Synthesis, characterization, cytotoxicity, apoptotic inducing activity, cellular uptake, interaction of DNA binding and antioxidant activity studies of ruthenium(II) complexes

Article abstract of DOI:10.1016/j.ica.2012.01.003

Two new ligands APIP, HAPIP and their relative ruthenium(II) complexes [Ru(bpy)₂(APIP)](ClO₄)₂ 1 and [Ru(bpy) ₂(HAPIP)](ClO₄)₂ 2 have been synthesized and characterized. The DNA binding con

Full text of DOI:10.1016/j.ica.2012.01.003

Inorganica Chimica Acta 387 (2012) 117–124
Contents lists available at SciVerse ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis, characterization, cytotoxicity, apoptotic inducing activity,
cellular uptake, interaction of DNA binding and antioxidant activity studies
of ruthenium(II) complexes
a
b
a
c
d,
Yun-Jun Liu a, , Zhen-Hua Liang , Xian-Lan Hong , Zheng-Zheng Li , Jun-Hua Yao , Hong-Liang Huang
⇑
⇑
a School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
b Department of Chemistry, Shaoguan University, Shaoguan 512005, PR China
c Instrumentation Analysis and Research Center, Sun Yat-Sen University, Guangzhou 510275, PR China
d School of Life Science and Biopharmaceutical, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new ligands APIP, HAPIP and their relative ruthenium(II) complexes [Ru(bpy)2(APIP)](ClO4)2 1 and
[Ru(bpy)2(HAPIP)](ClO4)2 2 have been synthesized and characterized. The DNA binding constants for
Received 28 January 2011
Received in revised form 7 October 2011
Accepted 4 January 2012
complexes 1 and 2 have been determined to be 6.08 ( 0.29) ꢀ 104 Mꢁ1 and 1.78 ( 0.30) ꢀ 105 Mꢁ1
.
The results suggest that these complexes intercalate between the base pairs of DNA. The cytotoxicity
of complexes has been evaluated by MTT assay. The apoptosis assay was carried out with AO/EB staining
methods. The cellular uptake was observed under fluorescence microscopy. The studies on the mecha-
nism of photocleavage demonstrate that superoxide anion radical (O2ꢀꢁ) and singlet oxygen (1O2) may
play an important role. The antioxidant activity against hydroxyl radical (ꢀOH) was also studied.
Ó 2012 Elsevier B.V. All rights reserved.
Available online 13 January 2012
Keywords:
Ruthenium(II) complexes
DNA-binding
Cytotoxicity
Apoptosis
Cellular uptake
Antioxidant activity
1. Introduction
external static electronic effects. The studies on the DNA-binding
behaviors of ruthenium complexes have attracted great attention,
In recent years, many researchers have focused on the studies of
interaction of small molecules with DNA [1–7]. DNA is generally
the primary intracellular target of anticancer drugs, so the interac-
tion between small molecules and DNA can cause DNA damage in
cancer cells, blocking the division of cancer cells, and resulting in
cell death [8,9]. The studies on the interaction of transition metal
complexes with DNA continue to attract the attention of researcher
due to their importance in design and development of synthetic
restriction enzymes, chemotherapeutic drugs and DNA foot print-
ing agents, DNA cleavage agents and DNA ‘‘molecular light switch’’
[10–14]. Generally, small molecule can interact with DNA through
the three non-covalent modes: groove binding, intercalation, and
and some of them exhibit interesting properties. However, little
is known about their in vitro behaviors. Only a few studies have
focused on their intracellular accumulation and antiproliferative
properties [15–19]. These studies found some complexes to
possess excellent antitumor activity and might be as potential
candidate for drugs. In fact, the activity of many anticancer, anti-
malarial and antibacterical agents finds its origin in intercalative
interactions with DNA [20]. In this study, we report the synthesis,
characterization, DNA-binding, cytotoxicity, apoptosis, cellular up-
take and antioxidant activity of two new ruthenium(II) polypyridyl
complexes [Ru(bpy)2(APIP)](ClO4)2 1 (bpy = 2,20-bipyridine, APIP =
2-(2-aminophenyl)imidazo[4,5-f][1,10]phenanthroline) and [Ru-
(bpy)2(HAPIP)](ClO4)2 2 (HAPIP = 2-(2-hydroxyl-5-aminophenyl)-
imidazo[4,5-f][1,10]phenanthroline, Scheme 1). The DNA-binding
behaviors were studied by spectroscopic titration, viscosity mea-
surements, thermal denaturation and photocleavage. The spectro-
scopic titration and viscosity changes of calf thymus DNA (CT DNA)
show these complexes interact with DNA through intercalative
mode. The cytotoxicity of complexes has been evaluated by MTT
(3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) as-
say. The apoptosis of BEL-7402 cells induced by Ru(II) complexes
was investigated. The retardation assay of pGL 3 plasmid DNA by
Abbreviations: APIP, 2-(2-aminophenyl)imidazo[4,5-f][1,10]phenanthroline;
HAPIP, 2-(2-hydroxyl-5-aminophenyl)imidazo[4,5-f][1,10]phenanthroline; CT DNA,
calf thymus DNA; bpy, 2,20-bipyridine; AO, acridine orange; EB, ethidium bromide;
DMSO, dimethylsulfoxide; RPMI, Roswell Park Memorial Institute; MLCT, metal to
ligand charge transfer; Tris, tris(hydroxymethyl)aminomethane; ES-MS, electrospray
mass spectroscopy; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide.
⇑
Corresponding authors.
E-mail addresses: lyjche@163.com (Y.-J. Liu), hhongliang@163.com (H.-L.
Huang).
