Ruthenium(II) complexes: Cellular uptake, cytotoxicity, DNA-binding, photocleavage and antioxidant activity studies

Article abstract of DOI:10.1016/j.molstruc.2011.06.011

Two new ruthenium(II) complexes [Ru(dmp)₂(DNPIP)]²⁺ 1 and [Ru(dmp)₂(DAPIP)]²⁺ 2 were synthesized and characterized. The DNA-binding behaviors of these complexes were investigated by absorption spectra, viscosity

Full text of DOI:10.1016/j.molstruc.2011.06.011

Journal of Molecular Structure 1001 (2011) 36–42
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Ruthenium(II) complexes: Cellular uptake, cytotoxicity, DNA-binding,
photocleavage and antioxidant activity studies
Zheng-Zheng Li , Zhen-Hua Liang , Hong-Liang Huang , Yun-Jun Liu a,
a
a
b,
⇑
⇑
a
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
School of Life Science and Biopharmaceutical, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
b
a r t i c l e i n f o
a b s t r a c t
Two new ruthenium(II) complexes [Ru(dmp) (DNPIP)]2 1 and [Ru(dmp) (DAPIP)] 2 were synthesized
+
2+
Article history:
Received 5 April 2011
Received in revised form 10 June 2011
Accepted 13 June 2011
Available online 17 June 2011
2
2
and characterized. The DNA-binding behaviors of these complexes were investigated by absorption spec-
tra, viscosity measurements and photocleavage. The DNA-binding constants for complexes 1 and 2 have
4
4
ꢁ1
been determined to be 6.24 (±0.11) ꢀ 10 and 1.64 (±0.4ꢀ) ꢀ 10 M . The results suggest that complexes
and 2 intercalate between the DNA base pairs. Binding stoichiometries were studied through a lumines-
cence-based Job plot. The major inflection points for complexes 1 and 2 at = 0.31 and = 0.51 were
observed. The data were consistent with 2:1 and 1:1 bp [complex]/[DNA] binding mode. Complex 1
shows higher activity than complex 2 against the selected tumor cell lines. In addition, the cellular uptake
and antioxidant activity on hydroxyl radical were also explored.
1
v
v
Keywords:
Ruthenium complexes
DNA-binding
Cytotoxicity
Cellular uptake
Antioxidant activity
Crown Copyright Ó 2011 Published by Elsevier B.V. All rights reserved.
1
. Introduction
dmp = 2,ꢀ-dimethyl-1,10-phenanthroline) and [Ru(dmp)
2
(DA-
PIP)](ClO 2 (DAPIP = 2-(2,4-diaminophenyl)imidazo[4,5-f][1,10]
4 2
)
Ruthenium complexes are emerging as promising candidates
phenanthroline, Scheme 1). The DNA-binding behaviors of these
complexes were investigated by viscosity measurements,
electronic absorption titration, and photoactivated cleavage. The
cytotoxicity of these complexes was evaluated by MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
The cellular uptake was investigated and the experiments on
antioxidant activity of these complexes against hydroxyl radical
for novel cancer therapies [1]. Two Ru-based drugs are in clinical
development: (ImH)[trans-RuCl (DMSO)(Im)] (Im = imidazole)
NAMI-A) is effective against lung metastases [2–6], and KP101ꢀ
(IndH)[trans-RuCl (Ind) ]) (Ind = indazole)] is active against colon
4
(
[
4
2
carcinomas [7–13] and DNA has been proposed as a possible
important target [12]. Thus, studies on the binding of Ru(II) com-
plexes with DNA have attracted great interest. A great progress
has been made in the research of ruthenium(II) complexes binding
with DNA in the last two decades. The results show that ruthe-
nium(II) complexes can bind DNA in a noncovalence such as elec-
trostatic binding, groove binding and intercalative mode. On the
other hand, many ruthenium (II) polypyridyl complexes exert
rather potent activities against selected tumor cells [14–16]. In
Å
( OH) was also explored.
2
. Experimental
2.1. Materials and methods
Calf thymus DNA (CT-DNA) was obtained from the Sino-Amer-
0
our previous work [17], we found complexes containing 2,2 -bipyr-
ican Biotechnology Company. pBR322 DNA was obtained from
Shanghai Sangon Biological Engineering & Services Co., Ltd.
Dimethyl sulfoxide (DMSO) and RPMI 1640 were purchased from
Sigma. Cell lines of hepatocellular (BEL-7402), hepatocellular
idine as ancillary have low cytotoxicity against the selected cell
lines. To observe the cytotoxic effect of ruthenium(II) complexes
containing different ancillary on the same tumor cell lines, in this
article, we report the synthesis, characterization, DNA-binding,
cytotoxicity, cellular uptake in vitro and antioxidant activity of
(
Hepg-2) and breast cancer (MCF-7) were purchased from
American Type Culture Collection, agarose and ethidium bromide
were obtained from Aldrich. RuCl O was purchased from
two new ruthenium(II) complexes [Ru(dmp)
2 4 2
(DNPIP)](ClO ) 1
3
ꢂxH
2
(
DNPIP = 2-(2,4-dinitrophenyl)imidazo[4,5-f][1,10]phenanthroline,
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(hydrox-
ymethylaminomethane)–HCl, 50 mM NaCl, pH = 7.2). A solution
of calf thymus DNA in the buffer gave a ratio of UV absorbance
⇑
Corresponding authors.
