Synthesis, characterisation, interaction with DNA, cytotoxicity, and apoptotic studies of ruthenium(ii) polypyridyl complexes

Article abstract of DOI:10.1071/CH13321

We report the synthesis and characterisation of two new ruthenium(ii) polypyridyl complexes containing monodentate ancillary ligands [Ru(L)4(4HEPIP)], where L≤4-aminopyridine (1) or pyridine (2) and 4HEPIP≤2-(4-hydroxy-3- ethoxyphenyl)-¹H-imida

Full text of DOI:10.1071/CH13321

CSIRO PUBLISHING
Aust. J. Chem. 2014, 67, 225–233
Full Paper
http://dx.doi.org/10.1071/CH13321
Synthesis, Characterisation, Interaction with DNA,
Cytotoxicity, and Apoptotic Studies of Ruthenium(II)
Polypyridyl Complexes
A
B
A
C. Shobha Devi, Penumaka Nagababu, V. Venkat Reddy,
A
A
A C
,
V. Sateesh, A. Srishailam, and S. Satyanarayana
A
Department of Chemistry, Osmania University, Hyderabad, 500007, India.
Institute of Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan.
B
C
Corresponding author. Email: ssnsirasani@gmail.com
We report the synthesis and characterisation of two new ruthenium(II) polypyridyl complexes containing monodentate
ancillary ligands [Ru(L)4(4HEPIP)], where L ¼ 4-aminopyridine (1) or pyridine (2) and 4HEPIP ¼ 2-(4-hydroxy-
3-ethoxyphenyl)-1H-imidazo[4,5-f][1,10](phenanthroline). These complexes were characterised by elemental analysis
and ultraviolet-visible, infrared, and 1H NMR spectroscopy. The binding properties of the two complexes towards calf
thymus (CT)-DNA were investigated with different spectrophotometric methods, viscosity measurements, and salt
dependent studies. Experimental results indicated that the complexes interact with CT-DNA base pairs by intercalation.
Upon irradiation at 365 nm, these complexes efficiently cleave pBR322 DNA from super coiled form I to nicked form II.
Their cytotoxicity on different cancer cell lines such as A549, Du145, and HeLa was investigated. The IC50 values are 39.5,
28.3, and 27.3 mM for complex 1, and 55, 67.9, and 47.9 mM for complex 2 respectively. Cellular uptake and apoptosis
induced by these complexes was also studied.
Manuscript received: 9 April 2013.
Manuscript accepted: 11 September 2013.
Published online: 2 October 2013.
Introduction
in particular in the DNA-binding behaviours as well as spectral
properties of the resulting ruthenium complexes.
Ruthenium(II) polypyridyl complexes have vast applications
due to their photochemical, photophysical, and DNA binding
properties in anticancer drugs, photocleavage agents, DNA
structure probes,[1–3] molecular light switches,[4] and photo-
dynamic therapy (PDT).[5] Complexes which have different
binding capabilities with DNA can show changes in absorption
and emission spectra.
Many important applications of these complexes require an
intercalative mode of binding with DNA. Many octahedral
ruthenium(II) complexes have been proved to bind with DNA
through an intercalative mode.[6–8] Octahedral complexes con-
taining an intercalating ligand binds to DNA in three dimen-
sions; however, its ancillary ligand can also be modified or
functionalised to tune the DNA binding. Recently, the effects
of some bidentate ancillary ligands such as bipyridine (bpy),
1,10-phenanthroline (phen), dimethyl bipyridine (dmb), and
diphenyl bipyridine (dip) have been investigated by many
research groups, including ours.[9–11] Studies on the biological
activity of these complexes have been paid great attention.[12–14]
However, there are only a few reports on ruthenium(II) com-
plexes with monodentate ancillary ligands.[15–18]
Previously, we have shown that RuII polypyridyl complexes
with monodentate ancillary ligands can also bind to DNA by
intercalation.[17,18] In order to investigate the DNA binding and
biological activities of the complexes, herein we report the
synthesis and characterisation of two new ruthenium poly-
pyridyl complexes containing monodentate ancillary ligands:
[Ru(L)4(4HEPIP)]2þ where L is 4-aminopyridine or pyridine
and 4HEPIP is 2-(4-hydroxy-3-ethoxyphenyl)-1H-imidazo
[4,5-f][1,10](phenanthroline). Their interactions with DNA
were investigated by electronic absorption, emission-quenching
studies, viscosity measurements, and salt dependent studies.
