Detailed Biological Profiling of a Photoactivated and Apoptosis Inducing pdppz Ruthenium(II) Polypyridyl Complex in Cancer Cells

Article abstract of DOI:10.1021/acs.jmedchem.5b00451

Ruthenium polypyridyl complexes show great promise as new photodynamic therapy (PDT) agents. However, a lack of detailed understanding of their mode of action in cells poses a challenge to their development. We have designed a new Ru(II) PDT candidate that efficiently enters cells by incorporation of the lipophilic aromatic pdppz ([2,3-h]dipyrido[3,2-a:2′,3′-c]phenazine) ligand and exhibits photoactivity through incorporation of 1,4,5,8-tetraazaphenanthrene ancillary ligands. Its photoreactivity toward biomolecules was studied in vitro, where light activation caused DNA cleavage. Cellular internalization occurred via an energy dependent mechanism. Confocal and transmission electron microscopy revealed that the complex localizes in various organelles, including the mitochondria. The complex is nontoxic in the dark, with cellular clearance within 96 h; however, upon visible light activation it induces caspase-dependent and reactive-oxygen-species-dependent apoptosis, with low micromolar IC₅₀ values. This investigation greatly increases our understanding of such systems in cellulo, aiding development and realization of their application in cancer therapy.

Full text of DOI:10.1021/acs.jmedchem.5b00451

Article
pubs.acs.org/jmc
Detailed Biological Profiling of a Photoactivated and Apoptosis
Inducing pdppz Ruthenium(II) Polypyridyl Complex in Cancer Cells
Suzanne M. Cloonan,†,∥,Δ Robert B. P. Elmes,‡,∥,Ψ MariaLuisa Erby,† Sandra A. Bright,†
Fergus E. Poynton,‡ Derek E. Nolan,† Susan J. Quinn,§ Thorfinnur Gunnlaugsson,*,‡
and D. Clive Williams*,†
†School of Biochemistry and Immunology and Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland
‡School of Chemistry and Trinity Biomedical Sciences Institute, Centre for Synthesis and Chemical Biology, Trinity College Dublin,
Dublin 2, Ireland
§School of Chemistry and Chemical Biology, University College Dublin, Dublin 2, Ireland
S
* Supporting Information
ABSTRACT: Ruthenium polypyridyl complexes show great promise as new
photodynamic therapy (PDT) agents. However, a lack of detailed
understanding of their mode of action in cells poses a challenge to their
development. We have designed a new Ru(II) PDT candidate that efficiently
enters cells by incorporation of the lipophilic aromatic pdppz ([2,3-
h]dipyrido[3,2-a:2′,3′-c]phenazine) ligand and exhibits photoactivity through
incorporation of 1,4,5,8-tetraazaphenanthrene ancillary ligands. Its photo-
reactivity toward biomolecules was studied in vitro, where light activation
caused DNA cleavage. Cellular internalization occurred via an energy
dependent mechanism. Confocal and transmission electron microscopy
revealed that the complex localizes in various organelles, including the
mitochondria. The complex is nontoxic in the dark, with cellular clearance
within 96 h; however, upon visible light activation it induces caspase-
dependent and reactive-oxygen-species-dependent apoptosis, with low
micromolar IC50 values. This investigation greatly increases our understanding of such systems in cellulo, aiding development
and realization of their application in cancer therapy.
imaging agents without giving rise to cellular photodamage.
Kelly et al., have shown that [Ru(TAP)2dppz]2+ (TAP =
tetraazaphenanthrene), which contains π-deficient, electron
accepting ligands, causes photodamage of DNA through
oxidation of guanine.15−17 The related complex [Ru-
(TAP)2bpy]2+ has been shown to cause DNA damage through
photoadduct formation.30 Recently, crystallographic resolution
of enantiopure Ru(II)(dppz) complexes bound to DNA
sequences has been achieved by Kelly, Cardin, and co-
workers5,11 and Barton18 and co-workers. These structures
have provided detailed structural understanding of the binding
interactions that give rise to the light-switch and photodamage
phenomena.
Photodynamic therapy (PDT) is a well-recognized tumor
ablative modality for the efficacious treatment of various
cutaneous and deep tissue tumors.19 PDT involves the
administration of a “photosensitizer” either systemically or
locally and subsequent illumination with a low energy, tissue-
penetrable light. The interaction of the photosensitizer and
light results in the photochemical activation of molecules within
INTRODUCTION
■
The excellent photophysical properties of Ru(II) polypyridyl
complexes have been intensely investigated over the past 30
years with a view to varied applications.1 The biological activity
of Ru(II) complexes has been investigated since the early 1950s
when Dwyer and co-workers reported their antibacterial
activity. Since then a number of complexes have been
developed as potential anticancer agents.2−4 In particular,
their DNA binding affinity and phototriggered DNA damage
have presented them as potential cellular imaging and
therapeutic agents.5−12 The advantages of using such Ru(II)
polypyridyl complexes as cellular targeting agents lies in the fact
that the structural nature of the polypyridyl units will dictate
the overall function of the metal complex, which includes their
solubility, lipophilicity, charge, and importantly, their photo-
physical properties. The use of extended polypyridyl ligands
suchs as dipyrido[3,2-a:2′,3′-c]phenazine (dppz) allows strong
binding to DNA through intercalation.4 The [Ru-
(phen)2dppz]2+ (phen = 1,10-phenanthroline) acts as a light-
switch complex, which is nonluminescent in solution but
luminesces strongly when bound to DNA which can be applied
to DNA imaging. Recently, Ru(II) complexes developed by us
and others13,14 have been shown to act as effective cellular
Received: December 8, 2014
© XXXX American Chemical Society
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Figure 1. (a) Synthesis of 3 (free ligand) and corresponding Ru(II) complexes 4 and 5: (i) glyoxal, EtOH, reflux; (ii) H2O, 50 W. (b) X-ray crystal
−
structure of 5. PF6 counterions and solvent molecules are omitted for clarity (see Supporting Information for full structure), and ellipsoids are
shown at 40% probability. (c) Chemical structure of 6.
the cell (usually the production of activated oxygen species)
which results in its rapid destruction. The ideal photosensitizer
should be water-soluble, easily accumulate in a cancer cell,
possess no or very low dark toxicity, and be nonmutagenic.20
Importantly, it should be readily available and be able to induce
programmed cell death. A majority of PDT agents developed to
date have been porphyrin-based and as a result suffer from a
number of undesirable characteristics: hydrophobicity, poor
light absorption, lack of specificity, dark toxicity, and prolonged
skin sensitivity, to name but a few.21 Ru(II) polypyridyl
complexes have recently been recognized as a major class of
“new” types of PDT agents that can overcome some of the
above problems.22,23 While such systems often possess higher
energy absorptions than that seen for porphyrin-based systems,
these can often been “pushed” to longer wavelengths by ligand
design or by using two-photon excitation, the latter enabling
the addressing of the excited state properties of the complexes
at comparable or longer wavelengths than currently used in the
clinic.
