Investigations using fluorescent ligands to monitor platinum(iv) reduction and platinum(ii) reactions in cancer cells

Article abstract of DOI:10.1039/b821603g

Coordination of the aniline containing fluorophores, coumarin 120 (C120) and coumarin 151 (C151) at the non-leaving group positions of cisplatin analogues (giving cis-[PtCl₂(C120)(NH₃)] and cis-[PtCl₂(C151)(NH₃)

Full text of DOI:10.1039/b821603g

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Investigations using fluorescent ligands to monitor platinum(IV) reduction
and platinum(II) reactions in cancer cells
Elizabeth J. New, Ran Duan, Jenny Z. Zhang and Trevor W. Hambley*
Received 2nd December 2008, Accepted 21st January 2009
First published as an Advance Article on the web 6th March 2009
DOI: 10.1039/b821603g
Coordination of the aniline containing fluorophores, coumarin 120 (C120) and coumarin 151 (C151) at
the non-leaving group positions of cisplatin analogues (giving cis-[PtCl2(C120)(NH3)] and
cis-[PtCl2(C151)(NH3)]) resulted in partial and complete quenching of the fluorescence, respectively.
Oxidation of the coumarin 120 complex to the Pt(IV) form (cis,trans,cis-[PtCl2(OH)2(C120)(NH3)])
resulted in further quenching compared to that seen for the Pt(II) complex. The fluorescence profiles of
these coumarin complexes were collected to evaluate their suitability for studying the metabolism of
cisplatin-based anticancer drugs. C151 has the more suitable profile with a lower energy excitation peak
and a better separation between the excitation and emission spectra. The complete damping of
fluorescence on coordination to Pt(II) makes it unsuitable for monitoring the reduction process, but
does allow it to be used to monitor loss of the aniline type ligand. All of the coumarin complexes
revealed moderate cyotoxcities in the range 10–22 mM indicating that they are suitable models of
anticancer agents. DNA dampens the fluorescence of both Pt(II) complexes and that of C120 has a
much higher DNA binding affinity (10 000 M-1) than does the complex of C151 (300 M-1). Treatment of
A2780 human ovarian carcinoma cells with the Pt-coumarin complexes resulted in fluorescence visible
by confocal microscopy, and co-localisation studies with organelle specific dyes suggest they are
concentrated in the late endosomes or lysosomes. Cells treated with the Pt(IV) complex of C120 revealed
strong fluorescence and a somewhat different distribution to cells treated with the Pt(II) complex
indicating reduction following uptake.
and their potential for oral administration due to their kinetic
Introduction
inertness and often higher lipophilicity. In order to harness such
The earliest investigations of platinum compounds for their
properties, a greater understanding of the mode of action of Pt(IV)
antitumour activity by Barnett Rosenberg and his group1–3
complexes is required and, in particular, of the mechanisms by
identified both Pt(II) and Pt(IV) complexes as active. However,
which the complexes enter the cell and undergo reduction and
it was the Pt(II) drug cisplatin that was initially studied more
metabolism.
extensively. Recent work has seen a return to Pt(IV) complexes
Previous studies in our group have utilised X-ray fluorescence
in an attempt to circumvent the limitations of cisplatin-based
techniques such as synchrotron radiation-induced X-ray emission
drugs which lie in their toxicity4,5 and tumour resistance.6–10
A
(SRIXE), and X-ray absorption near edge spectroscopy (XANES)
to reveal subcellular localisation and reduction.20 While providing
some valuable information, such techniques are limited in their res-
olution, particularly by the limiting probe size. Another technique
that has been widely used for the study of platinum complexes
is fluorescence microscopy.21–29 While providing good resolution
and allowing for clear identification of subcellular organelles,
these fluorescence microscopy studies did not give any information
about oxidation or coordination state.
Here, we describe work aimed at assessing and utilising
coordination and oxidation state dependent fluorescence as a
technique for studying drug metabolism. It has been observed
that the binding of a number of transition metals to a fluorophore
can either increase or decrease its fluorescence properties.30,31
While neither Pt(II) nor Pt(IV) have been reported to induce such
fluorescence changes, the results from other d8 and d6 ions are
encouraging.32–36 It was hypothesized that changes in fluorescence
induced by ligand loss and/or changes in oxidation state could be
utilised in microscopy studies to observe the temporal and physical
location of these key events.
number of anticancer active Pt(IV) complexes have been developed,
including ormaplatin, or tetraplatin,11 iproplatin,12,13 and JM216
(satraplatin).14 In general, Pt(IV) complexes are octahedral, while
Pt(II) complexes exhibit a square planar ligand arrangement.15
Reduction of the complex from Pt(IV) to the Pt(II) state is therefore
accompanied by loss of the axial ligands.†
Pt(IV) drugs are believed to be first reduced to Pt(II), whether
intra- or extra-cellularly, before binding to proteins and DNA16
and acting in a manner similar to their Pt(II) counterparts.