Effects of light switching on and off were also studied. These
complexes can intercalate into DNA base pairs and cleave
pBR322 DNA upon irradiation. We have also tested their
anticancer activity, cellular uptake, and apoptosis inducing
activities.
Experimental Section
Materials and General Methods
Chen et al. reported that many RuII polypyridyl complexes
are less soluble in water due to their big hydrophobic ligands.[19]
This limits their applications as good probes of biological
systems or anticancer drugs. Varying the ancillary ligands such
as monodentate ligands can change physicochemical properties,
All reagents and solvents were of analytical grade and were used
as received unless otherwise noted. Ruthenium(III) chloride
trihydrate (RuCl3 ꢀ 3H2O), 1,10-phenanthroline monohydrate,
pyridine, and 4-aminopyridine were purchased from Merck.
CT-DNA (calf thymus DNA) was purchased from Aldrich,
Journal compilation Ó CSIRO 2014
www.publish.csiro.au/journals/ajc

226
C. S. Devi et al.
NaBr
Conc. HNO3
N
N
O
O
N
N
Conc. H2SO4
Reflux, 2 h
CH3
O
Glac. AcOH
NH4OAC
Reflux, 4 h
OH
OHC
CH3
CH3
CI
O
O
N
CI
CI
N
N
N
N
RuCl3·xH2O
1M HCI
OH
OH
Ru
N
H
N
H
N
CI
NH2
DMF,
DMF
Reflux, 8 h
Reflux, 8 h
N
N
4equiv.
4equiv.
NH2
2ϩ
2ϩ
CH3
CH3
H2N
N
N
O
O
11
12
12
N
N
N
N
11
N
N
N
10
7
6
7
13
13
OH
OH
Ru
N
Ru
N
6
10
N
H
N
H
5
N
5
4
8
9
N
8
9
4
3
1
1
3
N
2
2
14
15
14
15
H2N
16
16
NH2
1
2
Fig. 1. Synthetic route for ligand 4HEPIP and complexes 1 and 2.
supercoiled pBR322 plasmid DNA (stored at ꢁ208C) and
standard at 400MHz at room temperature. Microanalyses
(C, H, N) were carried out with a Perkin-Elmer 240 elemental
analyser. Fluorescence measurements were performed on a
Hitachi F-2500 spectrofluorimeter. Viscosity experiments were
carried out on an Ostwald viscometer immersed in a water bath
maintained at 30 ꢃ 0.18C. Gels were photographed in a Gel doc
system (Alpha InfoTech Corporation). A Bright Line haemo-
cytometer (Sigma Ltd), 96-well plates (Orange Scientific), and a
Multi Skan EX Elisa reader (Thermo Scientific) were used for the
MTT assay. A flow cytometer (Guava Easycyte 8HT, Millipore)
was used to study cellular uptake and apoptotic inducing activi-
ties. Confocal imaging was done with a LeicaTCSSP5 confocal
microscope (Leica Microsystems, Wetzlar, Germany) which was
equipped with an Ar-Kr laser (used to excite ruthenium(II)
complexes at 488nm excitation, 600–620 nm emission).
agarose were obtained from Genei. 3-(4,5-Dimethylthiazole)-
´
2,5-diphenyltetraazolium bromide (MTT), 4 ,6-diamidino-
2-phenylindole dihydro chloride (DAPI, nuclear stain), and
cisplatin (Sigma–Aldrich) were used as received. Ultra-pure
Milli-Q water (18.2 mO) was used in all experiments. Double
distilled water was used for preparing various buffers. DMSO
was obtained from Sigma Ltd. Various cell lines used for in vitro
cytotoxicity were procured from American Type Culture Col-
lection (Manassas, VA, USA). RPMI-1640 or DMEM cell
culture media, supplemented with 10 % (v/v) fetal bovine
serum (FBS), 2 mM glutamine, 4.5 g Lꢁ1 glucose, and 1 ꢂ non-
essential amino acids and antibiotics, were obtained from
Sigma–Aldrich.
Physical Measurements
Synthesis and Characterisation of Ligand and Complexes
UV-Visible (UV-Vis) spectra were recorded with an Elico
SL159 spectrophotometer. Infrared (IR) spectra were recorded
on KBr disks on a Perkin-Elmer FT-IR 1605 spectrometer.
