Targeting the mitochondrial VDAC in hepatocellular carcinoma using a polyclonal antibody-conjugated to a nitrosyl ruthenium complex

Article abstract of DOI:10.1007/s00775-018-1589-x

The rational design of anti-cancer agents includes a new approach based on ruthenium complexes that can act as nitric oxide (NO) donor agents against specific cellular targets. One of the most studied classes of those compounds is based on bis(bipyridine) ruthenium fragment and its derivative species. In this work, we present the chemical and cytotoxicity properties against the liver hepatocellular carcinoma cell line HepG2 of cis-[Ru^II(NO⁺)Cl(dcbpy)₂]^2? conjugated to a polyclonal antibody IgG (anti-VDAC) recognizing a cell surface marker. UV–visible bands of the ruthenium complex were assigned with the aid of density functional theory, which also allowed estimation of the structures that explain the biological effects of the ruthenium complex–IgG conjugate. The interaction of cis-[Ru^II(NO⁺)Cl(dcbpy)₂]^3? with mitochondria was evaluated due to the potential of these organelles as anti-cancer targets, and considering they interact with the anti-VDAC antibody. The cytotoxicity of cis-[Ru^II(NO⁺)Cl(dcbpy)₂]^3?-anti-VDAC antibody was up to 80% greater in comparison to the free cis-[Ru^II(NO⁺)Cl(dcbpy)₂]^3? complex. We suggest that this effect is due to site-specific interaction of the complex followed by NO release.

Full text of DOI:10.1007/s00775-018-1589-x

JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-018-1589-x
ORIGINAL PAPER
Targeting the mitochondrial VDAC in hepatocellular carcinoma using
a polyclonal antibody‑conjugated to a nitrosyl ruthenium complex
Loyanne C. B. Ramos1 · Fernando P. Rodrigues1 · Juliana C. Biazzotto1 · Sergio de Paula Machado2 ·
Leonardo D. Slep3 · Michael R. Hamblin4,5 · Roberto S. da Silva1,4,5
Received: 20 December 2017 / Accepted: 23 June 2018
© SBIC 2018
Abstract
The rational design of anti-cancer agents includes a new approach based on ruthenium complexes that can act as nitric
oxide (NO) donor agents against specifc cellular targets. One of the most studied classes of those compounds is based on
bis(bipyridine) ruthenium fragment and its derivative species. In this work, we present the chemical and cytotoxicity proper-
ties against the liver hepatocellular carcinoma cell line HepG2 of cis-[RuII(NO+)Cl(dcbpy)2]2− conjugated to a polyclonal
antibody IgG (anti-VDAC) recognizing a cell surface marker. UV–visible bands of the ruthenium complex were assigned
with the aid of density functional theory, which also allowed estimation of the structures that explain the biological efects
of the ruthenium complex–IgG conjugate. The interaction of cis-[RuII(NO+)Cl(dcbpy)2]3− with mitochondria was evaluated
due to the potential of these organelles as anti-cancer targets, and considering they interact with the anti-VDAC antibody.
The cytotoxicity of cis-[RuII(NO+)Cl(dcbpy)2]3−-anti-VDAC antibody was up to 80% greater in comparison to the free cis-
[RuII(NO+)Cl(dcbpy)2]3− complex. We suggest that this efect is due to site-specifc interaction of the complex followed by
NO release.
Keywords Nitrosyl ruthenium complexes · Conjugated ruthenium-antibody complex · Nitric oxide delivery agent
Introduction
In the last decade, there has been growing interest in the
Electronic supplementary material The online version of this
physiology of nitric oxide (NO) which has led to the devel-
article (https://doi.org/10.1007/s00775-018-1589-x) contains
opment of a variety of new NO donors [1–7]. Metal-based
compounds are one of the most promising classes of NO
donor agents. Among these compounds, nitrosyl ruthenium
complexes are the most studied and best understood species
[4, 5, 8–21].
supplementary material, which is available to authorized users.
* Roberto S. da Silva
silva@usp.br
Michael R. Hamblin
hamblin@helix.mgh.harvard.edu
In general, these complexes have the structure
{Ru–NO}3+, and they can act as NO delivery agents via (a) a
reduction process [12, 15–17], (b) via direct light irradiation
on the metal–ligand charge transfer band dπ(RuII)–π*(NO+)
[16, 17], or (c) by photoinduced electron transfer [20, 21].
Depending on the pH, {Ru–NO}3+ can be fully converted
to {Ru–NO2}+, which could act as an NO delivery agent
either by photolysis or by an oxygen transfer reaction, as
described in the literature [16, 22]. The dπ(RuII)–π*(NO+)
backbonding strongly infuences the formation of the nitro
compound [22].
1
Departamento de Física e Química, Faculdade de Ciências
Farmacêuticas de Ribeirão Preto, Universidade de São
Paulo, Ribeirão Preto, SP, Brazil
2
Instituto de Química, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, RJ 21941-590, Brazil
3
Departamento de Química Inorgánica, Analítica y Química
Física and INQUIMAE, CONICET, Facultad de Ciencias
Exactas y Naturales, Universidad de Buenos Aires, Pabellón
2, Ciudad Universitaria, C1428EHA Buenos Aires, Argentina
4
Wellman Laboratories of Photomedicine, Massachusetts
General Hospital, Boston, MA 02114, USA
For example, in the case of the complex cis-[Ru(NO2)
5
(bpy)2L]n+/0 (L=Cl−, pyridine, isonicotinamide, 4-picoline,
Department of Dermatology, Harvard Medical School,
Boston, MA 02115, USA
Vol.:(0123456789)
1 3