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2012.01.003

118
Y.-J. Liu et al. / Inorganica Chimica Acta 387 (2012) 117–124
Scheme 1. The structure of complexes.
complexes 1 and 2 was also explored. The cellular uptake shows
that complexes can easily enter into the cytoplasm and accumulate
in the nuclei. The antioxidant activity of ligands and complexes was
performed by hydroxyl radical scavenging method. The studies on
the mechanism of photocleavage reveal that singlet oxygen (1O2)
and superoxide anion radical (O2ꢀꢁ) may play an important role.
obtained was washed with cold ethanol and dried at 50 °C in vac-
cuo. Yield: 73%. Anal. Calc. for C19H13N5: C, 73.30; H, 4.21; N, 22.49.
Found: C, 73.16; H, 4.13; N, 22.38%. FAB–MS: m/z = 312 [M+1]+. 1H
NMR (500 MHz, DMSO-d6): 9.05 (d, 2H, Hc, J = 8.0 Hz), 8.98 (d, 2H,
Ha, J = 8.2 Hz), 8.02–8.05 (m, 2H, Hb), 7.83 (d, 1H, Hg, J = 8.0 Hz),
7.18–7.35 (m, 1H, He), 6.92 (d, 1H, Hf, J = 8.0 Hz), 6.74 (t, 1H, Hd,
J = 7.6 Hz), 3.36 (s, 2H, HNH2).
2. Experimental
2.2.2. 2-(2-Hydroxyl-5-aminophenyl)imidazo[4,5-f][1,10]phenan-
throline (HAPIP)
2.1. Materials and methods
This compound was prepared with the similar method de-
scribed for APIP, with 2-(2-hydroxyl-5-nitrophenyl)imidazo[4,5-
f][1,10]phenanthroline (0.179 g, 0.5 mmol) [24] in place of APIP.
Yield: 72%. Anal. Calc. for C19H13N5O: C, 69.72; H, 4.00; N, 21.39.
Found: C, 69.60; H, 4.13; N, 21.52%. FAB–MS: m/z = 328 [M+1]+.
1H NMR (500 MHz, DMSO-d6): 9.06 (d, 2H, Hc, J = 8.2 Hz), 8.98 (d,
2H, Ha, J = 7.5 Hz), 7.87 (dd, 2H, Hb, J = 8.0 Hz), 7.43 (d, 1H, Hd,
J = 8.0 Hz), 6.84 (d, 1H, Hf, J = 8.6 Hz), 6.73–6.75 (m, 1H, He), 4.78
(s, 1H, HOH), 3.39 (s, 2H, HNH2).
Calf thymus DNA (CT DNA) was obtained from the Sino-
American Biotechnology Company. pBR 322 DNA was obtained
from Shanghai Sangon Biological Engineering & Services Co., Ltd.
Dimethyl sulfoxide (DMSO) and RPMI 1640 were purchased from
Sigma. Cell lines of BEL-7402 (hepatocellular), HepG-2 (hepatocel-
lular) and MCF-7 (breast cancer) were purchased from American
Type Culture Collection, agarose and ethidium bromide were ob-
tained from Aldrich. RuCl3ꢂxH2O was purchased from Kunming
Institution of Precious Metals. 1,10-Phenanthroline was obtained
from Guangzhou Chemical Reagent Factory. Doubly distilled water
was used to prepare buffers (5 mM tris(hydroxymethylaminome-
thane)–HCl, 50 mM NaCl, pH 7.2). A solution of calf thymus DNA
in the buffer gave a ratio of UV absorbance at 260 and 280 nm of
ca. 1.8–1.9:1, indicating that the DNA was sufficiently free of pro-
tein [21]. The DNA concentration per nucleotide was determined
by absorption spectroscopy using the molar absorption coefficient
(6600 Mꢁ1 cmꢁ1) at 260 nm [22].
2.2.3. Synthesis of [Ru(bpy)2(APIP)](ClO4)2 (1)
[Ru(bpy)2(NPIP)](ClO4)2 (0.485 g, 0.5 mmol) [23] was com-
pletely dissolved in acetonitrile. Then the ethanol of 45 cm3, Pd/C
(0.20 g, 10% Pd) and NH2NH2ꢂH2O (8 cm3) were added in the above
solution and refluxed under argon for 8 h to give a clear red solu-
tion. Upon cooling, a red precipitate was obtained by dropwise
addition of saturated aqueous NaClO4 solution. The crude product
was purified by column chromatography on a neutral alumina with
a mixture of CH3CN-toluene (3:1, v/v) as eluant. The mainly red
band was collected. The solvent was removed under reduced pres-
sure and a red powder was obtained. Yield: 71%. Anal. Calc. for
Microanalysis (C, H, and N) was carried out with a Perkin-Elmer
240Q elemental analyzer. Fast atom bombardment (FAB) mass
spectra were recorded on
a VG ZAB-HS spectrometer in a
C
39H29N9Cl2O8Ru: C, 50.71; H, 3.16; N, 13.65. Found: C, 50.48; H,
3-nitrobenzyl alcohol matrix. Electrospray mass spectra (ES-MS)
were recorded on a LCQ system (Finnigan MAT, USA) using meth-
anol as mobile phase. The spray voltage, tube lens offset, capillary
voltage and capillary temperature were set at 4.50 kV, 30.00 V,
23.00 V and 200 °C, respectively, and the quoted m/z values are
for the major peaks in the isotope distribution. 1H NMR spectra
were recorded on a Varian-500 spectrometer. All chemical shifts
were given relative to tetramethylsilane (TMS). UV–Vis spectra
were recorded on a Shimadzu UV-3101PC spectrophotometer at
room temperature. Luminescence spectra were recorded on a
Shimadzu RF-2000 spectrophotometer at room temperature.