The potential advantages of this class of drugs lie in their
activity coupled with a low toxicity while in the Pt(IV) state,17
their activity in the treatment of cisplatin-resistant tumours,14,18,19
School of Chemistry, The University of Sydney, NSW 2006, Australia.
E-mail: t.hambley@chem.usyd.edu.au; Fax: +61 2 9351 3329; Tel: +61
2 9351 3320
† By convention, ligands that lie above and below the plane of the am(m)ine
ligands in Pt(IV) complexes are referred to as “axial” ligands, while
those that lie in the same plane as the am(m)ine ligands are designated
“equatorial” ligands.57,58
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Results and discussion
Synthesis
The rationale of the present study was to synthesise Pt(II)
and Pt(IV) analogues of cisplatin in which a ligand contains a
fluorescent moiety. It was anticipated that the fluorescence of this
group would be quenched upon coordination or upon oxidation,
with the result that the presence or absence of fluorescence would
be a marker of metabolic events within the cell such as reduction
and/or ligand loss. In the present study, the amine (non-leaving)
group of cisplatin was targeted.
Cisplatin-based Pt(II) and Pt(IV) complexes were synthesised
containing a fluorescent coumarin moiety in place of one of the
ammine groups (3–5, Scheme 1). The coumarins used were the
aniline group-containing compounds, coumarin 120 (C120) and
coumarin 151 (C151). The analogous aniline complexes (1 and 2)
were also synthesised for comparison.
Fig. 1 Fluorescence excitation (bold lines) and emission (fine lines;
excitation at 370 nm) spectra of 0.1 mM solutions of C120 (solid) and
C151 (dashed) in DMF.
the overlap of the excitation and emission spectra of C151 is less
than for the spectra of C120. This is an advantage for fluorescence
microscopy, where overlap of spectra results in excitation light
interfering with the collection of the emission image, decreasing
the image contrast.37
These differences in excitation and emission energies can be un-
derstood by a consideration of the electronics of the systems. The
general consensus from theoretical studies of 7-aminocoumarins is
thatthefirstthree(longestwavelength)excitationpeaksresultfrom
p–p* transitions38–40 while a fourth, smaller peak at higher energy
can be attributed to an n–p* transition.38,41 The p–p* transitions
will primarily be considered and various studies have suggested
that the excited p* energy levels arise from tautomerisation of
the coumarin.42,43 From studying the energy changes that resulted
from changing the substituents of 7-aminocoumarins, it was
concluded that the longest wavelength singlet–singlet transition
(S0–S1) results from an intramolecular charge transfer from a p
orbital associated with the benzene ring and nitrogen atom to
a p orbital of the pyrone ring and carbonyl group. There is no
similarly clear definition of the structures of S2 and S3, but they
are believed to differ in charge distribution and dipole moments.44
The electronic structure of the S1 state is of greatest interest as
the S0–S1transition gives rise to the highest wavelength excitation
absorption, which is the one that will be excited in confocal
microscopy and is the primary contributor to the emission.
The longer excitation wavelengths of C151 compared to C120
represent an elevation of the S1 energy compared to the S0 level.
This can be explained by the fact that replacement of the methyl
group in C120 with the trifluoromethyl in C151 results in decreased
electron density in the pyrone ring, thus decreasing the stability of
the excited state.
On binding of C120 to Pt(II) (3; Fig. 2a), a 2.5-fold quenching of
fluorescence was observed, with a further 7-fold decrease on oxi-
dation of the complex to Pt(IV) (4). This conclusively demonstrates
that binding of platinum to a fluorophore results in fluorescence
quenching. The effect is considerably more marked for C151
(Fig. 2b), where the Pt(II) complex 5 shows negligible fluorescence.
There was no such decrease in fluorescence in solutions containing
the Pt(II) or Pt(IV) ions and unbound coumarins, confirming that
this quenching is a coordination-mediated event.
The C120 system is likely to be of greater utility in studying
the reduction event, as the fluorescence change on oxidation
from 3 to 4 is greater than on binding of the coumarin. The
Pt(IV) complex 4 exhibits considerable fluorescence, and both
Scheme 1
Pt(II) complexes were synthesised by treatment of the ammine-
trichloroplatinate(II) anion with aniline or the respective
coumarin, to give complexes 1, 3 and 5. Oxidation of these Pt(II)
complexes to the Pt(IV) dihydroxo forms was less straight-forward
as it was necessary to avoid simultaneous oxidation of the ligand.
Heating with peroxide for a short period of time led to successful
oxidation of 1 and 3, but was unsuccessful for 5. Oxidation of 5 in
other solvents resulted in ligand release and oxidation, suggesting
weaker binding of this ligand. This is consistent with the greater
electron-withdrawing ability of the trifluoromethyl group, which
is expected to result in weaker basicity of the amine group.
Fluorescence properties
The aim of this work was to synthesise complexes suitable for
study in cells by fluorescence confocal microscopy. Excitation and
emission spectra of C120 and C151 (Fig. 1) show that C151 is
a more suitable fluorophore for this purpose, as there is greater
excitation at 400 nm, the typical minimum excitation wavelength
for single-photon confocal microscopy experiments.37 In addition,
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Fig. 3 Fluorescence changes following treatment of 4 with ascorbic acid.
