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Enhanced cellular uptake and photochemotherapeutic potential of a lipophilic strained Ru(ii) polypyridyl complex.
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Cite this: RSC Adv., 2019, 9, 17254
Enhanced cellular uptake and
photochemotherapeutic potential of a lipophilic
strained Ru(II) polypyridyl complex†
Stephanie Mehanna,ab Najwa Mansour,ab Hassib Audi,a Kikki Bodman-Smith,b
Mohamad A. Mroueh,c Robin I. Taleb, a Costantine F. Daher a
and Rony S. Khnayzer *a
The use of ruthenium complexes as chemotherapeutic agents has been recently explored as one of the
alternatives to conventional treatments. In the present study, two Ru(II) polypyridyl complexes were
synthesized and characterized: a strained [Ru(bipy)2(BC)]Cl2 (complex 1) where [bipy ¼ 2,20 -bipyridine
and BC ¼ bathocuproine] along with the unstrained control [Ru(bipy)2(phen)]Cl2 (complex 2) where
[phen ¼ 1,10-phenanthroline]. The photophysical and photochemical analyses proved that unlike the
photostable complex 2, complex 1 ejected both bipy and BC ligands at a ratio of 3 : 1 respectively.
Results showed that the activity of complex 1 was significantly enhanced upon photoactivation. The
response was however particularly significant in B16-F10 melanoma cells where phototoxicity index (PI
¼ IC50 dark/IC50 light) was >900. When compared to cisplatin, the photoproducts were more potent
against all tested cell lines, implying that the complex acquired significant chemotherapeutic potential
upon irradiation. Cellular uptake of complex 1 and the free BC ligand were found to be significantly
facilitated as evidenced by 400–600 fold increase in concentration of the compounds inside the cells
relative to the extracellular culture medium. Complex 2 exhibited 35 times lower cellular concentration
Received 8th April 2019
Accepted 27th May 2019
relative to complex 1. Flow cytometry and plasmid DNA gel electrophoresis measurements showed that
complex 1 interacts with DNA inducing apoptosis in the dark and either late-apoptosis or necrosis upon
DOI: 10.1039/c9ra02615k
irradiation. These findings corroborate the importance of lipophilic ligands such as BC to enhance
rsc.li/rsc-advances
uptake and subsequently improve the photochemotherapy potential of Ru(II) polypyridyl complexes.
Introduction
The accidental discovery of cisplatin as a chemotherapeutic
drug paved the way to the extensive research on metal-based
anticancer agents. Metal complexes have distinct properties
and diverse mechanisms of action that might differ from other
chemotherapies.1 Cisplatin, oxaliplatin and carboplatin are
platinum complexes used clinically for the treatment of many
types of cancer such as head, neck, lung, ovarian and bladder
cancers.2 However, their clinical application has been limited by
adverse side effects, toxicity and development of resistance by
cancer cells.3 To overcome these constraints, research has been
a
Department of Natural Sciences, Lebanese American University, Chouran, Beirut
1102-2801, Lebanon. E-mail: rony.khnayzer@lau.edu.lb
b
Faculty of Health and Medical Sciences, Department of Microbial and Cellular
Sciences, University of Surrey, UK
c
School of Pharmacy, Department of Pharmaceutical Sciences, Lebanese American
University, Lebanon
† Electronic supplementary
10.1039/c9ra02615k
information
17254 | RSC Adv., 2019, 9, 17254–17265
(ESI)
available.
See
DOI:
directed towards platinum derivatives as well as other metalbased chemotherapeutic agents.4
More recently, ruthenium has gained a lot of interest mainly
due to distinct features that could potentially convey selectivity
towards cancer cells.5 The advantage of these drugs could mostly
be attributed to their potential selectivity to cancer cells through
intrinsic or extrinsic mechanisms, such as photochemotherapy.
Advantageous properties of ruthenium-based drugs include (i) the
ability of some complexes to achieve different oxidation states in
biological media,5 (ii) the possibility to design Ru(III) prodrugs that
can be selectively reduced to the active Ru(II) drugs by the hypoxic
environment of cancer cells,6 (iii) the possible exchange between Oand N- ligands which can allow binding to biological targets like
nucleic acids in a way similar to cisplatin,5 and (iv) intracellular
transport by transferring receptors that are overexpressed in cancer
cells.7
Selectivity of Ru(II) complexes has been further exploited by the
use of light-mediated activation of Ru(II) prodrugs through mechanisms including photodynamic therapy (PDT) and photoactivated chemotherapy (PACT).8 Unlike PDT, the mechanism of
PACT is oxygen-independent which is an advantage since the
environment of cancer cells is known to be hypoxic.9 Ru(II)
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polypyridyl complexes are particularly attractive for PACT because
of their tunable photophysical and photochemical properties.10
While most of these complexes are photostable, some can undergo
ligand photosubstitution reactions that result from distortions in
the octahedral geometry.11 Photodissociation typically occurs
following the population of the 3d–d state which is thermal
accessed from the 3MLCT excited state.12 PACT exploits several
mechanisms to induce cytotoxicity depending on the photoactivation strategy employed.8 Ejection of the straining ligand
upon irradiation oen leads to the formation of aqua complexes
that can interact with DNA similarly to cisplatin.8 We have recently
reported the quantitative ejection of the non-straining ligand bipy
in [Ru(bipy)2dpphen]2+ [where bipy ¼ 2,20 -bipyridine and dpphen
¼ 2,9-diphenyl-1,10-phenanthroline] and the formation of aquated
photoproduct.13 Other groups evaluated the photochemistry and
photobiology of similar Ru(II) bipyridine complexes in water where
aqua complexes like [Ru(bipy)2(H2O)2]2+ and [Ru(bipy)2(H2O)(OH)]+ were produced upon dissociation of the straining ligand.
Photoproducts were proved to damage DNA by variable mechanisms including DNA binding, intercalation, or cleavage.12,14–16
Other ligand frameworks with similar binding potential to DNA
have also been investigated.17–19 The cytotoxic species in Ru-based
PACT is not necessarily the aqua complex formed. In this respect,
our group demonstrated that the dissociating ligand 2,9-dimethyl1,10-phenanthroline (dmphen) in [Ru(bipy)2(dmphen)]Cl2 exhibited higher toxicity on ML-2 acute myeloid leukemia cells as
compared to [Ru(bipy)2(H2O)2]2+ and cisplatin.20 Likewise, Bonnet
and coworkers conrmed that the phototoxicity of [Ru(bipy)2(dmbipy)]2+ was primarily attributed to the ejected ligand 6,60 dimethyl-2,20 -bipyridine (dmbipy) rather than the ruthenium bisaqua complex which was proved to be less potent.21 Turro et al.
also exploited the ability of a Ru(II) complex to act as a caging
molecule for the active ligand 5-cyanouracil (5CNU) which
inhibited pyrimidine catabolism when photoejected from the
Ru(II) center.22 Importantly, we have recently demonstrated that
4,7-diphenyl-1,10-phenanthroline ligands signicantly enhance
the uptake and biological activity of Ru(II) complexes.23
In the current study, a sterically strained Ru(II) bipyridyl
complex [Ru(bipy)2(BC)]Cl2 (complex 1) [where BC ¼ bathocuproine] has been synthesized along with the unstrained
control [Ru(bipy)2(phen)]Cl2 (complex 2), Fig. 1. The aim of
introducing the BC ligand was to create steric strain around the
Fig. 1
Structures of complexes 1 and 2 (bipy ¼ 2,20 -bipyridine).