E-mail addresses: honglianghuangcn@hotmail.com (H.-L. Huang), lyjche@163.
com (Y.-J. Liu).
0
022-2860/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2011.06.011

Z.-Z. Li et al. / Journal of Molecular Structure 1001 (2011) 36–42
37
Scheme 1. The structure of complexes.
at 260 and 280 nm of ca. 1.8–1.ꢀ:1, indicating that the DNA was
sufficiently free of protein [18]. The DNA concentration per nucle-
otide was determined by absorption spectroscopy using the molar
red powder was obtained. Yield: 71%. Anal. Calcd. for
12Ru: C, 51.1ꢀ; H, 3.11; N, 12.70; Found: C, 51.43;
47 34 2
C H N10Cl O
+
H, 3.02; N, 13.11%. ES-MS [CH
3
CN, m/z]: ꢀ02.5 ([M–2ClO
4
–H] ),
ꢁ1
ꢁ1
2+
1
absorption coefficient (6600 M cm ) at 260 nm [1ꢀ].
451.6 ([M–2ClO
4
]
). H NMR (500 MHz, DMSO-d ): d 8.ꢀ0 (d, 1H,
6
Microanalysis (C, H, and N) was carried out with a Perkin-Elmer
40Q elemental analyzer. Fast atom bombardment (FAB) mass
J = 8.5 Hz), 8.60 (d, 2H, J = 8.5 Hz), 8.44 (dd, 4H, J = 6.5 Hz,
J = 4.5 Hz), 8.23 (d, 2H, J = 8.5 Hz), 7.ꢀ7 (d, 1H, J = 8.2 Hz), 7.37 (d,
1H, J = 8.5 Hz), 7.31 (dd, 4H, J = 7.5 Hz, J = 5.5 Hz), 7.24 (t, 2H,
J = 5.5 Hz), 7.14–7.18 (m, 4H), 2.50 (s, 6H), 2.30 (s, 6H). 13C NMR
2
spectra were recorded on a VG ZAB-HS spectrometer in a 3-nitro-
benzyl alcohol matrix. Electrospray mass spectra (ES-MS) were re-
corded on a 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
6
(DMSO-d , ppm): 167.71 C (2,ꢀ), 166.0ꢀ C (a), 148.ꢀ7 C (k),
148.23 C (i), 147.ꢀ4 C (e), 144.ꢀ6 C (g), 137.81 C (11), 136.47 C
(h), 130.48 C (c), 12ꢀ.ꢀ7 C (4,7), 12ꢀ.3ꢀ C (l), 128.84 C (d), 128.14
C (m), 127.16 C (f), 126.40 C (10), 125.76 C (5,6), 125.45 C (3,8),
124.18 C (b), 118.ꢀ3 C (j), 24.44 C (Me).
2
00 °C, respectively, and the quoted m/z values are for the major
1
13
peaks in the isotope distribution. H NMR and C 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 lumi-
nescence spectrometer at room temperature.
2
.2.3. Synthesis of [Ru(dmp)
Ru(dmp) (DNPIP)](ClO
dissolved in minimum amount of acetonitrile, then the Pd/C
2
(DAPIP)](ClO
) (0.552 g, 0.5 mmol) was completely
4 2
4 2
) (2)
[
2
3
3
(
0.20 g, 10% Pd), NH
2
NH
2
2
ꢂH O (8 cm ) and ethanol (20 cm ) were
added in the above solution and refluxed under argon for 8 h.
The hot solution was filtered and evaporated under reduced pres-
sure to remove the solvent to 6 cm . Upon cooling, a red precipitate
2
2
.2. Synthesis and characterization
3
.2.1. Synthesis of ligand (DNPIP)
The ligand DNPIP was synthesized according to literature [17].
