Production of reactive oxygen and nitrogen species by light irradiation of a nitrosyl phthalocyanine ruthenium complex as a strategy for cancer treatment

Article abstract of DOI:10.1039/c3dt52217b

Production of reactive oxygen species has been used in clinical therapy for cancer treatment in a technique known as Photodynamic Therapy (PDT). The success of this therapy depends on oxygen concentration since hypoxia limits the formation of reactive oxygen species with consequent clinical failure of PDT. Herein, a possible synergistic effect between singlet oxygen and nitric oxide (NO) is examined since this scenario may display increased tumoricidal activity. To this end, the trinuclear species [Ru(pc)(pz)₂{Ru(bpy) ₂(NO)}₂](PF₆)₆(I) (pc = phthalocyanine; pz = pyrazine; bpy = bipyridine) was synthesized to be a combined NO and singlet oxygen photogenerator. Photobiological assays using (I) at 4 × 10^-6 M in the B16F10 cell line result in the decrease of cell viability to 21.78 ± 0.29% of normal under light irradiation at 660 nm. However, in the dark and at the same concentration of compound (I), viability was 91.82 ± 0.37% of normal. The potential application of a system like (I) in clinical therapy against cancer may be as an upgrade to normal photodynamic therapy.

Full text of DOI:10.1039/c3dt52217b

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PAPER
Production of reactive oxygen and nitrogen species
by light irradiation of a nitrosyl phthalocyanine
ruthenium complex as a strategy for cancer
treatment†
Cite this: Dalton Trans., 2014, 43,
4021
Tassiele A. Heinrich,a Antonio Claudio Tedesco,b Jon M. Fukutoc and
Roberto Santana da Silva*a
Production of reactive oxygen species has been used in clinical therapy for cancer treatment in a tech-
nique known as Photodynamic Therapy (PDT). The success of this therapy depends on oxygen concen-
tration since hypoxia limits the formation of reactive oxygen species with consequent clinical failure of
PDT. Herein, a possible synergistic effect between singlet oxygen and nitric oxide (NO) is examined since
this scenario may display increased tumoricidal activity. To this end, the trinuclear species [Ru(pc)(pz)2-
{Ru(bpy)2(NO)}2](PF6)6 (I) (pc = phthalocyanine; pz = pyrazine; bpy = bipyridine) was synthesized to be a
combined NO and singlet oxygen photogenerator. Photobiological assays using (I) at 4 × 10−6 M in the
B16F10 cell line result in the decrease of cell viability to 21.78 0.29% of normal under light irradiation at
660 nm. However, in the dark and at the same concentration of compound (I), viability was 91.82 0.37%
of normal. The potential application of a system like (I) in clinical therapy against cancer may be as an
upgrade to normal photodynamic therapy.
Received 13th August 2013,
Accepted 14th December 2013
DOI: 10.1039/c3dt52217b
www.rsc.org/dalton
a hypoxic event usually results in diminished formation of
reactive oxygen species and consequent clinical failure of
Introduction
The importance of oxygen to living organisms and its essenti- PDT.12,13 The use of other classes of reactive species has been
ality to life is clear. Significantly, variation in the concentration proposed as an attempt to improve PDT, but the outcome
of this element is generally associated with the etiology of remains uncertain. Nitric oxide (NO) is one such proposed
some diseases.1–4 For example, tumors have often been radical molecule, since it displays tumoricidal character
described as being relatively low in oxygen concentration. The depending on the NO concentration14–17 and also could give
main reason for this hypoxia lies in the imbalance between the characteristic of vasodilation improving blood flow with a
oxygen supply and consumption. Insufficient supply of this consequent increase of oxygen concentration. The key to the
vital element is normally attributed to the abnormal structure successful application of NO in anticancer therapy is to
and impaired function of the microvessels responsible for achieve control of the NO concentration while ensuring that
blood vessel nutrition.5–7 The occurrence of hypoxia could the target tissue is reached. Several NO donor agents have
negatively affect oxygen-based therapies against cancer as is been studied for this purpose,18–22 including transition metal
the case of Photodynamic Therapy (PDT), which requires nitrosyl species. Owing to the specific features of the nitrosyl
the combination of light irradiation and the presence of ligand, ruthenium complexes are one of the most extensively
dioxygen.8–11 Indeed, when it comes to cancer treatment, the investigated systems.23–26 Among them, [RuL5NO]n+ complexes,