JBIC Journal of Biological Inorganic Chemistry
or 4-acetylpyridine), and where bpy is bipyridine (a strong
π-acceptor ligand), cis-[RuII(NO+)(bpy)2L]n+1 is formed
when the complex is dissolved in aqueous solution at
pH≤4.0 [5]. This may pave the way for the application of
this kind of system in bioassays, which are usually conducted
in physiological pH. Regarding in vitro and in vivo assays,
NO delivery agents based on ruthenium complexes have
been used for a number of biological purposes [4] including
experiments on vasorelaxation [22], Chagas disease [23–25],
and cytotoxicity [12–15]. Maybe the most appealing fea-
ture is the possibility to control the amount of NO release
based on the uptake of ruthenium species. For example, in
cancer cells, NO has been suggested to modulate several
diferent events [26, 27]. It seems to show dual functional-
ity: NO can be anti- or pro-tumorigenic depending on its
local concentration [26]. NO levels between 50 and 100 nM
activate signal transduction pathways that elicit tumorigenic
efects; above this level, NO exerts antitumoral functions
[26]. The key to maximize the efcacy and/or reduce the
side efects associated with NO, is better targeted delivery of
these agents to tumor sites. Several strategies have been used
in this way mainly focus in drug delivery system [28–34].
Although targeting NO delivery agents can promote an
increase in cellular uptake, it may reduce overall cytotoxic-
ity since certain threshold levels of NO over large areas may
be required for activation of signaling pathways. Efective
targeted NO delivery agents based on ruthenium complexes
may improve local NO concentration, which could increase
the cytotoxic efect of the nitric oxide derivative ruthenium
complex. Compounds containing ruthenium(II) and some
unsaturated ligands “L” show good stability mainly due to
existing backbonding dπ(RuII)–π*(L), and have been used as
models in kinetic and photochemical studies [35, 36]. One
class of compounds, for example, [RuII(bpy)2L(NO+)]n+ has
been studied due to its thermal stability, and also its ability
to release NO under external stimulation [4, 9, 10, 13, 14,
37–40]. This suggests these compounds might fnd applica-
tions as NO delivery agents and possible anti-cancer agents.
One possible target within cancer cells is mitochondria. In
addition to providing cellular energy, these organelles play
an important role in cell signaling events, including those
involving cell death [41]. The voltage-dependent anion chan-
nel (VDAC) is a protein in the mitochondrial outer mem-
brane, controlling cross-talk between mitochondria and the
remainder of cell. VDAC plays a central role in mitochon-
dria-mediated apoptosis by means of reactive oxygen spe-
cies (ROS) generation, and it should be noted that NO can
increase ROS levels [41]. Therefore, VDAC is a potential
target for NO donor agents based on ruthenium complexes.
To achieve this goal, one approach to improve the molecu-
lar specifcity of the ruthenium complex is by attaching it to
a ligand that recognizes a surface marker that is preferentially
expressed in cancer cells. Such strategy has been used for
some ruthenium complexes [42, 43]. Hence, this work aimed
to evaluate the cytotoxic efect of polyclonal antibody IgG,
anti-VDAC conjugated to the nitrosyl ruthenium complex cis-
[Ru(NO)Cl(dcbpy)2]3− where dcbpy is 2,2′-bipyridine-4,4′-
dicarboxylic acid as a NO delivery agent against the hepato-
cellular carcinoma cell line (HepG2).
Materials and methods
Chemical and reagents
RuCl3·nH2O, sodium nitrite, triethanolamine, N,N′-
dicyclohexylcarbodiimide (DCC), and N-hydroxysuccinim-
ide (NHS) were purchased from Sigma-Aldrich Chemicals
(Brazil) as high purity reagents. VDAC 1/2/3, FL-238, and
rabbit polyclonal IgG (200 µg mL−1) were acquired from
Santa Cruz Biotechnology (Santa Cruz, USA). Doubly dis-
tilled water was used in all experiments. Nitric oxide was
prepared by the reaction of nitric oxide with copper turnings
and was purifed according to the literature method [37].
Apparatus
The ultraviolet–visible (UV–Vis) spectra were recorded
on an Agilent 8453 spectrophotometer; the samples were
placed in a quartz cell with an optical path length of 1 cm.
The infrared spectra were acquired on a Prestige 21 Schi-
madzu FTIR spectrometer; the samples were pressed into
KBr pellets.
The pH was measured with a DM 20 pH meter (Digimed).
The photochemical experiments were carried out in aqueous
solution containing the ruthenium complex at 1.0×10−4 M.
A 447-nm Diode Laser Colibri (Quantum Tec®) was
employed. The electrochemical studies were performed in
aqueous solution using an AUTOLAB PGSTAT 30 potentio-
stat/galvanostat. A conventional three-electrode cell arrange-
ment consisting of a glassy carbon disk working electrode, a
platinum wire auxiliary electrode, and an Ag/AgCl reference
electrode was employed. The supporting electrolyte was KCl
at 0.1 mol L−1.
NO was measured on a Dual Channel Nitric Oxide Meas-
uring system inNO-T equipped with a selective amperomet-
ric sensor amiNO-600 from Innovative Instruments, Inc.
Mass spectrometry was accomplished on an ULTROTOFQ-
ESI-TOF Mass Spectrometer (Bruker Daltonics, Billerica,
USA) operating in the positive mode.
Computational methodology
Density functional theory (DFT) computations were
employed to fully optimize the ground state geometries
of all the species described in this work. The calculations
1 3

JBIC Journal of Biological Inorganic Chemistry
were done with Gaussian 09 using Becke’s three-parameter
hybrid functional with the correlation functional of Lee,
Yang and Parr formalized as the B3LYP hybrid functional
[44–47] and the efective core potential basis set LanL2DZ
[48–51], which proved to be suitable for geometry predic-
tions in coordination compounds containing metals of the
second row of the transition elements in the periodic table.
Diferent degrees of protonation of the carboxylate groups
in the dcbpy ligand were explored. The non-equivalency
of the –CO2− groups of the molecule renders four diferent
possibilities for monoprotonation and six possible combina-
tions for diprotonation. In addition, the carboxylato groups
can be engaged in solute–solvent hydrogen-bonding specifc
interactions, which have to be taken into account to avoid
artifacts in the computations arising from the excess of nega-
tive charge on the CO2− substituents. For this purpose, four
explicit water molecules were incorporated in the geometry
optimizations and all subsequent computations. All the dif-
ferent cases of mono- and diprotonated structures plus the
fully deprotonated molecule were fully optimized in vacuo.
Tight SCF convergence criteria and default setting were used
along the geometry optimizations. In all cases, vibrational
analyses prove that the stationary points reached along the
optimizations correspond to local minima in the potential
energy surface. The computations suggest that whereas the
monoprotonated stage is dominated by one single species,
several isomers might contribute in signifcant proportions
to the diprotonated species. The analysis of the electronic
structure was complemented with (TD)DFT computations
including at least 90 states of the same multiplicity than the
ground state. The spectra were computed at the gas phase
geometry. Apart from the explicit water molecules solvation
efects in aqueous solution were incorporated in the compu-
tations employing the PCM approximation, as implemented
in Gaussian 09.
was collected by fltration, washed with 30 mL of diethyl
ether, and stored under vacuum in the dark. Yield: 59%
(Anal. calc. for C24H22N5O13ClRu: C, 39.76; H, 3.06; N,
9.66. Found: C, 39.31; H, 2.96; N, 9.51%). m/z = 652.95
({[Ru(NO)Cl(dcbpy-H)2]}+).
Isolation of rat liver mitochondria
Mitochondria were isolated by standard diferential cen-
trifugation [52]. Male Wistar rats weighing approximately
200 g were killed by decapitation, according to research pro-
tocols approved by CEUA-USP, Rib. Preto. Livers (10–15 g)
were immediately removed, sliced in medium (50 mL)
consisting of 250 mM sucrose, 1 mM ethyleneglycol-
bis(β-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA), and
10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES)–KOH, pH 7.2, and homogenized three times for
15 s, at 1-min intervals, with a Potter–Elvehjem homog-
enizer. Homogenate was centrifuged (580g, 5 min), and
the resulting supernatant was centrifuged further (10,300g,
10 min). Pellets were then suspended in medium (10 mL)
consisting of 250 mmol L−1 sucrose, 0.3 mM EGTA, and
10 mM HEPES–KOH, pH 7.2, and centrifuged (3400g,
15 min). The fnal mitochondrial pellet was suspended in
medium (1 mL) consisting of 250 mM sucrose and 10 mM
HEPES–KOH, pH 7.2, and used within 3 h. Mitochondrial
protein content was determined by the Biuret reaction. For
the assays, mitochondria (0.5 or 1 mg protein mL−1) were
energized with 5 mM potassium succinate (plus 2.5 µM rote-
none) in a standard medium consisting of 125 mM sucrose,
65 mM KCl, and 10 mM HEPES–KOH, pH 7.4, at 30 °C.
Direct detection of NO release from complex
(Ru‑DCBPY)
NO release from complex (Ru-DCBPY) (100 µM) after
incubation with succinate-energized mitochondria (1 mg
protein mL−1) in standard medium was assessed with a
Selective Sensor Electrode (AmiNO 700) in the dark.
Synthesis of cis‑[Ru(NO)Cl(dcbpy‑H)2]
The complex cis-[Ru(NO)Cl(dcbpy-H)2] (Ru-DCBPY)
was synthesized according to a previously similar published
procedure [21]. The complex cis-[RuCl2(dcbpy-H2)2] was
the starting material; it was prepared in the same way as
cis-[RuCl2(bpy)2]·2H2O, described in [38]. Briefy, cis-
[RuCl2(dcbpy-H2)2]·2H2O (0.100 g, 0.14 mmol) was dis-
solved in 25 mL of milli-Q H2O and degassed with argon
for 30 min. Gaseous NO was then generated by reaction
between nitric acid solution (50% V/V) and metallic cop-
per. The produced gaseous mixture was passed through a
concentrated NaOH solution to eliminate NO2 traces. The
nitric oxide was then bubbled through the cis-[RuCl2(dcbpy-
H2)2]·2H2O complex solution for 40 min to give a reddish-
brown solution. This procedure was repeated three times for
24 h under constant stirring. The resulting brown precipitate
Mitochondrial assays
Mitochondrial oxygen consumption was monitored in the
standard medium, in an oxygraph equipped with a Clark-
type oxygen electrode (Hansatech instruments, oxytherm
electrode unit, UK). Mitochondrial membrane potential
was estimated spectrofuorimetrically; 10 µM of cationic dye
safranine O was used as probe and fuorescence was meas-
ured at 495/586 nm excitation/emission wavelength pair
[53]. ROS/RNS levels were evaluated by monitoring oxida-
tion of dichlorodihydrofuorescein diacetate (H2DCFDA)
(Molecular Probes, Eugene) at the 503/528 nm excitation/
emission wavelength pair [54]. The fuorimetric experiments
1 3