1H NMR spectra were recorded on a Bruker spectrometer with
[D6]DMSO as solvent and tetramethylsilane as an internal
1,10-Phenanthroline-5,6-dione,[20] 4HEPIP,[21] and [Ru(L)4
Cl2][22] were synthesised according to literature procedures.
The synthetic route for ligand 4HEPIP and complexes 1 and 2
are given in Fig. 1.

Ruthenium(II) Polypyridyl Complexes
227
Synthesis of [Ru(4HEPIP)Cl4]
sufficiently free of protein.[23] Concentration of CT-DNA was
determined by UV absorbance at 260 nm; extinction coefficient
The complex was synthesised by mixing RuCl3 ꢀ 3H2O
(0.53 g, 2 mmol) and 4HEPIP (0.89 g, 2.5 mmol) in 1 M HCl
(50 mL). The resulting mixture was stirred for ,30 min under
nitrogen, and then allowed to lay under a nitrogen atmosphere
for 10 days. The insoluble product [Ru(4HEPIP)Cl4] (65 %
yield) was collected by vacuum filtration and washed with
water, acetone, and diethyl ether and then dried under vacuum.
was 6600 Mꢁ1 cmꢁ1 [24] Stock solutions were stored at 48C and
.
used within five days.
Fluorescence titrations were performed at room temperature
to determine binding affinity between DNA and the complex.
The titration experiments were performed at a fixed ruthenium
concentration of 10 mM, to which increments of CT-DNA
solution was added until no emission was detected. Steady-state
emission quenching experiments using varying amounts of
[Fe(CN)6]4ꢁ as the quencher were performed in absence,
presence, and excess of DNA. [Fe(CN)6]4ꢁ was varied from
0–5 mM. The quenching constant was determined according to
the classical Stern–Volmer equation: [25]
Synthesis of [Ru(4-aminopyridine)44HEPIP]
(ClO4)2 ꢀ 2H2O (1)
This complex was synthesised by mixing [Ru(4HEPIP)Cl4]
(0.3 g, 0.5 mmol) and 4-aminopyridine (0.2 g, 2.0 mmol) in
DMF (20 mL). The resulting mixture was heated at reflux under
nitrogen atmosphere for 8 h. After cooling to room temperature,
the solution was filtered to remove small amounts of insoluble
components. The filtrate was reduced to 5 mL and then diluted
with 15 mL water. Upon dropwise addition of a saturated
solution of aqueous sodium perchlorate, a red precipitate
formed. The red solid was collected and washed with 2 mL of
ice cooled ethanol before being dried under vacuum and purified
by column chromatography on alumina using acetonitrile/
toluene (3/1) as eluent (72 % yield). (Found: C 45.90, H 4.24,
N 15.65. Anal. Calc. for C41H44Cl2N12O12Ru: C 45.98, H 4.29,
N 15.70 %). nmax(KBr)/cmꢁ1 3445 br (n, N–H), 1654 (n, C¼N),
1540 (n, C¼C), 550 (Ru–N). dH ([D6]DMSO) 1.41 (t, J ¼ 7.5,
3H, CH3), 4.23 (q, J ¼ 7.9, 2H, -OCH2), 6.61 (s, 1H, OH), 6.75
(t, J ¼ 6.8, 2H, H2), 7.1 (t, J ¼ 7.6, 8H, H15), 7.31 (s, 1H, H12),
7.42 (d, J ¼ 6.5, 1H, H8), 7.5 (d, J ¼ 7.5, 1H, H9), 7.75
(d, J ¼ 7.5, 2H, H3), 7.8 (d, J ¼ 7.7, 8H, H14), 7.97 (d, J ¼ 7.8,
2H, H1), 8.95 (s, 8H, NH2).
I0=I ¼ 1 þ Ksv½Qꢄ
where I0 and I are the luminescence intensities in the absence
and presence of quencher [Fe(CN)6]4ꢁ respectively, Ksv is a
linear Stern–Volmer quenching constant depending on the
ratio of the bound concentration of the complex to the concen-
tration of DNA, and [Q] is the concentration of the quencher
[Fe(CN)6]4ꢁ. In the plot of I0/I versus [Q], the linear Stern–
Volmer quenching constant Ksv is given by the slope.