(a) Emission spectra (with excitation at 370 nm) of 4 collected at various
intervals following the addition of ascorbic acid (10-fold excess). (b) Plot of
fluorescence intensity at maxima in emission spectra following the addition
of ascorbic acid.
Cytotoxicity
The cytotoxicities of the ligands and complexes were determined
using the spectrofluorimetric MTT assay. The concentrations of
complexes and ligands that inhibited cloned cell survival by 50%
(IC50) are shown in Table 1.
Interestingly, while cisplatin has
a 5-fold greater cyto-
toxicity than its Pt(IV) dihydroxo analogue, cis,trans,cis-
[PtCl2(OH)2(NH3)2], there is very little difference between the
cytotoxicities of the Pt(II) and Pt(IV) complexes of aniline and
C120. This may indicate that the Pt(IV) complexes are being
rapidly reduced in vivo to their Pt(II) forms. An increased reduction
rate is consistent with the fact that 2 (-568 mV vs. NHE) and 4
(-513 mV vs. NHE) have less negative reduction potentials than
cis,trans,cis-[PtCl2(OH)2(NH3)2] (-660 mV vs. NHE). Another
possible explanation is that these Pt(IV) complexes are acting via
an alternative mechanism, with comparable overall efficacy. Also,
a greater cellular uptake of the Pt(IV) complexes 2 and 4 could
contribute to the higher than expected cytotoxicity.
While the primary aim of this investigation was to attach a
fluorophore for use in microscopy, the Pt-C120 complex was
expected to have antitumour activity in its own right, based
on the high activity of similar ammine/amine platinum(II) and
platinum(IV) complexes.9,47 The fact that these complexes do
indeed exhibit cytotoxicity confirms that they are meaningful
models of active anticancer complexes. If, for example, a much
lower cytotoxicity was observed, any subsequent conclusions
Fig. 2 Excitation (heavy lines) and emission (fine lines; excitation at
370 nm) spectra of coumarins and complexes: (a) C120 (solid), 3 (short
dashes) and 4 (long dashes); (b) C151 (solid) and 5 (dashes).
complexes should be visible by fluorescence microscopy. However,
use of an excitation wavelength in the 380–400 nm range should
allow for reduction to be monitored. The shape of the excitation
spectrum of the coumarin changes substantially on coordination
to Pt(II) and further on oxidation to Pt(IV). Coordination leads
to the two primary peaks moving closer together in wavelength
and oxidation increases this effect to the point where the peaks
are indistinguishable or results in almost complete loss of one
of the peaks. As a result negligible excitation of 4 is observed
when using excitation wavelengths longer than 380 nm. The C151
system, with the almost complete quenching of fluorescence on
binding, would allow for greater differentiation of coordination
by microscopy. However, successful oxidation would be likely to
yield a similarly non-fluorescent Pt(IV) form, so no information
about the reduction event could be obtained.
Given the differences in fluorescence intensities observed for
Pt(II) and Pt(IV) complexes, a solution of 4 that had been treated
with the cellular reductant ascorbate was monitored over a period
of 2 d to observe whether the reduction of the complex to
3 resulted in a concomitant increase in fluorescence. A slight
but steady increase in fluorescence was observed over this time
(Fig. 3). A solution of 4 alone showed no fluorescence changes
over the 2 d period, implicating a reduction event or ligand
exchange as being responsible for the fluorescence increase. The
observed slow increase in fluorescence suggests slow reduction
of the Pt(IV) complex and indicates that it will be resistant
to reduction in the medium prior to cellular uptake. This is
consistent with the measured negative reduction potential of 4
(-513 mV vs. NHE) and with observations that trans-Pt(IV)-
dihydroxo complexes are particularly resistant to reduction in
comparison to Pt(IV) complexes with other axial ligands.45,46
Table 1 Cytotoxicity data for various complexes and ligands in A2780
cells following 72 h incubation
Complex/ligand
IC50 (mM) S.D.a
Cisplatin
cis,trans,cis-[PtCl2(OH)2(NH3)2]
Aniline
3.2 0.1
17.0 2.4
69 26
1
9.3 3.1
15.3 4.3
250 47
21.6 0.6
21.2 1.8
14.1 0.4
12.1 0.6
2
C120
3
4
C151
5
a Values are means of data from three independent experiments with
quadruplicate readings in each experiment.
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about the cellular location of the complex would be less applicable
to other complexes with known anticancer activity.
of its very low fluorescence intensity when excited at 405 nm
(Fig. 3). Thus, the observation of substantial fluorescence is
consistent with the rapid reduction of various Pt(IV) complexes
in vitro, regardless of their reduction potentials, with reduction of
cis,trans,cis-[PtCl2(OH)2(NH3)2] being complete within 4 h.50
In comparison to the C120 complex (3), the fluorescence of 5
is negligible (Fig. 2(b)), and the fact that fluorescence is observed
in cells treated with 5 (Fig. 4e) is most consistent with metabolism
of the complex to yield the free ligand. While cells treated with
3 clearly show organelle localised fluorescence, in cells incubated
with 5 the fluorescence is more generally distributed throughout
the cytoplasm and is indistinguishable from that produced by
C151, consistent with rapid loss of this ligand. These contrasting
observations for 3 and 5 are consistent with the lower stability of
the latter encountered in the synthetic work.