JBIC Journal of Biological Inorganic Chemistry
were accomplished on a Hitachi Model F-4500 spectro-
fuorimeter (Tokyo, Japan). Mitochondrial swelling was
estimated from the decrease in the apparent absorbance at
540 nm [55].
MTT cell viability assay
The viability of the HepG2 cells was assessed by means of
the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide] (MTT) colorimetric assay. The cells were plated on
96-well microtiter cell culture plates at 2×104 cells/well for
24 h. After the indicated treatment, the cells were incubated
at 37.0 °C for 3 h in a culture medium containing 10 mol L−1
MTT in phosphate-bufered saline (PBS). The blue MTT
formazan precipitate was then dissolved in 50 L of DMSO,
and the absorbance was measured at 570 nm with a multi-
well plate reader [57]. The cell viability was expressed as
the percentage of the absorption values in the treated cells
relative to the non-treated (control) cells. The data are pre-
sented as the means the standard error of three independent
experiments.
Synthesis of cis‑[RuCl(dcbpy)2(NO2)]4−‑anti‑VDAC
The porin antibody anti-VDAC was bound to the (Ru-
DCBPY) forming cis-[RuCl (dcbpy)2(NO2)]4−-anti-VDAC
via amide bond formation using a procedure similar to the
procedure described by Terpetsching et al. [56]. In this
protocol, DCC (0.230 g, 1.11 mM) and NHS (0.119 g,
1.030 mM) were dissolved in dimethylformamide (3.0 mL),
under stirring, and cooled in an ice bath. Next, (Ru-DCBPY)
(0.200 g, 2.53 mM) was added, and the mixture was stirred
for 2 h. The precipitate was removed by fltration through
a syringe flter (Millipore-Millex 0.22 µm). The fltrate
(500 μL) containing the active Ru complex was added to an
IgG solution in 0.2 M carbonate bufer pH 8.3 (1.0 mL, 1%
IgG) and stirred for 12 h. Purifcation of the labeled protein
was performed by gel fltration chromatography on Sepha-
dex G-25 coupled to a peristaltic pump (GILSON, miniplus
3) at 1 mL min−1 fow; 0.1 M phosphate bufer pH 7.2 was
used. The fractions were collected in a Fraction Collector
LKB, BROMA.
Statistical analysis
Statistical analysis was performed by two-way ANOVA.
Equality of variance was assumed with Bonferroni’s post
hoc test for pairwise comparisons. Results with P < 0.05
were considered statistically signifcant.
Results and discussion
NO bubbling through an aqueous cis-[RuCl2(dcbpy-H2)2]
solution afforded the nitrosyl ruthenium complex (Ru-
DCBPY) in high yield. Elemental analyses of complex
(Ru-DCBPY) as well as mass spectrum analysis (Supple-
mentary material: Figure 1) agreed with the proposed molec-
ular formula. Complex (Ru-DCBPY) displayed electronic
absorption spectrum with poor defned bands at 323 nm (log
ε = 3.97) and 305 nm (log ε = 4.13), which was assigned
based on theoretical calculations. Figure 1 provides a sche-
matic representation of the (Ru-DCBPY) complex, with the
carboxylato groups labeled to assist in the discussion that
follows. Table 1 compiles the most relevant DFT-computed
structural parameters (Ru–N and N–O distances, Ru–N–O
angle and IR frequencies) for the diferent cis-[RuII(NO+)
Cl(dcbpy-Hx)2]n (n vary of 2+ to −2) species explored in
this work.
Antibody detection (western blotting) and (UV–Vis)
assessment of fractions
IgG (anti-VDAC) was determined in (aliquot) fractions by
western blotting. The mitochondria harvested from aliquots
obtained from the Sephadex column were re-suspended in
CelLytic™M (Sigma, Brazil) lysis bufer for 2 min, on ice,
and centrifuged at 10,000g for 5 min. The resulting super-
natants were subjected to 12% SDS-PAGE, transferred to
a nitrocellulose membrane, and probed with antibodies
against cytochrome c (BD Pharmigen, purifed mouse anti-
cytochrome c) followed by horseradish peroxidase-coupled
detection.
HepG2 cell culture
The metric parameters are within the expectations for this
kind of compounds, with slight diferences depending on the
presence or absence of explicit water molecules along the
optimization procedure. In all cases, the N–O bond length at
ca. 1.19 Å and the almost linear Ru–N–O moiety is consist-
ent with a predominant RuII–NO+ character. The computa-
tions predict NO stretching frequencies (νNO) be insensi-
tive to the protonation degree of the complex. Although the
trans NO ligand has ofered efect on the nitrosyl stretching
frequency [58, 59], it seems to be not the case for nitrosyl
The HepG2 cells were obtained from the American Type
Culture Collection. The cell line was cultured in Dulbecco’s
medium supplemented with 10% fetal bovine fetal serum,
100 IU mL−1 penicillin G, 100 mg mL−1 streptomycin, and
1 µg mL−1 amphotericin. The cells were plated in 6, 12, 24
and 96-well plates and cultured until 75–90% confuence
was achieved (depending on the assay) in a humidifed incu-
bator at 37.0 °C with 5.0% CO2.
1 3

JBIC Journal of Biological Inorganic Chemistry
bipyridine ruthenium complexes, in which a net efect for
the σ-donor and π-acceptor character of diferent forms
of carboxybipyridine ligand does not show significant
change on the νNO stretching frequency. Bipyridine nitrosyl
ruthenium(II) complexes present νNO stretching bands in the
1800–2000 cm−1. The peak intensity varies from medium
to strong [4, 5, 10, 12–16, 20, 23, 37–40, 58–60]. In the
FTIR spectrum of (Ru-DCBPY) complex, νNO was found
in 1945 cm−1 indicating that the complex is of {RuII-NO+}6
type. In addition, the spectrum exhibits peaks at 1610,
1560 and 1460 cm−1, which is characteristic of the pyri-
dine rings. The carboxylate group is evidenced by a peak
at 1690 cm−1. The calculated νNO stretching frequencies for
(Ru-DCBPY) in this work were 1800 cm−1, which are lower
than the experimental ones. It is in part due to a tendency of
DFT methods with standard functionals to provide too delo-
calized molecular and electronic structures due to inherent
self-interaction errors [61–63]. However, the inclusion of
explicit water molecules at hydrogen-bonding distance of the
carboxylato groups brings the νNO values closer to the exper-
imental ones. In the absence of these specifc interactions,
Fig. 1 Schematic representation of the molecular structure of cis-
[Ru(NO)Cl(dcbpy)2]2−
Table 1 Structural parameters,
IR frequencies and relative
energies for the diferent
cis-[Ru(NO)Cl(dcbpy)2Hn]n−2
species computed in this
computations. The labels
employed to identify the
protonation sites correspond to
the ones in Fig. 1
N
Species
Protonation
pattern
dRu–N(O) (Å)
dN–O (Å)
νaNO
Fractionb
Ru–N–O
Isolated system
0
1
1
2
3
4
5
6
7
8
9
–
a
b
c
1.771
1.790
1.786
1.774
1.783
1.802
1.787
1.794
1.783
1.792
1.790
1.192
1.194
1.193
1.190
1.193
1.194
1.189
1.192
1.188
1.192
1.192
173.2
174.3
175.2
175.2
174.0
173.4
175.3
174.1
174.6
175.4
175.0
1808
1790
1797
1811
1795
1785
1808
1798
1815
1797
1799
100
78.8
2.5
0.8
d
18.0
80.1
0.4
1.1
0.0
0.5
17.9
2
a–b
a–c
a–d
b–c
b–d
c–d
10
11
With explicit water molecules
0
1
1
2
3
–
a
b
1.771
1.780
1.777
1.768
1.774
1.793
1.782
1.786
1.779
1.785
1.780
1.189
1.189
1.188
1.187
1.188
1.191
1.187
1.189
1.186
1.189
1.189
173.2
173.4
175.0
175.1
173.7
175.3
175.1
174.1
174.6
175.5
175.9
1819
1814
1820
1829
1819
1801
1819
1812
1826
1813
1816
100
54.0
9.4
4
5
c
d
4.1
32.5
53.0
4.5
8.4
1.5
11.0
21.6
2
6
7
8
9
10
11
a–b
a–c
a–d
b–c
b–d
c–d
aComputed for the optimized geometries in vacuo, values in cm−1
bComputed on the basis of the zero-point corrected energies arising from the PCM computations, neglect-
ing entropic contributions
1 3

