Cellular uptake, cytotoxicity, apoptosis, antioxidant activity and DNA binding of polypyridyl ruthenium(II) complexes

Article abstract of DOI:10.1016/j.jorganchem.2011.04.020

Ruthenium(II) polypyridyl complexes [Ru(phen)₂(APIP)](ClO ₄)₂ 1 and [Ru(phen)₂(HAPIP)](ClO ₄)₂ 2 have been synthesized and characterized. The DNA-binding behaviors were investigated by elec

Full text of DOI:10.1016/j.jorganchem.2011.04.020

Journal of Organometallic Chemistry 696 (2011) 2728e2735
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Cellular uptake, cytotoxicity, apoptosis, antioxidant activity and DNA binding
of polypyridyl ruthenium(II) complexes
,
a
a
b
c,
Yun-Jun Liu a , Zhen-Hua Liang , Zheng-Zheng Li , Jun-Hua Yao , Hong-Liang Huang
*
*
a School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
b Instrumentation Analysis and Research Center, Sun Yat-Sen University, Guangzhou 510275, PR China
c 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:
Ruthenium(II) polypyridyl complexes [Ru(phen)2(APIP)](ClO4)2 1 and [Ru(phen)2(HAPIP)](ClO4)2 2 have
been synthesized and characterized. The DNA-binding behaviors were investigated by electronic
absorption titration, luminescence spectra, viscosity measurements, thermal denaturation and photo-
activated cleavage. The DNA-binding constants Kb for complexes 1 and 2 were determined to be 3.38
(ꢀ0.42) ꢁ 105 Mꢂ1 (s ¼ 1.48) and 3.93 (ꢀ0.60) ꢁ 105 Mꢂ1 (s ¼ 3.14), respectively. The studies on the
photocleavage demonstrated that the effects of cleavage are concentration-dependent. The results
showed that complexes 1 and 2 interact with CT-DNA by intercalative mode. The cytotoxicity of
complexes 1 and 2 has been evaluated by MTT method. The apoptosis assay was carried out with acridine
orange/ethidium bromide (AO/EB) staining methods. The cellular uptake showed that complexes can
enter into the cytoplasm and accumulate in the nuclei. The antioxidant activity studies suggested that
the ligands and complexes may be potential drugs to eliminate the radical.
Received 10 February 2011
Received in revised form
14 April 2011
Accepted 19 April 2011
Keywords:
Ruthenium(II) complex
Cytotoxicity
Apoptosis
Cellular uptake
DNA binding
Crown Copyright Ó 2011 Published by Elsevier B.V. All rights reserved.
1. Introduction
them demonstrate remarkable anticancer activity [9e12]. Complex
[Ru(bpy)2(dppn)]2þ can effectively inhibit the proliferation of MCF-
The intercalation of transition metal polypyridyl complexes into
DNA has been a topic major bioinorganic interest in the past two
decades and continues to receive much attention [1e3]. As an
important target of anticancer drugs, DNA plays a central role in
replication, transcription, and regulation of genes. The presence of
metal-binding sites in DNA structure make different type of inter-
actions possible such as intercalation between base pairs, minor
groove binding, and major groove binding [4]. Complexes
[Ru(bpy)2(dppz)]2þ [5] and [Ru(bpy)2(ppd)]2þ [6] bind to DNA with
large affinities and may act as “molecular light switch”. Which
show no luminescence in aqueous buffer, but after bound to DNA by
intercalation they can luminescence brightly. On the other hand,
Ruthenium complexes are regarded as promising alternatives to
platinum complexes and several ruthenium complexes have now
been proposed as potential anticancer substances [7,8], some of
7 cells with a low IC50 (3.3 ꢀ 1.2
m
M) [13]. [Ru(phen)2(paip)]2þ can
effectively induce apoptosis of BEL-7402 cell line [14], and [(h6
-
C6Me6)RuCl(dppn)]2þ, bearing an arene ligand, that facilitates
diffusion through cell membrane and thereby enhances cellular
uptake, shows a high cell uptake of 1054.7 ng Ru/mg protein on HT-
29 (colon cancer) cells [15]. Our previous work showed that
complexes formed by intercalative ligands containing amino-group
possessed high antitumor activity [14,16]. In this article, two new
ligands, APIP, and HAPIP bearing amino-group and their Ru(II)
complexes [Ru(phen)2(APIP)](ClO4)2 1 [Ru(phen)2(HAPIP)](ClO4)2 2
(Scheme 1) have been synthesized and characterized by elemental
analysis, ES-MS, and 1H NMR. Their DNA-binding behaviors were
investigated by absorption titration, viscosity measurements,
thermal denaturation and photocleavage. The results showed that
complexes 1 and 2 interact with CT-DNA by intercalative mode. The
cytotoxicity of complexes 1 and 2 has been evaluated by MTT
(MTT
¼
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Abbreviations: APIP, 2-(20-aminophenyl)imidazo[4,5-f][1,10]phenanthroline;
HAPIP,
bromide)) assay. The apoptosis of BEL-7402 cells induced by Ru(II)
complexes was also studied. The study on the cellular uptake with
BEL-7402 cells was carried out and the results were observed under
fluorescence microscopy. The antioxidant activity of these
complexes was explored by hydroxyl radical (ꢃOH) scavenging
method in vitro.
