution was transferred to Eppendorf tubes and
layer on glass cover slips was gently washed (three times) with 0.5 mL of trypsin was added. To detach the cell from the
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surface of the plate, cells were incubated for 5 minutes under
the same conditions described before. Subsequently, 0.5 mL of
fresh culture medium was added to neutralize trypsin. Cell
suspensions together with first solutions collected were trans-
ferred to Eppendorf tubes and were mixed together. 100 µL of
the cell suspension was dissolved in 10 mL of CASY ton solu-
tion and measurements were performed. The positive control
of cells grown without complexes was also added. All exper-
iments were repeated at least three times.
Results and discussion
Synthesis
Fig. 2 Absorption (solid traces) and emission spectra (dashed traces) in
CH2Cl2 at room temperature and emission spectra in a CH2Cl2–MeOH
1 : 3 glassy matrix at 77 K (dotted traces) for the complexes PtL1Cl (black
traces), PtL1NCS (red traces) and PtL2Cl (blue traces). Emission spectra
were recorded at λexc = 300 nm.
The proligands HL1 and HL2 were synthesized from the com-
mercially available 3,5-dibromophenol and the appropriate
hydrophilic chain activated as tosylate, prepared according to
the literature31 (Scheme 1). Complexes PtL1Cl and PtL2Cl were
synthesized by a reaction of K2PtCl4 with HL1 and HL2, respect-
ively, in a CH3COOH–H2O (9 : 1 v/v) mixture placed in a micro-
wave reactor at 160 °C for 45 minutes, as previously described
for other Pt(II) complexes.32 The PtL1Cl complex was readily
converted into PtL1NCS upon treatment with sodium thio-
cyanate in methanol/dichloromethane at room temperature.13
The new proligands HL1 and HL2 and their Pt(II) derivatives
were fully characterized by elemental analysis, mass spectro-
metry, and NMR spectroscopy.
assigned to singlet-manifold spin-allowed ligand centered
(1LC) excitation processes mainly involving the 1,3-di(2-
pyridyl-benzene) cyclometallating ligand.33 At lower energy
(λabs = 370–440 nm), the samples show much weaker bands
with molar extinction coefficients in the range of 4–6 × 103
1
M−1 cm−1 and attributed to mixed transitions possessing LC
and metal-to-ligand charge transfer, 1MLCT, character.33
Upon excitation at 300 nm, dilute CH2Cl2 solutions of all
the three investigated complexes displayed bright structured
luminescence in the green-yellow region of the visible electro-
magnetic spectrum. The emission energies are only slightly
dependent on the ancillary ligand (λem = 542, 540 and 546 nm
for derivatives PtL1Cl, PtL1NCS and PtL2Cl, respectively), and
the luminescence of the complexes displays vibronic pro-
gression attributable to the intraligand modes, on the basis of
extensive studies reported for closely-related N^C^N platinum
complexes.34,35
Photophysical properties
In order to evaluate the potential of the novel neutral platinum
complexes as luminescent labels for bio-imaging purposes, we
have investigated their photophysical properties in solution.
For all three compounds, the absorption and emission spectra
are displayed in Fig. 2 and recorded for the sample concen-
tration of 1.0 × 10−5 M in dichloromethane solution at room
temperature, while the corresponding photophysical data are
listed in Table 1.
The CH2Cl2 solutions of the complexes (1.0 × 10−5 M)
showed electronic absorption features in the UV-visible range
typical of this class of compounds bearing a 1,3-di(2-pyridyl-
benzene), N^C^N, chromophoric ligand as firstly reported by
Williams and co-workers.33 In the region between 250 and
300 nm, the absorption spectra are characterized by rather
intense (ε = 1.3–2.3 × 104 M−1 cm−1) electronic transitions
While the emission profile does not appear to be depen-
dent on the presence of the quenching dioxygen molecules,
after a degassing procedure all the samples showed a much
more intense emission with PLQY that reached values as high
as 0.49, 0.46, and 0.66 for PtL1Cl, PtL1NCS, and PtL2Cl,
respectively, versus 0.01–0.02 obtained for the corresponding
air-equilibrated samples. Such an enhancement of the PLQY
Table 1 Most meaningful photophysical data of the complexes PtL1Cl, PtL1NCS, and PtL2Cl in a dilute CH2Cl2 solution at room temperature and in
a CH2Cl2–MeOH 1 : 3 glassy matrix at 77 K
λem [nm]
PLQY
τ [μs]
PLQY
τ [μs]
λem [nm]
τ [μs]
77 K
Complex
λabs [nm] (ε × 10−3/[M−1 cm−1])
Air-equilibrated
Degassed
PtL1Cl
293 (23), 326 sh (6), 378 (6), 438 (7)
293 (13), 332 sh (2), 374 (2), 430 (4)
542, 574 (sh)
540, 571 (sh)
0.01
0.02
0.27
0.39
0.49
0.46
12.4
11.8
529, 568, 610 (sh)
530, 571, 615
13.6
PtL1NCS
13.6a, 13.1 (20%),
3.8 (80%)b
14.4
PtL2Cl
293 (20), 361 sh (3), 378 (5), 436 (6)
546, 574 (sh),
0.02
0.28
0.66
12.1
531, 572, 615
a Recorded at λem = 520 nm. b Recorded at λem = 625 nm. The abbreviation “sh” denotes a shoulder.