was obtained by dropwise addition of saturated aqueous NaClO
solution. The crude product was purified by column chromatogra-
phy on a neutral alumina with a mixture of CH CN–toluene (3:1, v/
4
A mixture of 1,10-phenanthroline-5,6-dione (0.315 g, 1.5 mmol)
20], 2,4-dinitrobenzaldehyde (0.2ꢀ4 g, 1.5 mmol), ammonium
3
[
v) as eluent. The red band was collected. The solvent was removed
under reduced pressure and a red powder was obtained. Yield:
3
acetate (2.31 g, 30 mmol) and glacial acetic acid (20 cm ) was re-
fluxed with stirring for 2 h. The cooled solution was diluted with
water and neutralized with concentrated aqueous ammonia. The
precipitate was collected and purified by column chromatography
on silica gel (60–100 mesh) with ethanol as eluent to give the com-
70%. Anal. Calcd. for C47
H
38
N
10Cl
2
O
8
Ru: C, 54.13; H, 3.67; N,
CN, m/z]:
). H NMR (500 MHz,
): d 8.ꢀ1 (d, 2H, J = 8.5 Hz), 8.77 (dd, 4H, J = 8.5 Hz,
13.43; Found: C, 53.ꢀ4; H, 3.45; N, 13.82%. ES-MS [CH
3
+
2+
1
842.3 ([M–2ClO
4 4
–H] ), 421.8 ([M–2ClO ]
DMSO-d
6
pound as yellow powder. Yield: 80%. Anal. Calcd. for C1ꢀ
H
10
N
6
O
4
: C,
J = 8.5 Hz), 8.24 (d, 2H, J = 8.5 Hz), 7.ꢀ8 (d, 4H, J = 8.5 Hz), 7.62 (d,
2H, J = 8.0 Hz), 7.48 (t, 1H, J = 6.0 Hz), 7.38 (d, 4H, J = 8.5 Hz), 7.31
(d, 1H, J = 4.0 Hz), 6.01 (d, 1H, J = 5.5 Hz), 5.42 (s, 4H), 1.ꢀ4 (s,
5
ꢀ.07; H, 2.61; N, 21.75; Found: C, 5ꢀ.01; H, 2.54; N, 21.53%. FAB-
+
1
MS: m/z = 387 [M + H] . H NMR (500 MHz, DMSO–d
J = 8.2 Hz), 8.ꢀ1 (d, 2H, J = 8.0 Hz), 8.7ꢀ (d, 1H, J = 8.5 Hz), 8.75 (d,
H, J = 8.0 Hz), 8.44 (d, 2H, J = 8.5 Hz), 7.87 (dd, 2H, J = 5.5 Hz,
6
): ꢀ.08 (d, 1H,
1
3
6
6H), 1.70 (s, 6H). C NMR (DMSO-d , ppm): 167.87 C (2,ꢀ),
1
166.30 C (a), 155.13 C (e), 151.46 C (k), 14ꢀ.61 C (i), 148.85 C (g),
147.77 C (11), 145.14 C (c, 4,7), 138.00 C (m,d), 136.5ꢀ C (f,10),
12ꢀ.45 C (5,6), 127.31 C (3,8), 126.45 C (b), 124.8ꢀ C (h), 103.ꢀ3
C (l), ꢀ8.64 C (j), 24.45 C (Me).
1
3
J = 5.0 Hz). C NMR (DMSO-d
6
, ppm): 148.24 C (a), 147.0ꢀ C (k),
44.5ꢀ C (i), 143.ꢀ0 C (e), 132.11 C (g), 131.22 C (h), 130.11 C (c),
28.ꢀ3 C (l), 127.14 C (d), 126.0ꢀ C (m), 124.16 C (f), 123.41 C
1
1
(
b), 11ꢀ.84 C (j).
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
.2.2. Synthesis of [Ru(dmp)
2 4 2
(DNPIP)](ClO ) (1)
A mixture of cis-[Ru(dmp)
2
Cl O (0.312 g, 0.5 mmol) [21]
2
]ꢂ2H
2
3
and DNPIP (0.1ꢀ3 g, 0.5 mmol) in ethylene glycol (20 cm ) was re-
fluxed under argon for 8 h to give a clear red solution. Upon cool-
ing, a red precipitate was obtained by dropwise addition of
2.3. DNA binding and photoactivated cleavage
The DNA-binding and photoactivated cleavage experiments
saturated aqueous NaClO
by column chromatography on a neutral alumina with a mixture of
CH CN–toluene (3:1, v/v) as eluent. The main red band was col-
lected. The solvent was removed under reduced pressure and a
4
solution. The crude product was purified
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 titra-
tion and viscosity measurements. Buffer B (50 mM Tris–HCl,
3

3
8
Z.-Z. Li et al. / Journal of Molecular Structure 1001 (2011) 36–42
1
8 mM NaCl, pH 7.2) was used for DNA photocleavage
reference solution, then the cuvette was irradiated for 30 min with
the monochromatic UV lamp (k = 365 nm), its absorbance (A) at
3ꢀꢀ nm was taken immediately. In the same way, the A and A of
10 M complex 1 and 2, as well as the A and A of 100 M DMA
experiments.
The absorption titrations of the complex in buffer were per-
0
formed using a fixed concentration (20
l
M) for complex to which
l
0
l
increments of the DNA stock solution were added. Ru-DNA solu-
tions were incubated for 5 min before the absorption spectra were
recorded. The intrinsic binding constants K, based on the absorp-
tion titration, were measured by monitoring the changes of
absorption in the metal-to-ligand transfer (MLCT) band with
increasing concentration of DNA using the following equation [22].
were measured with the solvent DMF as a blank. All the records
were made three times and expressed as the average value.
2.6. Cell culture and cytotoxicity assay in vitro
Standard 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium
bromide (MTT) assay procedures were used [28]. Cells were placed
½
DNAꢃ
½DNAꢃ
1
3
¼
þ K
ð1Þ
and
in ꢀ6-well microassay culture plates (8 ꢀ 10 cells per well) and
e
a
ꢁ
e
f
e
b
ꢁ
e
f
b
ð
e
b
ꢁ
e
f
Þ
2
grown overnight at 37 °C in a 5% CO incubator. The complexes
tested were dissolved in DMSO and diluted with RPMI 1640 and
where [DNA] is the concentration of DNA in base pairs,
e
a
,
e
f
e
b
then added to the wells to achieve final concentrations ranging
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
form, respectively. In plots of [DNA]/(
by the ratio of slope to the intercept.
–
6
–4
from 10 to 10 M. Control wells were prepared by addition of
culture medium (100 L). Wells containing culture medium with-
out cells were used as blanks. The plates were incubated at 37 °C in
a 5% CO incubator for 72 h. Upon completion of the incubation,
stock MTT dye solution (20
After 4 h, buffer (100 L) containing N,N-dimethylformamide
50%) and sodium dodecyl sulfate (20%) was added to solubilize
l
a f b
e –e ) versus [DNA], K is given
2
ꢁ1
l
L, 5 mg mL ) was added to each well.