2-(20-hydroxyl-4-aminophenyl)imidazo[4,5-f][1,10]phenanthroline;
CT-DNA, calf thymus DNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide; Phen, 1,10-phenanthroline; AO, Acridine orange; EB, ethidium
bromide; DMSO, dimethylsulfoxide.
* Corresponding authors. Tel.: þ86 20 39352122; fax: þ86 20 39352128.
E-mail addresses: lyjche@163.com (Y.-J. Liu), honglianghuangcn@hotmail.com
(H.-L. Huang).
0022-328X/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2011.04.020

Y.-J. Liu et al. / Journal of Organometallic Chemistry 696 (2011) 2728e2735
2729
Found: C, 73.16; H, 4.13; N, 22.38%. FAB-MS: m/z ¼ 312 [Mþ1]þ. 1H
NMR (500 MHz, DMSO-d6): 9.05 (d, 2H, J ¼ 8.0 Hz), 8.98(d, 2H,
J ¼ 8.2 Hz), 8.02e8.05 (m, 2H), 7.83 (d, 1H, J ¼ 8.0 Hz), 7.18e7.35 (m,
1H), 6.92 (d, 1H, J ¼ 8.0 Hz), 6.74(t, 1H, J ¼ 7.6 Hz), 3.36 (s, 2H, HNH2).
2.3.2. 2-(2-Hydroxyl-5-aminophenyl)imidazo [4,5-f][1,10]
phenanthroline (HAPIP)
This compound was prepared in a manner identical to that
described for APIP, with 2-(2-hydroxyl-5-nitrophenyl)imidazo [4,5-
f][1,10]phenanthroline [20] in place of 2-(2-nitrophenyl)imidazo
[4,5-f][1,10]phenanthroline. Yield: 73%. Anal. Calcd. 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 ¼ 328 [Mþ1]þ. 1H NMR (500 MHz, DMSO-d6): 9.06 (d, 2H,
J ¼ 8.2 Hz), 8.98(d, 2H, J ¼ 7.5 Hz), 7.87 (dd, 2H, J ¼ 8.0 Hz), 7.43 (d,
1H, J ¼ 8.0 Hz), 6.84 (d, 1H, J ¼ 8.6 Hz), 6.73e6.75 (m, 1H), 3.39 (s,
2H, HNH2).
Scheme 1. The structure of complexes.
2. Experimental
2.1. Reagents and materials
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.
Dimethylsulfoxide (DMSO) and RPMI 1640 were purchased from
Sigma. Cell lines of BEL-7402 (hepatocellular), HepG-2 (hepato-
cellular) and MCF-7 (breast cancer) were purchased from
American Type Culture Collection, agarose and ethidium
bromide were obtained from Aldrich. RuCl3$xH2O was purchased
from Kunming Institution of Precious Metals. 1,10-Phenanthro-
line was obtained from Guangzhou Chemical Reagent Factory.
Doubly distilled water was used to prepare buffers (5 mM
Tris(hydroxymethylaminomethane)eHCl, 50 mM NaCl, pH ¼ 7.2).
A solution of CT-DNA in the buffer gave a ratio of UV absorbance
at 260 and 280 nm of ca. 1.8e1.9:1, indicating that the DNA was
sufficiently free of protein [17]. The DNA concentration per
nucleotide was determined by absorption spectroscopy using the
molar absorption coefficient (6600 Mꢂ1 cmꢂ1) at 260 nm [18].