success of this therapy depends on oxygen concentration, and where L is a nitrogen-type ligand, have been shown to be
remarkable as NO deliver agents from a pharmacological
viewpoint.27–32 The vasorelaxation properties of these
complexes33–35 and some cytotoxicity properties also imply
aFaculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo,
that these species can be potentially useful in clinical
therapy.36–39 Therefore, the aim of this work was to
evaluate the synergistic effect elicited by nitric oxide and
singlet oxygen produced by external stimulation of the same
molecule as a function of time. To this end, the trinuclear
species [Ru(pc)(pz)2{Ru(bpy)2(NO)}2](PF6)6 (I) (Fig. 1) was
Av. do Café s/n, Ribeirão Preto, SP 14040-903, Brazil. E-mail: silva@usp.br
bDepartamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão
Preto, Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto,
SP 14040-900, Brazil
cDepartment of Chemistry, Sonoma State University, Rohnert Park, CA 94928, USA
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c3dt52217b
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Synthesis of ruthenium complexes
[Ru(pc)(pz)2]. The synthesis of [Ru(pc)] (pc = phthalo-
cyanine) was prepared following a published procedure.41 The
[Ru(pc)(pz)2] (pz = pyrazine) complex was synthesized with
slight modifications of a well established procedure.40 Briefly,
0.600 g (1.60 × 10−3 mol) of [Ru(pc)] complex was melted with
0.016 g (2.00 × 10−4 mol) of pyrazine at 90.0 °C. The reaction
flask was kept for 48 h and [Ru(pc)(pz)2] was obtained by
addition of methanol. The solid was separated by vacuum fil-
tration; washed three times with concentrated acetic acid,
water, ethanol, and diethyl ether; and finally dried under
vacuum. Yield: 40.0%. The synthesized [Ru(pc)(pz)2] complex
was characterized with respect to the UV-visible spectrum and
compared to published data.40
[{Ru(NO)(bpy2)}2RuPc(pz)2](PF6)6. The cis-[Ru(NO2)(bpy)2-
(NO)](PF6)2 complex was prepared according to a method pre-
viously described in the literature.42 The cis-[Ru(NO2)-
(bpy)2(NO)](PF6)2 salt (0.100 g, 1.92 × 10−4 mol) was dissolved
in acetone (20 mL). An equimolar amount of NaN3 (0.013 g)
was then dissolved in methanol (5 mL) and added dropwise to
the above solution. After 20 min, 1.00 × 10−4 of [Ru(pc)(pz)2]
previously dissolved in acetone (5 mL) was added to the stirred
solution and the reaction was allowed to proceed for 17 h.
After that, 0.5 mL of concentrated HPF6 was added to the
stirred solution to produce a blue-green precipitate. The solu-
tion was filtered off, and the solid washed three times with
concentrated acetic acid, water, ethanol, and diethyl ether, and
finally dried under vacuum. Yield 45.0%. (Anal. calc. for
Ru3C80H58N22O2P6F36: C, 37.94; H, 2.31; N, 12.17. Found: C,
41.22; H, 2.40; N, 12.14.)
Fig. 1 Molecular structure of [Ru(phthalocyanine)(pz)2{Ru(bpy)2NO}2]6+
.
synthesized and photobiological properties are described in
B16F10 cells.
Experimental section
Apparatus
The ultraviolet-visible (UV-Vis) spectra were recorded on an
Agilent 8753 Diode Array UV/Vis Spectrophotometer. Infrared
spectra were obtained with a Prestige-21 Schimazur 02190
FTIR spectrophotometer, at a resolution of 4 cm−1, using solid
samples pressed in KBr pellets. The pH measurements were
performed by means of a DM-20 pH meter from Digimed.
Singlet oxygen was indirectly measured using 1,3-diphenyl-
isobenzofuran (DPBF) as a probe as previously described.36,40
1
DPBF reacts with O2 irreversibly, leading to a decrease in the
band at 415 nm. The solutions containing DPBF in ethanol
(8.0 × 10−3 M) and the sensitizers (OD = 0.2 at the irradiation
wavelength) in PBS were prepared on the day of the analysis
and stored in the dark. A volume of 40 μL of the DPBF solution
was added to 2.0 mL of a solution of the phthalocyanine ruthe-
nium complex, which was then irradiated at 660 nm with a
diode laser (Colibri Quantum Tec). The irradiation period, con-
trolled by a shutter, was selected in such a way that around 5%
of the DPBF would be bleached in the first cycle (5–30 s
depending on the employed sensitizer). This procedure was
repeated at least 15 times. The DPBF absorption decay at
415 nm was recorded after each cycle. The measured absor-
B16F10 cell culture
B16F10 cells were cultured in 75 cm2 flasks with RPMI
medium (Gibco) containing 10% defined supplement fetal
bovine serum plus 100 IU mL−1 penicillin G, 100 mg mL−1
streptomycin, and 1 μg mL−1 amphotericin. Cells were seeded
until 75–90% confluence in 96-well plates, depending on the
applied assay, and cultured in a humidified incubator at
37.0 °C with 5.0% CO2.