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ruthenium center due to the methyl groups at the 2,9-positions
and increase lipophilicity of the drug by introducing phenyl
groups at the 4,7-positions of the 1,10-phenanthroline ligand.
Here, the photochemistry of complex 1 was studied and
photolysis products were analyzed.
The in vitro cytotoxicity in the dark and upon light-activation
was then evaluated on a panel of cell lines and compared to the
control (complex 2). The cellular uptake of complexes 1, 2 and
the BC ligand were assessed using LC-MS/MS and rationalized
based on their lipophilicity. Finally, the apoptotic potential of
complex 1 was investigated using Annexin V/PI staining ow
cytometry and plasmid DNA gel electrophoresis experiments.
Experimental
Instrumentation
The instrumental parameters used for 1H NMR, 13C NMR,
MALDI-TOF MS, LC-MS/MS and irradiation were previously reported.13 Elemental analysis and HR-ESI MS were performed at
Atlantic Microlab and Michigan State University respectively.
Cell death was assessed by Annexin V/PI staining, using a C6
ow cytometer (BD Accuri, Ann Arbor, MI) and data were
analyzed on FL1-H versus FL2-H scatter plot.
Materials
cis-Bis(2,20 -bipyridine)dichlororuthenium(II)
hydrate
[Ru(bipy)2Cl2$2H2O], bathocuproine (BC), 1,10-phenanthroline
(phen), dowex 22 chloride, stationary phase material (Sephadex
LH 20), DMEM medium, penicillin G sodium salt, FBS, and all
other chemicals and solvents were purchased from Aldrich.
Formic acid and LC-MS grade water were from Fisher Chemical.
WST-1 reagent was from Roche©. Annexin V-Fluorescein Isothiocyanate (Annexin V-FITC) and Propidium Iodide (PI)
apoptosis/necrosis detection kit was from Abcam (Cambridge,
MA) and all cell lines were received from ATCC.
Synthesis of [Ru(bipy)2(BC)](PF6)2 (complex 1)
Synthesis was performed following a slightly modied published method.24 Briey, Ru(bipy)2Cl2$2H2O (100 mg, 0.19
mmol) and BC (74.25 mg, 0.21 mmol, MW 360.45 g mol1) were
mixed in 8 mL ethylene glycol and the solution was degassed for
1 h under argon and reuxed in a pressure vessel (260 C for 6
h). The product was then cooled at room temperature and
ltered through micropores (PVDF sterile syringe lters, 33
mm, 0.45 mm, Millipore® Millex®). The hexauorophosphate
(PF6) salt was precipitated by adding a saturated aqueous
solution of KPF6 dropwise and purication was achieved by
column chromatography on sephadex LH 20 column (methanol). Yield: 179.7 mg, 89%. 1H NMR (CD3CN, 500 MHz) d ¼
8.54 (d, J ¼ 10 Hz, 2H), 8.47 (d, J ¼ 10 Hz, 2H), 8.07 (dd, J ¼
12.5 Hz, 10 Hz, 2H), 8.04 (s, 2H), 7.99 (dd, J ¼ 12.5 Hz, 10 Hz,
2H), 7.76 (dd, J ¼ 5 Hz, J ¼ 1 Hz, 2H), 7.73 (dd, J ¼ 5 Hz, J ¼ 1 Hz,
2H), 7.63–7.58 (m, 12H), 7.36 (dd, J ¼ 10 Hz, J ¼ 5 Hz, 2H), 7.28
(dd, J ¼ 10 Hz, J ¼ 5 Hz, 2H), 1.97 (s, 6H) (Fig. S1†). 13C NMR
(CD3CN, 500 MHz) d ¼ 167.36, 158.62, 158.34, 153.86, 152.76,
150.40, 150.23, 138.77, 138.60, 136.73, 130.66, 130.55, 130.03,
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128.78, 128.42, 128.32, 128.24, 125.78, 125.46, 125.37, 26.39
(Fig. S2†). Elemental anal calcd for C46H36F12N6P2Ru: C, 51.94;
H, 3.41; N, 7.9. Found: C, 51.69; H, 3.57; N, 7.73. HRMS (ESI/
QTOF) for C46H34N6Ru [M]2+: m/z calcd 387.1029; found:
387.1034 (Fig. S3†). MS (MALDI/TOF) for C46H36F12N6P2Ru [M]+:
773.1 (Fig. S4†). UV/vis (ACN): lmax(3 M1 cm1) 454 nm
(22 000). Conversion of the complex to chloride salt was performed as previously reported, using dowex beads.13
Synthesis of [Ru(bipy)2(phen)]Cl2 (complex 2)
Synthesis was performed according to a published procedure.25
Study of the photochemistry of complexes 1 and 2 by 1H NMR
and LC-MS/MS
The photochemistry of complex 1 (PF6 salt) was evaluated by
1
H NMR in deuterated acetonitrile (CD3CN) and LC-MS/MS
was performed on the chloride form of the complex to
establish whether the photochemical transformation is
similar in water. Complex 2 was assessed by LC-MS/MS and
all analyses were conducted with and without irradiation. For
1
H NMR, photoactivation was performed at an output of 100
mW cm2 for 6 h (high concentration) as compared to 35 min
for LC-MS/MS (low concentration). BC and bipy ligands were
also independently inserted into the MS to enable their
identication in the spectra. The dissociation ratio of BC and
bipy was determined by 1H NMR analysis of the photolyzed
complex as well as LC-MS/MS using a 1 : 1 bipy : BC solution
as a standard.
Comparison of the stability of complex 1 between dark and
light conditions by UV/vis spectroscopy
The chloride salt of complex 1, [Ru(bipy)2(BC)]Cl2] was dissolved in water and irradiated with blue LED light (460 nm,
100 mW cm2) or incubated in the dark for different durations. UV/vis absorption spectra were then recorded at each
time point using the same parameters for all conditions
tested.
Lipophilicity
log P values were measured for complexes 1 and 2 according to
a modied procedure.26 Briey, 0.5–2 mg of the chloride form of
the complex were dissolved in 1 mL water saturated with octanol. 1 mL octanol saturated with water was then added and the
mixture was shaken at 1400 rpm for 1 h at room temperature.
Complete separation of the aqueous and organic phases was
obtained by centrifugation at 4300 rpm for 10 min. The lower
aqueous phase was then aspirated using a glass syringe while
degassing through the octanol phase to prevent any traces of
octanol from contaminating the needle. The two phases were
sequentially aspirated, and UV-vis absorption spectra were
recorded. log P values were computed using the following
formula:
log P ¼ log (Coctanol/Cwater)
17256 | RSC Adv., 2019, 9, 17254–17265
where C is concentration as determined by photometric
measurements.
Quantication of cellular uptake by LC-MS/MS
Preparation of calibration standards. Cellular uptake of
complexes 1 and 2 as well as the free ligand BC was measured
by LC-MS/MS based on external standards. Briey, 3, 6, 9, 12,
15, and 37.5 mM working stock solutions were prepared from
primary stock solutions and diluted with cell extract (A549
cells extracted with acetonitrile) to obtain 0.02, 0,04, 0.06,
0.08, 0.1, and 0.25 mM solutions. These samples were used for
the calibration curves which were obtained by plotting the
peak areas (measured by LC-MS/MS) against concentrations.
The LC-MS/MS conditions are listed in Table 1.