2.3.3. [Ru(phen)2(APIP)](ClO4)2$2H2O (1)
[Ru(phen)2(NPIP)](ClO4)2 (0.509 g, 0.5 mmol) [19] was
completely dissolved in acetronitrile. Then the ethanol of 50 cm3,
Pd/C (0.20 g, 10% Pd) and NH2NH2$H2O (8 cm3) were added in the
above solution and 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 eluent. The main red band
was collected. The solvent was removed under reduced pressure
and a red powder was obtained. Yield: 70%. Anal. Calcd. for
C43H29N9Cl2O8Ru$2H2O: C, 51.25; H, 3.30; N, 12.51; Found: C, 51.72;
H, 3.59; N, 12.94%. ES-MS [CH3CN, m/z]: 771.5 ([M ꢂ 2ClO4 ꢂ H]þ),
386.4 ([M ꢂ 2ClO4]2þ). 1H NMR (500 MHz, DMSO-d6):
d 8.78 (d, 4H,
J ¼ 8.2 Hz), 8.40 (s, 4H), 8.17 (d, 2H, J ¼ 8.2 Hz), 8.12 (d, 4H,
J ¼ 8.0 Hz), 8.08 (d, 2H, J ¼ 8.0 Hz), 7.98 (d, 2H, J ¼ 8.2 Hz), 7.75e7.78
(m, 5H), 7.20 (t, 1H, J ¼ 7.6), 6.91 (d, 1H, J ¼ 8.0 Hz), 6.74 (t, 1H,
J ¼ 7.8 Hz), 3.37 (s, 2H, HNH2).
2.2. Methods and instrumentation
2.3.4. [Ru(phen)2(HAPIP)](ClO4)2$2H2O (2)
Microanalysis (C, H, and N) was carried out with a PerkineElmer
240Q elemental analyzer. Fast atom bombardment (FAB) mass
spectra were recorded on a VG ZAB-HS spectrometer in a 3-nitro-
benzyl alcohol matrix. Electrospray mass spectra (ES-MS) were
recorded on an LCQ system (Finnigan MAT, USA) using methanol 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 ꢄC, 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 and emission spectra
were recorded on a Shimadzu RF-4500 luminescence spectrometer
at room temperature. The cellular uptake was observed under IX-50
inverted fluorescence microscopy (100ꢁ) with blue light as
excitation.
A mixture of cis-[Ru(phen)2Cl2]$2H2O [21] (0.280 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
CH3CNetoluene (3:1, v/v) as eluant. The main red band was
collected. The solvent was removed under reduced pressure and
a
red powder was obtained. Yield: 72%. Anal. Calcd. for
C43H29N9Cl2O9Ru$2H2O: C, 50.45; H, 3.25; N, 12.31; Found: C,
50.64; H, 3.63; N, 12.78%. ES-MS [CH3CN, m/z]: 788.3
([M ꢂ 2ClO4 ꢂ H]þ), 394.8 ([M ꢂ 2ClO4]2þ). 1H NMR (500 MHz,
DMSO-d6):
d
8.82 (d, 2H, J ¼ 8.0 Hz), 8.76 (d, 4H, J ¼ 8.0 Hz), 8.67 (d,
2H, J ¼ 8.5 Hz), 8.47 (d, 1H, J ¼ 8.0 Hz), 8.38 (s, 4H), 8.10 (t, 4H,
J ¼ 7.6 Hz), 7.74e7.78 (m, 6H), 7.60 (dd, 2H, J ¼ 7.8 Hz), 3.35 (s, 2H).
Caution: Perchlorate salts of metal compounds with organic
ligands are potentially explosive, and only small amounts of the
material should be prepared and handled with great care.
2.3. Synthesis of ligands and complexes
2.3.1. 2-(2-Aminophenyl)imidazo [4,5-f][1,10]phenanthroline (APIP)
2.4. DNA-binding assays
A
mixture of 2-(2-nitrophenyl)imidazo [4,5-f][1,10]phenan-
throline (0.171 g, 0.5 mmol) (NPIP) [19] was completely dissolved in
ethanol (50 cm3) with stirring for 1 h. Then the Pd/C (0.20 g, 10% Pd)
and NH2NH2$H2O (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
obtained was washed with cool ethanol and dried at 50 ꢄC in vacuo.