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on going from air-equilibrated to degassed solution is typical and it is identical to that used in the bio-imaging experiments
of luminescent compounds emitting from a triplet excited (see below). In this way it is possible to evaluate the sole effect
state and is accompanied by a concomitant prolongation of of the polarity of the media and the solvent/non-solvent ratio
the excited-state lifetime, being as long as 12.4, 11.8, and on the photophysical properties. Indeed, under these con-
12.1 μs for PtL1Cl, PtL1NCS, and PtL2Cl, respectively (see ditions, the dioxane is expected to play the role of a good solu-
Table 1). The high emission efficiency can be associated with bilizing solvent for the emissive complex, while the water acts
the rigidity of the terdentate cyclometallated ligand, which as the non-solvent. The absorption and emission spectra of
reduces the molecular distortions of the excited states, thus the five investigated solutions are displayed in Fig. 3 and 4,
disfavouring the pathway of nonradiative decays,35,36 and the respectively, and the photophysical data are summarized in
strong ligand field of the tridentate chelate increases the Table S1 of the ESI.†
energy of the metal centered (MC) d–d excited states, which
are common deactivation paths for the luminescent state.
Upon increasing the water content, a steady negative solvato-
chromic shift and decrease of the intensity of the lowest-
In order to further investigate the nature of the radiative lying band is clearly visible, which is in line with the attribu-
transition responsible for such a bright emission, we per- tion of the CT character of the electronic excitation in such a
formed photoluminescence studies of the complexes in a class of compounds.33,37 Also, such a finding implies the size-
CH2Cl2–MeOH 1 : 3 glassy matrix at low (77 K) temperature able decrease of the excited-state electric dipole moment with
(see Fig. 2 and Table 1). Upon lowering the temperature and respect to that of the ground state.37 A similar hypsochromic
rigidification of the environment, the glassy samples displayed
a
more structured and slightly hypsochromically shifted
(ca. 450 cm−1) emission spectra with a parallel yet minor pro-
longation of the excited state lifetime up to 13.6–14.4 μs. These
observations evidence the weak metal-to-ligand charge transfer
character of the lowest 3LC excited state.
It is noteworthy that, while samples of the two chloro-
containing derivatives do not show any additional band in the
spectra recorded at 77 K, for the derivative bearing the SCN−
as the ancillary ligand the emission spectrum clearly displays
a lower-energy lying band centered at ca. 625 nm, indicating
the presence of closely-interacting platinum complexes. The
excitation spectra recorded at both λem = 520 and 625 nm (see
Fig. S1 of the ESI†) are almost identical to the absorption
spectra recorded in dilute CH2Cl2 solution. We can therefore
conclude that, since no ground state aggregation is present,
the lower-energy band is due to an excited state arising from
the formation of excimers, as also reported by us for other
related (N^C^N)PtNCS derivatives.13 Furthermore, for both
complexes PtL1Cl and PtL2Cl the luminescent excited state
Fig. 3 Absorption spectra of complex PtL2Cl at a concentration of 5 ×
10−5 M in an air-equilibrated dioxane–water mixture at different ratios:
100 : 0 (black trace), 80 : 20 (red trace), 60 : 40 (blue trace), 40 : 60
(orange trace), and 20 : 80 (green trace).
decay follows
a
monoexponential kinetics with
τ
=
13.6–14.4 μs. On the other hand, for complex PtL1NCS, while a
similar lifetime (τ = 13.6 μs) was measured monitoring the
emission at λem = 520 nm, a bi-exponential decay was recorded
at λem = 625 nm, being τ1 = 13.1 μs (20%) and τ2 = 3.8 μs
(80%). Such a shorter component is ascribed to the emission
arising from excimeric species (see Table 1).