To reveal the subcellular location of the complexes, cells were
treated with nuclear or lysosomal stains following incubation with
C120 and its complexes. Examination of these images reveals that
the localisation of the Pt(II) and Pt(IV) complexes is similar in
most respects. Images taken from cells treated with Pt(II)-C120
or Pt(IV)-C120 and the syto21 nuclear stain (Fig. 5) reveal an
absence of fluorescence arising from the complexes in the nucleus.
This does not preclude the possibility that either complex is
present in the nucleus, as the fluorophore could interact with
DNA, resulting in quenching of its fluorescence or shifting of
the fluorescence out of the range being measured. Cells were
also incubated with both C120 and the syto21 nuclear stain to
determine the localisation of the fluorophore alone. These images
suggest that the ligand localises similarly to the complexes, with
no observable fluorescence in the nucleus.
The results indicate that, while they are cytotoxic, 1 and 3
are less cytotoxic than cisplatin, in accord with previous studies,
which have demonstrated that substitution of one or both ammine
ligands leads to decreased cytotoxicity.48,49 The high cytotoxicity
of C151 compared to that of C120 is somewhat surprising, but
may be accounted for by the higher reactivity resulting from the
presence of the trifluoromethyl group.
Confocal microscopy
Cells that had been treated with ligands and complexes were
visualised by confocal fluorescence microscopy, with excitation
at 405 nm. As shown in Fig. 4, incubation of A2780 cells with
C120, C151 and their complexes resulted in readily-detectable
fluorescence. From these images, there appears to be no fluores-
cence in the nucleus, with fluorescence possibly localised in other
organelles in the cytoplasm. When optical sections throughout the
cell were taken, this fluorescence could be observed in most layers.
As expected, untreated control cells showed no fluorescence.
The fluorescence observed in cells treated with 3 may be due to
this complex or to free C120. However, the C120 is attached to one
of the non-leaving group positions of the complex and the nature
of the fluorescence is different from that in cells treated with C120,
particularly in regard to the speed of photo-bleaching. Therefore,
it is most likely to be due predominantly to 3. At longer time
points, and following binding to thiol containing biomolecules, it
is to be expected that substantial amounts of C120 will be released
from 3.
The fluorescence observed in cells treated with 4 (Fig. 4(c))
is most unlikely to be due to the Pt(IV) complex (4) because
The green-blue colour observable in the superposition of images
obtained from co-staining with the complexes and the LysoTracker
Fig. 4 Confocal microscope images of A2780 cells treated for 4 h with 20 mM: (a) C120, (b) 3, (c) 4, (d) C151 and (e) 5. The brightness and contrast of
the image have been manually increased. Scale bars represent 10 mm.
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Fig. 5 Confocal microscope images of A2780 cells treated with complexes, C120 and nuclear stain. A2780 cells under confocal fluorescence microscope
4 h after treatment with (a) C120, (b) 3 and (c) 4 with 10 min staining with syto21 nuclear stain. Part (i) shows image collected through blue emission filter
(C120 and Pt complexes), (ii) image through green emission filter (syto21) and (iii) superposition of blue and green images. The brightness and contrast
of the image have been manually increased. Scale bars represent 10 mm.
Green lysosomal stain (Fig. 6) indicates co-localisation. However,
the blue fluorescence of the complexes is not restricted to the
regions showing green fluorescence, indicating that the complexes
have entered the lysosomes, but are also present elsewhere in the
cytoplasm, or in other organelles. In general, the Pt(II) treated
cells show more of the fluorescence localised in punctuate spots
corresponding to the lysosomes, whereas Pt(IV) treated cells have
a more diffuse distribution of the fluorescence. In some cells,
particularly noticeable in Fig. 6(c), the green fluorescence does
not correspond to distinct “spots” indicating lysosomes, but
appears to be relatively evenly spread throughout the cytoplasm.
This indicates that the stain was not completely specific for
the lysosomes. Since the stain has high specificity for acidic
organelles51 there could have been an aberration in the cells due
either to the nature of the cell line or to a generalised decrease
in pH within the cytoplasm resulting from treatment with the
complex. Given that the effect was most noticeable in Pt(IV)-
treated cells, it is more likely to be a result of treatment with the
complex. If the Pt(IV) complex is able to either avoid lysosomal
uptake or contribute to lysosomal destruction more so than its
Pt(II) counterpart, this could contribute to its higher than expected
relative cytotoxicity.
localisation by other means, to establish whether attaching a
coumarin moiety has affected the subcellular distribution of
the complex. X-Ray imaging of cisplatin in bone marrow cells
indicated that most drug was present in the cytoplasm.52 Later
electron microscopic analysis of cisplatin in A2780 cells, making
use of the electron-dense nature of platinum, detected platinum
on the plasma membrane and the nuclear envelope as well as
in the nucleus and cytoplasm, but there was no platinum within
other organelles.53 These results differ considerably from the
findings of the current work, in which the platinum complexes
have been found to localise in the lysosomes and perhaps in other
organelles, or in the cytoplasm. This suggests that the presence
of the coumarin does affect the localisation of the complex.