JBIC Journal of Biological Inorganic Chemistry
the high charge located on the –CO2− groups leads to several
anomalies, not only in the stretching frequencies but also
in the electronic structure picture and the predicted elec-
tronic spectroscopy. Even when isolated molecules have
been included in the table for completeness, the following
discussion will involve only hydrogen-bonded complexes.
There is no easy way to obtain by DFT reliable acidity
constant values, which could assist in the determination of
the degree of protonation in solution. The literature related
to other species holding the {Ru(dcbpy)2}2+ core suggests
that, at the pH employed in our experiments, the nitrosyl
species is probably best described as holding an average of
two protonated carboxylato sites. Simple enthalpic argu-
ments allow establishing that three out of the six possible
diprotonated species present in solution (labeled 6–11 in
Table 1) should predominate in acidic aqueous solution, with
still non-negligible amounts of the remaining ones. The elec-
tronic structure and the (TD)DFT predicted spectra for all
these species are in fact very similar, because the Ru–NO
interaction dominates the orbital ordering. Figure 2 displays
a MO diagram highlighting some relevant orbitals computed
for isomer 6, expected to be the most abundant in solution.
The antibonding LUMO and LUMO + 1 orbitals have
22 and 25% contribution of metal-centered atomic orbit-
als, refecting extensive mixing between the Ru dπ and the
NO π* orbitals. This composition is comparable to other
{RuNO}6 species and is consistent with a well-preserved
nitrosonium character of the coordinated NO fragment. The
high covalency of the Ru–NO bond results in a noticeable
stabilization of the dπ orbitals, which leads to a HOMO that
is mostly centered on the non-protonated carboxylato groups
of the dcbp ligand. The occupied MOs immediately below
the HOMO are also dcbpy, dcpyH2 or Cl− centered and show
little contribution from the Ru fragment. The HOMO-8 is
the frst MO with measurable dπ Ru-centered contribution,
and it is necessary to go as deep as HOMO-10 and HOMO-
18 to fnd the bonding MOs that result from the interaction
between the dπ set and the nitrosyl ligand. This electronic
picture results in an electronic spectrum, which is shifted to
the high energy end of the visible region of the spectrum. In
aqueous acidic medium, the Ru-DCBPY was found to be
light yellow in color exhibiting band at 305 nm. The spec-
trum feature was similar to the data reported in the literature
for nitrosyl ruthenium bipyridine [5, 14, 17, 21, 22, 37]. Fig-
ure 3 compares the experimental and computed electronic
spectra.
The individual six possible diprotonated species spec-
tra show very subtle variations. Nevertheless, the difer-
ences in the absorption maxima for the six cases are within
the error for the computational methodology, allowing to
focus the analysis on one particular example without loss
of generality. For the species number 6 (TD)DFT predicts
the most intense absorption in the 250–800 nm to be cen-
tered at 315 nm. This transition, which is mostly a ligand-to-
ligand charge transfer transition between dcbpyH2 and NO+
centered MO’s, accounts for the experimental absorption
Fig. 2 Molecular orbital diagram for species number 6
1 3

JBIC Journal of Biological Inorganic Chemistry
scan windows to isolate redox events of interest. The frst
redox couple has E1/2 =0.14 V (vs. Ag/AgCl) and is attrib-
uted to the redox process centered on the nitrosyl group
(NO+/0) when the CV was performed in a potential win-
dow of + 0.4 to − 0.2 V for the negative scan at the scan
rate of 100 mV s−1 at 25 °C. The ratio of the anodic (Epa)
and cathodic (Epc) current peak heights decreases in slow
scan rate consistent with a coupled chemical reaction. The
results are in line with the loss of NO following reduction
process of (Ru-DCBPY) as generally observed for nitrosyl
ruthenium complexes [4, 5, 10–16, 22, 58, 65, 66]. A sec-
ond peak attributed as irreversible process was observed at
E1/2 = − 0.42 V (vs. Ag/AgCl), due to NO0/−, when the CV
was performed in a potential window of + 0.4 to − 0.8 V.
In aqueous solution, E1/2 for (Ru-DCBPY) couples shifts
0.02 V to a higher potential in comparison to cis-[Ru(NO)
Cl(bpy)2]2+ due diferent σ-donor, π-acceptor character of
co-ligands. It is consistent with the cis-[Ru(NO)L(bpy)2]3+
series where the highest π-acceptor character of L changes
the electrochemical process centered on nitrosyl ligand to
higher values [67].
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.6
0.4
0.2
0.0
300
400
500
600
700
800
wavelength(nm)
Fig. 3 Experimental and (TD)DFT-computed spectra for [Ru(NO)
Cl(dcbpy-H)2]. The computed spectrum corresponds to species num-
ber 7
maximum at 305 nm. The shoulder at lower energies origi-
nates in a series of closely spaced low intensity absorptions
of similar origin involving any of the three dcbpy, dcpyH2
or Cl− co-ligands. These transitions overlap with the usual
π–π* absorptions due to the bipyridinic moieties. Finally,
the experimental weak absorption band in the 450–500 nm
region seems to involve transitions between orbitals in the
range HOMO-7 to HOMO-14 and the NO-centered LUMO
and LUMO + 1 orbitals and have mixed ligand to ligand
and metal to ligand charge transfer character, revealing the
existence of low lying excited states with Ru(III)–NO(0)
character.
Chemical reduction of (Ru-DCBPY) in 0.015 M ascor-
bic acid produced cis-[RuCl(dcbpy-H)2(H2O)]− as judged
by NO-sensor measurements (inset Fig. 4). Conducted
spectroelectrochemistry at 0.1 V vs. Ag/AgCl in 0.01 M
KCl pH = 3.0 controlled by the addition of HCl solution
(Fig. 4) helped us to infer the electro-reduction process of
the (Ru-DCBPY) species (Scheme 2).
The well-established electrochemical mechanism of
(Ru-DCBPY) in aqueous solution may help to explain
some biological results related to its use as NO delivery
agent. Interaction sites for (Ru-DCBPY) in biological
medium are cell/organelles membranes, which are also
considered potential targets of anti-cancer drugs [16, 29,
41]. Among them, mitochondrial plays a critical role in
numerous biochemical processes, particularly oxidative
phosphorylation, process in which ATP synthesis is driven
by the transfer of electrons from NADH and FADH2 along
the respiratory chain in the inner mitochondrial membrane
(IMM), to molecular oxygen. This process generates elec-
trochemical proton gradient across the IMM used to drive
the ATP synthesis via F1F0-ATP synthase. During this
process, mitochondrial reactive oxygen species (ROS)
are produced and may elicit the mitochondrial permeabil-
ity transition (MPT) in association with Ca2+. MPT is a
cyclosporine-(CsA)-sensitive well-documented phenom-
enon mediated by the opening mitochondrial transition
pores, which is related with cellular necrosis and cellular
apoptosis [68–71]. Mitochondrial parameters, oxidative
and bioenergetics provide perspectives for anti-cancer
strategies. In this context, we have explored the efects of
NO generated from ruthenium nitrosyl (Ru-DCBPY) on
mitochondria.
In physiological pH, Ru-DCBPY was slowly converted to
cis-[RuCl(dcbpy)2(NO2)]4− by nucleophilic attack of hydrox-
ide ion (Scheme 2); kobs was 5.35 × 10−4 2.47 × 10−5 s−1
at 25.0 0.1 °C (Supplementary Material: Figure 2). In
pH ≤ 2.0, a precipitate starts to form, probably due proto-
nation of all carboxylate substituent. In that condition, we
are not being able to measure the pKa of bonded 4,4′-dicar-
boxilic acid. As judged by published pKa for some cis-
[Ru(dcbpy-H2)2L2]n+ [64], it is reasonable to assume three
equilibriums depending on pH for cis-[Ru(NO)Cl(dcbpy-
H2)2]2+ complex (Scheme 1). In physiological pH, the best
assumption for the molecular structure of the nitrosyl ruthe-
nium complex discussed in this work is the existence of the
equilibrium constant described in Eq. 3.
The electrochemistry of nitrosyl ruthenium complexes
has been extensively studied in both aqueous and nonaque-
ous media [37, 39, 65, 66]. In general, these complexes
undergo two one-electron processes between −1.0 and 1.0 V
vs. Ag/AgCl centered on the nitrogen oxide derivative ligand
[58].
Cyclic voltammogram (CV) measurements of (Ru-
DCBPY) in aqueous solution KCl 0.1 and 0.01 M ace-
tate bufer solution was made separately in two diferent
1 3