A closer look at the molecular structure of the complex
PtL2Cl reveals its amphiphilic nature, where the tridentate
“platinum-Cl” core is the hydrophobic head and the triethyle-
neglycol chain is the hydrophilic tail. In order to further inves-
tigate the photophysical properties of PtL2Cl and the
possibility of establishing metallophilic Pt⋯Pt and π–π stack-
ing interactions, with subsequent modulation of both exci-
tation and emission properties,19,20,27 aggregation studies were
performed in an air-equilibrated dioxane–water mixture at
different ratios ranging from pure dioxane to dioxane–water
1 : 4. During these experiments, the concentration of the
Fig. 4 Emission spectra of complex PtL2Cl at a concentration of 5 ×
10−5 M in an air-equilibrated dioxane–water mixture at different ratios:
100 : 0 (black trace), solution 1, 80 : 20 (red trace), 60 : 40 (blue trace),
40 : 60 (orange trace), 20 : 80 (green trace). The samples were excited at
λexc = 400 nm (100 : 0, 80 : 20, 60 : 40) and at λexc = 420 nm (40 : 60 and
amphiphilic complex PtL2Cl was kept constant (5 × 10−5 M), 20 : 80).
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shift upon the increase of the solvent polarity has been
observed in the photoluminescence spectra, indicating signifi-
cant variation, and in particular a decrease, of the excited state
dipole moment. Nonetheless, despite the amphiphilic nature
of the molecules, the rather low emission intensity observed
(PLQY = 1–2%) at any dioxane–water content rules out the
presence of supramolecular organized structures in which the
hydrophobic platinum cores are arranged in such a way to
protect themselves from the surrounding quenching dioxygen
and water molecules.38 Furthermore, the absence of lower-
lying bands in both absorption and emission spectra indicates
the lack of any ground-state metallophilic Pt⋯Pt and π–π stack-
ing interactions. On the other hand, time-resolved emission
decays revealed bi-exponential kinetics with a shorter (τ1 = 4–5
ns) and a longer component (τ2 of a few hundreds of ns),
where the relative weight of the former increases upon increas-
ing the water content of the mixture (see Table S1 of the ESI†).
Although such a very short decay would point towards the sub-
stitution of the ancillary Cl− ligand with a water molecule at
first glance, we believe that this should not be the case here
due to the fact that the compound is prepared in a boiling
acetic acid–water mixture. Yet, such a short lifetime com-
ponent could rather unravel the establishment of specific
solvent–solute interactions (e.g., H-bonding between H2O and
chlorine ancillary ligand) which might favour efficient de-
activation processes via radiationless pathways.
Fig. 5 Fluorescence confocal microscopy images of the internalization
of three different platinum complexes at 50 μM into HeLa cells with <1%
DMSO/PBS as the incubating medium. (a) PtL1Cl, (b) PtL1NCS, and (c)
PtL2Cl. The emission spectra recorded from different cellular regions are
displayed (λexc = 405 nm, black traces) for (d) PtL1Cl, (e) PtL1NCS, and (f)
PtL2Cl, and compared to the corresponding emission recorded in dilute
CH2Cl2 (λexc = 300 nm, red traces).
traces) shows negligible differences, confirming the similar
nature of the emitting excited state for the two compared con-
ditions and ruling out an emission arising from aggregated or
interacting complexes through Pt⋯Pt or π–π interactions (see
also Photophysical properties).
In order to confirm the effective internalization of the plati-
num complexes inside the cells, z-stack experiments were also
carried out on cells stained with Phalloidin Alexa Fluor® 647,
as the F-actin and membrane label, by means of confocal
microscopy. The obtained results, including the orthogonal
views, are shown in Fig. 6a for complex PtL1Cl, and undoubt-
edly confirm that cellular uptake of the novel platinum deriva-
tives took place. The results of the z-stack experiments for
PtL1NCS and PtL2Cl are displayed in Fig. S2a and S3a of the
ESI,† respectively.
Bioimaging studies
In order to investigate the internalization and bio-imaging pro-
perties of the novel Pt(II) complexes as molecular probes, cellu-
lar uptake experiments were carried out on the living human
cervical carcinoma, HeLa, cell line under normal biological
conditions (37 °C, 5% CO2) by varying the staining solution in
terms of platinum complex concentration and the buffer used.
The results of the internalization experiments upon incubation
of HeLa cells with the three reported complexes, namely
PtL1Cl, PtL1NCS and PtL2Cl, at a concentration of 50 μM in
less than 1% DMSO/phosphate buffer saline (PBS) are dis-
played in Fig. 5.