This localisation pattern is similar to those of other platinum–
fluorophore complexes.24,27 While this limits the usefulness of
C120- and C151-based complexes in providing information about
the localisation and ligand exchange of cisplatin itself, there is still
much to be learnt about the bioaccumulation and metabolism of
other classes of platinum complexes.
If the complexes are not reaching the nucleus, they must be
killing the cells by an alternative mechanism, since cytotoxicity
data (Table 1) confirmed their role in inducing cell death. One
possible mechanism is the disruption of lysosomes, which has long
been identified as a trigger for cell death,54 and it is worth noting
It is interesting to compare the localisation results of this
experiment with the results of studies that determined the cellular
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Fig. 6 Confocal microscope images of A2780 cells treated with complexes and lysosomal stain. (a) Two A2780 cells and (b) multiple cells after 4 h
treatment with 3; (c) two cells and (d) multiple cells after four h treatment with 4. All cells were treated for 1.5 h with LysoTracker Green lysosomal stain.
Part (i) shows image collected through blue emission filter (Pt complexes), (ii) image through green emission filter (LysoTracker) and (iii) superposition
of blue and green images. Blue-green colour indicates co-localisation. The brightness and contrast of the image have been manually increased. Scale bar
represents 10 mm.
in this context that the great bulk of the platinum that enters the
cell does not reach the nucleus.55 More recently it has been found
that disruption of lysosomes and the resultant release of their
acidic contents into the cytoplasm causes disruption of the plasma
membrane, and thus the death of the cell.56 Lysosomal disruption
is a putative cause of the nephrotoxicity of cisplatin57 and has
also been identified as a necrosis-inducing pathway triggered for
other platinum complexes.24 As mentioned, such lysosomally-
induced apoptosis could be an explanation for the unusually high
cytotoxicity of 4 and would also contribute to the more diffuse
fluorescence distribution.
the voltage was set at the highest possible value while for Pt(IV)-
C120 it was set at approximately 93–95% of maximum. This could
be due to a higher cellular accumulation of the Pt(IV) complex,
a hypothesis that would have to be confirmed by performing
cellular uptake studies since uptake of Pt(IV) complexes is generally
lower than that of their Pt(II) analogues.58 It was also noticed
that the fluorescence of cells treated with uncomplexed C120
was weaker still than that of the Pt(II) complex, which could be
due to greater fluorescence bleaching, or to decreased cellular
uptake.
In addition to gaining an understanding of the cellular lo-
calisation of the complexes, these results allow a comparison
between the Pt(II) and Pt(IV) complexes. While they appear to
have similar localisation, the Pt(IV) complex gave rise to more
fluorescence than its Pt(II) counterpart. This difference can be
approximately quantified by comparing the photomultiplier tube
voltage levels required to give comparable images; for Pt(II)-C120
DNA-binding studies
The effect of DNA-binding on coumarin fluorescence was evalu-
ated as an explanation for the observed lack of fluorescence in the
nucleus. Titration of DNA into a solution of coumarin or complex
resulted in a decrease in fluorescence intensity across the spectrum
as shown in Fig. 7.
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Methods and instrumentation
Reagents
All solvents used were laboratory grade and were used without
further purification unless otherwise stated. Silver nitrate was
obtained from Merck. Hydrogen peroxide (30% v/v) was obtained
from Ajax Chemicals and stored in the dark at 5 ◦C. The Advanced
DMEM for cellular studies was sourced from Gibco, and
Leibovitz’s L-15 Medium from Invitrogen. Tetrapropylammon-
ium perchlorate was obtained from the Eastman Kodak Company.
Cisplatin, (SP-4-2)-diamminedichloroplatinum(II), was synthe-
sised from K2PtCl4 obtained from Aithaca Chemical Corporation.
All other reagents were acquired from Sigma Aldrich. For
cytotoxicity and confocal studies, the cell line used was the human
ovarian carcinoma cell line A2780. Cells were maintained in
exponential growth as monolayers at 37 ◦C in 5% CO2 in Advanced
DMEM supplemented with 2.5 mM glutamine and 2% fetal calf
serum.
Fig. 7 Changes in the fluorescence emission spectrum of 3 on the addition
of DNA (lex = 370 nm).
The addition of DNA resulted in a linear decrease in the
fluorescence of all solutions tested, demonstrating that DNA
quenches the fluorescence of these systems. While quenching
was incomplete under the conditions studied, it is feasible that
extrapolation to the higher DNA concentrations found in the
nucleus would result in complete quenching of fluorescence.