3.28; N, 13.59%. ESI-MS [CH3CN, m/z]: 723.4 ([M–2ClO4–H]+),
362.3 ([M–2ClO4]2+). 1H NMR (500 MHz, DMSO-d6): d 8.91 (dd,
4H, H3,3 , J = 8.4, J = 8.5 Hz), 8.80 (d, 2H, Hc, J = 8.2 Hz), 8.21–8.27
(m, 4H, H4,4 ), 8.14 (t, 2H, J = 7.5 Hz), 7.85 (d, 4H, H6,6 , J = 6.0 Hz),
7.58–7.66 (m, 2H, Hb), 7.36 (t, 4H, H5,5 , J = 7.6 Hz), 7.20 (t, 1H,
Hd, J = 7.5 Hz), 6.98–7.03 (m, 1H, Hf), 6.92 (t, 1H, He, J = 7.8 Hz),
6.72 (t, 1H, Hg, J = 7.8 Hz), 3.37 (s, 2H, HNH2).
0
0
0
0
2.2.4. Synthesis of [Ru(bpy)2(HAPIP)](ClO4)2 (2)
A mixture of cis-[Ru(bpy)2Cl2]ꢂ2H2O [25] (0.260 g, 0.5 mmol)
and HAPIP (0.164 g, 0.5 mmol) in ethanol (30 cm3) was refluxed
under argon for 8 h to give a clear red solution. Upon cooling, a
red precipitate was obtained by dropwise addition of saturated
aqueous NaClO4 solution. The crude product was purified by
column chromatography on a neutral alumina with a mixture of
CH3CN-toluene (3:1, v/v) as eluant. The mainly red band was
collected. The solvent was removed under reduced pressure
and a red powder was obtained. Yield: 70%. Anal. Calc. for
2.2. Synthesis of ligands and complexes
2.2.1. 2-(2-Aminophenyl)imidazo[4,5-f][1,10]phenanthroline (APIP)
A mixture of 2-(2-nitrophenyl)imidazo[4,5-f][1,10]phenanthro-
line (0.171 g, 0.5 mmol) (NPIP) [23] was completely dissolved in
ethanol (50 cm3) with stirring for 1 h. Then the Pd/C (0.20 g, 10%
Pd) and NH2NH2ꢂH2O (8 cm3) were added in the above solution
and refluxed for 6 h. The hot solution was filtered and evaporated
to remove the solvent under reduced pressure. The red compound
C
39H29N9Cl2O9Ru: C, 49.85; H, 3.11; N, 13.42. Found: C, 50.28; H,
3.54; N, 13.48%. ESI-MS [CH3CN, m/z]: 739.7 ([M–2ClO4–H]+),

Y.-J. Liu et al. / Inorganica Chimica Acta 387 (2012) 117–124
119
370.4 ([M–2ClO4]2+). 1H NMR (500 MHz, DMSO-d6): d 8.88 (t, 4H,
and the solution was then irradiated at room temperature with a
UV lamp (365 nm, 10 W). The samples were analyzed by electro-
phoresis for 1.5 h at 80 V on a 1.0% agarose gel in TBE (89 mM
Tris–borate acid, 2 mM EDTA, pH 8.3). The gel was stained with
0
H
3,3 , J = 5.0 Hz), 8.84 (d, 2H, Hc, J = 8.0 Hz), 8.21 (t, 2H, H4,
J = 7.0 Hz), 8.11 (t, 2H, H4’, J = 7.0 Hz), 7.88 (d, 2H, Ha, J = 8.0 Hz),
0
7.80 (d, 2H, H6, J = 7.8 Hz), 7.74 (d, 2H, H6 , J = 7.7 Hz), 7.65 (d,
0
1H, Hf, J = 7.5 Hz), 7.59 (t, 4H, H5,5 , J = 5.0 Hz), 7.36 (t, 2H, Hb,
1 lg/ml ethidium bromide and photographed on an Alpha Inno-
J = 6.0 Hz), 6.63 (d, 1H, He, J = 8.5 Hz), 6.48 (d, 1H, Hh, J = 7.5 Hz),
4.52 (s, 1H, HOH), 3.37 (s, 2H, HNH2).
tech IS-5500 fluorescence chemiluminescence and visible imaging
system.
Caution: Perchlorate salts of metal compounds with organic li-
gands are potentially explosive, and only small amounts of the
material should be prepared and handled with great care.
2.4. Cytotoxicity assay
Cytotoxicity of APIP, HAPIP, complexes 1 and 2 was evaluated
used standard MTT (3-(4,5-dimethylthiazole)-2,5-diphenyltetra-
azolium bromide) assay [31]. Cells were placed in 96-well microas-
say culture plates (8 ꢀ 103 cells per well) and grown overnight at
37 °C in a 5% CO2 incubator. Compounds tested were then added
to the wells to achieve final concentrations ranging from 6.25 to
2.3. DNA-binding studies
The DNA-binding and photoactivated cleavage experiments
were performed at room temperature. Buffer A (5 mM tris-
(hydroxymethyl)aminomethane (Tris) hydrochloride, 50 mM NaCl,
pH 7.0) was used for absorption titration, luminescence titration
and viscosity measurements. Buffer B (50 mM Tris–HCl, 18 mM
NaCl, pH 7.2) was used for DNA photocleavage experiments. Buffer
C (1.5 mM Na2HPO4, 0.5 mM NaH2PO4, 0.25 mM Na2EDTA, pH 7.0)
was used for thermal DNA denaturation experiments. Buffer D
(0.9% of physiological saline) was used for retardation assay of
pGL 3 plasmid DNA.
400
lM. Control wells were prepared by addition of culture med-
ium (100
lL). Wells containing culture medium without cells were
used as blanks. The plates were incubated at 37 °C in a 5% CO2
incubator for 48 h. Upon completion of the incubation, stock MTT
dye solution (20
4 h incubation, buffer (100
l
L, 5 mg mLꢁ1) was added to each well. After
lL) containing N,N-dimethylformamide
(50%) and sodium dodecyl sulfate (20%) was added to solubilize the
MTT formazan. The optical density of each well was then measured
on a microplate spectrophotometer at a wavelength of 490 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. Each experiment was repeated at least three times to get
the mean values. Three different tumor cell lines were the subjects
of this study: BEL-7402 (hepatocellular), HepG-2 (hepatocellular)
and MCF-7 (breast cancer) (purchased from American Type Culture
Collection).