JBIC Journal of Biological Inorganic Chemistry
Scheme 1 Efect of pH on
the chemical composition of
cis-[Ru(NO)Cl(dcbpy-H2)2]2+
complex
First, we have determined the capability of succinate-
energized isolated mitochondria induce NO release from
(RU-DCBPY) using a selective electrode. Figure 5, in
the gray area, shows time-course of direct determina-
tion of NO release suggesting that ruthenium complex
underwent one-electron reduction centered on nitrogen
oxide derivative ligand site, followed by NO release in the
presence of mitochondrial NADH coenzyme. Previously,
we demonstrated two similar mechanisms of NO release
from ruthenium complexes of this same class, exposed
to NADH [12, 16]. NADH would bind directly to metal
centers of ruthenium and ruthenium complexes, which
suggests applicability of coenzyme.
1 3

JBIC Journal of Biological Inorganic Chemistry
NO released from complex (Ru-DCBPY) induced
Ca2+-dependent/CsA-sensitive mitochondrial swelling at
10–100 µM, which attested to NO involvement in the classic
mitochondrial permeability transition (MPT) (Fig. 7), a pro-
cess usually associated with ROS/RNS generation [71–74].
CsA almost completely inhibited swelling induced by com-
plex (Ru-DCBPY) at 50 µM. The inhibition of swelling by
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-
3-oxide (cPTIO), a NO scavenger, suggests that NO was the
MPT inducer. We propose that in the presence of superoxide
anion (O·2−) from mitochondria, NO generates peroxynitrite,
which in turn, inhibits respiratory chain and elicits MPT
[15, 16].
9000
8400
7800
7200
6600
6000
5400
1.5
1.0
0.5
0.0
0
150
300
450
600
Time (s)
300
400
500
600
700
800
Wavelength
Efects of NO released from complex (Ru‑DCBPY)
on mitochondrial membrane potential
and mitochondrial respiration
Fig. 4 Spectroelectrochemistry measurements of (Ru-DCBPY)
(7.4×10−5 M) in aqueous 0.01 M KCl solution, pH=3.0. Inset:
Chronoamperometry with NO sensor of aqueous (Ru-DCBPY)
(7.4×10−5 M) containing ascorbic acid (0.015 M)
Respiratory control ratio (RCR: state 3/state 4 respira-
tion rates) indicates the degree of mitochondrial coupling.
Table 2 shows that complex (Ru-DCBPY) at 10 and 50 µM
decreased RCR values, whilst at 100 µM it increased the
values. High NO concentrations inhibited respiration prob-
ably because reaction between NO and superoxide gener-
ated peroxynitrite, an irreversible respiratory chain inhibi-
tor [72–75]. In agreement with the RCR results, complex
(Ru-DCBPY) decreased the mitochondrial membrane
potential in a dose–response manner (10–100 µM) (Fig. 8).
Mitochondria efciently induced NO release from complex
(Ru-DCBPY) in a controlled way, via an oxidation–reduc-
tion mechanism. We propose that, once released, NO inhib-
ited respiratory chain and raised ROS levels inducing MPT;
ROS levels could be even increased by MPT. According to
Efects of complex (Ru‑DCBPY) on mitochondrial
reactive oxygen/nitrogen species (ROS/RNS) levels
and mitochondrial swelling
The production of ROS/RNS by HepG2 cells after incu-
bation with (Ru-DCBPY) was examined to provide evi-
dence on cell death mechanisms involving mitochondria.
Figure 6 shows increased ROS/RNS levels in succinate-
energized mitochondria, incubated as indicated, through
oxidation of the nonspecifc probe H2DFDA [54]. The
mitochondrial permeability transition inhibitor cyclo-
sporine A (CsA) partially inhibited the effect of NO
released from complex (Ru-DCBPY) at 100 µM (higher
concentration).
Scheme 2 Control reduction potential electrolysis of (Ru-DCBPY) specie at 0.1 V vs. Ag/AgCl in pH 3.0
1 3