Interestingly, while a number of Ru(II) based complexes
have been shown to successfully induce DNA photocleavage
following light activation in vitro,2,7,22,23 only a limited number
of such complexes have shown promise as potential PDT
agents by demonstrating photoactivation induced cytotoxicity
at a cellular level,3,12,24 while there has only been one report to
date into the biological mechanism of action of cell death
behind such Ru(II) polypyridyl complex-induced PDT within
cells.25 It is clear that in order to realize the therapeutic and
diagnostic potential of these complexes, a thorough under-
standing of the internalization properties and activity is
essential. Ideally, this requires mapping the path from uptake
to localization to activity and toxicity and finally the means and
extent of clearance, and while a number of studies have
explored individual aspects, a full picture has yet to be captured.
Thomas and co-workers have demonstrated that various Ru(II)
polypyridyl complexes can be taken up into cells by
nonendocytotic active transport. Using noncytotoxic Ru(II)
complexes, the group showed that such complexes could
accumulate within cells and directly image nuclear DNA.26
Indeed, a dinuclear Ru(II) polypyridyl compound was found to
enter living cells, accumulated in the nucleus (and other
organelles), and could directly image nuclear DNA utilizing the
MLCT “light-switch” property of the compound. Several other
researchers have shown similar effects, but often the delivery of
such complexes has required the use of structures possessing
polyamino acid conjugates as delivery vehicles.27−30
In this study we report a comprehensive profile of the
biological activity of a new Ru(II) polypryridyl anticancer
phototherapy agent, [Ru(TAP)2pdppz]2+ (pdppz = [2,3-
h]dipyrido[3,2-a:2′,3′-c]phenazine) 5. The key design features
of this complex are the combination of (a) an extended
“hooked” dipyridophenazine ligand for enhanced intercalation
and DNA binding with (b) a π-deficient ligand network for
phototriggered damage. In addition, we present results for the
control complex [Ru(phen)2pdppz]2+ 4 which also possesses
the “hooked” dipyridophenazine ligand but is not expected to
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cause significant photodamage due to the absence of the TAP
ligands. Rapid cellular uptake of these complexes followed by
localization within mitochondria is observed. This is followed
by perinuclear clustering of the mitochondria, which results in
changes in cellular appearance with the formation of a concave
or bean-shaped nucleus. 5 is found to give rise to minimal dark
toxicity and recovery of the cell upon removal and elimination
of the complex. Then, upon photoactivation, programmed
cellular death is turned on leading to rapid cellular death.
Furthermore, we demonstrate part of the mechanism of action
of this novel Ru(II) PDT agent.
similar behavior to that observed for Ru(TAP)2(dppz)23 which
can be justified if the lowest excited MLCT state is due to a
charge transfer to one of the two TAP ligands. As expected, no
MLCT based emission was observed for the control complex 4.
In the first step to profile the biological activity of 5 the DNA
binding affinity was investigated. A series of DNA titrations
were carried out in 10 mM phosphate buffer at pH 7.4. The
addition of salmon testes (st) DNA to 4 and 5 resulted in
significant changes to their absorption spectra which are
summarized in Supporting Information. These showed that in
the case of 4 a 53% hypochromism was observed for the pdppz
band (located at 308 nm), while the MLCT band experienced a
30% hypochromism. For structure 5, a hypochromism of 43%
and 30% was seen for these two transitions, respectively. These
defined hypochromicities would indicate intercalation of the
pdppz ligand between the stacked bases. The intrinsic binding
constant (K) and binding site stoichiometry (n) were
determined from these spectroscopic results using the model
of Bard et al.37 (see Supporting Information), which showed
that 4 and 5 had high affinity for DNA, with K = 1.2 × 107 M−1
( 0.3) and n = 1.45 ( 0.03) for 4, while for 5 K = 5.4 × 106
M−1 ( 0.5) and n = 1.55 ( 0.02). In the presence of 100 mM
NaCl, the resulting K values were found to be in the region of
106 M−1 (Supporting Information), indicating that binding
strength is only moderately sensitive to salt concentration. In
agreement with the ground state studies, significant changes
were also seen in the emission spectra of 4 and 5 upon binding
to DNA (see Supporting Information). Compound 4 is best
described as a “light switch” for DNA, as it showed no
significant luminescence in aqueous buffer solution (as has been
demonstrated for other phen based polypyridyl Ru complexes
containing one or more dppz structures), as upon addition of
st-DNA, intense MLCT-based photoluminescence was ob-
served, centered at 620 nm (Supporting Information).56
Complex 5 exhibits opposite behavior upon addition of st-
DNA, where the intense luminescence of 5 in aqueous buffer is
effectively quenched upon addition of DNA, behavior which is
also characteristic of the excited MLCT Ru−TAP state where
the excited state is localized on the TAP ligand. [Ru-
(TAP)2(L)]2+ complexes (L = phen or bpy)16,38 have been
shown to photo-oxidize guanine containing nucleotides.
Therefore, the emission of 5 was next investigated in the
presence of [poly(dA-dT)]2 and [poly(dG-dC)]2.57 For the
former, a marked 57% increase in the MLCT emission intensity
was observed, induced by the protection afforded upon
intercalation into the double helix from oxygen based
quenching. Conversely, in the case of [poly(dG-dC)]2, a
dramatic 98% luminescence decrease was observed most likely
due to the aforementioned photooxidation process. Analysis of
the binding of these complexes by linear dichromism (LD)
spectroscopy further supported the intercalative nature of
binding of both complexes to DNA (see Supporting
Information).