The absorption titrations of the complex in buffer were per-
formed using a fixed concentration (20 lM) for complex to which
increments of the DNA stock solution were added. Ru-DNA solu-
tions were allowed to incubate for 5 min before the absorption
spectra were recorded. The intrinsic binding constants K, based
on the absorption titration, were measured by monitoring the
changes of absorption in the MLCT band with increasing concen-
tration of DNA using the following equation [26]:
½DNAꢃ=ðea
ꢁ
ef Þ ¼ ½DNAꢃ=ðeb
ꢁ
ef Þ þ 1=½Kbðeb
ꢁ
ef Þꢃ
ð1Þ
where [DNA] is the concentration of DNA in base pairs, ea
;
ef and eb
correspond to the apparent absorption coefficient Aobsd/[Ru], the
extinction coefficient for the free ruthenium complex and the
extinction coefficient for the ruthenium complex in the fully bound
2.5. Apoptosis study
Apoptosis studies were performed with a staining method
utilizing acridine orange (AO) and ethidium bromide (EB) [32].
According to the difference in membrane integrity between necro-
tic and apoptosis. AO can pass through cell membrane, but EB
cannot. Under fluorescence microscope, live cells appear green.
Necrotic cells stain red but have a nuclear morphology resembling
that of viable cells. Apoptosis cells appear green, and morphologi-
cal changes such as cell blebbing and formation of apoptotic bodies
will be observed.
form, respectively. In plots of [DNA]/(ea
given by the ratio of slope to the intercept.
ꢁ
ef ) versus [DNA], Kb is
Thermal denaturation studies were carried out with a Perkin-
Elmer Lambda 35 spectrophotometer equipped with a Peltier
temperature-controlling programmer ( 0.1 °C). The melting
temperature (Tm) was taken as the mid-point of the hyperchromic
transition. The melting curves were obtained by measuring the
absorbance at 260 nm for solutions of CT-DNA (80
lM) in the ab-
sence and presence of the Ru(II) complex (30 M) as a function
l
A monolayer of BEL-7402 cells was incubated in the absence or
of the temperature. The temperature was scanned from 40 to
90 °C at a speed of 1 °C minꢁ1. The data were presented as
(A ꢁ A0)/(Af ꢁ A0) versus temperature, where A, A0, and Af are the
observed, the initial, and the final absorbance at 260 nm,
respectively.
presence of complex 2 at concentration of 50
CO2 for 48 h. After 48 h, each cell culture was stained with AO/EB
solution (100
g mlꢁ1 AO, 100 g mlꢁ1 EB). Samples were observed
lM at 37 °C and 5%
l
l
under a fluorescence microscope.
Viscosity measurements were carried out using an Ubbelodhe
viscometer maintained at a constant temperature at 25.0 ( 0.1)
°C in a thermostatic bath. DNA samples approximately 200 base
pairs in average length were prepared by sonicating in order to
minimize complexities arising from DNA flexibility [27]. Flow time
was measured with a digital stopwatch, and each sample was mea-
sured three times, and an average flow time was calculated. Rela-
tive viscosities for DNA in the presence and absence of complexes
2.6. Cellular uptake
Cells were placed in 24-well microassay culture plates (4 ꢀ 104
cells per well) and grown overnight at 37 °C in a 5% CO2 incubator.
Complexes tested were then added to the wells. The plates were
incubated at 37 °C in a 5% CO2 incubator for 48 h. Upon completion
of the incubation, the wells were washed three times with phos-
phate buffered saline (PBS), after removing the culture media in
the wells. The cells were visualized by fluorescent microscopy.
were calculated from the relation
g
= (t ꢁ t0)/t0, where t is the ob-
served flow time of the DNA-containing solution and t0 is the flow
time of buffer alone [28,29]. Data were presented as (
g/g
)
1/3 versus
2.7. Scavenger measurements of hydroxyl radical (ꢀOH)
binding ratio [30], where is the viscosity of DNA in the presence
of complexes and g0 is the viscosity of DNA alone.
For the gel electrophoresis experiment, supercoiled pBR 322
DNA (0.1 lg) was treated with the Ru(II) complexes in buffer B,
g
Scavenger measurements of hydroxyl radical (ꢀOH) were carried
out in aqueous media. The hydroxyl radical (ꢀOH) was generated by
the Fenton system [33]. The solution of the tested complexes was

120
Y.-J. Liu et al. / Inorganica Chimica Acta 387 (2012) 117–124
prepared with DMF (N,N-dimethylformamide). The 5 ml of assay
mixture contained following reagents: safranin (28.5 M), EDTA-
Fe(II) (100 M), H2O2 (44.0 M), the tested compounds (0.5–
4.5 M) and a phosphate buffer (67 mM, pH 7.4). The assay mix-
tures were incubated at 37 °C for 30 min in a water bath. After
which, the absorbance was measured at 520 nm. All the tests were
run in triplicate and expressed as the mean. Ai was the absorbance
in the presence of the tested compound; A0 was the absorbance in
the absence of tested compounds; Ac was the absorbance in the
absence of tested compound, EDTA-Fe(II), H2O2. The suppression
absorbance in the MLCT band with increasing concentration of
CT DNA. The values of K are 6.08 ( 0.29) ꢀ 104 Mꢁ1 and 1.78
( 0.29) ꢀ 105 Mꢁ1 for 1 and 2. It is obvious that the K values of
complexes 1 and 2 are comparable with those of other known
DNA-intercalative complexes: [Ru(NH3)4(dppz)]2+ (1.24 ꢀ 105)
l
l
l
l
[39], [Ru(bpy)2(PPIP)]2+ (4.30 ꢀ 104 Mꢁ1
) [40] and [Ru(dmb)2-
(DBHIP)]2+ (8.64 0.16 ꢀ 104 Mꢁ1) [41], but is not as large as that
of classical DNA-intercalator [Ru(bpy)2(dppz)]2+ (4.90 ꢀ 106 Mꢁ1
)
[42].
ratio (ga) was calculated on the basis of (Ai ꢁ A0)/(Ac ꢁ A0) ꢀ 100%.