JBIC Journal of Biological Inorganic Chemistry
activate mitochondrial pathway to apoptosis. RNS-induced
MPT may induce both apoptosis and necrosis. Various
mechanisms by which NO acts to kill diferent types of can-
cer, such as breast, cervical, gastric, colorectal, and head and
neck, have been addressed. However, local NO concentration
is the main factor involved.
(Ru‑DCBPY)–IgG conjugate
Nitrosyl ruthenium complexes may constitute useful NO
delivery agents in anti-cancer therapy because they allow for
control of the local NO concentration. We hypothesized that
conjugating the nitrosyl ruthenium complex (Ru-DCBPY)
with antibody, in basic medium, might improve the specifc-
ity of the complex for the target action site. Antibody–drug
conjugates (ADCs) have been extensively studied as a prom-
ising class of anti-cancer agents. Cell recognition by drugs
and consequent binding of the drug to target cells poten-
tially increase drug accumulation in the desired tissues and
organs. In this proof-of-concept study, we considered a novel
system involving polyclonal antibody IgG anti-VDAC, the
mitochondrial voltage-dependent anion-selective channel,
conjugated to complex (Ru-DCBPY). The aim is targeting
drugs to mitochondria, an organelle with high levels of the
reducer agent NADH. Complex (Ru-DCBPY) covalently
bound to the IgG immunoglobulin through a coupling reac-
tion illustrated in Scheme 3; a previously published proce-
dure was employed [56].
Fig. 5 Release of NO from (Ru-DCBPY), evaluated by mitochon-
dria-dependent oxy-reduction systems, detected directly through
selective electrode. (Ru-DCBPY) (100 µM) was incubated with mito-
chondria in standard medium. The plot, representative of three inde-
pendent experiments, shows NO release. Gray area shows time course
after mitochondria addition
600
100 μM
50 μM
400
200
100 μM + CsA
10 μM
Control
0
0
100
200
300
400
500
600
Time (s)
Fig. 6 Time course of mitochondrial ROS generation in the pres-
ence of NO released from complex (Ru-DCBPY), as assessed with
H2DCFDA. Mitochondria (0.5 mg protein mL−1) were incubated in
the standard medium described in Fig. 1, at 30 °C, in the presence of
5 µM succinate+2.5 µM rotenone. Plots are representative of at least
three experiments with diferent mitochondrial preparations
The resulting complex (Ru-DCBPY)–IgG conjugate was
purifed in a Sephadex G-25 column. UV–Vis spectroscopy
helped to identify the separated fractions containing the
complex (Ru-DCBPY)–IgG conjugate. The eluted conju-
gates were analyzed by Western blotting. Figure 9 depicts
the comparative cytotoxicity on the Hepg2 cell line induced
by free VDAC, complex (Ru-DCBPY) and complex (Ru-
DCBPY)–IgG conjugate. The viability of HepG2 cell
decreased in the presence of selected fractions from the col-
umn separation, containing the conjugates (fractions 14–17)
as compared to the control (cell culture medium only), free
VDAC, cis-[RuCl2(dcbpy-H)2] and free (Ru-DCBPY). Cel-
lular uptake measured for aqueous complex (Ru-DCBPY)
was in the order of 10−8 mol L−1, which contained the (Ru-
DCBPY)–IgG conjugate. Cytotoxicity was probably a con-
sequence of interactions between the anti-VDAC antibody
conjugate and the mitochondrial VDAC protein [72, 73].
In principle, the remaining complex (Ru-DCBPY) in the
complex (Ru-DCBPY)–IgG conjugate underwent chemical
reaction in the cancer cell membrane. This process could be
attributed to the reduction of conjugated (Ru-DCBPY) or
oxygen transfer reaction of cis-[RuCl(dcbpy)2(NO2)]4− gen-
erating NO, which may have reacted with superoxide to
give peroxynitrite, a species that inhibits the mitochondrial
respiration process by several biochemical pathways [16].
CsA; EGTA; CsA + 50 μM
Control
10 μM
50 μM
2min
100 μM
Fig. 7 Time course of mitochondrial swelling induced by NO
released from complex (Ru-DCBPY). Mitochondria (0.5 mg pro-
tein mL−1) were incubated in the standard medium described in
Fig. 1, at 30 °C, in the presence of 5 µM succinate+2.5 µmol L−1
rotenone. Data are representative of at least three experiments with
diferent mitochondrial preparations
Brown and Borutaite, NO-induced cell necrosis occurs via
respiratory chain inhibition at site I and/or IV and via RNS-
or oxidant-induced signal transduction. DNA damage may
1 3

JBIC Journal of Biological Inorganic Chemistry
Table 2 Efects of NO released
Mitochondrial parameters
dcbpy (µM)
Control
from complex (Ru-DCBPY)
on respiratory parameters of
succinate-energized isolated
rat liver mitochondria (1 mg
protein mL−1) in standard
medium
10
50
100
V3 (ng of O atoms min−1
)
)
231.5 6.3
31.3 2.2
245.1 7.4
6.4 1.4
197.0 11.3
34.5 2.1
189.1 5.3*
5.8 1.5
202.2 1.4
48.3 8.1
250.4 8.9
4.3 0.5
123.6 7.7
120. 8.3*
255.1 4.6
1.3 0.9*
1.3 0.4*
V4 (ng of O atoms min−1
VCCCP
RCR
ADP/O
2.0 0.2
1.7 0.2
1.7 0.3
*Values are presented as mean SEM (n = 3), P < 0.05 vs. control
Fig. 8 Time course of membrane potential induced by NO released
from complex (Ru-DCBPY) in succinate-energized mitochon-
dria. Mitochondria (1 mg of protein mL−1) were incubated with
10–100 µM of (Ru-DCBPY) in the standard medium, in the presence
of 5 µM succinate (plus 2.5 µmol L−1 rotenone), at 30 °C. Plots are
representative of at least three experiments with diferent mitochon-
drial preparations
Fig. 9 MTT cell viability assay. Efects on HepG2 cell viability of
follow treatments: control, DMEM cell culture medium; VDAC
(IgG anti-VDAC antibody solubilized in DMEM medium); com-
plex (I), ruthenium nitrosyl complex Ru-DCBPY not conjugated
with IgG anti-VDAC antibody, e.g., Ru-DCBPY; Fractions 14, 15,
16, and 17 were from the conjugate purifcation on a Sephadex col-
umn, as described in “Materials and methods”, (Ru-DCBPY)–IgG.
The experiments were performed in triplicate, independently, and
expressed as the mean and standard deviation
Typically, these mitochondrial events are involved in cell
death and can potentially kill tumor cells.
Conclusions
which could be explained by the (Ru-DCBPY) complex tar-
geting the mitochondrial VDAC where it undergoes reduc-
tion and NO release. Alterations of the NAD+/NADH redox
The use of an antibody–nitrosyl ruthenium complex conju-
gate was shown to be able to kill the HepG-2 cancer cell line.
Local NO release accounted for the observed cytotoxicity,
Scheme 3 Chemical synthesis of the NHS-ester of (Ru-DCBPY) ligation, in basic medium, with IgG anti-VDAC antibody (protein)
1 3