Absorption titrations were performed in Tris buffer using
a fixed complex concentration (20 mM) to which increments of
the DNA stock solution was added. Solutions were allowed
to incubate for 5 min before the absorption spectra were
recorded. The intrinsic binding constant Kb of these complexes
to DNA were calculated by a non-linear least-square method
using Eqn 1:[26]
1=2
ðea ꢁ ef Þ=ðeb ꢁ ef Þ ¼ ðb ꢁ ðb2 ꢁ 2K2Ct½DNAꢄ=sÞ Þ=2KCt
Synthesis of [Ru(pyridine)44HEPIP](ClO4)2 ꢀ 2H2O (2)
ð1aÞ
This complex was synthesised by mixing [Ru(4HEPIP)Cl4]
(0.30 g, 0.5 mmol) and pyridine (0.02 mL, 2.0 mmol) in DMF
(20 mL). The resulting mixture was heated at reflux under
nitrogen atmosphere for 8 h. After cooling to room temperature,
the solution was filtered to remove small amounts of insoluble
components. The filtrate was reduced to 5 mL and then diluted
with 15 mL water. Upon drop wise addition of a saturated
aqueous solution of sodium perchlorate, a red precipitate
formed. The red solid was collected and washed with 2 mL of
ice cooled ethanol before being dried under vacuum and purified
by column chromatography on alumina using acetonitrile/
toluene (3/1) as eluent (70 % yield). (Found: C 49.10, H 3.52,
N 11.05. Anal. Calc. for C41H40Cl2N8O12Ru: C 49.00, H 3.50,
N 11.15 %). nmax(KBr)/cmꢁ1 3328 br (n, O–H, N–H), 1615
(n, C¼N), 1511, 1536 (n, C¼C), 532 (Ru–N). dH ([D6]DMSO)
1.45 (t, J ¼ 7.4, 3H, CH3), 4.2 (q, J ¼ 7.7, 2H, -OCH2), 6.68
(s, 1H, OH), 6.71 (t, J ¼ 6.7, 2H, H2), 6.93 (t, J ¼ 6.9, 8H, H15),
7.21 (s, 1H, H12), 7.33 (t, J ¼ 7.5, 4H, H16), 7.4 (d, J ¼ 7.6, 1H,
H8), 7.5 (d, J ¼ 7.5, 1H, H9), 7.55 (d, J ¼ 7.8, 2H, H3), 7.77
(t, J ¼ 7.8, 2H, H1), 7.8 (d, J ¼ 7.6, 8H, H14).
b ¼ 1 þ KCt þ K½DNAꢄ=2s
ð1bÞ
where [DNA] is the concentration of DNA, ea is the extinction
coefficient observed for the absorption band at given DNA
concentration, ef is the extinction coefficient of the free
complex without DNA, eb is the extinction coefficient of the
complex fully bound to DNA. K is the equilibrium binding
constant in Mꢁ1, Ct is the total metal concentration and s is the
binding site size. In plots of (eaꢁef)/(ebꢁef) versus [DNA], Kb is
given by the ratio of the slope to the intercept.
Viscosity measurements of complexes were carried out in
BPE-buffer (water bath maintained at 30 ꢃ 0.18C). Flow time
was measured with a digital stopwatch and every sample was
tested three times to get an average calculated time. The data are
presented as (Z/Z0)1/3 vs the concentration of [RuII]/[DNA],
where Z and Z0 are the viscosities of DNA in the presence and
absence of complex.
For the gel electrophoresis experiment, supercoiled pBR 322
DNA (0.1mg) was treated with the RuII complexes in TAE-buffer
(pH 8.0; 40mM Tris, 20mM acetic acid, 1 mM EDTA), and the
solution was then irradiated at room temperature with a UV lamp
(365nm, 10W). The samples were analysed by electrophoresis
for 1.5 h at 60V on a 0.8 % agarose gel in TAE-buffer. The
gel was stained with 1 mg mLꢁ1 ethidium bromide.
DNA Binding Assays
The DNA-binding experiments were performed at room
temperature. Buffer (5 mM Tris, 50 mM NaCl, pH ¼ 7.0; Tris ¼
tris(hydroxymethyl)aminomethane) was used for absorp-
tion and luminescence titrations and BPE-buffer (6 mM
Na2HPO4, 2 mM NaH2PO4, 1 mM Na2EDTA, pH ¼ 7; EDTA ¼
ethylenediaminetetraacetate) was used in viscosity measurements.