3.3. Luminescence spectra
3. Results and discussion
The luminescence spectra have been confirmed to be effective
for characterizing the binding mode of the metal complexes to
DNA [43,44]. Complexes 1 and 2 can emit luminescence in Tris–
HCl buffer at ambient temperature with a maximum appearing
at 599 and 600 nm. The enhancements in the emission intensity
of complexes 1 and 2 with increasing CT DNA concentrations are
given in Fig. 3. Upon addition of DNA, the emission intensities of
complexes 1 and 2 increase about 1.63 and 1.88 times larger than
the original and saturate at a ration of [DNA]/[Ru] = 15.4 and 14.6,
respectively. The phenomenon was related to the extent to which
the complexes penetrate into the hydrophobic environment inside
the DNA, thereby avoiding the quenching effect of solvent water
molecules. The binding constants of the complexes interacting
with DNA from the emission spectra can be derived using the lumi-
nescence titration method. The binding data obtained from the
emission spectra were fitted using the McGhee and von Hippel
equation [45] to acquire the binding parameters. The intrinsic
binding constants Kb of 2.91 ( 0.22) ꢀ 104 (s = 1.01) and 3.19
( 0.18) ꢀ 105 Mꢁ1 (s = 1.05) for complexes 1 and 2 were deter-
mined. Comparing with that obtained from absorption spectra,
the binding constants obtained from luminescence titration with
McGhee–von Hippel method are the same magnitude with that ob-
tained from absorption titration method.
3.1. Viscosity measurements
To investigate the DNA-binding mode of complexes 1 and 2, vis-
cosity measurements on solutions of CT DNA incubated with the
complexes were carried out. It is well known that a classical inter-
calation of a ligand into DNA is known to cause a significant in-
crease in the viscosity of a DNA solution due to an increase in
the separation of the base pairs at the intercalative site and, hence,
an increase in the overall DNA molecular length [29]. Fig. 1 shows
the change in the relative viscosity of CT DNA on addition of com-
plexes 1 and 2 together with those of [Ru(bpy)3]2+. It is well known
complex [Ru(bpy)3]2+ to bind with DNA only through electrostatic
mode. No obvious effect of [Ru(bpy)3]2+ on the relative viscosity of
the DNA solution was observed. On increasing the amounts of
complexes 1 and 2, the relative viscosity of CT DNA solution in-
crease steadily. These results suggest that complexes 1 and 2 inter-
calate between the base pairs of DNA. Similar results were also
observed for the other complexes [34–36].
3.2. Absorption spectra titration
Electronic absorption spectroscopy is one of the most useful
techniques for DNA-binding studies of metal complexes [37,38].
The absorption spectra of the complexes 1 and 2 (20 lM) in the
3.4. Thermal denaturation study
presence of CT DNA are shown in Fig. 2. As the DNA concentration
is increased, the MCLT (metal-to-ligand charge transfer) transition
bands of complexes 1 at 458 and 2 at 459 nm exhibit hypochro-
mism of about 30.48 and 17.87%, and bathchromism of 2 and
3 nm, respectively. These spectral characteristics may suggest a
mode of binding that involves a stacking interaction between the
aromatic chromophore and the DNA base pairs. In order to
elucidate the binding strength of the complexes, the DNA-binding
constants K were determined by monitoring the changes of
Generally, the melting temperature of DNA increases when me-
tal complexes bind to DNA by intercalation, as intercalation of
complexes between DNA base pairs can cause stabilization of base
stacking and hence raises the melting temperature of double-
strand DNA. As is shown in Fig. 4, in the absence of any added com-
plexes, the thermal denaturation carried out for DNA gave a Tm of
61.59 0.5 °C under our experimental conditions. In the presence
of complexes 1 and 2, the Tm is 66.66 0.5 and 68.23 0.5 °C.
The melting point increased by +5.07 for complex 1 and +6.64 °C
for complex 2. The increase in Tm of DNA with the two Ru(II) com-
plexes are comparable to those observed for classical intercalators
[46,47].
3.5. Photocleavage by Ru(II) complexes
It is well known that DNA cleavage is controlled by relaxation of
supercoiled circular conformation of pBR 322 DNA to nicked circu-
lar and/or linear conformations. When electrophoresis is applied to
circular plasmid DNA, the fastest migration will be observed for
DNA of closed circular conformation (Form I). If one strand is
cleaved, the supercoil will relax to product a slower moving nicked
conformation (Form II). If both strands are cleaved, a linear confor-
mation (Form III) will be generated that migrates in between. As
shown in Fig. 5, no obvious cleavage was observed for the control
in which metal complexes was absent (DNA alone), or incubation
of the plasmid with the Ru(II) complexes in the dark. With increas-
ing concentration of complexes, the Form I decrease and Form II
Fig. 1. Effect of increasing amounts of complexes [Ru(bpy)3]2+ (N), 1 (j) and 2 (d)
on the relative viscosity of calf thymus DNA at 25 ( 0.1) °C. [DNA] = 0.25 mM.

Y.-J. Liu et al. / Inorganica Chimica Acta 387 (2012) 117–124
121
Fig. 2. Absorption spectra of complex 1 (a) and 2 (b) in Tris–HCl buffer upon addition of CT-DNA. [Ru] = 20
concentration. Plots of [DNA]/(ea f ) versus [DNA] for the titration of DNA with Ru(II) complexes.
lM. Arrow shows the absorbance change upon the increase of DNA
ꢁ
e
Fig. 3. Luminescence spectra of complexes 1 (a) and 2 (b) in Tris–HCl buffer upon addition of CT DNA. [Ru] = 5 lM. Arrow shows the intensity change upon the increase of
DNA concentration. Inset: Scatchard plot of each complex.
of DNA was improved. Partial inhibition of DNA cleavage was ob-
served in presence of histidine, suggesting that 1O2 is likely to be
the reactive species responsible for the cleavage reaction. These re-
sults demonstrate that superoxide anion radical (O2ꢀꢁ) and singlet
oxygen (1O2) may play important role in the cleavage of the plas-
mid DNA.