RESULTS AND DISCUSSION
■
Synthesis and Spectral Characteristics of Ru(II)
Complexes. The ligands and Ru(II) complexes employed in
the present studies are shown in Figure 1a. In the design of 4
and 5, we anticipated that further extension of the flat, planar
well-known dppz structure31 would increase DNA binding
ability, which would be concomitantly felt in modulation of
their photophysical properties. This was informed from our
recent study of the interaction of the quaternarized pdppz
ligand with DNA.31 In the case of 5 it was envisaged that
incorporation of a “TAP-like” moiety on the ligand in tandem
with variation of the ancillary ligands would confer both
increased DNA binding and photocleavage ability with possible
formation of DNA−photoadducts, as has been seen previously
with Ru(II) complexes containing TAP ligands.32,33 The
synthetic pathway of 4 and 5 is shown in Figure 1a (see
Supporting Information for experimental data). In short, the
synthesis of 3 was achieved by condensation of 5,6-
diaminoquinoxaline34 1 with 1,10-phenanthroline-5,6-
dione31,35 2 by reflux in EtOH, yielding 3 as a gray solid in
95% yield. The microwave irradiation of 3 in the presence of
the appropriate ruthenium bispolypyridyl dichloride36 for 15
min followed by precipitation from water using excess
ammonium hexafluorophosphate yielded the crude complexes
4 and 5 in 69% and 52% yield, respectively, after purification.
Synthesized as their chloride salts, all complexes are water-
soluble, and their photophysical properties were investigated in
10 mM phosphate buffered aqueous solutions at pH 7.4.
Red prism shaped crystals were obtained of the hexafluor-
ophosphate complex of 5 by slow evaporation from acetonitrile.
The X-ray crystal structure of 5 is shown in Figure 1b and
confirms the incorporation of two TAP ligands and the
extended planar pdppz ligand around the Ru(II) center. The
complex crystallized in a triclinic system with space group P1,
̅
and the unit cell contains two complexes (the δ and λ
enantiomers) and four hexafluorophosphate counteranions.
Complex 5 exhibits a distorted octahedral geometry, with Ru−
N bond distances lying in a narrow range between 2.060(3) and
2.086(3) Å. [Ru(TAP)2dppz]2+, 6, was synthesized according
to a modification of the literature procedure reported by
Kirsch-De Mesmaeker et al.,23 and its chemical structure is
shown in Figure 1c.
Having demonstrated the high affinity of 5 for DNA, we next
considered the DNA photocleavage ability of the complex. In
order to evaluate this for 4 and 5, agarose gel electrophoresis of
pBR322 plasmid DNA was undertaken (Figure 2). When
incubated in the dark, neither complex showed any DNA
cleavage. However, 5 showed extremely efficient photocleavage
after 30 min of irradiation under aerobic conditions at a DNA
phosphate to Ru(II) dye (P/D) ratio of 100. Furthermore, at
P/D = 50 the complex showed complete conversion of the
supercoiled DNA to both nicked and linear forms. As expected,
The characteristic UV−vis absorbance spectra together with
the excitation and emission spectra of 4 and 5 are shown in the
Supporting Information, showing transitions that are typical of
related compounds such as dipyrido[3,2-a:2′,3′-c]phenazine
(dppz). 5 possesses a band at 308 nm due to the pdppz ligand
and a broad structured band centered at 415 nm which is
attributed to the MLCT transitions of the Ru(II) center.
Excitation of 5 at 415 nm in aqueous pH 7.4 buffered solution
gave rise to MLCT based emission with λmax at 630 nm. This is
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intense luminescent spot (at the resolution of confocal
microscopy) was observed after 8 h associated near the nucleus
for complex 5 as demonstrated in Figure 3a. Concomitant with
this single location was the observation of a concave or bean-
shaped nucleus. After 24 h compound 5 appeared to localize
around the nucleus. An enlargement of a representative cell
treated with 5 is shown for illustrative purposes in Figure 3b. It
should be noted that the difference between the emission
properties of 4 and 5 could account for the lower visualization
of 4 in the cytoplasm at earlier time points. The fact that
cellular uptake was found to be temperature-dependent
suggests that these compounds are not membrane-permeable
and require active or facilitated uptake (Figure 3c).
Figure 2. Agarose gel electrophoresis of pBR322 DNA (1 mg/mL)
after irradiation (2 J cm−2) at λ > 390 nm in 10 mM phosphate buffer,
pH 7. 4. Lane 1: plasmid DNA control. Lane 2: [Ru(bpy)3]2+ (P/D of
100). Lanes 3−4: 5 (P/D of 100, 50). Lane 5: 5 in the dark (P/D of
100). Lanes 6 and 7: 4 (P/D of 100, 50).
The control compound 6 was similarly observed to localize
in the cytoplasm of HeLa cells; however, uptake appeared to be
slower with the compound only clearly visible within cells after
24 h. Previous studies have shown that extending the size of the
polypyridyl ligand confers lipophilic character to the complexes
and results in enhanced uptake into cells2,18 (Supporting
Information).
Uptake of Compounds into Isolated Organelles. As
these compounds were shown to bind to st-DNA but did not
appear to localize within the nucleus of whole cells, organelles
were isolated from rat liver tissue and incubated for 20 min with
complexes 4 and 5. Both compounds were shown to be taken
complex 4 did not show a marked increase in the formation of
either linear or nicked DNA strands.