The UV absorbance of the CT-DNA at 260 and 280 nm in Tris-
buffer had a ratio of about ,1.9 : 1 indicating that the DNA was
Cell Culture and Cytotoxicity Assay
Standard MTT assay procedures were used,[27] to study the
effect of complexes on cell growth. A549, Du145, and HeLa cell

228
C. S. Devi et al.
16
14
12
10
8
lines growing exponentially were added to 96–well plates at a
density of 3 ꢂ 103 per well. After incubation for 24 h cells were
exposed to the tested compounds of serial dilutions. The com-
pounds were dissolved in dimethyl sulfoxide and diluted with
RPMI-1640 or DMEM to the required concentrations before
use. Growth inhibition assays were carried out over a 48 h
continuous exposure period. MTT was added to each well to
yield a working concentration of 0.4 mg mLꢁ1 and the plates
were kept in an incubator for 2 h. After this time the medium
was aspirated, 200 mL of DMSO was added to each well, and the
wells were agitated gently for 5 min before measuring
the absorbance at 620 nm. The IC50 values were determined
by plotting the percentage viability versus concentration on a
logarithmic graph and reading off the concentration at which
50 % of cells remain viable relative to the control.
140
120
100
80
6
4
2
0
0.4 0.5 0.6 0.7 0.8 0.9 1.0
r
60
40
20
590
600
610
620
630
Wavelength [nm]
Cellular Uptake and Apoptosis
Fig. 2. Emission spectra of 2 in Tris-buffer at 258C upon addition of
CT-DNA, [Ru] ¼ 20 mM, [DNA] ¼ 0–120 mM. The arrow shows the
increase in intensity upon increasing CT-DNA concentrations.
HeLa cells in growth medium were seeded in 35 mm tissue
culture dishes (Corning) and incubated at 378C under a 5 % CO2
atmosphere until 70 % confluency. The culture medium was
removed and replaced with fresh medium containing the
ruthenium(II) complexes at 10 mM (final DMSO concentration,
1 % v/v). After incubation for 1 h, the cell layer was trypsinised
and washed twice with cold phosphate buffered saline (PBS).
The samples were rinsed in 500 mL of cold PBS and analysed by
a flow cytometer immediately. The samples were collected
in FL2 channel (excitation at 488 nm and emission at
585–620 nm). For confocal imaging, HeLa cells were cultured
on coverslips (Corning, 22 ꢂ 50 mm), until they reached 70 %
confluency. The cells were incubated with complexes at 50 mM
concentration for 2 h, washed with PBS, and then photographed
with a confocal microscope. Confocal microscopy imaging was
carried out on one set without any staining and one set was
stained with DAPI 0.1 mg mLꢁ1 to identify localisation of the
complexes.
spectra using the modified Scatchard equation.[28] The binding
data obtained from the emission spectra were fitted using a plot
of r/Cf vs r, where r is the Cb/[DNA] and Cf is the concentration
of the free complex. The intrinsic binding constants K
5.91 ꢃ 0.3 ꢂ 104 Mꢁ1 for complex 1 and 5.02 ꢃ 0.25 ꢂ 104 Mꢁ1
for complex 2 were determined. Comparing these binding
constants we can see that complex 1 binds to DNA more avidly
than complex 2.
Steady-state emission quenching experiments using
K4[Fe(CN)6] as quencher may provide further information
about complexes binding to DNA, but cannot determine the
mode of binding. We performed these experiments at ambient
temperature, using a similar method as that described by
Satyanarayana et al.[29] As illustrated in Fig. 3, in the absence
of CT-DNA complex was efficiently quenched by [Fe(CN)6]4ꢁ
,
resulting in a linear Stern–Volmer plot. However, in the pres-
ence and excess of CT-DNA, it is difficult to be quenched, which
may be explained by the fact that the bound cation of RuII is
protected from the anionic water-bound quencher by the array of
negative charges along the DNA phosphate backbone.[30]
Results and Discussion
Synthesis and Characterisation
The ligand 4HEPIP was prepared by the condensation of 1,10-
phenanthroline-5,6-dione with 4-hydroxy-3-ethoxybenzaldehyde
and ammonium acetate in glacial acetic acid in 73 % yield.
The corresponding ruthenium(II) complexes were synthesised
by a direct reaction of [Ru(4HEPIP)Cl4] with the appropriate
precursor ligands and the desired complexes 1 and 2
(Fig. 1.) were synthesised as their perchlorate salts in 72 % yield
after purification by column chromatography. Spectral charac-
terisation of these complexes is given in the Experimental
Section.