3.6. Condensation of pGL 3 plasmid DNA by Ru(II) complexes
When the positively charged cationic metal complexes meet
the negatively charged plasmid DNA, adduct of complex-DNA is
formed by the condensation process between the metal complexes
and DNA. The abilities of cationic metal complexes to condense
plasmid DNA into particular structure is a primary prerequisite
for efficient gene transfection and cellular uptake. In order to
evaluate the abilities of complexes 1 and 2 to condense DNA, the
retardation of pGL 3 plasmid DNA was performed. Fig. 7 shows
when the concentrations of complexes 1 and 2 are 1 and 2 mM,
both complexes cannot condense the DNA, however, at high con-
centrations of 6 mM for complex 1, or 4 and 6 mM for complex
2, the effects of condensation of DNA were obviously observed.
Fig. 4. Thermal denaturation of CT DNA in the absence (j) and presence of
complexes 1 (d) and 2 (N). [Ru] = 30 M, [DNA] = 80 M.
l
l
increase gradually. At the concentration of 16
can completely cleave the plasmid DNA.
lM, both complexes
To investigate the role of radicals in the DNA cleavage by the
complexes, reactions were carried out by incubating the complexes
1 and 2 with DNA in presence of hydroxyl radical scavengers
(D
-mannitol and DMSO), singlet oxygen scavenger (
L-histidine),
3.7. Cytotoxicity assay in vitro
and a superoxide scavenger (superoxide dismutase, SOD). Fig. 6
shows that the DNA cleavage of the plasmid by complexes 1 and
2 was not inhibited in the presence of hydroxyl radical (ꢀOH) scav-
engers such as mannitol and DMSO, which indicated that hydroxyl
radical was not likely to be the cleaving agent. In the presence of
SOD, a facile superoxide anion radical (O2ꢀꢁ) quencher, the cleavage
The potential antiproliferative effects of complexes 1 and 2 on
the viability of tumor cell lines (BEL-7402, HepG-2, and MCF-7)
were detected by the MTT assay. Cisplatin was used as a positive
control. The concentrations which showed 50% (IC50) inhibition
of the cell viability were calculated and the results were listed in

122
Y.-J. Liu et al. / Inorganica Chimica Acta 387 (2012) 117–124
Fig. 5. Photoactivated cleavage of pBR 322 DNA in the presence of different concentrations of Ru(II) complexes after irradiation at 365 nm for 30 min.
Fig. 6. Photoactivated cleavage of supercoiled pBR 322 DNA by complexes 1 and 2 (20 lM) in the absence and presence of different inhibitors [100 mM mannitol, 200 mM
dimethyl sulfoxide (DMSO), 1000 U mlꢁ1 superoxide dismutase (SOD), 1.2 mM distidine] after irradiation at 365 nm for 30 min.
Fig. 7. Agarose gel electrophoresis retardation of pGL3 plasmid DNA in the presence different concentrations of complex 1 and 2. [DNA] = 0.5 lg.
to remove individual cells that are no longer needed or that func-
tion abnormally [48]. Apoptosis assays were performed with a
staining method utilizing acridine orange (AO) and ethidium bro-
mide (EB). The AO/EB staining is sensitive to DNA and was used
to assess changes in nuclear morphology. Apoptotic cells will show
apoptotic features such as nuclear shrinkage, chromatin condensa-
Table 1
The IC50 values for complexes 1, 2 and cisplatin against selected cell lines.
Complex
IC50 (mM)
BEL-7402
HepG-2
MCF-7
1
2
0.492
0.197
0.019
0.313
0.097
0.025
0.215
0.118
0.012
tion. In the absence of complex 2 (50 lM), the living cells were
Cisplatin
stained bright green in spots (Fig. 8A). However, apoptotic and ne-
crotic cells can be distinguished from one another using fluores-
cence microscope after being stained with AO/EB solution. After
treatment of BEL-7402 cell line with complex 2, green apoptotic
cells containing apoptotic bodies, as well as red necrotic cells, were
observed (Fig. 8B). Similar result for complex 1 was also observed.
The percentage (%) of necrotic and apoptotic cell of BEL-7402 cells
in the presence of different concentration of complexes 1 and 2.
The results were depicted in Fig. 9. As increasing concentrations
of complexes 1 and 2, the ratios of apoptotic versus necrotic cells
are 0.13, and 0.14 for 1, 1.59 and 4.07 for 2. The results showed
the number of apoptotic cells increased obviously with increasing
concentration of complex 2.
Table 1. The IC50 values for complex 2 (0.197, 0.097 and 0.118 mM
on BEL-7402, HepG-2 and MCF-7, respectively) are far lower than
that of complex 1 (0.492, 0.313 and 0.215 mM) under the identical
condition. It is clear that complex 2 is more sensitive against the
selected tumor cell lines than complex 1, but these complexes all
exhibit relatively lower in vitro cytotoxicity against the selected
cell lines than cisplatin. The results showed that the cytotoxicity
of complexes 1 and 2 was found to be concentration dependent,
and the cell viability decreased with increasing the concentrations
of ruthenium(II) complexes.
3.8. Apoptotic activity
3.9. Cellular uptake studies
Apoptosis plays a major role during the development and
homeostasis. Apoptosis is a form of programmed cell death which
occurs through the activation of the cell-intrinsic suicide machin-
ery. And apoptosis is primarily a physiological process necessary
The uptake of the complexes 1 and 2 by cells was studied with
Human hepatocellular carcinoma cell line (BEL-7402). Complex 2
(50
l
M) was added to the wells (4 ꢀ 104 cells per well). The plates

Y.-J. Liu et al. / Inorganica Chimica Acta 387 (2012) 117–124
123
Fig. 8. BEL-7402 cells were stained by AO/EB and observed under fluorescence microscopy. BEL-7402 cells without treatment (A) and in the presence of complex 2 (B)
incubated at 37 °C and 5% CO2 for 48 h. cells in a, b and c are living, apoptotic and necrotic cells, respectively.