Viscosity measurements were carried out using an Ubbelodhe
viscometer maintained at a constant temperature at 25.0 (±0.1)
l
(
°C in a thermostatic bath. DNA samples about 200 bp in average
the MTT formazan. The optical density of each well was then mea-
sured on a microplate spectrophotometer at a wavelength of
length were prepared by sonication to minimize complexities aris-
ing from DNA flexibility [23]. Flow time was measured with a dig-
ital 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 calculated from the
4
ꢀ0 nm. The IC50 values were determined by plotting the percent-
age viability versus concentration on a logarithmic graph and read-
ing 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, Hepg-2 and MCF-7 (pur-
chased from American Type Culture Collection).
0
0
relation
g = (t–t )/t , where t is the observed flow time of the
0
DNA-containing solution and t is the flow time of buffer alone
1/3
[
24,25]. Data are presented as (
g
/g )
0
versus binding ratio [26],
where
g is the viscosity of DNA in the presence of complexes and
g
0
is the viscosity of DNA alone.
For the gel electrophoresis experiment, supercoiled pBR322
2.7. Celluar uptake study
DNA (0.1 lg) was treated with the Ru(II) complexes in buffer B,
4
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 0.8% agarose gel in TBE (8ꢀ mM
Tris–borate acid, 2 mM EDTA, pH = 8.3). The gel was stained with
Cells were placed in 24-well microassay culture plates (4 ꢀ 10
2
cells per well) and grown overnight at 37 °C in a 5% CO incubator.
Compounds tested (50
l
M) were then added to the wells. The
plates were incubated at 37 °C in a 5% CO
2
incubator for 48 h. Then,
1
l
g/ml ethidium bromide and photographed on an Alpha Inno-
the wells were washed three times with phosphate buffered saline
(PBS), and after removing the culture media, the cells were visual-
ized by fluorescent microscopy.
tech IS–5500 fluorescence chemiluminescence and visible imaging
system.
2.4. Continuous variation analysis
2.8. Antioxidant activity
Å
Binding stoichiometries were obtained for complexes 1 and 2
The hydroxyl radical ( OH) in aqueous media was generated by
with CT DNA using the method of continuous variation [27]. The
concentrations of both complex and DNA were varied, while the
the Fenton system [2ꢀ]. The solution of the tested complexes was
prepared with DMF (N,N-dimethylformamide). The assay mixture
sum of the reactant concentrations was kept constant at 50
in terms of base pairs for the DNA). Solutions of complexes and
DNA were prepared in Tris–HCl buffer (pH = 7.4). In the sample
solutions, the mole fraction of complex was varied from 0 to
.0 in 0.1 ratio steps. The fluorescence intensities of these mixtures
l
M
(5 mL) contained following reagents: safranin (28.5
Fe(II) (100 M), (44.0 M), the tested compounds
(0.5–4.5 M) and a phosphate buffer (67 mM, pH = 7.4). The assay
mixtures were incubated at 37 °C for 30 min in a water bath. Then
the absorbance was measured at 520 nm. All the tests were run in
lM), EDTA-
(
l
H
2
O
2
l
l
v
1
were measured at 25 °C using an excitation wavelength of 460 nm.
The intensity in fluorescence was plotted versus the mole fraction
triplicate and expressed as the mean. A
presence of the tested compound; A was the absorbance in the ab-
sence of tested compounds; A was the absorbance in the absence
of tested compound, EDTA-Fe(II), H . The suppression ratio (
)/(A –A
) ꢀ 100%.
i
was the absorbance in the
0
v
of complex to generate a Job plot. Linear regression analysis of
c
the data was performed in the software of Origin 7.0.
2
O
2
a
g )
was calculated on the basis of (A
i
–A
0
c
0
2.5. Detecting the formation of singlet oxygen photo-induced by
complexes
3. Results and discussion
A
3 ml N,N-dimethylformamide (DMF) solution containing
3.1. Electronic absorption titration
complexes (10 lM) and ꢀ,10-dimethylanthracene (DMA, 100 lM)
was prepared in a quartz cuvette, and the initial absorbance (A
at 3ꢀꢀ nm of this solution was recorded on a Shimadzu UV-
101PC spectrophotometer with 10 M complexes 1 and 2 as
0
)
The electronic absorption spectra of complexes 1 and 2 mainly
consist of two or three resolved bands. The band below 300 nm is
3
l
attributed to intraligand (IL)
p
?
p
ꢄ transition, the band at 345–

Z.-Z. Li et al. / Journal of Molecular Structure 1001 (2011) 36–42
3ꢀ
3
50 nm is attributed to
p
?
p
ꢄ transition, and the low energy
absorption band centered at 455–465 nm is assigned to metal-
to-ligand charge transfer (MLCT) transition by comparison with
the spectrum of other polypyridyl Ru(II) complexes [30–32].