Electronic Absorption Spectra Studies
Electronic absorption spectroscopy serves as the most common
means to study the interactions between metal complexes and
DNA.[31,32] A complex binding to DNA through intercalation
usually results in hypochromism and bathochromism, due to the
intercalation mode involving a strong stacking interaction
between an aromatic chromophore and the base pairs of DNA.
Absorption spectra of complex 1 in presence and absence of
DNA is shown in Fig. 4 (given in Fig. S2 for complex 2).
The hypochromism (H%) of the metal-to-ligand charge
transfer (MLCT) bands in complexes 1 and 2, defined as
H% ¼ 100 ꢂ (AfreeꢁAbound)/Afree, where A ¼ absorption, was
determined to be about 8.7 ꢃ 0.05 and 7.5 ꢃ 0.05 %, respec-
tively. In order to compare the DNA-binding affinities of the two
complexes quantitatively, their intrinsic binding constants Kb
were obtained by monitoring the changes to intra ligand (IL)
bands at 272, 279 nm for both complexes respectively.
Fluorescence Studies
In the absence of DNA, both complexes 1 and 2 can emit
luminescence in Tris buffer at ambient temperature, with
maxima appearing at 604 and 605 nm, respectively. The results
of the emission titrations for complex 2 in the absence and
presence of increasing concentration of DNA is illustrated in
Fig. 2 (given in Fig. S1 for complex 1). The enhancement of
emission intensity is an indication of binding of the complexes
to the hydrophobic pocket of DNA, and complexes can be
protected efficiently by the hydrophobic environment inside the
DNA helix. We have also calculated the binding constants of
the two complexes interacting with DNA from the emission
The calculated binding constants Kb are 5.81 ꢃ 0.15 ꢂ 104
and 5.01 ꢃ 0.22 ꢂ 104, for complex 1 and 2, revealing greater
DNA-binding affinity of complex 1 compared with 2. Since the

Ruthenium(II) Polypyridyl Complexes
229
Complex 1
Complex 2
1.9
1.9
(a)
(a)
1.7
1.5
1.3
1.1
1.7
1.5
1.3
1.1
0.9
0.7
(b)
(c)
(b)
(c)
0.9
0.7
0.5
0.5
0.0003
0.0008
0.0013
0.0018
0.0023
0.0003
0.0008
0.0013
0.0018
0.0023
K4[Fe(CN)]6 [mol LϪ1
]
K4[Fe(CN)]6 [mol LϪ1
]
Fig. 3. Emission quenching of complexes 1 and 2 with K4[Fe(CN)6] in the absence (a), presence (b) of [Ru] ¼ 20 mM, and with excess DNA (c).
5.0
0.60
4.5
0.55
0.50
0.45
2.2
2.0
1.8
4.0
3.5
3.0
0.40
1.6
2.5
2.0
0.35
0.14
1.4
1.2
1
2
0.16 0.18
0.20 0.22
[DNA]ϫ 105
1.5
1.0
0.5
1.0
0.8
0.6
0.4
0.2
.
Ϫ2.0
Ϫ1.5
Ϫ1.0
Ϫ0.5
Log[Naϩ]
225
300
375
450
525
Fig. 5. Salt dependence of the equilibrium binding constants for com-
plexes 1 and 2. Slopes are ꢁ1.21 and ꢁ1.30 respectively.
Wavelength [nm]
Fig. 4. Absorption spectra of complex 1 in Tris-buffer upon addition of
CT-DNA in absence (top) and presence (lower) of CT-DNA; [complex]
¼ 20 mM. The arrow shows change in absorption with increasing DNA
concentration. Inset: Plot of (eaꢁef)/(ebꢁef) versus [DNA] for the titration of
DNA with RuII complex.
complexes.[33] The results obtained from absorption spectra are
comparable to those obtained from emission binding constants:
both sets of binding constants show that complex 1 binds to DNA
more avidly than complex 2.
two complexes have the same intercalating ligand, the differ-
ence comes mostly from the ancillary ligands, where amino
groups in complex 1 are favourable to form H-bonding interac-
tions with the DNA. The two complexes in the current study
werefoundtohave smallerKb valuesthanthe reportedcomplexes
[Ru(L)4PIP]2þ ([Ru(4-APy)4(PIP)]2þ Kb ¼ 7.23ꢂ 104 Mꢁ1, [Ru
(Py)4PIP]2þ ¼ 6.5 ꢂ 104 Mꢁ1, where 4-APy ¼ 4-aminopyridine,
Py¼ pyridine, and PIP ¼ 2-phenyl,1H-imidazo[4,5-f][1,10]
(phenanthroline)),[18] since the intercalator for complexes 1 and
2 is (4HEPIP), which has additional groups such as hydroxyl and
ethoxy which may exert some steric hindrance. These steric
clashes then prevent the complexes from intercalating effectively
and thus cause a decrease of the intrinsic binding constants.