Yield: 73%. Anal. Calcd. for C19H13N5: C, 73.30; H, 4.21; N, 22.49;
The DNA-binding and photoactivated cleavage experiments
were performed at room temperature. Buffer A (5 mM Tris, 50 mM
NaCl, pH 7.0) was used for absorption titration, luminescence
titration and viscosity measurements. Buffer B (50 mM TriseHCl,
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

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Y.-J. Liu et al. / Journal of Organometallic Chemistry 696 (2011) 2728e2735
D (0.9% of physiological saline) was used for retardation assay of
pGL 3 plasmid DNA.
in a 5% CO2 incubator. Compounds tested were then added to the
wells to achieve final concentrations ranging from 6.25 M to
400 M. Control wells were prepared by addition of culture
medium (100
m
The absorption titrations of the complex in buffer were per-
m
formed using a fixed concentration (20
mM) for complex to which
mL). Wells containing culture medium without cells
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 metal-to-ligand charge transition (MLCT) band
with increasing concentration of DNA using the following equation
[22].
were used as blanks. The plates were incubated at 37 ꢄC in a 5% CO2
incubator for 48 h. Upon completion of the incubation, stock MTT
dye solution (20
incubation, buffer (100
m
L, 5 mg mLꢂ1) was added to each well. After 4h
mL) containing N,N-dimethylformamide
(50%) and sodium dodecyl sulfate (20%) was added to solubilize the
MTT formazan. The culture medium and cisplatin act as the nega-
tive and positive control, respectively. 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 loga-
rithmic 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 (BEL-7402, HepG-2 and MCF-7) were the subjects
of this study.
ꢀ
ꢁ.ꢀ
ꢁ
ꢀ
ꢀ
ꢁꢁ
1=2
3a
ꢂ
3f
3b
ꢂ
3f
¼
b ꢂ b2 ꢂ 2K2Ct½DNAꢅ=s
=2KCt
(1a)
b ¼ 1 þ KCt þ K½DNAꢅ=2s
(1b)
where [DNA] is the concentration of CT-DNA in base pairs, the
apparent absorption coefficients 3a 3f and 3b correspond to Aobsed/
,
[Ru], the extinction coefficient for the free ruthenium complex, and
the extinction coefficient for the ruthenium complex in fully bound
form, respectively. K is the equilibrium binding constant in Mꢂ1, Ct
is the total metal complex concentration in nucleotides and s is the
binding size.
Thermal denaturation studies were carried out with a Perkine
Elmer Lambda 35 spectrophotometer equipped with a Peltier
temperature-controlling programmer (ꢀ0.1 ꢄC). The melting
temperature (Tm) was taken as the mid-point of the hyperchromic
transition. The melting curves were obtained by measuring the
2.6. Apoptosis study
Apoptosis studies were performed with a staining method
utilizing acridine orange (AO) and ethidium bromide (EB) [27].
According to the difference in membrane integrity between
necrotic and apoptosis. AO can pass through cell membrane of
living or early apoptotic cells, while staining by EB indicates loss of
membrane integrityt. 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 morphological changes such as cell blebbing and
formation of apoptotic bodies will be observed.
absorbance at 260 nm for solutions of CT-DNA (80
mM) in the
absence and presence of the Ru(II) complex (30 M) as a function of
m
the temperature. The temperature was scanned from 50 to 90 ꢄC at
a speed of 1 ꢄC minꢂ1. The data were presented as (A ꢂ A0)/(Af ꢂ A0)
versus temperature, where A, A0, and Af are the observed, the initial,
and the final absorbance at 260 nm, respectively.
A monolayer of BEL-7402 cells was incubated in the absence or
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 mlꢂ1 AO, 100 g mlꢂ1 EB). Samples were observed
m
M at 37 ꢄC and 5%
m
m
Viscosity measurements were carried out using an Ubbelodhe
viscometer maintained at a constant temperature at 25.0 (ꢀ0.1) ꢄC
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 [23]. Flow time was
measured with a digital stopwatch, and each sample was measured
three times, and an average flow time was calculated. Relative
viscosities for DNA in the presence and absence of complexes were
under a fluorescence microscope.
2.7. Cellular uptake
Cells were placed in 24-well microassay culture plates (4 ꢁ 104
cells per well) and grown overnight at 37 ꢄC in a 5% CO2 incubator.
Complexes tested were then added to the wells. The plates were
incubated at 37 ꢄC 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 medium, the
cells were visualized by fluorescent microscopy.
calculated from the relation
h
¼ (t ꢂ t0)/t0, where t is the observed
flow time of the DNA-containing solution and t0 is the flow time of
buffer alone [5,24]. Data were presented as (
ratio [25], where is the viscosity of DNA in the presence of
h/h0)
1/3 versus binding
h
complexes and h0 is the viscosity of DNA alone.