Under these conditions, as can be seen by analyzing the
fluorescence confocal images, staining solutions containing
each of the three compounds show rapid (within 10 minutes)
internalization of the platinum complex into HeLa cells
(Fig. 5). In order to gain deeper insights into the chemical
nature (either a monomeric compound or an aggregated
species) of the emissive species responsible for the photo- Fig. 6 Fluorescence confocal microscopy images of the distribution of
PtL1Cl (50 μM in less than 1% DMSO containing PBS) inside HeLa cells
luminescence of the stained cells, we recorded photo-
luminescence spectra of cellular compartments upon
platinum complex uptake and the results are shown in
and co-localization experiments showing the presence of compound
PtL1Cl inside the cell nucleus, nucleoli, and cytoplasmic parts of the cell.
(a) Orthogonal views of the image showing PtL1Cl signals (green)
Fig. 5d–f (black traces). For all the investigated complexes, an
coming from inside the cytoplasmic and nuclear regions of the cells.
intense and broad emission band has been observed. The The cells are stained with Phalloidin Alexa Fluor® 647 (red). (b) PtL1Cl,
bright-field (BF) image of HeLa cells, DAPI staining of the nucleus, and
recorded emission spectra are centered at around 560 nm for
all three complexes, namely PtL1Cl, PtL1NCS and PtL2Cl.
overlay (OL) of the three panels. (c) SYTO® RNASelect™ green stains the
RNA inside cells including nucleoli; BF image, PtL1Cl, and overlay of the
Furthermore,
a comparison of the emission profile
three panels. The excitation wavelength for DAPI and PtL1Cl was
405 nm, while SYTO® RNASelect™ and Phalloidin Alexa Fluor® 647
obtained from living cells with the photoluminescence spectra
recorded in dilute dichloromethane solution (Fig. 5d–f, red were excited at 488 and 633 nm, respectively.
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Upon internalization, the complexes are distributed
throughout cellular cytoplasmic compartments and, more
interestingly, they are also present in the nuclear region, inde-
pendently of their molecular structure or their more hydro-
phobic/hydrophilic nature.
To further investigate the uptake and accumulation of the
complexes (50 μM <1% DMSO/PBS), co-localization experi-
ments were carried out using 4′,6-diamidino-2-phenylindole-
6-carboxamidine (DAPI), SYTO® RNASelect™ and Alexa
Fluor® 647 Phalloidin as the nucleus, the nucleoli and the
F-actin/membrane staining agent, respectively. The corres-
ponding microscopy images obtained after 20 minutes incu-
bation are displayed in Fig. 6b and c for complex PtL1Cl and in
Fig. S2b-c–S3b-c of the ESI† for derivatives PtL1NCS and
PtL2Cl, respectively. Such co-staining experiments clearly con-
firmed the ability of the three reported platinum complexes to
cross the nuclear membrane and localize inside the nuclear
region as well. Indeed, a rather good overlap (overlap coeffi-
cient = 0.9) can also be observed when signals arising from the
SYTO® RNASelect™ and platinum complexes are compared,
confirming the presence of the complex inside nucleoli as pre-
viously reported using N^N^N tridentate platinum com-
plexes,19,20 and observed for a related N^C^N derivative.22,23 It
is noteworthy that the presence of an SCN− group as the ancil-
lary ligand instead of the Cl− (PtL1Cl vs. PtL1NCS) as well as
the introduction of a more hydrophilic triethyleneglycol chain
into the cyclometallating phenyl ring (PtL1Cl vs. PtL2Cl) do not
significantly affect the uptake behaviour in terms of cellular
sub-localization, as shown in Fig. 6 and Fig. S2 and S3 of the
ESI.†
In order to monitor real-time events under normal cellular
conditions, an ideal bio-imaging probe should not influence,
strongly interact, or alter the correct cellular functioning and
bio-chemical processes. Thus, luminescent molecules used as
staining agents should preferentially be present at concen-
trations as low as possible. To this end, we performed concen-
tration effect and kinetics studies in living HeLa cells. By
lowering the concentration of the staining platinum complexes
down to 5 μM in <1% DMSO/PBS as the incubating media,
cellular uptake was found to be very rapid as well, and the emis-
sion from the platinum complexes inside the cells appeared
just one minute after addition of the platinum(II) complex to
the cells. The staining pattern of the cytoplasmic and nuclear
regions was found to increase over time, reaching a plateau
after 10 minutes of incubation, as shown in Fig. 7 and Fig. S4
and S5 of the ESI† for complexes PtL1Cl, PtL1NCS and PtL2Cl,
respectively. Such rapid internalization kinetics might be due
to an efficient diffusion-controlled uptake mechanism, even
though a parallel energy-driven mechanism cannot be ruled
out at this stage.