Fig. 11. Scavenging effect of the ligands and complexes on hydroxyl radicals.
Fig. 9. The percentage (%) of necrotic (N) and apoptotic (A) cell of BEL-7402 cells
Experiments were performed in triplicate.
incubated with different concentrations of complexes 1 and 2 for 24 h by flow
cytometry.
3.10. Antioxidant activity against hydroxyl radical (ꢀOH)
were incubated at 37 °C in a 5% CO2 incubator for 48 h. Upon com-
pletion of the incubation, the wells were washed three times with
phosphate buffered saline (PBS), after removing the culture media,
the cells were observed under fluorescent microscope. As shown
in Fig. 10A and B, the bright red fluorescent spots in the images
were observed. The results show that complexes 2 can be uptaken
by cells, and they can enter into the cytoplasm and accumulate in
Various oxygen species occur during the metabolizable process
of biological systems. Superoxide anion radical (O2ꢀꢁ) and hydroxyl
radical (ꢀOH) are presumed to play an important role in a number
of biological phenomena such as aging, coronary heart disease,
rheumatoid arthritis, cancer and other pathological conditions.
The hydroxyl radical (ꢀOH) is by far the most potent and therefore
the most dangerous oxygen metabolite, elimination of this radical
is one of the major aims of antioxidant administration [49]. The
the nuclei. Similar result for complex
observed.
1 (25 lM) was also
Fig. 10. BEL-7402 cells incubated with complex 2 (50 lM) for 48 h imaged by fluorescence microscopy. (A) Imaged under fluorescence, (B) imaged under fluorescence and
visible light. Note that the cytoplasm is extensively stained with the Ru(II) complexes.

124
Y.-J. Liu et al. / Inorganica Chimica Acta 387 (2012) 117–124
Table 2
ꢀ
Scavenging ratio (%) of ligands and complexes against OH.
ꢀ
Comp
Average inhibition (%) for OH
0.5 (l
M)
1.0 (l
M)
1.5 (l
M)
2.0 (l
M)
2.5 (l
M)
3.0 (l
M)
3.5 (l
M)
4.0 (l
M)
4.5 (lM)
APIP
HAPIP
1
6.59
4.96
1.13
3.56
10.85
14.54
1.88
19.76
36.52
6.04
25.19
47.52
17.36
22.88
33.72
61.35
23.39
24.50
47.28
66.67
35.47
29.74
52.92
68.76
41.88
36.27
60.46
73.05
67.17
48.74
68.99
76.60
80.38
73.85
2
3.59
14.70
hydroxyl radical (ꢀOH) in aqueous media was generated by the Fen-
ton system. The antioxidant activity of ligands, APIP and HAPIP and
their complexes 1 and 2 were investigated. Fig. 11 depicts the
inhibitory effect of ligands and complexes on ꢀOH. The average sup-
pression ratio against ꢀOH (Table 2) valued from 6.59% to 72.86% for
APIP, 4.96% to 82.27% for HAPIP, 1.13% to 93.96% for complex 1, and
3.59% to 77.45% for complex 2. The antioxidant activity against hy-
droxyl radical of complex 1 appeared higher than that of complex 2
[3] Y.J. Liu, C.H. Zeng, Z.H. Liang, J.H. Yao, H.L. Huang, Z.Z. Li, F.H. Wu, Eur. J. Med.
Chem. 45 (2010) 3087.
[4] S. Shi, X.T. Geng, J. Zhao, T.M. Yao, C.R. Wang, D.J. Yang, L.F. Zheng, L.N. Ji,
Biochimie 92 (2010) 370.
[5] J. Tan, B.C. Wang, L.C. Zhu, Bioorg. Med. Chem. 17 (2009) 614.
[6] M. Roy, R. Santhanagopal, A.R. Chakravarty, Dalton Trans. (2009) 1024.
[7] Q. Wang, Z.Y. Yang, G.F. Qi, D.D. Qin, Biometals 22 (2009) 927.
[8] G. Zuber, J.C. Quada, S.M. Hecht Jr., J. Am. Soc. Chem. 120 (1998) 9368.
[9] S.M. Hecht, J. Nat. Prod. 63 (2000) 158.
[10] A. Sigel, H. Sigel (Eds.), vol. 33, Marcel Dekker, New York, 1996, p. 177.
[11] L.N. Ji, X.H. Zou, J.G. Liu, Coord. Chem. Rev. 216–217 (2001) 513.
[12] V.G. Vaidyananthan, B.H. Nair, Dalton Trans. (2005) 2842.
[13] P.P. Pelligrini, J.P. Aldrich-Wright, Dalton Trans. (2003) 176.
[14] P.U. Maheswari, M. Palaniandavar, Inorg. Chim. Acta 357 (2004) 901.
[15] P. Lincoln, B. Norden, Int. Pat., WO 99/15535, 1999.
[16] J. Liu, X.H. Zou, Q.L. Zhang, W.J. Mei, J.Z. Liu, L.N. Ji, Met.-Based Drugs 7 (2000)
343.
at the concentration range (from 3.0 to 4.5 lM) under the same
experimental condition. For complex 1, the hydroxyl radical scav-
enging ability can be enhanced when ligand (APIP) bonds metal
center to form complex at high concentrations. However, the anti-
oxidant activity of ligand (HAPIP) is weakened when it bonds metal
center to form complex 2. These results suggest that ligands and
their complexes may be potential drugs to eliminate the hydroxyl
radical.
[17] C.A. Puckett, J.K. Barton, J. Am. Chem. Soc. 129 (2007) 46.