Therefore, the absence of fluorescence in the nucleus might be
explained by association with DNA.
In addition, there was a dramatic difference between the DNA
binding affinities of the coumarins, which were calculated to be
300 for C151 and 10 000 for C120. These values suggest that the
addition of the trifluoromethyl group interferes with the DNA
binding ability of the coumarin. The fact that no shift in the
position of the fluorescence maximum of C151 was observed on
addition of DNA is consistent with non-intercalation. The fact
that C151 is more cytotoxic than C120 (Table 1), despite this
apparent lack of interaction with DNA suggests that it may be
acting by an alternative mechanism of toxicity.
Physical measurements
Infrared spectra were collected as diffuse reflectance infrared
transform spectra (DRIFTS) using a Bio-Rad FTS-40 spectropho-
tometer equipped with Win-IR Windows 3.1 based software. KBr
was used as both the background and the matrix over the range
500–4000 cm-1. 195Pt NMR spectra were collected by Dr I. Luck
using a Bruker AMX 400 MHz spectrometer. Spectra were col-
lected at 298 K and chemical shifts were referenced to Na2[PtCl6].
1H and 13C NMR spectra were collected at 300 K on a Bruker
300 MHz spectrometer using commercially available deuterated
solvents. For spectra recorded in D2O, TSP was used as an internal
standard, while for other solvents, isotopic impurities were used as
internal reference signals. Microanalysis (C, H, N) was performed
by Chemical and Micro Analytical Services, Belmont, Victoria
and the Microanalytical Unit, Australian National University,
Australian Capital Territory. Mass spectra were collected using a
Finnigan LCQ MS detector. Microfiltration of aqueous solutions
was achieved using a Minisart filter unit (0.2 mm pore size) with a
disposable 1 mL syringe (Becton-Dickinson).
Absorption spectra were collected using a Varian Cary Eclipse
1E UV-vis spectrophotometer, with a 1 cm ¥ 1 cm silica cuvette
(Starna). Scans were run on 0.03 mM DMF solutions at a scan
speed of 120 nm min-1 and absorbances collected between 250–
600 nm. Fluorescence measurements were performed using a
Varian Cary Eclipse fluorescence spectrophotometer, with a 1 cm ¥
1 cm silica cuvette (Starna). Scans were run at a scan speed of
120 nm min-1 with excitation and emission slit widths of 5 nm.
Excitation spectra were obtained using a 430–1100 nm emission
filter. For in situ reduction experiments, emission spectra were
recorded at regular time intervals after the addition of ascorbic
acid (ten molar equivalents, 29 mM) or cysteine (ten molar
equivalents, 45 mM). Parameters were the same as described
previously, and emission spectra were collected over the range
375–600 nm with excitation at 370 nm.
Conclusions
Our studies confirm that coordination-sensitive fluorescence is a
valuable technique for the study of the cellular metabolism of
platinum complexes. It was hoped that a Pt(II) complex with
a fluorophore in the non-leaving group position would show
appreciable fluorescence that would be quenched on oxidation
to Pt(IV). In this way, the appearance of fluorescence would
indicate that the reduction event had occurred. While this was
the case for the C120 complexes, the use of C151 in an attempt
to optimise the fluorescence properties of the fluorophore resulted
in almost complete quenching of fluorescence on coordination to
Pt(II). Even if the Pt(II) complex could be successfully oxidised,
it is unlikely that this would result in further useful fluorescence
changes. Rather than providing information about the reduction
event, therefore, the C151 complex can be used to study loss of
the amine ligand and this is seen to be substantial. The C120
complexes provided valuable insights into differences between the
uptake of the Pt(II) and Pt(IV) complexes and suggested that the
Pt(IV) complex interacts differently with the lysosomes. However, a
coumarin with fluorescence properties intermediate between those
of C120 and C151 may allow an even clearer distinction between
the Pt(II) and Pt(IV) states, enabling the reduction process to be
followed in live cells.
Electrochemical measurements were performed using a BAS
100B/W Electrochemical Analyser. Cyclic voltammagrams were
collected at room temperature at a scan rate of 100 mV sec-1, using
a glassy carbon working electrode, a platinum auxiliary electrode
and a silver/silver chloride reference electrode. Complexes were
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dissolved in acetone containing 0.1 M tetrapropylammonium
perchlorate to a final concentration of 1 mM. Solutions were
degassed with argon for ten min prior to measurement. Ferrocene
was added to each sample as an internal reference.
Images were collected and processed using Leica acquisition and
analysis software.
Syntheses
(NH4)+[PtCl3(NH3)]-. Synthesis was performed according to
the procedure of Cai et al.61 Cisplatin (1.6 g, 5.3 mmol), tetraethy-
lammonium chloride (1.13 g, 6.4 mmol) and ammonium chloride
(0.08 g, 1.5 mmol) were dissolved in N,N -dimethylacetamide
(60 mL). The reaction mixture was stirred at 100 ◦C under a
slow stream of N2 for 9.5 h, during which time the colour of
the solution changed from yellow to orange. The final solution
volume was approximately 10 mL. Hexane–ethyl acetate (1 : 1,
140 mL) was added, and the mixture kept overnight at -20 ◦C.