2.8. Scavenger measurements of hydroxyl radical (ꢃOH)
For the gel electrophoresis experiment, supercoiled pBR 322
DNA (0.1 mg) was treated with the Ru(II) complexes in buffer B, and
Scavenging measurements on hydroxyl radical (ꢃOH) were
carried out in aqueous media. The hydroxyl radical (ꢃOH) was
generated by the Fenton system [28]. The solution of the tested
complexes was prepared with DMF (N,N-dimethylformamide). The
assay mixture (5 mL) contained following reagents: safranin
the solution was then irradiated at room temperature with a UV
lamp (365 nm, 10 W). The samples were analyzed by electropho-
resis for 1.5 h at 80 V on a 1.0% agarose gel in TBE (89 mM Tris-
borate acid, 2 mM EDTA, pH ¼ 8.3). The gel was stained with
1 mg/ml ethidium bromide and photographed on an Alpha Innotech
(28.5
mM), EDTA-Fe(II) (100
mM), H2O2 (44.0
mM), the tested
IS-5500 fluorescence chemiluminescence and visible imaging
system.
compounds (0.5e3.5
mM) and
a
phosphate buffer (67 mM,
pH ¼ 7.4). The assay mixtures were incubated at 37 ꢄC 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 ratio (ha) was calculated on the basis of (Ai ꢂ A0)/
(Ac ꢂ A0) ꢁ 100%.
2.5. Cytotoxicity assay
Cytotoxicity of complexes 1 and 2 was evaluated by using
standard MTT (3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium
bromide) assay [26]. Cells were placed in 96-well microassay
culture plates (8 ꢁ 103 cells per well) and grown overnight at 37 ꢄC

Y.-J. Liu et al. / Journal of Organometallic Chemistry 696 (2011) 2728e2735
2731
3. Results and discussion
3.1. Synthesis and characterization
Complexes 1 and 2 were prepared by heating of ligands with cis-
[Ru(phen)2Cl2]. The ligands, APIP and HAPIP, were synthesized with
a method similar to that described by Steck and Day [29]. The
structures of the ligands and their complexes were confirmed by
elemental analysis, FAB-MS, ES-MS, and 1H NMR. The chemical
shifts of protons on the nitrogen atoms of imidazole ring were not
observed, probably because the protons are very active and easy to
be exchanged between the two nitrogens of the imidazole ring in
solution. In the ES-MS spectra for the complexes 1 and 2, as
expected, the intense signals for [M
ꢂ
2ClO4
ꢂ
H]þ and
[M ꢂ 2ClO4]2þ were observed, the obtained molecular weights
were consistent with the expected values.
3.2. Absorption spectral studies
Fig. 2. Effect of increasing amounts of complexes 1 (-) and 2 (C) on the relative
viscosity of CT-DNA at 25 (ꢀ0.1) ꢄC. [DNA] ¼ 0.25 mM.
Upon increasing amounts of calf thymus DNA (CT-DNA) to
complexes 1 and 2 in buffer A, the changes in absorbance at MLCT
transition bands (455 nm for 1 and 460 nm for 2) are observed. The
observed hypochromisms are 23.07 and 28.95% with red shifts of 2
and 1 nm for 1 and 2, respectively (Fig. 1). Therefore these spectral
characteristics suggest that these complexes interact with DNA
most likely through a mode that involves a stacking interaction
between the aromatic chromophore and the base pairs of DNA. The
intrinsic constants Kb were determined by monitoring the changes
of absorbance in the MLCT band with increasing concentration of
CT-DNA. The values of Kb are 3.38 (ꢀ0.42) ꢁ 105 Mꢂ1 (s ¼ 1.48) and
3.93 (ꢀ0.60) ꢁ 105 Mꢂ1 (s ¼ 3.14) Mꢂ1 for 1 and 2, respectively.
These are of the same order as that reported for [Ru(bpy)2(PIP)]2þ
of the base pairs at the intercalation site and, hence, an increase in
the overall DNA molecular length [35]. The effects of complexes 1, 2
on the viscosity of rod-like DNA are shown in Fig. 2. Upon
increasing the amounts of complexes 1 and 2, the relative viscosity
of CT-DNA solution increases steadily. The increased degree of
viscosity, which may depend on its affinity to DNA, follows the
order 2 > 1. These results suggest that complexes 1 and 2 can bind
to CT-DNA through intercalation mode.