Addition of CT DNA to buffered aqueous solution of the complexes
produces distinctive changes in their absorption spectra. The
absorption spectra of complexes 1 and 2 in the absence and pres-
ence of CT DNA are given in Fig. 1. As the DNA concentration is in-
creased, the MLCT transition bands of complexes 1 at 468 and 2 at
4
67 nm exhibit hypochromism of about 38.41 and 31.85%, and
bathochromism of 2 nm, respectively. The intrinsic constants K
b
for complexes 1 and 2 were determined by monitoring the changes
in absorbance at the MLCT band with increasing concentration of
4
CT DNA. The values of K
(
b
are 6.24 (±0.11) ꢀ 10 and 1.64
4
ꢁ1
±0.4ꢀ) ꢀ 10 M for complexes 1 and 2, respectively. The DNA-
binding constant of complex 1 is larger than that of complex 2,
which is caused by the electron-withdrawing substituent (–NO
in DNPIP) on the intercalative ligand improving the DNA-binding
affinity, and the electron-pushing substituent (–NH in DAPIP)
Fig. 2. Effect of increasing amounts of complexes 1 (j) and 2 (d) on the relative
viscosity of calf thymus DNA at 25 (±0.1) °C. [DNA] = 0.25 mM.
2
2
the concentration of CT DNA increased, the emission intensities
of complexes 1 (at 5ꢀ7 nm) and 2 (at 5ꢀ6 nm) were about 6.ꢀ8
and 4.ꢀꢀ times larger than the original, respectively. This clearly
indicates that complex 1 is in a more hydrophobic environment
in the presence of DNA when compared to complex 2.
decreasing the DNA affinity. Similar results were observed for
other ruthenium(II) complexes [33]. These values are comparable
(maip)]2 (3.23 ꢀ 10 M ) and
+
4
ꢁ1
to that of complexes [Ru(dmp)
2
2
+
4
ꢁ1
[
Ru(dmp)
2
(paip)] (4.34 ꢀ 10 M ) [34], but is not as strong as
+ 6
ꢁ1
that of [Ru(bpy)
2
(dppz)]2 (4.ꢀ ꢀ 10 M , dppz = dipyrido[2,3-
0
0
a:3 ,2 -c]phenazine) [35].
3.4. Continuous variation analysis
3.2. Viscosity measurements
Binding of complexes 1 and 2 to CT DNA was examined at 25 °C
in Tris–HCl buffer by the method of continuous variation analysis
to determine the overall stoichiometries. Fig. 4 shows normalized
Job plots for DNA. The point of the intersection of two best fit lines
in the Job plots for the complexes 1 and 2 with DNA are 0.31 and
It is well-established that intercalation results in a lengthening
of DNA, thus producing increases in relative specific viscosity of
solution of DNA [23,36]. Therefore, to probe the nature of the inter-
action between the ruthenium complexes and DNA, the effect of
the addition of the complexes 1 and 2 on the viscosity of aqueous
CT DNA solutions was studied. Fig. 2 shows the changes in the rel-
ative viscosity of CT DNA on addition of complexes 1 and 2. Upon
increasing the amounts of complexes 1 and 2, the relative viscosity
of CT DNA solution increase steadily, which confirms that all these
complexes interact with CT DNA through intercalative mode. The
increased degree of viscosity depends on complex-DNA affinities,
which followed the order of complex 1 > 2.
0
1
.51 that correspond to complex/DNA stoichiometries of 1:2 and
:1, respectively.
3.5. Photoactivated cleavage of pBR322 DNA
It is well known when circular plasmid DNA is subjected to
electrophoresis, relatively fast migration will be observed for the
intact supercoiled form (Form I); If scission occurs on one strand
(
nicked), the supercoiled will relax to generate a slower-moving
3
.3. Luminescence studies
open circular form (Form II) [37]. As shown in Fig. 5a, No obvious
DNA cleavage was observed for the control in which metal com-
plex was absent (DNA alone), or incubation of the plasmid with
the Ru(II) complexes in darkness. With increasing concentration
of complexes, the Form I decrease and Form II increase gradually.
Under the same experimental condition, complex 1 exhibits more
effective DNA cleavage activity than complex 2. The different
Emission intensity of complexes 1 and 2 from their MLCT ex-
cited states upon exicitation at 468 and 467 nm is found to depend
on DNA concentration. For each titration of CT DNA, luminescence
enhancements occur within minutes of DNA addition, indicating
that association rates are relatively rapid. As shown in Fig. 3, as
Fig. 1. Absorption spectra of complex 1 (a) and 2 (b) in Tris–HCl buffer upon addition of CT-DNA. [Ru] = 20
concentration. Plots of [DNA]/(e –e ) versus [DNA] for the titration of DNA with Ru(II) complexes.
lM. Arrow shows the absorbance change upon the increase of DNA
a
f

4
0
Z.-Z. Li et al. / Journal of Molecular Structure 1001 (2011) 36–42
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 concentrations.
Table 1
Absorbance at 3ꢀꢀ nm for detecting the formation of singlet oxygen in the presence of
complexes 1 and 2.
A
0
A
D
A
DMA complex
DA –DA
DMA
1
DMA + 1
2
DMA + 2
1.156
0.216
1.080
0.206
1.041
0.451
0.1ꢀ8
0.470
0.185
0.485
0.705
0.018
0.610
0.021
0.556
Complex
0.0ꢀ5
0.14ꢀ
Fig. 4. Job plot using luminescence data for complexes 1 (a) and 2 (b) with CT DNA
in Tris–HCl buffer, pH = 7.0.