Hydroxyl and ethoxy are electron donor groups; this makes the
intercalating ligand more electron dense, hence interaction
between DNA and the complex decreases. These results are in
good agreement with the reported analogous ruthenium
Salt Dependent Studies
The clear dependence of the binding constants for these com-
plexes upon Naþ binding to DNA may be analysed by Record’s
polyelectrolyte theory.[34] From this theory, the slope of the lines
in Fig. 5 provide an estimate of Zc, where Z is the charge on the
complex and c is the fraction of counter ions[35] associated with
each DNA phosphate (c ¼ 0.88 for double-stranded B-form
DNA). The data in Fig. 5 show that the slopes of the lines are
greater than 1, being 1.41 and 1.36 for complexes 1 and 2
respectively. These values are less than the theoretically
expected value of Zc ¼ 2 ꢂ 0.88 ¼ 1.76. Lower values could
arise from coupled anion release (from the ligand) or from
changes in ligand or DNA hydration upon binding. By
increasing the Naþ concentrations,[35] the relative binding
affinities of the complexes decreased similar to that of proven
intercalators like ethidium bromide.[29]

230
C. S. Devi et al.
Viscosity Measurements
after incubation with with complexes and irradiation at 365 nm;
super coiled form (form I) was cleaved, generating the elec-
trophoretically slower-moving open circular form (form II).
With increasing concentrations of the complexes (Fig. 7, lanes
a-c and e-g), the amount of Form I DNA diminished gradually,
whereas form II increased. Further, a second unirradiated con-
trol solution confirmed that the complexes did cause photo-
sensitised cleavage: in the presence of the singlet oxygen (1O2)
scavenger histidine (lanes d and h), the activity of both the
complexes decreases, indicating that singlet oxygen is likely to
be the reactive species responsible for the cleavage reaction.
Similar results were observed for other ruthenium(II) poly-
pyridyl complexes.[11,38]
Though photophysical studies are quite useful in determining
binding constants of metal complexes to DNA,[29,36] to further
elucidate the binding mode of the present complexes, viscosity
measurements were carried out. A classical intercalation model
demands that the DNA helix lengthens as base pairs are sepa-
rated to accommodate the binding ligand, hence this leads to
an increase in the viscosity of DNA.[37] Ethidium bromide,
a well known DNA intercalator, increases the relative viscosity
strongly by lengthening the DNA double helix through inter-
calation. Similarly, upon increase in the amounts of complexes 1
and 2, the relative viscosity of DNA increases steadily. The
increased degree of viscosity, which may depend on the binding
affinity to DNA, follows the order EB .1 .2 (Fig. 6). These
results also suggest that both complexes intercalate between the
base pairs of DNA and parallel the results obtained by absorp-
tion, fluorescence, and quenching measurements. Based on the
binding data and the viscosity experiment we conclude that
these complexes bind to DNA by intercalation, but they do not
intercalate as strongly as proven intercalators like ethidium
bromide,[29] or analogous RuII complexes containing bidentate
polycyclic heteroaromatic ligands.[9–11]
Cytotoxicity Assay
The potential antiproliferative effects of complexes 1 and 2 on
the viability of tumour cell lines (A549, Du145, and HeLa) were
assessed by the MTT assay. Cisplatin was used as a positive
control. The concentrations which showed 50 % (IC50) inhibi-
tion of the cell viability were calculated and the results showed
that IC50 values are 39.5, 28.3, and 27.3 mM for 1, and 55, 67.9,
and 47.9 mM for 2, respectively. It is clear that the selected
tumour cells are more sensitive to complex 1 than complex 2;
however these complexes exhibit relatively lower in vitro
cytotoxicity than cisplatin (12.3, 6.5, and 19.2 respectively). The
cytotoxicity of 1 and 2 was found to be concentration dependent,
DNA Photocleavage
The potential of the present complexes to cleave plasmid DNA
was studied by gel-electrophoresis using super coiled pBR322
DNA. No DNA cleavage was observed for control (unirradiated
DNA) in which the complexes were absent (Fig. 7, lane 0). Gel
electrophoresis separation of pBR322 DNA was undertaken
Control
3.0
Complex 2
Complex 1
EtBr
2.5
1
2.0
2
1.5
1.0
0.5
0.013
0.033
0.053
0.073
0.093
0.113
RuII/[DNA]
100
101
102
103
104
Red fluorescence after 1 h
Fig. 6. Effect of increasing amounts of ethidium bromide (EtBr) and
complexes 1 and 2 on relative viscosity of CT-DNA at 30 ꢃ 0.18C. The total
concentration of DNA is 0.25 mM.