JBIC Journal of Biological Inorganic Chemistry
oxide donors: novel biomedical applications and perspectives, 1st
edn. Elsevier, New York, pp 1–24
couple have also been described as a new mechanism for
anti-cancer drug activity, and may be involved in the cyto-
toxicity of the (Ru-DCBPY)–IgG conjugate.
15. Toledo JC, de França Lopes LG, Alves AA, da Silva LP, Franco
DW (2002) Release of NO by a nitrosyl complex upon activa-
tion by the mitochondrial reducing power. J Inorg Biochem
89(3):267–271
Acknowledgements This work was supported by CNPq, CAPES,
FAPESP Grant 2016/08851-0 and scholarship 2009/11471-9 (Loyanne
Ramos). MRH was supported by US NIH Grants R01AI050875 and
R21AI121700, Air Force Ofce of Scientifc Research Grant FA9550-
13-1-0068, by US Army Medical Research Acquisition Activity Grant
W81XWH-09-1-0514, and by US Army Medical Research and Mate-
riel Command Grant W81XWH-13-2-0067.
16. Rodrigues FP, Pestana CR, Polizello AC, Pardo-Andreu GL,
Uyemura SA, Santos AC, Alberici LC, da Silva RS, Curti C
(2012) Release of NO from a nitrosyl ruthenium complex through
oxidation of mitochondrial NADH and efects on mitochondria.
Nitric Oxide 26(3):174–181
17. Pereira ADC, Ford PC, da Silva RS, Bendhack LM (2011)
Ruthenium-nitrite complex as pro-drug releases NO in a tissue
and enzyme-dependent way. Nitric Oxide 24(4):192–198
18. Fry NL, Mascharak PK (2011) Photoactive ruthenium nitrosyls as
NO donors: how to sensitize them toward visible light. Acc Chem
Res 44(4):289–298
References
19. Smith NA, Sadler PJ (2013) Photoactivatable metal complexes:
from theory to applications in biotechnology and medicine. Philos
Trans R Soc A 371:20120519
1. Miller MR, Megson IL (2007) Recent developments in nitric oxide
donor drugs. Br J Pharmacol 151(3):305–321
20. da Silva RS, Marchesi MS, Khin C, Lunardi CN, Bendhack LM,
2. Bonavida B (ed) (2010) Nitric oxide (NO) and cancer: prognosis,
prevention, and therapy. Springer Science and Business Media,
New York
Ford PC (2007) Photoinduced electron transfer between the cati-
2+
onic complexes [Ru(NH
3)5pz] and trans-[RuCl([15]aneN4)
NO]+ mediated by phosphate ion: visible light generation of nitric
oxide for biological targets. J Phys Chem B 111(24):6962–6968
21. Cicillini SA, Prazias ACL, Tedesco AC, Serra OA, da Silva RS
(2009) Nitric oxide and singlet oxygen photo-generation by light
irradiation in the phototherapeutic window of a nitrosyl ruthenium
conjugated with a phthalocyanine rare earth complex. Polyhedron
28(13):2766–2770
3. Serafim RA, Primi MC, Trossini GH, Ferreira EI (2012)
Nitric oxide: state of the art in drug design. Curr Med Chem
19(3):386–405
4. Tfouni E, Truzzi DR, Tavares A, Gomes AJ, Figueiredo LE,
Franco DW (2012) Biological activity of ruthenium nitrosyl com-
plexes. Nitric Oxide 26(1):38–53
5. de Lima RG, Silva BR, da Silva RS, Bendhack LM (2014)
Ruthenium complexes as NO donors for vascular relaxation
induction. Molecules 19(7):9628–9654
22. Da Rocha ZN, Marchesi MSP, Molin JC, Lunardi CN, Miranda
KM, Bendhack LM, Ford PC, da Silva RS (2008) The inducing
NO-vasodilation by chemical reduction of coordinated nitrite ion
in cis-[Ru(NO2)L(bpy)2]+ complex. Dalton Trans 32:4282–4287
23. Sarniguet C, Toloza J, Cipriani M, Lapier M, Vieites M,
Toledano-Magaña Y, García-Ramos JC, Ruiz-Azuara L, Moreno
V, Maya JD, Azar CO, Gambino D, Otero L (2014) Water-soluble
ruthenium complexes bearing activity against protozoan parasites.
Biol Trace Elem Res 159(1–3):379–392
6. Szabo C (2016) Gasotransmitters in cancer: from patho-
physiology to experimental therapy. Nat Rev Drug Discovery
15:185–203
7. Lundberg JO, Gladwin MT, Weitzberg E (2015) Strategies to
increase nitric oxide signalling in cardiovascular disease. Nat
Rev Drug Discov 14(9):623–641
8. Paulo M, Banin TM, de Andrade FA, Bendhack LM (2014)
Enhancing vascular relaxing efects of nitric oxide-donor ruthe-
nium complexes. Future Med Chem 6(7):825–838
24. Sesti-Costa R, Carneiro ZA, Silva MC, Santos M, Silva GK, Mil-
anezi C, da Silva RS, Silva JS (2014) Ruthenium complex with
benznidazole and nitric oxide as a new candidate for the treatment
of chagas disease. PLoS Negl Trop Dis 8(10):3207
9. Liu J, Duan Q, Wang J, Song Z, Qiao X, Wang H (2015) Pho-
tocontrolled nitric oxide release from two nitrosylruthenium
isomer complexes and their potential biomedical applications.
J Biomed Opt 20(1):015004
25. Iniguez E, Sánchez A, Vasquez MA, Martínez A, Olivas J, Sat-
tler A, Sánchez-Delgado RA, Maldonado RA (2013) Metal–drug
synergy: new ruthenium (II) complexes of ketoconazole are highly
active against Leishmania major and Trypanosoma cruzi and
nontoxic to human or murine normal cells. J Biol Inorg Chem
18(7):779–790
10. Carneiro ZA, Biazzotto JC, Alexiou AD, Nikolaou S (2014)
Nitric oxide photorelease from a trinuclear ruthenium nitrosyl
complex and its in vitro cytotoxicity against melanoma cells. J
Inorg Biochem 134:36–38
26. Ridnour LA, Thomas DD, Switzer C, Flores-Santana W, Isenberg
JS, Ambs S, Roberts DD, Wink DA (2008) Molecular mechanisms
for discrete nitric oxide levels in cancer. Nitric Oxide 19(2):73–76
27. Thomas DD, Ridnour LA, Isenberg JS, Flores-Santana W, Switzer
CH, Donzelli S, Colton C, Harris CC, Roberts DD, Wink DA
(2008) The chemical biology of nitric oxide: implications in cel-
lular signaling. Free Radic Biol Med 45(1):18–31
11. Castro PFDS, Andrade DL, Reis CDF, Costa SHN, Batista AC,
Silva RS, Rocha ML (2016) Relaxing efect of a new ruthe-
nium complex nitric oxide donor on airway smooth muscle of
an experimental model of asthma in rats. Clin Exp Pharmacol
Physiol 43(2):221–229
12. Rodrigues FP, Carneiro ZA, Mascharak P, Curti C, da Silva
RS (2016) Incorporation of a ruthenium nitrosyl complex into
liposomes, the nitric oxide released from these liposomes and
HepG2 cell death mechanism. Coord Chem Rev 306:701–707
13. Ramos LCB, Marchesi MSP, Callejon D, Baruf MD, Lunardi
CN, Slep LD, Bendhack LM, da Silva RS (2016) Enhanced
antitumor activity against melanoma cancer cells by nitric oxide
release and photosensitized generation of singlet oxygen from
ruthenium complexes. Eur J Inorg Chem 22:3592–3597
14. Negri LB, Martins TJ, Ramos LCB, da Silva RS (2017) Nitric
oxide derivative ruthenium compounds as NO-based chemother-
apeutic and phototherapeutic agents. In: Sebara AB (ed) Nitric
28. Stevens EV, Wells A, Shin JH, Liu J, Der CJ, Schoenfsch MH
(2010) Nitric oxide-releasing silica nanoparticle inhibition of
ovarian cancer cell growth. Mol Pharm 7(3):775–785
29. Hirst D, Robson T (2007) Targeting nitric oxide for cancer ther-
apy. J Pharm Pharmacol 59(1):3–13
30. Xiang HJ, Deng Q, An L, Guo M, Yang SP, Liu JG (2016)
Tumor cell specifc and lysosome-targeted delivery of nitric
oxide for enhanced photodynamic therapy triggered by 808 nm
near-infrared light. Chem Commun 52(1):148–151
1 3