[18] D.L. Ma, C.M. Che, F.M. Siu, M. Yang, K.Y. Wong, Inorg. Chem. 46 (2007) 740.
[19] G.L. Pascu, A.C.G. Hotze, C. Sanchez-Cano, B.M. Kariuki, M.J. Hannon, Angew.
Chem. 119 (2007) 4452.
[20] T.R. Krugh, J.W. Neely, Biochemistry 12 (1973) 4418.
[21] J. Marmur, J. Mol. Biol. 3 (1961) 208.
4. Conclusion
[22] M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1954)
3047.
[23] Y. Xiong, X.F. He, X.H. Zou, J.Z. Wu, X.M. Chen, L.N. Ji, R.H. Li, J.Y. Zhou, K.B. Yu, J.
Chem. Soc., Dalton Trans. 1 (1999) 19.
Two new ligands APIP, HAPIP and relative two ruthenium(II)
complexes [Ru(bpy)2(APIP)]2+ and [Ru(bpy)2(HAPIP)]2+ were syn-
thesized and characterized. The DNA-binding behaviors show
these complexes interact with CT DNA by intercalation. Complexes
1 and 2 can inhibit the proliferation of the selected cell lines in dif-
ferent degree. Apoptotic assay suggests that complexes 1 and 2 can
induce apoptosis. The cellular uptake shows these complexes can
enter into the cytoplasm and accumulate in the nuclei. Upon irra-
diation at 365 nm, complexes 1 and 2 can cleave the plasmid DNA.
The studies on the mechanism of photocleavage demonstrate that
superoxide anion radical (O2ꢀꢁ) and singlet oxygen (1O2) may play
an important role. At high concentration, complexes 1 and 2 can
effectively condense the pGL 3 DNA. The antioxidant experiments
against hydroxyl radical show the ligands and complexes possess
excellent antioxidant abilities.
[24] J.G. Liu, B.H. Ye, H. Chao, Q.X. Zhen, L.N. Ji, Chem. Lett. 28 (1999) 1085.
[25] B.P. Sullivan, D.J. Salmon, T.J. Meyer, Inorg. Chem. 17 (1978) 3334.
[26] A. Wolf, G.H. Shimer Jr., T. Meehan, Biochemistry 26 (1987) 6392.
[27] J.B. Chaires, N. Dattagupta, D.M. Crothers, Biochemistry 21 (1982) 3933.
[28] A.E. Friedman, J.C. Chambron, J.P. Sauvage, N.J. Turro, J.K. Barton, J. Am. Chem.
Soc. 112 (1990) 4960.
[29] S. Satyanaryana, J.C. Dabrowial, J.B. Chaires, Biochemistry 31 (1993) 9319.
[30] G. Cohen, H. Eisenberg, Biopolymers 8 (1969) 45.
[31] T.J. Mosmann, Immunol. Methods 65 (1983) 55.
[32] D.L. Spector, R.D. Goldman, L.A. Leinwand, In cells: a laboratory manual, vol. 1,
Cold Spring Harbor Laboratory Press, New York, 1998 (Chapter 15).
[33] C.C. Winterbourn, Free Radic. Biol. Med. 3 (1987) 33.
[34] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 32 (1993) 2573.
[35] H.L. Huang, Y.J. Liu, C.H. Zeng, L.X. He, F.H. Wu, DNA Cell Biol. 29 (2010) 261.
[36] Q. Wang Q, Z.Y. Yang, G.F. Qi, D.D. Qin, Eur. J. Med. Chem. 44 (2009) 2425.
[37] Y.J. Liu, C.H. Zeng, J.H. Yao, F.H. Wu, L.X. He, H.L. Huang, Chem. Biodivers. 7
(2010) 1770.
[38] J.K. Barton, A. Danishefsky, J. Goldberg, J. Am. Chem. Soc. 106 (1984) 2172.
[39] X.Q. He, Q.Y. Lin, R.D. Hu, Spectrochim. Acta, Part A 68 (2007) 184.
[40] R.B. Nair, E.S. Teng, S.L. Kirkland, C.J. Murphy, Inorg. Chem. 37 (1998) 139.
[41] L.F. Tan, H. Chao, Y.J. Liu, B. Sun, W. Wei, L.N. Ji, J. Inorg. Biochem. 99 (2005)
513.
Acknowledgements
[42] Y.J. Liu, Z.H. Liang, Z.Z. Li, C.H. Zeng, J.H. Yao, H.L. Huang, F.H. Wu, Biometals 23
(2010) 739.
[43] C. Zhao, H. Lin, S. Zhu, H. Sun, Y. Chen, J. Inorg. Biochem. 70 (1998) 219.
[44] M. Howe-Grant, K.C. Wu, W.R. Bauer, S.J. Lippart, Biochemistry 15 (1976) 4339.
[45] J.D. McGhee, P.H. von Hippel, J. Mol. Biol. 86 (1974) 469.
[46] M.J. Waring, J. Mol. Biol. 13 (1965) 269.
This work was supported by the National Nature Science Foun-
dation of China (Nos. 31070858 and 30800227) and Guangdong
Pharmaceutical University for financial supports.
[47] G.A. Neyhart, N. Grover, S.R. Smith, W.A. Kalsbeck, T.A. Fairly, M. Cory, H.H.
Thorp, J. Am. Chem. Soc. 115 (1993) 4423.
References
[48] D. Vinatier, P.H. Dufour, D. Subtil, Eur. J. Obstet. Gyn. RB 67 (1996) 85.
[49] U. Udilova, A.V. Kozlov, W. Bieberschulte, K. Frei, K. Ehrenberger, H. Nohl,
Biochem. Pharm. 65 (2003) 59.
[1] U. Schatzschneider, J. Niesel, I. Ott, R. Gust, H. Alborzinia, S. Wölfl,
ChemMedChem 3 (2008) 1104.
[2] L.F. Tan, J.L. Shen, X.J. Chen, X.L. Liang, DNA Cell Biol. 28 (2009) 461.