Cellular Uptake of Ru(II) Polypyridyl Complexes. The
uptake and cellular localization of 4 and 5 in the dark (no
photoactivation) in a cervical cancer cell line (HeLa) were
investigated. Confocal fluorescence microscopy was used to
track the red emission arising from the complexes18 over a
period of 4, 8, and 24 h. Complexes appeared to move from the
outside of the cell toward the nucleus in a time-dependent
manner. Here discrete “packets” of luminescence in the
cytoplasm were observed at all time points with complex 4
and at short time points (4 h) for complex 5. A large single
Figure 3. Time dependent localization of 4 and 5 in HeLa cells. 0.5 × 105 HeLa cells were treated as required, washed twice, incubated with DAPI
(Blue nuclear stain), and viewed using an Olympus FV1000 point scanning microscope with a 60× oil immersion lens with an NA of 1.42. (a) Cells
were treated with 100 μM 4 or 5 for 4, 8, or 24 h. (b) Cells were treated with 100 μM 5 for 24 h; enlargement of representative cells for illustrative
purposes. (c) Cells were treated with 100 μM 5 for 4 h at 37 or 4 °C, and the percentage of cells with compound fluorescence was expressed over the
total amount of cells (approximately 50) per field of view. Data points represent the mean SEM.
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the subcellular localization of the compounds in whole cells
using confocal fluorescence microscopy and examining the
colocalization of 5 with mitochondria (Figure 5a), lysosomes
(Figure 5b), and the endoplasmic reticulum (Figure 5c). The
results of these experiments suggest that 5 appears to colocalize
to mitochondria and/or lysosomes (Figure 5a−d).
Further analysis of Ru(II) complex localization using
transmission electron microscopy (TEM) of HeLa cells
revealed that 5 accumulates in mitochondria as indicated by
the dense staining of HeLa cell mitochondria treated with 5
(Figure 6a). Previously, dinuclear Ru(II) polypyridyl complexes
prepared by the Keene group have been observed to also
accumulate in the mitochondria.3 The TEM imaging experi-
ments also confirm the clustering of loaded mitochondria near
the nucleus, also known as perinuclear mitochondrial clustering.
Compound 5 was also shown to reduce the mitochondrial
membrane potential (MMP) of HeLa cells in vitro after 30 min
but with apparent recovery after 4 h, showing the reduction in
MMP to be time-dependent and further confirming the
accumulation of 5 in mitochondria (Figure 6b,c). Compound
4 also reduced the MMP of HeLa cells (Figure 6d) but, unlike
that seen for 5 above, without recovery even after 8 h.
In order to evaluate the cytotoxic anticancer potential of 4
and 5, both were tested for cytotoxicity in five cancer cells lines:
two mesothelioma cell lines, the cervical cancer cell line, HeLa,
and two Burkitt’s lymphoma cell lines. As these complexes
could potentially exhibit “light-switch” cytotoxicity inside the
cells upon light activation, both compounds were “photo-
activated” using low intensity light (λ ≥ 400 nm, ∼18 J cm−2)
upon incubation in cells and assessed. Of the two complexes, 4
showed no light dependent cytotoxicity at high concentrations
in the various cell lines (Table 1). In contrast to these results,
compound 5 displayed light-dependent cytotoxicity in HeLa
and mesothelioma cells (CRL5915) in a concentration
dependent manner (Table 1) with minimal dark toxicity
which was observed only at higher concentrations. These
results demonstrate that the activity of the complex is achieved
by simple light activation as is clear from determining the IC50
Figure 4. Rat liver nuclei show uptake of compounds 4 and 5.
Nuclear-rich fractions isolated from rat liver tissue were treated with
100 μM 4 or 5 for 20 min before organelles were washed with PBS
and viewed by confocal microscopy. Isolated nuclei stained with (a)
DAPI and (b) Ru(II) complex 4 or 5.
up by isolated, intact nuclei (Figure 4a,b). Compound 5 was
shown to have a decreased luminescence intensity when
compared with compound 4, as was predicted based on the
observed quenching of 5 when bound to DNA, as discussed
above. The compounds stained the nucleus in a similar manner
to DAPI.
Investigation into the Subcellular Localization and
Light Activation of Compounds 4 and 5. The results listed
above confirm the ability of the compounds to bind not only to
isolated st-DNA but also to DNA in its natural environment
within a mammalian nucleus. However, when these complexes
are incubated with whole cells, the compounds fail to reach the
nucleus. In order to investigate why this occurred, we looked at
Figure 5. Subcellular location of 5. Assessment of colocalization of 5 with mitochondria (a, d), lysosomes (b, d), and endoplasmic reticulum (ER) (c,
d).
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Figure 6. Effect of Ru(II) complexes on mitochondria. TEM of HeLa cells untreated (control), or treated with 100 μM 5 at different magnifications.
Shown are the effects of 5 (b, c) and 4 (d) on TPMP accumulation, a direct measure of MMP: N, nucleus; arrowheads, mitochondria. Statistical
analysis was performed using a one-way ANOVA: ∗, p < 0.05. Data points represent the mean SEM.
structural modifications can dictate the function of the resulting
Ru(II) polypyridyl complex. The control 6 was also assessed for
light activation, and these results demonstrated comparable
photoinduced toxicity to 5 (Supporting Information).
Table 1. Effects of Ru(II) Complexes 4 and 5 on Malignant
Cell Lines with or without Light Activation
a
IC50 (μM)
4, dark
>100
4, light
>100
5, dark
5, light
Photoactivation of 5 (100 μM) in vitro was monitored using
real-time confocal microscopy (image taken every 10 min for
12 h) and resulted in cells with an apoptotic phenotype. Using
light irradiation alone, in the absence of compound 5, or using
compound 5 alone in the absence of light irradiation, there was
no effect on HeLa cells. Photoactivated cell death was also
found to be time-dependent as demonstrated in Figure 7a.
Further analysis of the mechanism of cell death elicited by this
compound using PI (propidium iodide) FACS (fluorescent
activated cell sorter) analysis, which detects the formation of
apoptotic bodies, revealed the induction of light-induced
apoptosis in a dose- and time-dependent manner, shown in
Figure 7b and Figure 7c, respectively. Light-induced cell death
was rapid and potent (70−80% cell death at 100 μM) with the
dark death effect being minimal (6−8% cell death at 10 and 100
μM). Recent investigations into the anticancer effects of Ru(II)
polypyridyl complexes document IC50 values of between 5 and
500 μM in the absence of light, implying a weak “dark” toxic
effect.39 Other Ru(II) complexes have been previously
examined as PDT agents, with Ru(II) phthalocyanine, Ru(II)
2,3-naphthalocyanines having been shown to display photo-
toxicity in the low micromolar range30,40 which is comparable
to that found in the present study. Previous literature on the
efficacy of nonporphyrin PDT agents report IC50 values of
HeLa
70 6.3
>100
8.8 2.9
CRL 5915
One58
Mutu-I
DG-75
73.3 17.7
53.2 11.5
>100
92.3 19.2
62.2 9.3
>100
42.8 2.6
38.3 5.6
17.6 1.4
42.5 0.8
>100
40.2 3.5
>100
>100
90 13.8
a
(1−5) × 103 cells/well were seeded in a 96-well plate and treated
with the respective drug for 24 h irradiation with 18 J cm−2 of light.