The colourless supernatant was then decanted, and the solid
extracted with acetonitrile (3 ¥ 15 mL), giving a yellow solid (un-
reacted cisplatin and NH4Cl) and an orange solution containing
(NH4)+[PtCl3(NH3)]-. These were separated by filtration and the
solid washed with water to recover unreacted cisplatin (138 mg,
0.46 mmol). To the orange acetonitrile solution, water (10 mL)
was added and the acetonitrile removed under reduced pressure,
resulting in an orange aqueous solution of (NH4)+[PtCl3(NH3)]-
(4.8 mmol, 91% based on unreacted cisplatin). The solution was
made up to 100 mL (48 mM) with water and stored at 4 ◦C.
Reduction in situ
Emission spectra of a 0.1 mM solution of Pt(IV)-C120 in DMF
were recorded at regular time intervals after the addition of
ascorbic acid (ten molar equivalents, 19 mM). Parameters were the
same as described previously, and emission spectra were collected
over the range 375–600 nm with excitation at 370 nm.
DNA fluorescence quenching and binding studies
DNA binding studies and the determination of binding constants
were carried out as described previously.59 The complexes were
made up in 1% DMF (C120) or 5% (C151) solutions at 20 mM
concentrations and aliquots of 1.75 mM (C120) or 3.36 mM
(C151) DNA were added.
Cytotoxicity assays
Solutions of cisplatin, transplatin, cis-Pt(II)-azaindole, trans-
Pt(II)-azaindole and 7-azaindole were prepared as 0.5 mM solu-
tions (in 100 mM aqueous KCl) immediately prior to the assay. For
the platinum complexes 1% DMF was added to ensure solubility.
IC50 values were determined using the MTT assay, as described by
Carmichael et al.,60 which makes use of the conversion of MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) to
a purple formazan product by the mitochondrial dehydrogenase
of viable cells. This insoluble formazan was quantified spec-
trophotometrically upon dissolution in DMSO. Approximately
2 ¥ 104 A2780 cells in 100 mL DMEM were seeded into each
well of flat-bottomed 96-well plates (Corning) and allowed to
attach overnight. Complexes were diluted in medium to seven
concentrations spanning a 4-log range, and 10 mL of each drug
solution was added to quadruplicate wells to produce the final
desired concentrations (final DMF concentrations were limited to
0.2%). Following 72 h incubation, MTT (1.0 mM) was added to
each well, and the plates incubated for a further 4 h. The culture
medium was removed, and DMSO (200 mL) was added. The
plates were shaken for 5 seconds and the absorbance measured
immediately at 600 nm in a Victor3V microplate reader (Perkin
Elmer). IC50 values were determined as the drug concentration
required to reduce the absorbance to 50% of that in the untreated,
control wells.
(SP-4-3)-ammineanilinedichloroplatinum(II) (1). To a solution
of (NH4)+[PtCl3(NH3)]- prepared above (25 mL, 2.27 mmol),
aniline (182 mL, 186 mg, 2.0 mmol) was added. After the solution
was stirred at room temperature for 2 d, a yellow-brown precipitate
had formed. This was isolated by vacuum filtration and washed
with diethyl ether, giving Pt(II)-aniline (660 mg, 1.75 mmol, 87.5%).
IR (KBr): 3069 (NH3), 1684, 1596 (NH2, NH3) cm-1. 1H NMR
(DMF-d7): d 7.451 (d, 2H, J = 7.96 Hz, aromatic H), 7.389 (broad,
2H, NH2) 7.333 (t, 2H, J = 7.75 Hz, aromatic H) 7.197 (t, 1H,
J = 6.80 Hz, aromatic H), 4.184 (broad, 3H, NH3). 195Pt NMR
(DMF-d7): d -2068. Elemental analysis, found: C 17.61, H 3.01,
N 6.83. Calculated for C6H10N2Cl2Pt: C 17.57, H 2.95, N 6.83.
(OC-6-43)-ammineanilinedichlorodihydroxoplatinum(IV)
(2).
(1) (50 mg, 0.13 mmol) was suspended in 15 mL of distilled water,
and heated to 80 ◦C. To this suspension was added a 30% aqueous
solution of H2O2 (1 mL), and the mixture was stirred at 80 ◦C for
40 min. After this time, the dark brown precipitate was filtered
off and washed with diethyl ether, giving Pt(IV)-aniline (20 mg,
0.049 mmol, 37%).
IR (KBr): 3457 ((Pt-)O–H), 3102 (NH3), 1700, 1634, 1559 (NH2,
NH3) cm-1. Elemental analysis, found: C 19.18, H 2.65, N 7.46.
Calculated for C6H12N2O2Cl2Pt: C 19.16, H 2.68, N 7.45.