3.4. Thermal denaturation
(4.7
ꢀ
0.20
ꢁ
105 Mꢂ1
)
[30] and [Ru(phen)2(HPIP)]2þ
(9.10 ꢁ 105 Mꢂ1) [31], [Ru(phen)2(PPIP)]2þ (1.10 ꢀ 0.30 ꢁ 105 Mꢂ1
)
It is well known that complexes which intercalate between the
base pairs of DNA can stabilize the duplex structure. The increase in
absorbance at 260 nm was recorded from 40 to 90 ꢄC, and Tm was
then determined from the denaturation curve. The thermal dena-
turation experiments carried out on DNA in the absence of
complexes revealed that the Tm for the duplex is 60.6 ꢀ 0.5 ꢄC under
our experimental conditions. As shown in Fig. 3. The observed
[32], but is smaller than those of [Ru(phen)2(dppz)]2þ
(5.1 ꢁ106 Mꢂ1) [33] and [Ru(pdto)(dppz)]2þ (3.0 ꢀ 0.01 ꢁ106 Mꢂ1
)
[34].
3.3. Viscosity measurements
In order to clarify the binding mode of ruthenium(II) complexes
1 and 2 with DNA, viscosity of DNA solutions containing varying
amount of added complexes were measured. A classical intercala-
tion of a ligand into DNA is known to cause a significant increase in
the viscosity of a DNA solution due to an increase in the separation
change of melting temperatures (DTm) in the presence of
complexes 1 and 2 are 7.8 and 11.3 ꢄC, respectively. The large
change in Tm suggests the binding affinities of both complexes with
DNA are very strong. The binding constants of complexes 1 and 2 to
CT-DNA at Tm were determined by McGee’s equation [22].
Fig. 1. Absorption spectra of complexes 1 (a) and 2 (b) in TriseHCl buffer upon addition of increasing concentrations of CT-DNA. [Ru] ¼ 20
upon the increase of DNA concentration. Plots of (3a 3f)/(3b 3f) versus [DNA] for the titration of DNA with Ru(II) complexes.
mM. Arrow shows the absorbance change
ꢂ
ꢂ

2732
Y.-J. Liu et al. / Journal of Organometallic Chemistry 696 (2011) 2728e2735
Fig. 5. Photoactivated cleavage of pBR 322 DNA in the presence of different concen-
trations of Ru(II) complexes after irradiation at 365 nm for 30 min.
changes of standard enthalpy, standard free energy and standard
entropy of binding of the complex to CT-DNA. The values of D
D
G0T , H0,
and
D
S0 were ꢂ31.54 kJ molꢂ1, ꢂ47.86 kJ molꢂ1 and ꢂ54.76 J molꢂ1 Kꢂ1
for complex 1, and ꢂ31.92 kJ molꢂ1
,
ꢂ33.79 kJ molꢂ1 and
ꢂ6.27 J molꢂ1 Kꢂ1 for complex 2.
Fig. 3. Thermal denaturation of CT-DNA in the absence (-) and presence of complexes
3.5. Luminescence spectra studies
1 (C) and 2 (:). [Ru] ¼ 30
m
M, [DNA] ¼ 80
mM.
Complexes 1 and 2 can luminescence in Tris buffer at ambient
temperature, with a maximum appearing at 590 and 592 nm for 1
and 2, respectively. The luminescence spectra and the change of
emission intensity are shown in Fig. 4. When the ratio of [DNA]/[Ru]
reached 12.46 for 1 and 13.35 for 2, the emission intensities of
complexes 1 and 2 increase to 1.13 and 1.75 times of the original
intensities, respectively. This implies that these complexes can
interact with DNA and be protected by the hydrophobic environ-
ment inside the DNA helix efficiently.
1
n
1
Tm0
1
Tm
R
Hm
ꢂ
¼
lnð1 þ KLÞ
(2)
D
where Tm0 and Tm are the melting temperature of CT-DNA alone and
in the presence of complex, respectively. ΔHm is the enthalpy of
DNA melting (per bp), R is the gas constant, K is the DNA-binding
constant at Tm, L is the concentration of free Ru(II) complex, and
n is the binding site size.
The value of ΔHm ¼ 6.9 kcal molꢂ1 was determined by different
scanning calorimetry [36]. On the basis of the absorption spectra
titration experiment and the neighbor-exclusion principle, the
values of n for both complexes were assumed to be 2.0 base pairs.
The binding constants K for complex 1 and 2 at Tm were calculated
to be 2.90 ꢁ 104 and 6.15 ꢁ 104 Mꢂ1, respectively. According to the
van’t Hoffs equations (2)e(4) [37]
3.6. Agarose gel electrophoresis
There is a particular ongoing interest in DNA cleavage reactions
photoactivated by metal complexes [38,39]. The cleavage reaction
on plasmid DNA can be monitored by agarose gel electrophoresis.