After 24 h, each well was then treated with 20 μL of Alamar Blue and
left to incubate at 37°C in the dark for 4−6 h. Fluorescence was read
at 590 nm (excitation 544 nm). The background fluorescence of the
media without cells + Alamar Blue was taken away from each group,
and the control untreated cells represented 100% cell viability. Data
represent the mean SEM.
values for the cytotoxicity of 5 in these cell lines when exposed
to light which were found to be between 8.8 and 43 μM,
compared to the IC50 values of between 40.2 μM and greater
than 100 μM for the cell lines tested for the cytotoxicity of 5 in
the dark. The low cytotoxicity observed for 4 suggests that this
complex may be used as a luminescent cellular probe within
these cell lines, whereas complex 5 may have more applications
as a novel PDT agent. This clearly demonstrates the enormous
scope that such complexes have within biology, where simple
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Figure 7. Ru(II) complex 5 induces programmed cell death of HeLa cells. (1−5) × 103 cells/well were seeded and treated with the respective drug
for 24 h irradiation with ∼18 J cm−2 of light followed by incubation for the indicated time-points or a further 24 h. Confocal microscopy was used
to assess cells for the appearance of apoptotic cells (a). PI staining was used to assess the effect of concentration (b), time (c), ROS inhibition (e),
and caspase inhibition (f) on Ru(II) complex 5 light-induced toxicity. Intracellular DNA strand breakage was identified via the comet assay (d), and
cleavage of the enzyme PARP was identified through Western blotting (g) following treatment with 5. Statistical analysis was performed using a one-
way ANOVA: ∗, p < 0.05. Data points represent the mean SEM.
between 63 nM and 8 μM in the light compared to between 27
μM and >100 μM in the dark.41,42
pathways of Ru(II) (TAP) complexes with biomolecules have
also been reported.43,44
In our study, cells treated with 5 and light irradiation showed
a significant degree of single stranded DNA migration and
damage compared to dark controls as demonstrated by the
single cell gel electrophoresis comet assay (Figure 7d). Cells
treated with 4 and irradiation also underwent a small amount of
DNA damage; however, this was in no way to the same extent
as was observed for 5-treated cells, as illustrated by shorter
comet tails.
As some other PDT agents elicit their cell death effects
through the activation of reactive oxygen species (ROS), we
utilized the antioxidant N-acetylcysteine (NAC) to investigate
the involvement of ROS in photoinduced programmed cell
death mediated by 5. HeLa cells preincubated with NAC (5
mM) for 1 h, treated with 5, and activated with light were not
found to undergo the same amount of cell death as those cells
without NAC as illustrated in Figure 7e. These results suggest
that ROS are involved in the photoinduced cell death and that
complex 5 may induce a “classical” PDT response in cells as
observed with other PDT agents. The involvement of ROS,
which are formed in mitochondria, again supports the
involvement of 5 with mitochondria. However, it is important
to point out that non-oxygen dependent photoreaction
The cell death induced by 5 was inhibited by pretreating the
cells for 4 h with 40 μM general caspase inhibitor z-VAD-fmk
(Figure 7f). Caspases are intracellular cysteine proteases
involved in forms of programmed cell death, and these results
further confirmed the cells were undergoing apoptosis that was
light induced. Caspases are sometimes known to cleave the
DNA repair enzyme PARP (poly(ADP-ribose) polymerase)
from a 113 kDa molecule into 89 and 24 kDa fragments during
apoptosis. However, this was not found to be the case with 5, as
no induced PARP cleavage was observed (Figure 7g).
Treatment with paclitaxel was used as a positive control for
PARP cleavage. These overall results clearly demonstrate the
importance of the presence of the TAP ligand in 5, which is
absent in 4, and the direct mechanistic effect 5 has on cellular
viability. Finally, the ability of a photosensitizer to enter a cell
and be eliminated without harming the cell is essential for any
effective PDT agent. In this study we examined the effects of 5
on HeLa cells over a 48−96 h time frame using PI FACS
analysis and confocal microscopy. Confocal microscopy
demonstrated that after 48 and 96 h, 5 is no longer visible in
the cell. PI FACS analysis also showed that 5 had no obvious
apoptotic effect after 48 h, theoretically reducing any potential
G
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side effects in vivo (Supporting Information). Collectively,
these results support the clinical advancement of complex 5 as a
potential PDT agent. This complex will be used as a platform
for the development of improved structures with higher
wavelength absorptions and improved efficacy. A higher
absorption wavelength would eliminate any absorption by
hemoglobin or other blood proteins in vivo and could also be
achieved by using two-photon microscopy instead of conven-
tional confocal (or one-photon) microscopy while still
maintaining the same emission spectrum as recently demon-
strated with similar Ru polypyridyl compounds.45−47 Also of
clinical importance, ruthenium compounds are known to bind
to high and low density lipoproteins, serum proteins, and
albumin in vivo, which have been shown to enhance drug
accumulation into the tumor tissue;48,49 interestingly, however,
a recent study by Pernot et al. on arene ruthenium porphyrin
PDT compounds demonstrated that fluence rate in PDT was
more important than the photosensitizer concentration.50
Conventional chemotherapy targets rapidly dividing cells as
opposed to tumor cells, resulting in serious side effects, and
while targeted therapies overcome this limitation, they are
expensive and only available for a limited number of cancers
with specific, well-defined mutations. In contrast, the potential
use of complex 5 as a PDT agent would overcome these
obstacles, as the very nature of PDT agents ensures that only
complexes at the site of interest, a tumor, would be
photoactivated to induce cell death in vivo. While nontumor
cells found in the tumor region would also likely experience
PDT-induced toxicity, the lack of toxicity with nonphotoacti-
vated complexes found in the rest of the body together with the
observed clearance of nonphotoactivated complexes from
healthy HeLa cells also highlights the development potential
of complex 5.