(SP-4-3)-ammine(7-amino-methylcoumarin)dichloroplatinum(II)
(3). C120 (350 mg, 2.0 mmol) was dissolved in ethanol (200 mL)
and slowly added (over a period of 3 h) to a solution of
[PtCl3(NH3)]- (2.27 mmol) in water (25 mL). The solution was
stirred at room temperature in the dark for 3 d, after which time a
pale yellow precipitate had formed. This was isolated by vacuum
filtration and washed with diethyl ether, giving Pt(II)-C120
Confocal microscopy
A2780 cells were seeded (4 mL of approximately 5 ¥ 105 cells
mL-1) in 6-well plates (Iwaki) on glass cover slips and allowed to
grow to 40–60% confluence, at 37 ◦C in 5% CO2. At this stage,
cells were treated with 12 mM drug for 24 or 72 h. The cover
slips were then washed with phosphate-buffered saline (pH 7.4),
mounted on slides and sealed with nail varnish. Confocal images
were acquired using a Leica TCS SPII multiphoton microscope.
For all images an x63/1.20 water objective was used. Two-photon
excitation at 767 nm was provided by a Ti-Sapphire laser and
excitation at 633 nm for transmittance images, by a He/Ne laser.
(797 mg, 1.74 mmol, 77%).
IR (KBr): 3239 (NH3), 1700 (C O) 1623, 1558 (NH2, NH3) cm .
-1
=
1H NMR (DMF-d7): d 7.745 (d, 1H, J = 8.50 Hz, aromatic H),
7.432 (dd, 1H, J = 8.53, 2.03 Hz, aromatic H), 7.354 (d, 1H,
J = 2.00 Hz, aromatic H), 6.334 (d, 1H, J = 1.20 Hz, CH), 4.266
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(broad, 3H, NH3), 3.917 (broad, 2H, NH2), 2.484 (d, 3H, J = 1.10,
CH3).195Pt NMR (DMF-d7): d -2054. Elemental analysis, found:
C 27.05, H 2.93, N 6.25. Calculated for C10H12N2O2Cl2Pt: C 26.21,
H 2.64, N 6.11.
14 L. R. Kelland, G. Abel, M. J. McKeage, M. Jones, P. M. Goddard,
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(OC-6-43)-ammine(7-amino-4-methylcoumarin)dichloro-dihy-
droxoplatinum(IV) (4). (3) (20 mg, 0.044 mmol) was suspended
in water (15 mL) and a 30% aqueous solution of H2O2 (500 mL)
was added. The mixture was stirred in the dark at 70 ◦C for 1 h.
The resulting dark brown solid was collected by vacuum filtration,
giving Pt(IV)-C120 (15 mg, 69%).
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=
IR (KBr): 3598 ((Pt-)O–H), 3239 (NH3), 1732 (C O) 1617 (NH2,
1
NH3) cm-1. H NMR (DMF-d7): d 7.927 (d, 1H, J = 6.37 Hz,
aromatic H), 7.653 (broad, 3H, NH3) 7.561 (dd, 1H, J = 6.36,
1.56 Hz, aromatic H), 7.515 (d, 1H, J = 1.48 Hz, aromatic H),
6.513 (d, 1H, J = 0.95 Hz, CH), 4.117 (broad, 2H, NH2), 2.796
(broad, 2H, OH), 2.678 (d, 3H, J = 2.68, CH3).195Pt NMR (DMF-
d7): d 789.
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and J. Reedijk, J. Biol. Inorg. Chem., 2004, 9, 414–422.
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29 R. A. Alderden, PhD Thesis, The University of Sydney, 2006.
30 A. W. Czarnik, in Fluorescent Chemosensors for Ion and Molecule
Recognition, ed. A. W. Czarnik, American Chemical Society,
Washington, DC, 1993, pp. 1–9.
(SP-4-3)-ammine(7-amino-trifluoromethylcoumarin)dichloro-
platinum(II) (5). C151 (183 mg, 0.80 mmol) was dissolved in
ethanol (200 mL) and slowly added (over a period of 3 h) to
a [PtCl3(NH3)]- solution prepared as described above (20 mL,
0.80 mmol). The solution was stirred at room temperature for 3 d,
after which time a yellow-grey precipitate had formed. This was
isolated by vacuum filtration and washed with diethyl ether, giving
Pt(II)-C151 (197 mg, 49%).
IR (KBr): 3277, 3205 (NH2, NH3), 1749, 1622 (NH2, NH3) cm-1.
1H NMR (DMF-d7): d 7.735 (d, 1H, J = 8.04 Hz, aromatic H),
7.661 (broad, 2H, NH2), 7.482 (d, 1H, J = 10.02 Hz, aromatic H),
7.438 (s, 1H, aromatic H), 6.971 (s, 1H, aromatic H), 3.991 (broad,
3H, NH3). 195Pt NMR (DMF-d7): d -2087. Elemental analysis,
found: C 23.31, H 1.80, N 5.53. Calculated for C10H9N2F3Cl2Pt:
C 23.45, H 1.77, N 5.47.
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