Cellular uptake
Human alveolar adenocarcinoma cells (A549) were seeded in
6-well plates at a nal concentration of 1.2 105 cells per
well and allowed to adhere overnight at 37 C/5% CO2. 3 mM
of complex 1, complex 2, or BC were then added, and the
medium was decanted immediately, or else plates were le in
the dark for 1, 3, 6, 12, or 24 h at 37 C/5% CO2. Aer
removing the supernatant, cells were washed with PBS containing 1.5% DMSO (3 times) and harvested by scraping in
the presence of 1.5 mL acetonitrile (used as the extraction
solvent). Cells were then completely lysed using an ultrasonic
homogenizer (model 150VT; continuous mode at medium
power for 1 min) and compounds were extracted in the
supernatant by centrifugation at 13 000 rpm for 10 min.
Concentrations were obtained by LC-MS/MS based on the
calibration curves designed above and results were reported
as mean SEM (n ¼ 4).
Isolation of mesenchymal stem cells (MSCs) from rat bone
marrow
Animals. A single, 12 weeks old rat was provided by the
animal facility at the Lebanese American University. The animal
was maintained under optimal laboratory conditions and
received food and water ad libitum. All experiments were
approved by the University's Animal Care and Use Committee
(ACUC) and complied with the Guide for the Care and Use of
Laboratory Animals.27–30
Harvesting and culture of MSCs. MSCs were isolated from rat
bone marrow according to a modied procedure.31 Briey, the
rat was sacriced by CO2 asphyxiation and both hind legs were
aseptically removed. Femoral and tibial bones were then isolated and washed with EtOH and 1% PBS. The bone marrows
were ushed out using a needle lled with DMEM containing
10% FBS and 1.5% pen-strep. The cells collected were then
cultured in a 75 cm2 ask and incubated at 37 C with 5% CO2.
MSCs were isolated following the classical adhesion method.32
In brief, aer 48 h incubation, cells were washed with 1% PBS to
remove non-adherent cells and the medium was changed
frequently until cells reached high conuency (around 90%).
MSCs were identied by their spindle-shaped morphology as
observed under inverted microscope (Nikon Eclipse TE300).31
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Table 1
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LC-MS/MS conditions used for the analysis of [Ru(bipy)2(BC)]Cl2 (complex 1), [Ru(bipy)2(Phen)]Cl2 (complex 2), and the ligand BC
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[Ru(bipy)2(BC)]Cl2 (complex 1)
Mobile phase
Precursor ion (m/z)
Product ions (m/z)
Collision energy (V)
Retention time (min)
Mass mode
Scan type
[Ru(bipy)2(Phen)]Cl2 (complex 2)
BC
Isocratic 5% A: H2O (0.1% formic acid), 95% B: MeOH (0.1% formic acid), ow rate (0.5 mL min1)
386.86
296.78
361.16
308.7, 361.16
181.1, 218.67
345.23, 359.16
27
27
52
0.40
0.38
0.50
ESI positive mode
ESI positive mode
ESI positive mode
Single reaction monitoring (SRM)
SRM
SRM
Cell viability assay
Statistical analysis
Cell survival assays were performed on human alveolar
adenocarcinoma A549, murine melanoma B16-F10, human
colon adenocarcinoma Caco-2, human colorectal adenocarcinoma HT-29 and triple negative human breast adenocarcinoma MDA-MB-231 cell lines treated with complexes 1 and
2, cisplatin, bipy, and BC, as previously described.13 Briey,
DMEM containing 10% FBS and 1.5% pen-strep at 37 C with
5% CO2 was used to maintain cells. The latter were seeded
and allowed to adhere at a concentration of 104 cells per well
in a 96 well-plate. Drugs were added to the media at different
concentrations using 3-fold serial dilution. Cells were incubated with drugs for 12 h followed by 35 min exposure to light
(LED Engin, 460 nm peak wavelength and 100 mW cm2
power output) or else the plates were le in the dark. Cell
survival was measured using WST-1 kit (Roche©) aer 72 h
incubation. Three independent experiments were performed,
each comprised of triplicate measurements. To test the
activity of complex 1 on normal cells, the isolated MSCs were
treated with the complex using the same conditions
described for cancer cells.
The results were analyzed for statistical signicance using oneway analysis of variance (ANOVA). Values of the different tested
parameters within each group are presented as mean SEM. All
data were analyzed with the statistical package SPSS 18, and
differences between groups were considered statistically
signicant if p-value < 0.05. The IC50 values were calculated
using the nonlinear regression curve with the use of Graph Pad
Prism version 5.0 soware for Windows.
Analysis of cell death by ow cytometry
The type of cell death was determined by ow cytometry using
a modied protocol.33,34 Briey, B16-F10 cells were treated with
complex 1 (10 mM) and irradiated as described above or le in
dark conditions. Aer 24 h incubation at 37 C/5% CO2, cells
were harvested and treated for 5 min with PI (5 mg mL1) and
FITC-conjugated annexin V antibody (5 mg mL1) at 37 C.
Annexin V/PI data were then analyzed by ow cytometry on FL1H versus FL2-H scatter plot.
Results and discussion
We report here the synthesis, characterization, and photochemotherapeutic activity of [Ru(bipy)2BC]2+. The incorporation
of methyl groups on the 2,9-positions of the 1,10-phenanthroline ligand near the Ru(II) coordination sphere imparts a steric
strain on the Ru(II) complex. The latter likely distorted the
octahedral geometry of the complex, making it photochemically
labile and thus prone to photolysis.
When the complex absorbed visible light (460 nm), ligand
ejection occurred. This can be attributed to the population of
the dissociative triplet metal-centered state (3*dd) from the
triplet metal-to-ligand charge transfer band (3*MLCT). The
MLCT absorption of complex 1 was rst assessed by UV/vis. The
band was centered around 454 nm whereas the average
extinction coefficient was 22 000 2460 M1 cm1 in acetonitrile ([Ru(bipy)2BC](PF6)2), Fig. 2. The maximum peak in the UV
range (287 nm) can be assigned to intra-ligand p / p*
Assessment of DNA damage by gel electrophoresis
The plasmid (pUC8) was added to 10 mM potassium phosphate
buffer (pH 7.4) in a 96-well plate followed by treatment with
complex 1 at 10 or 200 mM concentrations. Plates were then
irradiated at 460 nm (100 mW cm2) or le in the dark. Aer
12 h incubation, DNA suspensions were mixed with loading dye
and electrophoresis was performed in the presence of a negative
control (untreated pUC8) on a 2% agarose gel containing 0.1 mg
mL1 ethidium bromide (AppliChem, USA) and TAE buffer (Tris
base, acetic acid and EDTA). The gel was then visualized using
Bio-Rad ChemiDoc™ gel imaging system.
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Fig. 2 UV-vis absorption spectra of complex 1 as hexafluorophosphate salt (in acetonitrile) and chloride salt (in water).
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transition. An LED system with a peak wavelength of 460 nm
(light output ca. 100 mW cm2) was therefore utilized as the
excitation source for all photochemical and photobiological
studies.
UV-vis performed on complex 1 in the dark indicated that the
compound is thermally stable at room temperature. However,
upon exposure to blue light (100 mW cm2, 460 nm), complex 1
undergoes a photochemical ligand displacement reaction that
leads to the depletion of the starting material with MLCT
absorbance maximum at 455 nm and the formation of ruthenium aqua complexes with a red-shied absorption maximum
around 490 nm. The half-life of complex 1 under those irradiation conditions was 2 minutes, Fig. 3.