When circular plasmid DNA is subjected to electrophoresis, rela-
tively fast migration will be observed for the intact supercoiled
form (Form I); if scission occurs on one strand (nicked), the
supercoiled form will relax to generate a slower-moving open
circular form (Form II) [40]. As seen in Fig. 5, control photoreaction
with DNA alone or incubation with the ruthenium complexes in
darkness results in little DNA cleavage. With increasing concen-
trations of the Ru(II) complexes, the amount of Form I was gradu-
ally diminished, whereas Form II increased. Comparing the cleaving
effect of complexes 1 and 2, complex 1 exhibits more effective DNA
cleavage activity than complex 2, which is not consistent with the
DNA-binding affinity of two Ru(II) complexes.
ꢂ
ꢃ
ꢂ
ꢃ
K2
K1
D
H0
R
1
1
ln
¼
ꢂ
(3)
T1 T2
D
G0T ¼ ꢂRTlnK
(4)
(5)
D
GT0
¼
D
H ꢂ T
D
S0
where K1 and K2 are the DNA-binding constants of the complexes at the
temperature of T1 and T2, respectively.
H0, G0T and S0 are the
D
D
D
Fig. 4. Emission spectra of complexes a) 1 and b) 2 in TriseHCl buffer in the absence and presence of CT-DNA. Arrow shows the intensity change upon increasing DNA
concentrations.

Y.-J. Liu et al. / Journal of Organometallic Chemistry 696 (2011) 2728e2735
2733
for complexes 1 and 2 ranged from 35.17 to 235.14
mM, suggesting
that complexes 1 and 2 exhibited antitumor activity against the
selected cell lines in different degree. Comparing the cytotoxicity of
complexes 1 and 2, complex 1 displayed higher cytotoxicity than
complex 2, but these complexes all exhibit relatively lower cyto-
toxicity in vitro than cisplatin. Furthermore, we also found that the
coordination of the APIP and HAPIP to the Ru(II) metal center to
form complexes 1 and 2, the antitumor activity of two ligands was
obviously weakened. And the cytotoxicity of phen located between
APIP and HAPIP. Fig. 7 was the antiproliferative activity for ligands,
complexes 1 and 2 against the selected cell lines, with the
increasing concentration of these compounds, the proliferation of
tumor cells was effectively inhibited.
Fig. 6. Agarose gel electrophoresis retardation of pGL 3 plasmid DNA in the presence
different concentrations of complexes 1 and 2. Lane 0 (DNA alone); 1: (1) 1 mM; (2)
2
mM; (3)
4
m
mM; (4)
g.
6 mM. 2: (5) 1 mM; (6) 2 mM; (7) 4 mM; (8) 6 mM
[DNA] ¼ 0.5
Table 1
The IC50 values for APIP, HAPIP, complexes 1 and 2 against selected cell lines.
Compound
IC50 (mM)
3.9. Apoptosis induced by Ru(II) complexes
BEL-7402
HepG-2
MCF-7
PHEN
APIP
HAPIP
1
26.57 ꢀ 2.78
18.47 ꢀ 3.26
32.47 ꢀ 2.77
43.07 ꢀ 2.73
179.82 ꢀ 5.66
19.78 ꢀ 2.55
e
e
Apoptosis induced by compounds is one of the considerations in
drug development. The apoptotic cells usually show apoptotic
features such as nuclear shrinkage and chromatin condensation.
Apoptosis assay was carried out with a staining method using
acridine orange (AO) and ethidium bromide (EB). The AO/EB
staining is sensitive to DNA and was used to assess the changes in
nuclear morphology. Apoptotic and necrotic cells can be distin-
guished from one another using fluorescence microscopy. In the
presence of complex 2, the living cells were stained bright green in
spots (Fig. 8A). After treatment of BEL-7402 cell line with complex 2
for a period of 48 h, green apoptotic cells containing apoptotic
bodies stained by acridine orange, as well as red necrotic cells
stained by ethidium bromide, were also found (Fig. 8B). Similar
results for complex 1 were also observed.
42.98 ꢀ 2.54
45.35 ꢀ 3.25
35.17 ꢀ 2.78
146.25 ꢀ 6.43
25.48 ꢀ 3.15
21.14 ꢀ 3.13
33.13 ꢀ 3.42
44.35 ꢀ 3.12
235.14 ꢀ 6.47
12.24 ꢀ 2.55
2
Cisplatin
3.7. Retardation of pGL 3 plasmid DNA
DNA condensation is prerequisite for efficient gene delivery
using nonviral vectors. It is well known that polyamine can effec-
tively condense DNA [41,42]. In recent years, the small molecules to
condense DNA have been paid great attention. Complex
[Ru(bpy)2(PIPSH)]2þ [43] can condense DNA at a concentration of
80 m
M. And complex Cu2(egtb)(ClO4)2(H2O)2]2þ [44] can effectively
and rapidly condense free DNA. The abilities of complexes 1 and 2
to condense pGL 3 DNA were assessed by gel retardation assay.