other organelles such as the endoplasmic reticulum. These
results clearly highlight the influence the microenvironment of
the cell has on the complexes and the importance of performing
such experiments in live cells. Further evidence for the
mitochondrial localization of 5 was obtained by TEM studies
of HeLa cells, which show densely stained mitochondria
appearing to localize around the nucleus. Being lipophilic and
cationic in nature, we demonstrate that the complexes enter
mitochondria, most likely driven by the MMP, with 5 being
shown to reduce the MMP within 30 min of treatment with
recovery of the MMP after 4 h. The subsequent perinuclear
clustering of compound laden mitochondria may be suggestive
of a large degree of depolarization induced mitophagy as
described by Youle et al.52
The cellular photoreactivity of 5 was investigated by
assessing the antiproliferative/cytotoxicity in the presence and
absence of light irradiation of low intensity (18 J cm−2) in a
number of malignant cell lines. The complex showed potent
light-dependent toxicity in a range of cell lines (IC50 values of
between 8.8 and 43 μM) and was shown to induce dose and
time dependent programmed cell death in HeLa cells; the
critical involvement of caspases confirmed this form of
programmed cell death to be apoptosis. This could be
occurring via an electron transfer mechanism (which is
photoinduced driven), which could potentially involve binding
of 5 to DNA within the mitochondrion itself, though further
investigations would be required to confirm this. Moreover, the
light-dependent cytotoxicity of 5, which is consistent with ROS-
mediated apoptosis, was not observed for 4, and importantly, in
the absence of light activation, complex 5 was found to be
cleared from the cells without causing any damage. In addition,
compared to commercially available porphyrins, which often
consist of a mixture of products, complex 5 is pure. 5 can also
can be easily modified and will be used to further refine the
structure−activity relationship required for even more potent
PDT agents. Studies are also currently underway to develop
analogues of 5 that can be photoactivated by longer
wavelengths of light to allow for even better tissue penetration.
It should be noted that while other investigators have described
mitochondrial uptake of Ru(II) based polypyridyl com-
pounds,18,25,39 a vast majority of these complexes are not
photoactive, highlighting the novel importance of the current
study.
CONCLUSION
■
In this study, we report the synthesis of a new photodynamic
therapeutic agent 5 and the related control pdppz light-switch
complex 4. The luminescence of 4 significantly increases when
bound to DNA, while the luminescence of 5 decreases when
bound to DNA. While the structures of 4 and 5 are similar, they
have very different mechanisms of action on st-DNA in
aqueous solution. While both compounds have been shown to
bind DNA with high affinity, gel electrophoresis measurements
reveal that only complex 5 cleaves DNA. We further
demonstrate that the presence of the TAP ligand in 5 is
instrumental to the photoactivity of the complex within cells
(this we also show to be the case for 6). From our thorough
investigation of the biological properties of these complexes, a
number of important observations have been made, including
that the complexes are found to be actively transported to the
interior of the cells and accumulate with clear visualization
(600−700 nm range) within the cells after 8 h. Furthermore,
the increased lipophilicity of the extended pdppz ligand
employed in 4 and 551 is found to increase the rate of uptake
of the complexes compared to the corresponding dppz
containing complex 6. Moreover, the uptake of the complexes
4 and 5 into isolated nuclei indicates that the complexes are
capable of displaying MLCT luminescence when bound to
DNA within the nuclei, our results confirming that 4 is more
emissive compared to 5, as seen for the binding of these
complexes to isolated st-DNA. However, in live cell imaging,
the compounds are found to mostly localize within
mitochondria and lysosomes, with lesser amounts found in
In summary, complex 5 reveals it has promise for
development as a new photodynamic therapeutic. It is pure,
hydrophilic, easily accumulates in cancer cells, has little dark
toxicity, clears the cells within 96 h, can be easily photo-
activated, appears to have high singlet oxygen production, and
can induce programmed cell death. The increased under-
standing gained by a comprehensive biological profiling of the
activity of this complex brings us one step closer to the use of
Ru(II) polypyridyl complexes to turn on cytotoxicity in
cancerous cells, and this has not been demonstrated in such a
detailed manner before.
MATERIALS AND METHODS
■
Compounds. Synthesis of Ru(II) polypyridyl complexes and ICP-
MS sample preparation are described in the Supporting Information.
All new compounds were characterized using conventional methods
(see full characterization in Supporting Information), which included
the use of 600 MHz NMR and elemental analysis (CHN), both of
which confirmed that the purity of all compounds made was over 95%
(all 1H and 13C NMR spectra are shown in the Supporting
H
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Information). Cell culture reagents were obtained from Greiner Bio-
one, and all other chemicals were obtained from Sigma unless
otherwise stated.
irradiated for 30 min and phase-contrast images were taken every 10
min for 12 h.
Propidium Iodide Staining. For the detection of apoptotic
bodies by PI FACS analysis, 250 000 cells were treated with the
appropriate amount of compound and incubated for a specified time.
Cells were harvested by centrifugation at 300g for 5 min and washed
with 5 mL of ice-cold PBS. The pellet was resuspended in 200 μL of
PBS and 2 mL of ice-cold 70% ethanol, and cells were fixed overnight
at 4 °C. After fixation, the cells were pelleted by centrifugation at 300g
for 5 min and the ethanol was carefully removed. The pellet was
resuspended in 400 μL of PBS with 25 μL of RNase A (10 mg/mL
stock) and 75 μL of propidium iodide (1 mg/mL). The tubes were
incubated in the dark at 37 °C for 30 min. Cell cycle analysis was
performed using appropriate gates counting 10 000 cells and analyzed
using CELLQUEST software package. For mechanistic studies, cells
were preincubated with 40 μM z-VAD-fmk or 5 mM N-acetylcysteine
before treatment with 5 and irradiation, and PI FACS analysis was
then performed.