1
H NMR spectra were obtained to determine the photolysis
products of complex 1 and compare it to the spectra of the free
ligands bipy and BC. The peaks assigned to the starting material
(before irradiation) were absent in the photolysis products thus
proving that [Ru(bipy)2BC](PF6)2 underwent a complete
conversion in CD3CN upon irradiation. 1H NMR spectra were
examined before and aer irradiation to specify which of the
ligands dissociate from the metal center, Fig. 4. The spectrum of
the photolyzed solution included chemical shis assigned to
the free bipy and BC ligands which indicated that both ligands
were prone to ejection from the complex upon light activation.
Typically, the straining ligand is more likely to dissociate from
the complex due to the weaker ligand eld axis.24
However, previous studies have described a similar dissociative potential of bipy upon irradiation of the strained
Absorption spectra of complex 1 in water as a function of time
(a) in the dark (b) upon exposure to blue light (100 mW cm2, 460 nm);
inset: absorbance values at 455 nm as a function of time. Arrows
indicate the spectral changes trend throughout light exposure.
Fig. 3
17258 | RSC Adv., 2019, 9, 17254–17265
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Fig. 4 1H NMR spectra of bpy (a), BC (b), complex 1 after photolysis (c)
and complex 1 before photolysis (d). *Denotes peaks attributed to bipy,
and †represents peaks of BC.
complex [Ru(bipy)2(dpphen)]2+ [where dpphen ¼ 2,9-diphenyl1,10-phenanthroline]. Ejection of the bipy ligand could be
favored due to the free rotation around C2–C20 axis in addition to
the asymmetrical distortions of the octahedral complex.13,24
Peaks labeled as a and b (Fig. 4) refer to the methyl groups on
the [Ru(bipy)BC(CD3CN)x](PF6)2 (where x ¼ 1 or 2) and BC
respectively and are generated by different ligand substitution
pathways, thus allowing relative signal quantication. Upon
ejection of bipy, complex 1 undergoes photosubstitution in dacetonitrile to form [Ru(bipy)BC(CD3CN)x](PF6)2 (where x ¼ 1
or 2) and bipy. In this case, the two methyl groups of BC are not
equivalent and therefore produced signals with equal integration at different chemical shis (Fig. 5a). The ejection of BC led
to the formation [Ru(bipy)2(CD3CN)x](PF6)2 (where x ¼ 1 or 2)
and free BC ligand with a characteristic single peak in the
aliphatic region attributed to the two equivalent methyl groups
(letter b, Fig. 5). The 1H NMR aliphatic peaks pertaining to the
1
H NMR spectrum (aliphatic region) of photolyzed complex 1.
“a” represent the protons on the methyl groups of BC in [Ru(bpy)
BC(CH3CN)x](PF6)2 (where x ¼ 1 or 2), “b” denotes proton signals
attributed to the methyl groups of BC and “c” refers to solvent residual.
Fig. 5
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methyl substituents of the BC were integrated and relativelyquantied in the photolyzed complex, Fig. 5. The dissociation
ratio was found to be 3 : 1 bipy to BC.
LC-MS studies were conducted to probe the photochemical
products of [Ru(bipy)2BC]Cl2 in water. The ESI-MS spectrum of
the complex before photactivation (Fig. 6a) displayed only one
peak at m/z [M 2Cl]2+ ¼ 387.20 which corresponds to
[Ru(bipy)2BC]2+, whereas bipy, BC and Ru aqua photoproducts
([Ru(bipy)2(H2O)2]2+ and [Ru(bipy)BC(H2O)]2+) (Table 2) were
detected aer irradiation (Fig. 6b). Complex 1 is likely to
undergo aquation upon photochemical ligand dissociation in
water. However, the number of aqua or hydroxo ligands per Ru
center cannot be conrmed by our results because the electrospray ionization used in the LC-MS may alter the aquation
process. Upon photoactivation of [Ru(bipy)2(dmbipy)]2+ in
water, various products were reported such as [Ru(bipy)2(H2O)2]2+, [Ru(bipy)2(H2O)(OH)]+, and the ligand decient
[Ru(bipy)2]2+.12,14,21 Furthermore, we have detected [Ru(bipy)(dpphen)(OH)]2+ as a photoproduct of [Ru(bipy)2(dpphen)]
Cl2 in aqueous medium.13
To determine the bipy : BC dissociation ratio in water, a 1 : 1
mixture of the two ligands was prepared as a standard. The
bipy : BC ratio found by LC-MS was 3.5 : 1 as deduced from
the results of the averaged relative intensity of the peaks. LC-MS
data thus correlated with 1H NMR data and both suggested that
dissociation was favoured towards the substitution of bipy by
solvent molecules. 1H NMR (Fig. S1†), 13C NMR (Fig. S2†) and
LC-MS/MS (Fig. 6a) spectra revealed that complex 1 is kinetically
inert in dark conditions and thus can be stored for days without
decomposition when protected from light. As previously
demonstrated,13 complex 2 did not undergo any detectable
ligand substitution upon irradiation with the blue LED setup
used here.
The cellular uptake of complex 1 was measured and values
were used to determine the optimal incubation period with the
drug prior to photoactivation. Aer 6 to 12 hours of incubation
with A549 cells, complex 1 reached a maximum concentration
of nearly 2.2 nmol/106 cells (Fig. S9a†). Assuming that cells have
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Table 2 Analysis of ESI-MS data (Fig. 6b). Molecules (ligands and Ru
complexes) were assigned to the peaks by comparing the m/z found
to the theoretical one calculated using ChemDraw Pro 15.0
Molecule
m/z found
m/z calculated
Bipy
[Ru(bipy)2(H2O)(OH)]2+
[Ru(bipy)BC(OH)]2+
BC
Complex 1: [Ru(bipy)2BC]2+
157.13
224.60
317.67
361.20
387.20
[M + H+]+ 157.07
[M]2+ 224.53
[M]2+ 317.57
[M + H+]+ 361.17
[M]2+ 387.10
an average volume of 1.7 pL35 and that the complex is homogeneously distributed within the cell, the concentration of
complex 1 is around 1400 mM per cell (Fig. 7a), which is about
430 times the initial concentration (3 mM). Complex 2 also
reached an elevated cellular concentration of around 40 mM per
cell aer 12 h (Fig. 7b), yet was still approximately 35 times
lower than complex 1. Transport of complex 2 inside the cell
must similarly be facilitated but to a lesser extent. The ligand BC
tested independently reached a maximum concentration of
2000 mM per cell with similar kinetic prole as complex 1,
Fig. 7a, inset. Complex 1 is therefore considerably concentrated
in the cells suggesting a facilitated mode of transport which is
likely mediated through the presence of the lipophilic BC
ligand.
Puckett and Barton demonstrated that cellular uptake of
dppz complexes of Ru(II) was signicantly enhanced by the
presence of the lipophilic ligand 4,7-diphenyl-1,10phenanthroline, as evidenced by the increased photoluminescence intensity across the cell. Similarly, these reported
results imply that cellular transport of Ru(II) complexes was
facilitated by the lipophilic BC ligand.36,37
Studies on metal complexes including platinum- and
ruthenium-based chemotherapeutics suggested that cellular
uptake increased with the lipophilicity of the complexes.36,38 The
partition coefficient (log P) between the hydrophobic octanol
phase and the hydrophilic water phase was thus measured to
determine whether the extent of lipophilicity could explain the
Fig. 6 ESI-MS spectra of [Ru(bipy)2BC]Cl2 in water, (a) before photolysis and (b) after 35 min photolysis with blue LED (output: 100 mW cm2).