Fig. 6. Agarose gel electrophoresis retardation of pGL3 plasmid DNA by complexes
and 2. [DNA] = 0.5 g.
1
l
Table 2
The IC50 values for complexes 1 and 2 against selected cell lines.
Fig. 5a. Photoactivated cleavage of pBR 322 DNA in the presence of different
concentrations of Ru(II) complexes after irradiation at 365 nm for 30 min.
Complex
IC50(lM)
BEL-7402
HepG-2
MCF-7
1
2
36.38 ± 3.28
77.07 ± 3.61
1ꢀ.78 ± 2.55
32.7ꢀ ± 3.7ꢀ
50.63 ± 3.ꢀ1
25.48 ± 3.15
46.48 ± 3.24
ꢀ6.41 ± 3.1ꢀ
12.24 ± 2.55
Cisplatin
as mannitol [2ꢀ] and dimethylsulfoxide (DMSO) [38], which indi-
cated that hydroxyl radical was not likely to be the cleaving agent.
In the presence of superoxide dismutase (SOD), a facile superoxide
Åꢁ
anion radical (O ) quencher, the cleavage was obviously improved.
2
Fig. 5b. Photoactivated cleavage of supercoiled pBR 322 DNA by complexes 1 and 2
The DNA cleavage of the plasmid was inhibited in the presence of
the singlet oxygen ( O
gesting that O is likely to be the reactive species responsible for
2
(
2
20 lM) in the absence and presence of different inhibitors [100 mM mannitol,
ꢁ
1
1
00 mM dimethyl sulfoxide (DMSO), 1000 U ml
superoxide dismutase (SOD),
2
) scavenger histidine and NaN
3
[3ꢀ,40], sug-
1
1
.2 mM distidine] after irradiation at 365 nm for 30 min.
the cleavage reaction.
cleaving efficiency may be ascribed to the different binding affinity
of two Ru(II) complexes to DNA.
3.6. Detecting the generation of singlet oxygen
In order establish the reactive species responsible for the
photoactivated cleavage of the plasmid, the influence of different
potentially inhibiting agents was investigated. Fig. 5b shows that
the DNA cleavage of the plasmid by complexes 1 and 2 was not
1
To detect the production of O
2
under irradiation at 365 nm in
the presence of complexes 1 or 2, the experiments were performed
using ꢀ,10-dimethylanthracene (DMA) as a quencher of single oxy-
gen [41]. It is well known that DMA has a strong absorption at
Å
inhibited in the presence of hydroxyl radical ( OH) scavengers such

Z.-Z. Li et al. / Journal of Molecular Structure 1001 (2011) 36–42
41
Fig. 7. Cell viability of complexes 1 and 2 on cell lines: BEL-7402 (a), Hepg-2 (b) and MCF-7 (c) in vitro. Each data point is the mean ± standard error obtained from at least
three independent experiments.
oxygen is indeed occurred in the presence complexes 1 or 2 upon
irradiation.
3.7. Retardation of pGL 3 DNA by Ru(II) complexes
Several studies reported the polyamine can condense DNA [42–
4
4]. However, the studies of small molecules to condense DNA
have been paid less attention. The large DNA-binding affinities of
complexes 1 and 2 prompt us to consider if these complexes can
effectively condense DNA into compact structures. Based on this
hypothesis, the retardation of pGL 3 DNA by the two complexes
was carried out. The effect on retardation was analyzed by agarose
gel electrophoresis as shown in Fig. 6. when the concentrations of
complexes 1 and 2 are 1 and 2 mM, complexes 1 and 2 can not con-
dense the DNA, however, the concentrations reach 3 and 5 mM, the
effects on condensation of DNA were observed.
3.8. Cytotoxicity assay in vitro
The IC50 values of the Ru(II) complexes 1 and 2 in three cell lines
(
BEL-7402, Hepg-2 and MCF-7) are listed in Table 2 and the cell
viability is depicted in Fig. 7. The IC50 values for 1 and 2 range from
6.38 to ꢀ6.41 M. Comparing the IC50 values of complex 1 and 2,
Fig. 8. BEL-7402 cells incubated with complexes 2 (50 lM) for 48 h. A imaged
under fluorescence and visible light.
3
l
Complex 1 appeared to have higher cytotoxicity against all the se-
lected cells than complex 2, but cytotoxicity of the two Ru(II) com-
plexes was relatively low when compared with cisplatin. Fig. 7
showed that the cell viability decreased with increasing concentra-
tion of complexes 1 and 2. The results obtained showed that the
cytotoxicity for complexes 1 and 2 against the selected tumor cell
lines is consistent with the DNA-binding affinity.
3.9. Cellular uptake
4
Complex 2 (50
l
M) was added to the well (4 ꢀ 10 cells per
2
well). The plates were incubated at 37 °C in a 5% CO incubator
for 48 h. Then the well was washed three times with PBS, and
the cells were observed under fluorescent microscopy. In the con-
trol experiment, BEL-7402 cells was not stained (data no pre-
sented). However, in the presence of complex 2, the spots
stained by complex 2 were observed in the image (Fig. 8). The re-
sults show complex 2 was successfully uptaken by BEL-7402 cells.
Fig. 9. Scanvenging effect of complexes 1 and 2 on hydroxyl radicals. Experiments
were performed in triplicate.