Western Blotting. For the detection of PARP cleavage by Western
blot analysis 5 × 106 cells were harvested by centrifugation at 500g for
5 min and the pellet was washed with ice-cold PBS. Cells were
resuspended in 60 μL of PBS and 60 μL of lysis buffer (Laemmli
buffer; 62.5 mM Tris-HCl, 2% w/v SDS, 10% glycerol, 0.1% w/v
bromophenol blue supplemented with protease inhibitors). Samples
were prepared for SDS−PAGE resolved on an 8% loading gel and
transferred onto PVDF membranes. Membranes were probed with
anti-PARP (Calbiochem) (recognizes full length 113 kDa PARP as
well as the 85 kDa cleaved form) primary antibody followed by
incubation with the corresponding IgG HRP conjugated secondary
antibody. Membranes were developed using electrochemilumines-
cence detection.
Crystallographic Experimental Section. Diffraction data for all
compounds were collected on a Bruker APEX 2 DUO CCD
diffractometer using graphite-monochromatized Incoatec IμS Cu Kα
(λ = 1.541 78 Å) radiation. Crystals were mounted in a cryoloop/
MiTeGen micromount and collected at 100(2) K using an Oxford
Cryosystems Cobra low temperature device. Data were collected using
ω and φ scans and were corrected for Lorentz and polarization
effects.22 The structures were solved by direct methods with SHELXS
2013 and refined by full-matrix least-squares procedures on F2 using
SHELXL-2013 software.24 All non-hydrogen atoms were refined
anisotropically. Hydrogen atoms, with the exception of those of the
water molecule, were added geometrically in calculated positions and
refined using a riding model. Hydrogen bond analysis was used to
place the hydrogens of the water molecule, and their positions were
kept fixed. Details of the data collection and refinement are given in
the Supporting Information. CCDC 1012983 contains the supple-
mentary crystallographic data for this paper. These data can be
obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
Agarose Gel Electrophoresis. The DNA photocleavage studies
were carried out by treating pBR322 plasmid DNA (1 mg/mL) with
each of the complexes at varying ratios (P/D of 100, 50). The samples
were then subjected to 2 J cm−2 using a Hamamatsu L2570 200 W
HgXe arc lamp equipped with a NaNO2 filter before being separated
using 0.8% agarose gel electrophoresis in a TBE (90 mM Tris-borate, 2
mM EDTA, pH 8.0) buffer. Electrophoresis was carried out at 5 V/cm
(40 mA, 90 V). Visualization of the DNA was achieved by staining the
gel for 90 min with an aqueous solution of ethidium bromide, which
was then illuminated with a transilluminator (Bioblock 254 UV
illuminator).
Cell Culture. HeLa, ONE-58, and CRL5915 cells were grown in a
cell culture flask using low-glucose Dulbecco’s modified Eagle medium
supplemented with 10% fetal bovine serum and 50 μg/mL penicillin/
streptomycin at 37 °C in a humidified atmosphere of 5% CO2.
MUTU-I and DG-75 cells were cultured in RPMI medium with
supplements as listed above; however, MUTU-I cells also required the
addition of 1 mM HEPES, 100 mM sodium pyruvate, and 50 mM α-
thioglycerol in PBS with 20 μM bathocuprione disulfonic acid. For
photoactivation studies, cells were subjected to 18 J cm−2 using a
Hamamatsu L2570 200 W HgXe arc lamp equipped with a NaNO2
filter.
Viability Assay. For the Alamar blue cytotoxicity test involving
Ru(II) polypyridyl complexes, (1−5) × 103 cells/well were seeded in a
96-well plate and treated with the respective drug for 24 h
irradiation. After 24 h, each well was then treated with 20 μL of Alamar
blue (BioSource) (prewarmed to 37 °C) and left to incubate at 37 °C
in the dark for 4−6 h. Fluorescence was read using at 590 nm
(excitation 544 nm). The background fluorescence of the media
without cells + Alamar blue was taken away from each group, and the
control untreated cells represented 100% cell viability. Each compound
was screened over a 1 μM to 1 mM concentration range in triplicate
on two independent days with activity expressed as percentage cell
viability compared to vehicle treated controls. All data points
Colocalization Studies. 0.3 × 105 HeLa cells were seeded, left for
24 h, and transfected with an excitable (405 nm) GFP mitochondrial/
lysosomal or CFP-tagged ER marker. After 24 h, cells were treated
with 5 (100 μM) for 16 h, washed twice with fresh media, and
analyzed by live confocal microscopy. The sample was first excited
with a 488 nm laser diode, and the emission of drug was monitored
and captured at 600−700 nm. The sample was then excited with a 405
nm laser diode (GFP) or a green helium−neon laser (CFP), and the
emission of the excitable marker was monitored and captured at 495−
550 nm (GFP) or 470−500 nm (CFP). Both images were then
overlaid and analyzed using the Imaris 3D software analyzer
(Bitplane).
Membrane Potential. HeLa cells were incubated for the required
times with Ru(II) complexes followed by the addition of a final
concentration of 5 nM TPMP, 100 nCi/mL [3H]TPMP, and 5 nM
sodium tetraphenylboron (TPB) for 90 min with or without 1 μM
FCCP. After incubation, the cells were pelleted by centrifugation, 100
μL of the supernatant was removed and the cell pellet resuspended in
100 μL of 20% Triton X-100. The radioactivity in the pellet and
supernatant was quantitated using a liquid scintillation counter with
appropriate quench corrections. Accumulation ratio = [cpm/mg
(pellet)]/[cpm/μL (supernatant)]. MMP = accumulation ratio
without FCCP − accumulation ratio with FCCP.
Transmission Electron Microscopy. TEM was carried out as
previously described.53 In brief, 1 × 106 cells were treated with 100 μM
5 for 24 h, fixed with 4% glutaraldehyde for 1 h, washed in 0.5 M
phosphate, solidified in 2% warm agarose solution at 4 °C for 30 min,
and cut into small slices. Slices were further fixed in 2% osmium
tetroxide (O2O4) solution in 0.05 M potassium phosphate buffer and
dehydrated using an increasing alcohol series. Pellets were embedded
in a 50% resin solution for 2−3 h and a 100% epoxy. Ultrathin sections
were cut on an ultramicrotome and collected on copper grids and
counterstained with uranyl acetate and lead citrate. Ultrastructural
examination was carried out in a JEOL 1210 electron microscope.