Fig. 7 Confocal images of the kinetics experiments of HeLa cells incu-
bated with PtL1Cl at a concentration of 5 μM in <1% DMSO/PBS at
different incubation times: (a) 1 minute, (b) 5 minutes, (c) 10 minutes,
and (d) 20 minutes, showing the fast internalization of the compound.
The samples were excited at λexc = 405 nm. Scale bar is 10 μm.
longer triethyleneglycol chain slowed down the kinetics
slightly, as demonstrated by the lower intensity of the signal
recorded with the confocal microscope under identical exci-
tation power. Such an effect can be most likely ascribed to the
larger molecular size of the PtL2Cl when compared to PtL1Cl,
since the longer polar (i.e., triethyleneglycol) chain is expected
to impart a more amphiphilic character to the complex, thus
helping to cross the lipophilic cellular membranes.20,29,30
Moreover, it has been found that the derivative bearing the
NCS ligand, namely PtL1NCS, shows the slowest cellular
internalization kinetics. Indeed, for this complex the signal
observed after one minute of incubation was almost negligible
and photoluminescence started to appear after five minutes,
yet with low intensity. Afterwards, an internalization pattern
somewhat similar to that obtained for the staining solution at
50 μM started to appear for all three platinum derivatives (see
Fig. 7 and Fig. S4–S5 of the ESI,† boxes b–d).
Since the incubation medium can play an important role in
the uptake of platinum complexes and it is not always that the
change from PBS to cell culture medium leads to the same
results,20 we have also investigated the behaviour of the com-
pounds using the commercial DMEM. The switch from PBS to
DMEM, as the culture medium, gave similar uptake results for
the three investigated complexes, as shown in Fig. S6 of the
ESI† for complex PtL2Cl as an example.
By comparing the kinetic experiments performed at 5 μM of
the platinum complexes, we were able to evidence the effect
exerted by both the ancillary ligand and the longer triethylene-
glycol chain. Indeed, while the uptake of complex PtL1Cl by
living HeLa cells was found to be the fastest amongst the three
different platinum derivatives employed, introduction of a
Finally, it is worth noticing that even after 30 minutes of
incubation the cells possess a healthy appearance, and no
evident sign of apoptosis was observed during the imaging
experiments. This observation indicates a negligible degree of
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inside the nuclear region as well. They also suggest that the
introduction of a more hydrophilic triethyleneglycol chain into
the cyclometallating phenyl ring does not significantly affect
the uptake behavior in terms of cellular sub-localization.
Further studies are needed to gain a better understanding of
the relationship between the chemical structure of the coordi-
nation sphere of platinum(II) and the localization site inside
the cell.
In any case, it is worth pointing out that the introduction of
oligo-ethyleneglycol chains, which allow a very low cytotoxicity,
could be a tool to increase not only the aqueous solubility but
also the biocompatibility of highly luminescent Pt(II) complex
families for bio-imaging applications.
Fig. 8 Cellular viability studies after 30 minutes incubation with
different complexes at a concentration of 50 μM in <1% DMSO contain-
ing culture medium.
Acknowledgements
In Italy, this work was supported by the Ministero dell’Istru-
zione, dell’Università e della Ricerca (FIRB 2004:RBPR05JH2P
and PRIN 2008:2008FZK5AC_002).
L.D.C., M.M., F.F., and D.S. kindly acknowledge Université
de Strasbourg, CNRS, the Région Alsace, the Communauté
Urbaine de Strasbourg, the Département du Bas-Rhin, and the
Ministère de l’Enseignement Supérieur de la Recherche
(MESR) for funding the purchase of the confocal microscope.
ERC grant no. 2009-247365 is acknowledged for financial
support. L.D.C. is also grateful to AXA research funds.
toxicity of the internalized compounds inside the cells, which
is quite surprising since as already mentioned the compounds
are in the nucleus. To further quantify such a finding, viability
tests were performed by means of the CASY® equipment in
order to evaluate the cytotoxicity and the results are shown in
Fig. 8.
Interestingly, the number of viable cells after 30 minutes
incubation of the different complexes, a time period much
longer than that used for the imaging experiments, is found to
be close to the control experiments, strongly indicating the
low cytotoxicity of the complexes and their suitability as
efficient phosphorescent probes to be used in bio-imaging
applications.
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