Peaks are assigned in Table 2.
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LC-MS/MS analysis for the uptake of 3 mM of complex 1 (a), BC
(a, inset), and complex 2 (b) by A549 cells. Results are averages of four
different experiments (SEM) and are expressed in mM per cell, as
estimated from the calibration curves (Fig. S5–S7†).
Fig. 7
results of cellular uptake, Table 3. The dicationic complex 1
(log P ¼ 1.57) was found to be signicantly more lipophilic as
compared to complex 2 (log P ¼ 2.82). The lipophilic property
of complex 1, which is largely imparted by the coordinated BC
ligand (calculated log P ¼ 6.96), can explain the fast uptake of
complex 1 by the cells with accumulated concentration of 0.6
nmol/106 cells immediately aer incubation with the drug,
Fig. S9a.† The dicationic complex 2 proved to be rather hydrophilic (log P ¼ 2.82), which correlates well with the lower
uptake of the complex through the cell membrane.36 Although
cisplatin is only slightly more lipophilic than complex 2 (log P ¼
2.21), studies have recently emphasized the role of active
transport in the cellular uptake of this neutral Pt(II) complex.39
Complex 1 is a potential candidate for PACT owing to its
signicant uptake and photochemical properties. However for
efficient PACT, the compounds should be active predominantly
upon photolysis, thus inducing selective toxicity while minimizing side effects.40 This is commonly assessed by measuring
the difference in cytotoxicity in vitro between dark and light
conditions, known as the photoindex or PI (PI ¼ IC50 dark/IC50
light).41 Cellular uptake plateaued aer 12 h for complexes 1
and 2 (Fig. 7), thus cell survival assays were performed aer
a 12 h incubation period with the complexes. In all cell lines
tested, the potency of complex 1 improved upon photoactivation. White light, more specically blue light, was reported to induce cytotoxicity in vitro and in vivo as compared to
dark conditions. Bonnet and coworkers demonstrated that the
viability of certain types of cancer cells, like human malignant
melanoma (A375), A549 and MDA-MB-231 cells, decreased
when irradiated with blue light and thus highlighted the
necessity of including the adequate controls when studying
photoactivable anticancer agents.42 Likewise, Wang and
coworkers showed that blue light decreased the viability and
mediated the death of human promyelocytic leukemia cells
(HL60) in a time-dependent fashion. A 24 hour exposure lead to
overproduction of reactive oxygen species (ROS) and mitochondrial depolarization. When tested in vivo, irradiation with
blue light caused regression of HL60-xenograed tumors and
Western blot analyses proved that the effect resulted from the
activation of the mitochondrial apoptosis pathway.43 In our
irradiation experiments, blue light (l ¼ 460 nm, 35 min irradiation at 100 mW cm2) resulted in an average of 35% loss of
cell viability versus the dark control, which was consistent with
the results of Bonnet on the same cell lines.42 However cell
viability results, under photoexcitation, were assessed against
control cells exposed to light on the same plate.13,20,23,26 In that
perspective, the inuence of light, if any, was subtracted from
the data allowing the assessment of the effect of drugs. While
IC50 values ranged between 5.55 mM (in MDA-BD-231 cells) and
>100 mM (B16-F10 cells) in dark conditions, the values were as
Table 3 IC50 values (mM) of complexes 1 and 2 in dark versus light conditions and the calculated phototoxicity index (PI) on different cell lines.
Cytotoxicity of BC, bipy, and cisplatin was assessed only in the dark and all values reported are the mean SEM of three separate experiments,
each including samples in triplicates
IC50 valuesa (mM)
Treatment
A549
B16-F10
Caco-2
HT-29
MDA-MB-231
log P
Complex 1
Dark: 51.8a,c 18.8
Light: 4.96a 1.16
PI: 11.1
>100b
>100b
Dark: >100b,c
Light: >100b,c
PI: —
7.74b,c 1.00
Dark: >100a
Light: 0.11b 0.10
PI: >900
>100a
>100
Dark: >100a
Light: >100b
PI: —
7.70a 0.20
Dark: 29.8a 2.9
Light: 14.8a 0.81
PI: 2.0
>100b
>100
Dark: >100b
Light: >100b
PI: —
21.8b 0.54
Dark: 91.0a 5.90
Light: 12.0b 1.9
PI: 7.6
>100c
>100c
Dark: >100c
Light: >100c
PI: —
>100c
Dark: 5.55a 1.45
Light: 0.63a 0.90
PI: 8.9
>100b
37.5b 0.14
Dark: >100b
Light: >100b
PI: —
30.9b 0.82
1.57 0.01
BC
bipy
Complex 2b
Cisplatin
a
c
6.96c
1.88d
2.82 0.10
2.21c
Values in a column not sharing a common superscript a, b, or c are signicantly different (p < 0.05). b IC50 data previously reported by our group.13
log P values reported in the literature.26,47 d log P values estimated by ChemDraw Professional (v15.0, CambridgeSo).
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low as 0.11 mM (in B16-F10 cells) upon irradiation (Table 1). The
response was however particularly signicant in B16-F10 cells
where PI > 900, as compared to only 2–11 in the other cell lines
(Table 3, Fig. 8). Complex 1 exhibited substantial PACT potential on B16-F10 cells in view of the high PI which is comparable
to the highest reported in the literature44 and 70-fold lower IC50
than the prototypical cisplatin (where IC50 only reached
RSC Advances
a minimum of 7.70 0.20 mM in B16-F10 cells). The photoproduct of complex 1 was consistently more potent than
cisplatin in all cell lines tested indicating that this complex
acquired signicant chemotherapeutic properties upon irradiation. When tested on HT-29 cells, cisplatin exhibited a large
IC50 value > 100 mM, suggesting that these cells could have
developed resistance to cisplatin. This nding is consistent with
Fig. 8 Effect of complexes 1, 2, bipy, BC, and cisplatin on the survival of A549, B16-F10, Caco-2, HT-29, and MDA-MB-231 cells. Treatment was
performed with 3-fold dilutions of the complexes and ligands, starting at 120 mM. The cytotoxicity of complexes 1 and 2 was evaluated in both
dark and light conditions (blue LED at a power of 100 mW cm2) and all others were kept in the dark. Each experiment was repeated three times
and measurements were performed in triplicates. The graphs shown here correspond to a single representative trial.
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a published report conrming that colon cancer cells were less
responsive to cisplatin treatment possibly due to their potential
for DNA repair.45 Control cell viability experiments were done on
normal cells (isolated MSCs). The IC50 in the dark was 52 13
mM and decreased signicantly down to 0.024 0.002 mM upon
exposure to light, Fig. S10.† These results indicate photoactivatable potential of complex 1 on all tested cell lines, suggesting a lack of intrinsic mechanism of discrimination
between normal and cancerous cells. However, it is envisioned
that a delicate spatial and temporal control of light delivery can
infer selectively in PACT as demonstrated by the high PI of
complex 1, Table 3.
The low IC50 in the dark, which was most notable in MDAMB-231 cells (5.55 1.45 mM), could have resulted from the
geometry of the complex. When studying the binding geometry
of [Ru(bipy)2phen]2+ with DNA, Lincoln and Nordén found that
the phenanthroline ligand was oriented nearly perpendicular to
the DNA double helix and was able to achieve a stacking interaction with the nucleobases.46 The phenanthroline derivative,
BC, in complex 1 is most likely exposed, which could have led to
similar interactions with DNA thus mediating dark cytotoxicity.