Images were taken with a 1500−3000× objective (2 μM scale bars). A
number of images were obtained as a representative of each sample.
Comet Assay. HeLa cells were treated with 10 μM 4 or 20 μM 5
for 24 h irradiation and incubated for a further 6 h. Following that,
cells were trypinized and resuspended in low melting point agarose
(expressed as the mean
SEM) were analyzed using GraphPad
Prism (version 4) software (GraphPad software Inc., San Diego, CA).
Confocal Microscopy. HeLa cells were seeded at a density of 0.75
× 105 cells/2 mL, left for 24 h before treating with 4 or 5 for the
indicated length of time. Cells were washed ×2 with new media to
remove excess drug and analyzed by live confocal microscopy using an
Olympus FV1000 point scanning microscope with a 60× oil
immersion lens with an NA (numerical aperture) of 1.42. The
software used to collect images was FluoView, version 7.1 software.
For temperature dependent uptake studies, cells were placed at 4 °C
for 30 min before treatment with 5 for 4 h at 4 °C. Uptake was
assessed at the fluorescence per cell at 600−700 nm, carried out on
300 cells on 3 independent days. For the real-time confocal
microscopy experiments with photoirradiation, treated cells were
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(LMPA) and added to slides precoated with normal melting point
agarose (NPA). Slides were then lysed (2.5 M NaCl, 100 mM EDTA,
10 mM Tris, 1% (v/v) Triton X-100, pH 10) for 2 h at −20 °C and
transferred to an alkaline buffer (300 mM NaOH, 1 mM EDTA, pH
>13) for 20 min to allow for unwinding of DNA and expression of
alkali-labile damage. Slides were then subjected to electrophoresis at
24 V, 300 mA for 30 min. Samples were then neutralized in 0.4 M Tris,
pH 7.5, for 20 min and stained with PI. Slides were viewed using an
Olympus IX81 microscope with a 20× lens. The software Cell∧P was
used to collect images.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Additional experimental procedures, synthesis and character-
ization, NMR spectra, mass spectra, crystal data and structure
refinement results, UV−vis and fluorescence emission spectra,
calculation of binding constants, agarose gel electrophoresis
results, and a cif file of crystallographic information. The
Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.5b00451.
AUTHOR INFORMATION
■
Corresponding Authors
*T.G.: e-mail, gunnlaut@tcd.ie; fax, +353 1 6712826, phone,
+353 1 8963459.
*D.C.W.: e-mail, clive.williams@tcd.ie; fax, +353 1 8963130;
phone, +353 1 8963964.
Present Addresses
ΔS.M.C.: Division of Pulmonary and Critical Care Medicine,
Department of Medicine, Weill Cornell Medical College, A-337
Laboratory, 1300 York Avenue, New York, NY 10065.
ΨR.B.P.E.: Department of Chemistry, Maynooth University,
National University of Ireland, Maynooth, Ireland.
Author Contributions
∥S.M.C. and R.B.P.E. contributed equally.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Science Foundation Ireland (Grants SFI RFP 2009,
SFI 2010 PI, and 2013 PI), HEA PRTLI Cycle 4, The Irish
Research Council for Science, Engineering and Technology
(IRCSET Postgraduate Studentship to R.B.P.E.), Master and
Back Altaformazione Programma, 2009, Autonomous Region of
Sardinia, and TCD for financial support.
ABBREVIATIONS USED
■
dppz, dipyrido[3,2-a:2′,3′-c]phenazine; FACS, fluorescent
activated cell sorter; LD, linear dichroism; MCLT, metal
charge to ligand transfer; MMP, mitochondrial membrane
potential; NAC, N-acetylcysteine; TAP, tetraazaphenanthrene;
TEM, transmission electron microscopy; pdppz, [2,3-h]-
dipyrido[3,2-a:2′,3′-c]phenazine; PDT, photodynamic therapy;
phen, 1,10-phenanthroline; PI, propidium iodide; ROS, reactive
oxygen species; st-DNA, salmon testes DNA
J
DOI: 10.1021/acs.jmedchem.5b00451
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K
DOI: 10.1021/acs.jmedchem.5b00451
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(56) The initial addition of st-DNA (P/D of 0−4) resulted in an
immediate fluorescence increase; however, upon further addition of st-
DNA (P/D of 0−20) the emission spectrum of 4 exhibited a decrease
in photoluminescence with no changes being observed thereafter. We
attribute this behavior to biphasic interactions in which these bulky
complexes are initially efficiently packed along the DNA helix,
providing increased shelter from solvent quenching resulting in
increased fluorescence compared to the isolated complexes along the
helix. This is similar behavior that has been seen for related
systems.54,55 Similar behavior is observed with DNA homopolymers;
however, the initial fluorescence intensity is increased upon interaction
with [poly(dA-dT)]2.
(57) Further titrations using the homopolymers [poly(dA-dT)]2 and
[poly(dG-dC)]2 revealed that significant discrimination was observed
for both complexes, whereby 4 showed superior binding affinity with K
= 1.8 × 107 M−1 ( 0.4) and n = 1.71 ( 0.02) for [poly(dA-dT)]2 and
K = 8.0 × 106 M−1 ( 0.4) and n = 1.54 ( 0.08) for [poly(dG-dC)]2.
Similarly, 5 bound [poly(dA-dT)]2 with K = 1.1 × 107 M−1 ( 0.17)
and n = 1.85 ( 0.02), while the binding of 5 to [poly(dG-dC)]2 gave
K = 3.77 × 106 M−1 ( 0.2) and n = 1.33 ( 0.01). A full table of
binding constants and binding site stoichiometries is available in
Supporting Information.
L
DOI: 10.1021/acs.jmedchem.5b00451
J. Med. Chem. XXXX, XXX, XXX−XXX