Fig. 8. Flow cytometric results of HeLa cells incubated with untreated
cells (control) and complexes 1 and 2 at 378C for 1 h.
Complex 1 [μM]
Complex 2 [μM]
20
20
40
1ϩ His
40
2ϩ His
60
60
Form II
Form I
0
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Fig. 7. Photocleavage of pBR322 DNA in the presence of 1 and 2, after irradiation with UV light at 365 nm. Lane 0 is
control (unirradiated pBR322), lanes (a)–(c) and (e)–(g) include addition of 20, 40, and 60 mM solutions of complexes 1
and 2 respectively. Lanes (d) and (h) are complexes 1 and 2 in presence of histidine (2 mM).

Ruthenium(II) Polypyridyl Complexes
231
as cell viability decreased with increasing concentration of the
complexes. The IC50 values obtained for both 1 and 2 are in good
agreement with the reported ruthenium(II) polypyridyl
complexes.[11,14]
Cellular Uptake
The cellular uptake potential of both complexes was studied
in HeLa cells using flow cytometry and confocal micro-
scopy.[39–41] The uptake profile of complexes 1 and 2 in HeLa
DAPI stained
Bright
Control
cells
1,
50 μM
3 h
2,
50 μM
3 h
Fig. 9. Confocal images of control (untreated) cells, and cells treated with 1 and 2. DAPI stained, bright, and fluorescent images. Cells were incubated with a
50 mM solution of complex 1 and observed by confocal microscopy (excitation, 488 nm; emission, 600–620 nm).
104
103
104
103
Q1
0.32 %
Q2
3.59 %
Q1
0.38 %
Q2
4.97 %
102
102
101
100
101
100
Q4
91.6 %
Q3
4.54 %
Q4
86.1 %
Q3
9.56 %
100
101
102
103
104
100
101
102
103
104
Annexin V
Annexin V
CONTROL HeLa (UNTREATED) 14 h
COMPOUND 1 10 μM 14 h
Fig. 10. Nexin (Annexin V and 7-AAD) staining showing differentiated live (Q4), early apoptotic (Q3), late apoptotic (Q2), and dead (Q1) HeLa
cells after treatment with complex 1 for 14 h. The percent of apoptotic cells were detected by analysing with a flow cytometer using Express Pro
and Flow Jo softwares.

232
C. S. Devi et al.
cells incubated with 10 mM solutions of the complexes for 2 h is
shown in Fig. 8. The Median-red fluorescence intensity (MFI)
values for complexes 1, 2, and untreated cells (control)
were 19.5, 10.11, and 1.1, respectively. These results clearly
demonstrate that both complexes have efficient cellular
uptake properties, with complex 1 having better uptake than
complex 2. The cellular distribution was studied by confocal
microscopy. Complexes 1 and 2 (50 mM) gradually penetrated
into the interior of the nucleus and shown diffuse cytoplasm
and nuclear fluorescence after 2 h of incubation with HeLa
cells. Control cells exhibit homogeneous nuclear staining, and
treated cells display typical changes such as fragmented nuclei
or shrinkage indicating DNA cleavage. Confocal images of the
control (cells treated with vehicle 1 % DMSO) and treated with
complex (their fluorescent and DAPI stained) are given in
Fig. 9. The results show that both the complexes can be uptaken
by HeLa cells, and they enter into the cytoplasm and accu-
mulate in the nuclei.
Supplementary Material
Emission spectra of complex 1 and electronic absorption spectra
of complex 2 are available on the Journal’s website.
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