Control experiments showed no differences between the cytotoxicity of complex 1 when the supernatant containing the
compound was replaced with fresh medium or kept prior to the
photoactivation step. This emphasized the role of cellular
uptake in mediating cytotoxicity. In accordance with reported
results, complex 2 was neither potent in the dark nor upon light
irradiation on all cell lines tested (IC50 > 100 mM).12,13 Complex 2,
with similar characteristic MLCT absorption in the blue as
complex 1, is a valid control in PACT since it remains both
structurally unchanged and biologically inactive upon irradiation. Previous reports demonstrated that [Ru(bipy)2phen]2+
displayed an ordered binding with DNA.46 However, the poor
cellular uptake of complex 2 is likely the limiting factor
inducing low cytotoxicity and IC50 > 100 mM.
The photolysis products of complex 1 are consistently cytotoxic on all tested cell lines. Generally, it is expected that the
photochemically generated metal aqua complexes are responsible for the biological effect of the complex, similarly to
cisplatin.47 However, our group and others have demonstrated
that the ejected ligand could be potent.20–22 Both dissociating
ligands, bipy and BC, were therefore included in cell survival
studies to investigate their potential cytotoxic effect. While only
slightly toxic on MDA-MB-231 cells (IC50 ¼ 37.5 0.140 mM,
Table 3), bipy was not potent on all other cell lines tested (IC50 >
100 mM, Table 3). These results were expected since bipy is
known to form electrostatic interactions rather than intercalation with DNA.25 Likewise, BC was consistently inactive against
all cancer cells studied (IC50 > 100 mM), which, however, was not
anticipated since BC, unlike bipy, could intercalate DNA and
subsequently lead to cell death.48 To experimentally rule out the
lack of uptake, the cellular concentration of BC was measured in
cultured A549 cells at different time points. When compared to
complex 1 (maximum concentration of 1400 mM per cell aer
12 h, Fig. 7a), the optimal cellular uptake of BC was achieved
aer 3 h and it leveled off at a higher concentration of 2000 mM
per cell (Fig. 7a, inset). BC is therefore effectively transported
17262 | RSC Adv., 2019, 9, 17254–17265
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across the cell membrane. Owing to the high lipophilicity of
bipy and BC, with log P values of 1.88 and 6.96 respectively
(Table 3), cellular uptake should not have been compromised
for these free ligands. BC is a copper-specic chelator; in the
presence of endogenous Cu2+ or in a medium supplemented
with Cu2+, it could bind to the metal ion, forming a copper
chelate complex.49 Rabinovitz and coworkers previously
demonstrated that 2,9-dimethyl-1,10-phenanthroline (neocuproine), a similar copper chelator, was cytotoxic exclusively
when bound to Cu2+. Upon incubation of neocuproine with
either cells seeded in a medium enriched with Cu2+ in vitro or in
vivo, a signicant chemotherapeutic effect was observed. To
prove that the copper chelate was the active complex, they
oversaturated the medium with a copper-specic chelating
agent, bathocuproine sulfonate (BCS). This led to normal cell
growth and thus conrmed that neocuproine is dependent on
the presence of copper for inducing cytotoxicity.50,51 Typically
cuprous compounds, which result from the intracellular
reduction of cupric precursors, are signicantly more potent
and oen deemed the active species.52,53 McMillin and
coworkers have reported the interaction of Cu(BC)2+ with DNA
and described a nonclassical mode of binding involving
bridging structures that lead to DNA aggregation and elongation without intercalation.54–56 However, we recently tested the
cytotoxic effect of [Cu(BC)2]+ in the dark, and the IC50 values
obtained (21.8 and 25.5 mM in A549 and A375 cells, respectively)
reected moderate to low toxicity compared to cuprous neocuproine complex (IC50 of 0.9 and 1.8 mM on A549 and A375
cells, respectively). These results indicated that, unlike neocuproine, the activity of BC is not considerably enhanced by the
presence of copper.26 Therefore, the potency of complex 1
photoproducts was primarily attributed to the formed ruthenium aqua complexes and to a less extent the dissociating
ligands, bipy and BC. The photoproducts may possibly have
synergistic effect on the various tested cells, leading to the high
potency aer light irradiation. Numerous studies have indicated the role of metal aqua complexes in mediating cytotoxicity
through DNA interaction and/or modication.15,57
The in vitro mechanism of death induced by complex 1 was
determined on B16-F10 cells using Annexin V/PI staining. In
dark conditions, 26% of the cells stained positive for
annexin V and negative for PI whereas 21% of the cells stained
positively for both annexin V and PI (Fig. 9b). The precursor
appeared to induce apoptosis as cells are distributed between
living (annexin V and PI negative), early apoptosis (annexin V
positive and PI negative) and late apoptosis/necrosis (annexin V
and PI positive).58 Upon photoactivation of the complex, the
percentage of cells that were annexin V and PI positive
increased to 90% (Fig. 9c). Due to the lack of cells undergoing
early apoptosis in the lower right quadrant (annexin V positive
and PI negative), the mechanism of cell death of the photolysis
products could be through either late-apoptosis or necrosis. It is
worth mentioning that the photoactivation of the complex
considerably enhanced the percentage of cells undergoing cell
death, which is in accordance with the results of the cell survival
assay.
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Fig. 9 Analysis of the mechanism of complex 1-mediated cytotoxicity in B16-F10 cells using Annexin/PI staining on a flow cytometer. (a)
Control: no treatment, (b) cells treated with 10 mM of the complex and incubated for 24 h in the dark, (c) cells treated with 10 mM of the complex
and irradiated for 35 min using the blue LED system at an output of 100 mW cm2. FL1-H: Annexin staining, FL2-H: PI staining.
To further assess the effect of complex 1 on DNA, gel electrophoresis was performed on pUC8 plasmid, Fig. 10. At low
concentration of the precursor complex (in dark conditions,
lane 2), DNA migrated slower than the untreated pUC8 plasmid
control (lane 1). These results suggest binding of complex 1 to
plasmid DNA and possibly, intercalation, as observed with other
compounds like cisplatin.59 Similarly, EtBr intercalation
decreased at low concentrations of the photoactivated complex
(Fig. 10, lane 3) whereas EtBr signal was completely lost at high
concentrations of both the precursor and the photoproducts
(lanes 4 and 5 respectively). Such behavior strongly suggest
binding to DNA which likely changes in structure, charge, and
molecular weight while hindering the intercalation of EtBr.60
These results therefore imply that binding of the complex to
DNA is not exclusively photoinduced and that the precursor
molecule is capable of interacting with nucleic acids. This is
potentially due to the presence of BC ligand in complex 1, which
facilitates interaction with DNA nucleobases46 leading to the
dark cytotoxicity (Fig. 8, Table 3).
The cell death induced by complex 1 in both dark and light
conditions could correlate with the observed DNA interactions.
Apoptosis is typically one of the major mechanisms that lead to
cell inactivation in response to DNA damage.61 Many signaling
pathways are involved, including p53-mediated activation of
pro-apoptotic factors as a result of DNA double-stranded breaks,
MAP-kinase activation, and the capacity for DNA repair.62–64 It
was previously demonstrated that Ru(II) anticancer drugs can
trigger DNA damage which in turn activates apoptosis via the
intrinsic pathway.65 Zhang and coworkers reported the intercalative potential of Ru(II) polypyridyl complexes which were
shown to induce apoptosis in A549 cells, as concluded from ow
cytometric studies.66 Likewise, Xu and his colleagues found that
Ru(II) bipyridyl complexes were capable of DNA intercalation
and photo-induced DNA cleavage and described an apoptosismediated death of human hepatocellular carcinoma cells
(BEL-7402).67 Many other studies on Ru(II) anticancer drugs
have reported a similar intersection between DNA damage and
apoptosis.68–70 Further in vitro experiments are currently
underway to investigate the cascade of events and molecular
mechanisms of action associated with Ru(II) polypyridyl
complexes.