3.10. Antioxidant activity
3
ꢀꢀ nm, and this absorption will be photobleached upon chemical
1
quenching reaction of DMA with O
. For complexes 1 and 2 alone, the absorbance changed little be-
fore and after irradiation, whereas for the mixture containing both
DMA and complexes 1 or 2, the change of A was very obvious. The
2
. The data were listed in Table
In the last decade, a great deal of research has been devoted to
1
the study of different types of antioxidants (natural and synthetic)
which may at least minimize the deleterious effects induced by
reactive oxygen species (ROS) [45]. It is well known that hydroxyl
D
Å
decrease in absorbance at 3ꢀꢀ nm reflect the decrease in concen-
tration of DMA. These results suggest that the generation of single
radical ( OH) can result in cell membrane disintegration and
membrane protein damage. As shown in Fig. ꢀ and Table 3. the

4
2
Z.-Z. Li et al. / Journal of Molecular Structure 1001 (2011) 36–42
Table 3
Å
The scavenging ratios (%) of complexes against OH.
Å
Comp
Average inhibition (%) for OH
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5 (lM)
1
2
0.26
1.32
3.60
2.ꢀ1
15.17
3.6ꢀ
33.16
12.66
3ꢀ.07
20.32
42.85
27.ꢀ7
48.84
35.ꢀ4
53.47
60.ꢀ4
61.6ꢀ
65.17
Å
suppression ratio against OH ranged from 0.26% to 61.6ꢀ% for
[7] I. Bratsos, S. Jedner, T. Gianferrara, E. Alessio, Chimia 61 (2007) 6ꢀ2.
[
8] C.G. Hartinger, M.A. Jakupec, S. Zorbas-Seifried, M. Groessl, A. Egger, W. Berger,
H. Zorbas, P.J. Dyson, B.K. Keppler, Chem. Biodiversity 5 (2008) 2140.
ꢀ] F.T. Garzon, M.R. Berger, B.K. Keppler, D. Schmaehl, Cancer Chemother.
Pharmacol. 1ꢀ (1ꢀ87) 347.
complex 1, and 1.32% to 65.17% for complex 2. At low concentra-
tions (from 0.5 to 3.5 lM), the antioxidant activity of complex 1
is higher than complex 2. These results showed that the suppres-
sion ratio increased with increasing sample concentrations in the
range of 0.5–4.5 lM. It is believed that the information obtained
would be helpful to develop new potential antioxidants and ther-
apeutic agents for some diseases.
[
[
[
[
[
[
[
[
[
10] M.R. Berger, F.T. Garzon, B.K. Keppler, D. Schmaehl, Anticancer Res. ꢀ (1ꢀ8ꢀ)
61.
7
11] M.H. Seelig, M.R. Berger, B.K. Keppler, J. Cancer Res. Clin. Oncol. 118 (1ꢀ82)
1ꢀ5.
12] S. Kapitza, M. Pongratz, M. Jakupec, P. Heffeter, W. Berger, L. Lackinger, B.K.
Keppler, B. Marian, J. Cancer Res. Clin. Oncol. 131 (2005) 101.
13] M.A. Jakupec, M. Galanski, V.B. Arion, C.G. Hartinger, B.K. Keppler, Dalton
Trans. (2008) 183.
14] 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.
15] C.P. Tan, S.S. Lai, S.H. Wu, S. Hu, L.J. Zhou, Y. Chen, M.X. Wang, Y.P. Zhu, W.
Liang, Y.L. Peng, L.N. Ji, A.L. Xu, J. Med. Chem. 53 (2010) 7613.
16] M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1ꢀ54)
4
. Conclusion
2
Two new ruthenium(II) polypyridyl complexes [Ru(dmp) (DN-
PIP)]2 1 and [Ru(dmp)
+
(DAPIP)] 2 have been synthesized and
2+
2
characterized. The DNA-binding of these complexes with CT DNA
indicated that the two complexes can intercalate between DNA
base pairs. Both complexes can bring about cleavage of plasmid
DNA when irradiated at 365 nm for 30 min. The studies of mecha-
nism on photocleavage demonstrated that superoxide anion radi-
3047.
17] H.L. Huang, Z.Z. Li, Z.H. Liang, Y.H. Yao, Y.J. Liu, Eur. J. Med. Chem. (2011) doi:
10.1016/j.ejmech.2011.04.04ꢀ.
18] J. Marmur, J. Mol. Biol. 3 (1ꢀ61) 208.
[
[
1ꢀ] M. Yamada, Y. Tanaka, Y. Yoshimoto, S. Kuroda, I. Shimao, Bull. Chem. Soc. Jpn.
65 (1ꢀꢀ2) 1006.
Åꢁ
1
2
cal (O ) and singlet oxygen ( O ) may play an important role.
[20] J.B. Bolger, A. Gourdon, E. Ishow, J.P. Launay, Inorg. Chem. 35 (1ꢀꢀ6) 2ꢀ37.
2
[
[
[
21] J.B. Chaires, N. Dattagupta, D.M. Crothers, Biochemistry 21 (1ꢀ82) 3ꢀ33.
22] A. Wolf, G.H. Shimer Jr, T. Meehan, Biochemistry 26 (1ꢀ87) 63ꢀ2.
23] S. Satyanaryana, J.C. Dabrowial, J.B. Chaires, Biochemistry 31 (1ꢀꢀ2) ꢀ31ꢀ.