JBIC Journal of Biological Inorganic Chemistry
49. Hay PJ, Wadt WRJ (1985) Ab initio efective core potentials for
molecular calculations. Potentials for the transition metal atoms
Sc to Hg. J Chem Phys 82:270–283
31. Park J, Pramanick S, Kim J, Leeab J, Kim WJ (2017) Nitric
oxide-activatable gold nanoparticles for specific targeting
and photo-thermal ablation of macrophages. Chem Commun
53(81):11229–11232
50. Hay PJ, Wadt WRJ (1985) Ab initio efective core potentials for
molecular calculations. Potentials for K to Au including the out-
ermost core orbitals. J Chem Phys 82:299–310
32. Safdar S, Payne CA, Tu NH, Taite LJ (2013) Targeted nitric
oxide delivery preferentially induces glioma cell chemosensi-
tivity via altered p53 and O6-methylguanine-DNA methyltrans-
ferase activity. Biotechnol Bioeng 110(4):1211–1220
33. Li YH, Guo M, Shi SW, Zhang QL, Yang SP, Liu JG (2017) A
ruthenium-nitrosyl-functionalized nanoplatform for the target-
ing of liver cancer cells and NIR-light-controlled delivery of
nitric oxide combined with photothermal therapy. J Mater Chem
B 5(38):7831–7838
51. Wadt WR, Hay PJJ (1985) Ab initio efective core potentials for
molecular calculations. Potentials for main group elements Na to
Bi. J Chem Phys 82:284–298
52. Pedersen PL, Greenawalt JW, Reynafarje B, Hullihen J, Decker
GL, Soper JW, Bustamente E (1978) Preparation and characteriza-
tion of mitochondria and submitochondrial particles of rat liver
and liver-derived tissues. Methods Cell Biol 20:411–481
53. Åkerman KE, Wikström MK (1976) Safranine as a probe of the
mitochondrial membrane potential. FEBS Lett 68(2):191–197
54. Wrona M, Patel K, Wardman P (2005) Reactivity of 2′, 7′-dichlo-
rodihydrofuorescein and dihydrorhodamine 123 and their oxi-
dized forms toward carbonate, nitrogen dioxide, and hydroxyl
radicals. Free Radic Biol Med 38(2):262–270
34. Deng Q, Xiang HJ, Tang WW, An L, Yang SP, Zhang QL, Liu
JG (2016) Ruthenium nitrosyl grafted carbon dots as a fuores-
cence-trackable nanoplatform for visible light-controlled nitric
oxide release and targeted intracellular delivery. J Inorg Bio-
chem 165:152–158
35. Wyland KR, Hofman EE, Jain A (2017) DNA interaction stud-
ies and photoinduced ligand exchange kinetics of a sterically
strained Ruthenium(II) complex. Inorg Chim Acta 454:62–66
36. Campagna S, Puntoriero F, Nastasi F, Bergamini G, Balzani
V (2007) Photochemistry and photophysics of coordination
compounds: ruthenium. In: Balzani V, Campagna S (eds) Pho-
tochemistry and photophysics of coordination compounds I.
Springer, Berlin, pp 117–214
55. Blondin GA, Green DE (1967) The mechanism of mitochondrial
swelling. Proc Natl Acad Sci USA 58(2):612–619
56. Terpetschnig E, Szmacinski H, Malak H, Lakowicz JR (1995)
Metal–ligand complexes as a new class of long-lived fuorophores
for protein hydrodynamics. Biophys J 68(1):342–350
57. Mosmann T (1983) Rapid colorimetric assay for cellular growth
and survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 65:55–63
37. Sauaia MG, de Lima RG, Tedesco AC, da Silva RS (2005)
Nitric oxide production by visible light irradiation of aque-
ous solution of nitrosyl ruthenium complexes. Inorg Chem
44(26):9946–9951
58. Tfouni E, Krieger M, McGarvey BR, Franco DW (2003) Struc-
ture, chemical and photochemical reactivity and biological activ-
ity of some ruthenium amine nitrosyl complexes. Coord Chem
Rev 236(1–2):57–69
38. Meyer TJ, Godwin JB (1971) Preparation of ruthenium nitrosyl
complexes containing 2, 2′-bipyridine and 1, 10-phenanthroline.
Inorg Chem 10(3):471–474
59. Caramori GF, Frenking G (2007) The nature of the Ru–NO bond
in ruthenium tetraammine nitrosyl complexes. Organometallics
26(24):5815–5825
39. Sauaia MG, Oliveira FS, Tedesco AC, da Silva RS (2003) Con-
trol of NO release by light irradiation from nitrosyl-ruthenium
complexes containing polypyridyl ligands. Inorg Chim Acta
355:191–196
60. Videla M, Jacinto JS, Baggio R, Garland MT, Singh P, Kaim W,
Slep LD, Olabe JA (2006) Structure, spectroscopy, and electro-
philic and nucleophilic reactivities of bound nitrosyl. Inorg Chem
45:8608–8617
40. Ghosh K, Kumar S, Kumar R (2011) Synthesis and characteriza-
tion of a novel ruthenium nitrosyl complex and studies on photo-
lability of coordinated NO. Inorg Chem Commun 14(1):146
41. Elmore S (2007) Apoptosis: a review of programmed cell death.
Toxicol Pathol 35(4):495–516
61. Cohen AJ, Mori-Sánchez P, Yang W (2008) Insights into
current limitations of density functional theory. Science
321(5890):792–794
62. Coropceanu V, Malagoli M, André JM, Brédas JL (2002) Charge-
transfer transitions in triarylamine mixed-valence systems: a joint
density functional theory and vibronic coupling study. J Am Chem
Soc 124(35):10519–10530
42. Grau-Campistany A, Massaguer A, Carrion-Salip D, Barragán
F, Artigas G, López-Senín P, Moreno V, Marchán V (2013) Con-
jugation of a Ru(II) arene complex to neomycin or to guanidi-
noneomycin leads to compounds with diferential cytotoxicities
and accumulation between cancer and normal cells. Mol Pharm
10(5):1964–1976
63. Renz M, Kaupp M (2012) Predicting the localized/delocal-
ized character of mixed-valence diquinone radical anions.
Toward the right answer for the right reason. J Phys Chem A
116(43):10629–10637
43. Wang T, Zabarska N, Wu Y, Lamla M, Fischer S, Monczak K, Ng
DYW, Sven Rau S, Weil T (2015) Receptor selective ruthenium-
somatostatin photosensitizer for cancer targeted photodynamic
applications. Chem Commun 51:12552–12555
64. Shimidzu T, Iyoda T, Izaki K (1985) Photoelectrochemical prop-
erties of bis (2,2′-bipyridine)(4,4′-dicarboxy-2,2′-bipyridine)
ruthenium(II) chloride. J Phys Chem 89(4):642–645
65. Borges SDS, Davanzo CU, Castellano EE, Zukerman-Schpector J,
Silva SC, Franco DW (1998) Ruthenium nitrosyl complexes with
N-heterocyclic ligands. Inorg Chem 37(11):2670–2677
66. Calandreli I, Oliveira FD, Liang GG, da Rocha ZN, Tfouni E
44. Becke ADJ (1988) Density functional calculations of molecular
bond energies. J Chem Phys 84:4524–4529
45. Becke ADJ (1993) Density-functional thermochemistry. III. The
role of exact exchange. J Chem Phys 98:5648–5652
(2009) Synthesis and characterization of trans-[Ru(NO)Cl(L)
46. Lee C, Yang W, Parr RG (1988) Development of the Colle–Sal-
vetti correlation-energy formula into a functional of the electron
density. Phys Rev B 37(2):785–789
]
(PF6)2(L=isonicotinamide; 4-acetylpyridine) and related specie4s.
Inorg Chem Commun 12(7):591–595
47. Perdew JP (1986) Density-functional approximation for the cor-
relation energy of the inhomogeneous electron gas. Phys Rev B
33:8822–8824
67. Sauaia MG, da Silva RS (2003) The reactivity of nitrosyl ruthe-
nium complexes containing polypyridyl ligands. Transit Met
Chem 28(3):254–259
48. Dunning TH Jr, Hay PJ (1976) Modern theoretical chemistry. In:
Schaefer HF III (ed) Modern theoretical chemistry. Plenum, New
York, pp 1–28
68. Zoratti M, Szabò I (1995) The mitochondrial permeability transi-
tion. Biochim Biophys Acta 1241(2):139–176
69. Halestrap AP (2009) What is the mitochondrial permeability tran-
sition pore. J Mol Cell Cardiol 46(6):821–831
1 3

JBIC Journal of Biological Inorganic Chemistry
70. Petronilli V, Cola C, Massari S, Colonna R, Bernardi P (1993)
Physiological efectors modify voltage sensing by the cyclosporin
A-sensitive permeability transition pore of mitochondria. J Biol
Chem 268(29):21939–21945
72. Brown GC (2007) Nitric oxide and mitochondria. Front Biosci
12(6):1024–1033
73. Brown GC, Borutaite V (2001) Nitric oxide, mitochondria, and
cell death. IUBMB Life 52(3–5):189–195
71. Petronilli V, Costantini P, Scorrano L, Colonna R, Passamonti S,
Bernardi P (1994) The voltage sensor of the mitochondrial perme-
ability transition pore is tuned by the oxidation-reduction state of
vicinal thiols. Increase of the gating potential by oxidants and its
reversal by reducing agents. J Biol Chem 269(24):16638–16642
74. Kroemer G, Galluzzi L, Brenner C (2007) Mitochondrial mem-
brane permeabilization in cell death. Physiol Rev 87(1):99–163
75. Gogvadze V, Orrenius S, Zhivotovsky B (2009) Mitochondria as
targets for chemotherapy. Apoptosis 14(4):624–640
1 3