Conclusions
Agarose gel electrophoresis of pUC8 plasmid in 10 mM
potassium phosphate buffer (pH 7.4). Lane 1: pUC8 untreated, lanes 2
and 4: complex 1 in dark conditions, 10 mM and 200 mM respectively,
lanes 3 and 5: complex 1 in light conditions (blue LED at a power of 100
mW cm2), 10 mM and 200 mM respectively.
Fig. 10
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Owing to their tuneable photophysical and photochemical
properties, Ru(II) polypyridyl complexes and particularly those
having a distorted octahedral geometry are attractive for PACT.
Upon MLCT photoexcitation of complex 1, both bipy and BC
were ejected at a ratio of 3 : 1, respectively. The formed Ru aqua
complexes were likely the origin of the photocytotoxicity. The
potential of this complex in PACT appeared to be cell-line
dependent, as evidenced by disparate PI values. Cell death
mechanism with complex 1 is most likely occurring through
apoptosis in the dark and late-apoptosis or necrosis upon irradiation. Both the precursor and the photoproducts of complex 1
are likely to bind DNA leading to cellular damages. Complex 1,
on the other hand, proved to be photostable and was relatively
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less potent in both dark and light conditions, which could be
partly attributed to its low uptake into the cells. Finally, the
design of Ru(II) polypyridyl prodrugs with lipophilic ligands is of
prime importance to ameliorate cellular uptake, a cornerstone
towards efficient photochemotherapy.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
This work was funded by the School Research and Development
Council at the Lebanese American University and the Lebanese
National Council for Scientic Research (Ref: 05-06-14).
Notes and references
1 N. Muhammad and Z. Guo, Curr. Opin. Chem. Biol., 2014, 19,
144–153.
2 G. Y. Ho, N. Woodward and J. I. G. Coward, Crit. Rev. Oncol.
Hematol., 2016, 102, 37–46.
3 L. Kelland, Nat. Rev. Cancer, 2007, 7, 573–584.
4 N. P. E. Barry and P. J. Sadler, Chem. Commun., 2013, 49,
5106–5131.
5 A. Bergamo, C. Gaiddon, J. H. M. Schellens, J. H. Beijnen and
G. Sava, J. Inorg. Biochem., 2012, 106, 90–99.
6 P. Schluga, C. G. Hartinger, A. Egger, E. Reisner, M. Galanski,
M. A. Jakupec and B. K. Keppler, Dalton Trans., 2003, 2006,
1796–1802.
7 W. Guo, W. Zheng, Q. Luo, X. Li, Y. Zhao, S. Xiong and
F. Wang, Inorg. Chem., 2013, 52, 5328–5338.
8 C. Mari, V. Pierroz, S. Ferrari and G. Gasser, Chem. Sci., 2015,
6, 2660–2686.
9 A. L. Harris, Nat. Rev. Cancer, 2002, 2, 38–47.
10 S. Campagna, F. Puntoriero, F. Nastasi, G. Bergamini and
V. Balzani, in Photochemistry and Photophysics of
Coordination Compounds I, Springer, Berlin, Heidelberg,
2007, pp. 117–214.
11 B. Durham, J. V. Caspar, J. K. Nagle and T. J. Meyer, J. Am.
Chem. Soc., 1982, 104, 4803–4810.
12 B. S. Howerton, D. K. Heidary and E. C. Glazer, J. Am. Chem.
Soc., 2012, 134, 8324–8327.
13 N. Mansour, S. Mehanna, M. A. Mroueh, H. Audi,
K. Bodman-Smith, C. F. Daher, R. I. Taleb, M. El-Sibai and
R. S. Khnayzer, Eur. J. Inorg. Chem., 2018, 2524–2532.
14 T. N. Singh and C. Turro, Inorg. Chem., 2004, 43, 7260–7262.
15 R. N. Garner, L. E. Joyce and C. Turro, Inorg. Chem., 2011, 50,
4384–4391.
16 M. Cristina, P. Vanessa, R. Riccardo, P. Malay, J. Hess,
S. Bernhard, O. Luciano, S. Julia, I. Ott, S. Luca, F. Stefano
and G. Gilles, Chem.–Eur. J., 2014, 20, 14421–14436.
17 E. Wachter, D. K. Heidary, B. S. Howerton, S. Parkin and
E. C. Glazer, Chem. Commun., 2012, 48, 9649–9651.
18 T. Sainuddin, M. Pinto, H. Yin, M. Hetu, J. Colpitts and
S. A. McFarland, J. Inorg. Biochem., 2016, 158, 45–54.
17264 | RSC Adv., 2019, 9, 17254–17265
Paper
19 V. H. S. van Rixel, B. Siewert, S. L. Hopkins, S. H. C. Askes,
A. Busemann, M. A. Siegler and S. Bonnet, Chem. Sci.,
2016, 7, 4922–4929.
20 D. F. Azar, H. Audi, S. Farhat, M. El-Sibai, R. J. Abi-Habib and
R. S. Khnayzer, Dalton Trans., 2017, 46, 11529–11532.
21 J.-A. Cuello-Garibo, M. S. Meijer and S. Bonnet, Chem.
Commun., 2017, 53, 6768–6771.
22 M. A. Sgambellone, A. David, R. N. Garner, K. R. Dunbar and
C. Turro, J. Am. Chem. Soc., 2013, 135, 11274–11282.
23 H. Audi, D. F. Azar, F. Mahjoub, S. Farhat, Z. El Masri, M. ElSibai, R. J. Abi-Habib and R. S. Khnayzer, J. Photochem.
Photobiol., A, 2018, 351, 59–68.
24 A.-C. Laemmel, J.-P. Collin and J.-P. Sauvage, Eur. J. Inorg.
Chem., 1999, 1999, 383–386.
25 A. M. Pyle, J. P. Rehmann, R. Meshoyrer, C. V. Kumar,
N. J. Turro and J. K. Barton, J. Am. Chem. Soc., 1989, 111,
3051–3058.
26 C. A. Hageh, M. A. Assaad, Z. E. Masri, N. Samaan, M. ElSibai, C. Khalil and R. S. Khnayzer, Dalton Trans., 2018, 47,
4959–4967.
27 National Research Council (US), Committee for the Update of
the Guide for the Care and Use of Laboratory Animals, Guide for
the Care and Use of Laboratory Animals, National Academies
Press (US), Washington (DC), 8th edn, 2011.
28 N. Zeeni, C. Daher, G. Fromentin, D. Tome, N. Darcel and
C. Chaumontet, Stress, 2013, 16, 211–219.
29 L. E. Hayek, M. Khalifeh, V. Zibara, R. A. Assaad,
N. Emmanuel, N. Karnib, R. El-Ghandour, P. Nasrallah,
M. Bilen, P. Ibrahim, J. Younes, E. A. Haidar, N. Barmo,
V. Jabre, J. S. Stephan and S. F. Sleiman, J. Neurosci., 2019,
39, 2369–2382.