The data obtained from continuous variation analysis were consis-
tent with a 1:2 and 1:1 [complex]/[DNA] binding mode for com-
plexes 1 and 2, respectively. Cytotoxicity assay in vitro showed
that complex 1 displayed higher antitumor activity than complex
[24] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 32 (1ꢀꢀ3) 2573.
[
[
[
25] G. Cohen, H. Eisenberg, Biopolymers 8 (1ꢀ6ꢀ) 45.
26] M.T. Carter, M. Rodriguez, A. Bard, J. Am. Chem. Soc. 111 (1ꢀ8ꢀ) 8ꢀ01.
27] P. Job, Ann. Chim. (Paris) ꢀ (1ꢀ28) 113.
2
against selected tumor cell lines. The cellular uptake indicated
that complex 2 can enter into the cytoplasm and accumulate in
the nuclei. Antioxidant activity experiments showed that two
Ru(II) complexes exhibited good antioxidant activity against hy-
[28] C.C. Winterbourn, Biochem. J. 182 (1ꢀ7ꢀ) 625.
[2ꢀ] C.C. Cheng, S.E. Rokita, C.J. Burrows, Angew. Chem. Int. Ed. Engl. 32 (1ꢀꢀ3) 277.
[
[
30] Y.J. Liu, X.Y. Wei, W.J. Mei, L.X. He, Transition Met. Chem. 32 (2007) 762.
31] Y.J. Liu, C.H. Zeng, J.H. Yao, F.H. Wu, L.X. He, Huang HLChem Biodivers 7 (2010)
Å
droxyl radical ( OH).
1770.
[
[
32] L.F. Tan, X.H. Liu, H. Chao, L.N. Ji, J. Inorg. Biochem. 101 (2007) 56.
33] Y.J. Liu, X.Y. Wei, F.H. Wu, W.J. Mei, L.X. He, Spectrochim. Acta Part A 70 (2008)
Acknowledgements
171–176.
[
[
34] C.H. Zeng, Y.J. Liu, Z.Z. Li, Z.H. Liang, H.L. Huang, F.H. Wu, Transition Met. Chem.
35 (2010) 731.
35] A.E. Friedman, J.C. Chambron, J.P. Sauvage, N.J. Turro, J.K. Barton, J. Am. Chem.
Soc. 112 (1ꢀꢀ0) 4ꢀ60.
This work was supported by the National Nature Science Foun-
dation of China (Nos. 30800227 and 31070858) and Guangdong
Pharmaceutical University.
[
[
[
[
[
[
[
36] L.S. Lerman, J. Mol. Biol. 3 (1ꢀ61) 18.
37] J.K. Barton, A.L. Raphael, J. Am. Chem. Soc. 106 (1ꢀ84) 2466.
38] S.A. Lesko, R.J. Lorentzen, P.O. Ts’o, Biochemistry 1ꢀ (1ꢀ80) 3023.
3ꢀ] R. Nilsson, P.B. Merkel, D.R. Kearns, Photochem. Photobiol. 16 (1ꢀ72) 117.
40] A.K. Patra, M. Nethaji, A.R. Chakravarty, J. Inorg. Biochem. 101 (2007) 233.
41] X.L. Hong, H. Li, C.H. Peng, J. Mol. Struct. ꢀꢀ0 (2010) 1ꢀ7–203.
42] H.L. Huang, G.P. Tang, Q.Q. Wang, D. Li, F.P. Shen, J. Zhou, H. Yu, Chem.
Commun. (2006) 2382.
References
[
[
1] C.S. Allardyce, P.J. Dyson, Platinum Met. Rev. 45 (2001) 62.
2] J.M. Rademaker-Lakhai, D. Van Den Bongard, D. Pluim, J.H. Beijnen, J.H.M.
Schellens, Clin. Cancer Res. 10 (2004) 3717.
[
[
[
[
3] G. Sava, A. Bergamo, S. Zorzet, B. Gava, C. Casarsa, M. Cocchietto, A. Furlani, V.
Scarcia, B. Serli, B. Iengo, E. Alessio, G. Mestroni, Eur. J. Cancer 38 (2002) 427.
4] A. Bergamo, R. Gagliardi, V. Scarcia, A. Furlani, E. Alessio, G. Mestroni, G. Sava, J.
Pharmacol. Exp. Ther. 28ꢀ (1ꢀꢀꢀ) 55ꢀ.
5] G. Sava, I. Capozzi, K. Clerici, G. Gagliardi, E. Alessio, G. Mestroni, Clin. Exp.
Metastasis 16 (1ꢀꢀ8) 371.
[
[
[
43] V. Vijayanathan, T. Thomas, A. Shirahata, T.J. Thomas, Biochemistry 40 (2001)
13633.
44] T. Sarkar, A.S. Petrov, J.R. Virko, C.T. Santai, S.C. Harvey, I. Mukerji, N.V. Hud,
Biochemistry 48 (200ꢀ) 667.
45] C. Santini, M. Pellei, G.G. Lobbia, D. Fedeli, G. Falcioni, J. Inorg. Biochem. ꢀ4
(
2003) 348.
6] L. Morbidelli, S. Donnini, S. Filippi, L. Messori, F. Piccioli, P. Orioli, G. Sava, M.
Ziche Br, J. Cancer 88 (2003) 1484.