Fig. 6 showed when the concentrations of complexes 1 and 2 are 1
and 2 mM, complexes 1 and 2 cannot condense the DNA. At the
high concentrations of 4 and 6 mM, complexes 1 and 2 can effec-
tively condense DNA.
3.10. Celluar uptake study
The spectroscopic data and viscosity measurements showed
that complexes 1 and 2 can intercalate between the base pairs of
CT-DNA. This prompted us to consider whether these complexes
can cross the cell membrane and enter into the cytoplasm and
accumulate in the nuclei. In order to prove our hypothesis, the
cellular uptake with BEL-7402 cells was carried out. Complexes 1
3.8. Cytotoxicity assay in vitro
The cytotoxicity of RuCl3, PHEN, APIP, HAPIP and complexes 1
and 2 was evaluated by MTT with three cell lines: BEL-7402, hepG-2
and MCF-7. The concentrations of these compounds ranged from
(25
mM) and 2 (50
m
M) were added to the well (4 ꢁ 104 cells per
well). After treatment for 48 h, the cells were observed under
fluorescent microscopy. As shown in Fig. 9, for cells treated with
complexes 1 and 2, the bright red fluorescent spots in the images
were observed, whereas no any red fluorescent spots was observed
in the control (data not presented). These results indicated that
complex 1 and 2 can enter into the cytoplasm and accumulate in
the nuclei.
6.25 mM to 400 mM. The IC50 values obtained of these compounds
against the selected four tumor cell lines are listed in Table 1. The
resulting concentration-effect curves obtained with continuous
exposure for 48 h are depicted in Fig. 7. The results showed that
RuCl3 has no cytotoxicity against selected cell lines. The IC50 values
Fig. 7. Cell viability of APIP, HAPIP, complexes 1 and 2 on tumor BEL-7402 (a), HepG-2 (b) and MCF-7 (c) cell proliferation in vitro. Each data point is the mean ꢀ standard error
obtained from at least three independent experiments.

2734
Y.-J. Liu et al. / Journal of Organometallic Chemistry 696 (2011) 2728e2735
Fig. 8. BEL-7402 cell were stained by AO/EB and observed under fluorescence microscopy. BEL-7402 cell without treatment (a) and in the presence of complex 2 (b) incubated at
37 ꢄC and 5% CO2 for 24 h. Cells in a, b and c are living, apoptotic and necrotic cells, respectively.
3.11. Antioxidant activity against hydroxyl radical (ꢃOH)
As shown in Fig. 10 and Table 2, the inhibitory effect of the ligands
and their Ru(II) complexes on ꢃOH increased with increasing of the
The hydroxyl radical (ꢃOH) is one of the most reactive products of
reactive oxygen species (ROS), which can result in cell membrane
disintegration, membrane protein damage, DNA mutation and
initiate the development of many diseases. In this report, the
hydroxyl radical in aqueous media was generated by the Fenton
system. The antioxidant activity of the ligands APIP, HAPIP and
complexes 1 and 2 against hydroxyl radical (ꢃOH) were investigated.
sample concentration in the range of 0.5e3.5 mM. The suppression
ratio against ꢃOH valued from 6.59 to 49.29 for APIP, 4.96 to 69.15 for
HAPIP, 3.76 to 66.20 for complex 1 and 2.40 to 58.40 for complex 2,
respectively. The suppression ratio increased when APIP bonded
Ru(II) metal center to form complex 1, whereas the suppression ratio
lessened for HAPIP to form complex 2 under the same experimental
condition. The information obtained from the present work would
Fig. 9. BEL-7402 cells incubated with complexes 1 (25
mM, A and B) and 2 (50 mM, C and D) for 48 h imaged by fluorescence microscopy. A and C imaged under fluorescence, B and D
imaged under fluorescence and visible light. Note that the cytoplasm is extensively stained with the Ru(II) complexes.

Y.-J. Liu et al. / Journal of Organometallic Chemistry 696 (2011) 2728e2735
2735
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This work was supported by the National Nature Science
Foundation of China (Nos. 31070858, 30800227) and Guangdong
Pharmaceutical University for financial supports.