30 N. Karnib, R. El-Ghandour, L. E. Hayek, P. Nasrallah,
M. Khalifeh, N. Barmo, V. Jabre, P. Ibrahim, M. Bilen,
J. S. Stephan, E. B. Holson, R. R. Ratan and S. F. Sleiman,
Neuropsychopharmacology, 2019, 44, 1152.
31 A. Smajilagić, M. Aljičević, A. Redžić, S. Filipović and
A. C. Lagumdžija, Bosnian J. Basic Med. Sci., 2013, 13, 27–30.
32 M. Najar, H. Fayyad-Kazan, W. H. Faour, M. Merimi,
E. M. Sokal, C. A. Lombard and H. Fahmi, Inammation
Res., 2019, 68, 203–213.
33 G. Nasreddine, M. El-Sibai and R. J. Abi-Habib, Invest. New
Drugs, 2019, 37, 1–10.
34 E. Kassab, M. Darwish, Z. Timsah, S. Liu, S. H. Leppla,
A. E. Frankel and R. J. Abi-Habib, Transl. Oncol., 2013, 6,
25–32.
35 D. N. Wheatley, M. S. Inglis, M. A. Foster and J. E. Rimington,
J. Cell Sci., 1987, 88(pt 1), 13–23.
36 C. A. Puckett and J. K. Barton, J. Am. Chem. Soc., 2007, 129,
46–47.
37 C. A. Puckett, R. J. Ernst and J. K. Barton, Dalton Trans., 2010,
39, 1159–1170.
38 A. Ghezzi, M. Aceto, C. Cassino, E. Gabano and D. Osella, J.
Inorg. Biochem., 2004, 98, 73–78.
39 N. D. Eljack, H.-Y. M. Ma, J. Drucker, C. Shen,
T. W. Hambley, E. J. New, T. Friedrich and R. J. Clarke,
Met. Integr. Biometal Sci., 2014, 6, 2126–2133.
This journal is © The Royal Society of Chemistry 2019
View Article Online
Open Access Article. Published on 03 June 2019. Downloaded on 5/2/2026 2:22:39 AM.
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
Paper
40 M. Dickerson, Y. Sun, B. Howerton and E. C. Glazer, Inorg.
Chem., 2014, 53, 10370–10377.
41 C. Mari and G. Gasser, Chimia, 2015, 69, 176–181.
42 S. L. Hopkins, B. Siewert, S. H. C. Askes, P. Veldhuizen,
R. Zwier, M. Heger and S. Bonnet, Photochem. Photobiol.
Sci., 2016, 15, 644–653.
43 J. Zhuang, Y. Liu, Q. Yuan, J. Liu, Y. Liu, H. Li and D. Wang,
Oncol. Lett., 2018, 15, 6291–6296.
44 A. N. Hidayatullah, E. Wachter, D. K. Heidary, S. Parkin and
E. C. Glazer, Inorg. Chem., 2014, 53, 10030–10032.
45 K. Gharehbaghi, T. Szekeres, J. A. Yalowitz, M. FritzerSzekeres, Y. G. Pommier and H. N. Jayaram, Life Sci., 2000,
68, 1–11.
46 P. Lincoln and B. Nordén, J. Phys. Chem. B, 1998, 102, 9583–
9594.
47 F. Qu, S. Park, K. Martinez, J. L. Gray, F. S. Thowfeik,
J. A. Lundeen, A. E. Kuhn, D. J. Charboneau, D. L. Gerlach,
M. M. Lockart, J. A. Law, K. L. Jernigan, N. Chambers,
M. Zeller, N. A. Piro, W. S. Kassel, R. H. Schmehl, J. J. Paul,
E. J. Merino, Y. Kim and E. T. Papish, Inorg. Chem., 2017,
56, 7519–7532.
48 E. Grueso, G. López-Pérez, M. Castellano and R. Prado-Gotor,
J. Inorg. Biochem., 2012, 106, 1–9.
49 K. T. Spencer, P. D. Lindower, G. R. Buettner and
R. E. Kerber, J. Cardiovasc. Pharmacol., 1998, 32, 343–348.
50 A. Mohindru, J. M. Fisher and M. Rabinovitz, Biochem.
Pharmacol., 1983, 32, 3627–3632.
51 A. Mohindru, J. M. Fisher and M. Rabinovitz, Nature, 1983,
303, 64–65.
52 C. R. Kowol, P. Heffeter, W. Miklos, L. Gille, R. Trondl,
L. Cappellacci, W. Berger and B. K. Keppler, JBIC, J. Biol.
Inorg. Chem., 2012, 17, 409–423.
53 C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato and
C. Marzano, Chem. Rev., 2013, 114, 815–862.
This journal is © The Royal Society of Chemistry 2019
RSC Advances
54 F. Liu, K. A. Meadows and D. R. McMillin, J. Am. Chem. Soc.,
1993, 115, 6699–6704.
55 D. R. McMillin, F. Liu, K. A. Meadows, T. K. Aldridge and
B. P. Hudson, Coord. Chem. Rev., 1994, 132, 105–112.
56 D. R. McMillin and K. M. McNett, Chem. Rev., 1998, 98, 1201–
1220.
57 V. Brabec and O. Nováková, Drug Resist. Updates, 2006, 9,
111–122.
58 S. Abou Najem, G. Khawaja and S. Rizk, Clin. Lymphoma,
Myeloma Leuk., 2018, 18, S207.
59 E. Wachter, D. K. Heidary, B. S. Howerton, S. Parkin and
E. C. Glazer, Chem. Commun., 2012, 48, 9649–9651.
60 Y. Chen, X. Luo, L. Bai, X. Hu, J. Zhou, P. Zhang and Y. Yu,
New J. Chem., 2017, 41, 10225–10230.
61 S. Nowsheen and E. S. Yang, Exp. Oncol., 2012, 34, 243–254.
62 M. H. Hodroj, A. Jardaly, S. Abi Raad, A. Zouein and S. Rizk,
Cancer Manage. Res., 2018, 10, 1079–1088.
63 C. Khalil and W. Shebaby, Toxicol. Rep., 2017, 4, 441–449.
64 W. P. Roos and B. Kaina, Trends Mol. Med., 2006, 12, 440–
450.
65 L. Zeng, P. Gupta, Y. Chen, E. Wang, L. Ji, H. Chao and
Z.-S. Chen, Chem. Soc. Rev., 2017, 46, 5771–5804.
66 P. Zhang, J. Chen and Y. Liang, Acta Biochim. Biophys. Sin.,
2010, 42, 440–449.
67 L. Xu, N.-J. Zhong, Y.-Y. Xie, H.-L. Huang, G.-B. Jiang and
Y.-J. Liu, PLoS One, 2014, 9, e96082.
68 W. Li, G.-B. Jiang, J.-H. Yao, X.-Z. Wang, J. Wang, B.-J. Han,
Y.-Y. Xie, G.-J. Lin, H.-L. Huang and Y.-J. Liu, J. Photochem.
Photobiol., B, 2014, 140, 94–104.
69 A. Srishailam, Y. P. Kumar, P. Venkat Reddy, N. Nambigari,
U. Vuruputuri, S. S. Singh and S. Satyanarayana, J.
Photochem. Photobiol., B, 2014, 132, 111–123.
70 Y.-Y. Xie, H.-L. Huang, J.-H. Yao, G.-J. Lin, G.-B. Jiang and
Y.-J. Liu, Eur. J. Med. Chem., 2013, 63, 603–610.
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