Cell viability assay
bance was corrected by the absorbance of the sensitizer at the Cell proliferation was assessed using the classical [3-(4,5-
respective detection wavelength. The experiment was carried dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT)
out at least three times for each sensitizer. The DPBF quantum colorimetric assay, the MTT colorimetric assay, as described pre-
yields for each irradiation cycle were calculated as previously viously.43 To this end, 20 000 cells were incubated for 3 h in
described.40 The luminescence measurements of singlet 96-well cell culture plates containing the trinuclear species (I)
oxygen were performed in an apparatus consisting of an dissolved in DMSO solution and cells were incubated for 3 h at
Nd-YAG laser (Continuum, Santa Clara, CA) with excitation 37.0 °C in culture medium containing 10 μM MTT diluted in
(8 nm half life) provided by the third harmonic at 355 nm. The phosphate-buffered saline (PBS). The blue MTT formazan pre-
luminescent decay kinetics was detected by employing a cipitate was then dissolved in 200 μL DMSO, and the absorbance
germanium detector operating at liquid nitrogen temperature. was measured at 570 nm on an ELISA reader (Power Wave
A silicon filter was used to prevent any fluorescent signal from X equipped with KC4 Kineticalc for Windows). Cell viability is
interfering with the singlet oxygen measurements. NO release expressed as the percentage of these values in treated cells com-
was measured using an ISO–NOP NO meter from Word Pre- pared with the non-treated (control) cells. Data are presented as
cision Instruments.
the mean standard error of triplicate cultures.
4022 | Dalton Trans., 2014, 43, 4021–4025
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Table 1 Concentration of nitric oxide released (mol L−1) from (I) upon
photolysis. Correlations between pH and light irradiation wavelength
Phototoxicity assays
B16F10 cells were incubated for 4 h under the same conditions
as those used during the cellular viability assay. After incu-
bation with (I) in solution (4.0–0.04 × 10−6 M) the cells were
irradiated at 660 nm (5 J cm−2) using a diode laser (Quantum
Tech, São Carlos, Brazil). Immediately after light exposure, the
cells were rinsed with PBS and incubated again with RPMI for
4 h at 37.0 °C, for accomplishment of the MTT cell viability
assay. Control cells were incubated without (I) and irradiated
under the same conditions.
λirr
pH
377 nm
447 nm
660 nm
pH 3.0
pH 7.4
2.08 × 10−5
8.14 × 10−6
1.43 × 10−5
3.63 × 10−7
1.02 × 10−5
<10−7
[Ru(pc)(pz)2],40 the Q-band was shifted to low energy by
20 nm, probably as a result of the structural change undergone
by the phthalocyanine ring in the trinuclear complex (Fig. 2).
The luminescence spectrum of (I) in acetonitrile upon exci-
tation at the Q-band presented an emission band at 690 nm.
As for the NO measurement in complex (I), there was nitric
oxide (NO) release upon light irradiation at 377 nm (Table 1).
The photochemical pathway for the photolysis at high energy
was attributed to the electron transfer mechanism centered on
the MLCT band originating from the dπ(RuII)–π*(NO+) tran-
sition, by analogy with the similar photochemical behavior
observed for ruthenium bipyridine species,42 as depicted in
the equation below:
Student’s t-test and GraphPad Prism software were
employed for statistical analysis of the cell assays.
Results and discussion
The synthesis of the trinuclear species (I), as far as we know, is
the first example of two bonded nitrosyl ruthenium complexes
in the photosensitized phthalocyanine moiety. The complex (I)
is stable in aqueous solution in the dark, but upon irradiation
with visible light, nitric oxide was selectively photodissociated
from the nitrosyl ruthenium complex, as inferred by UV-vis
spectroscopy and NO-sensor measurement.
The FTIR spectrum of (I) displayed an usual NO frequency
at 1940 cm−1, characteristic of nitrosyl stretching41 (Fig. 1,
ESI†). The UV-visible spectrum in dimethylsulfoxide (DMSO)
(Fig. 2) consisted of three distinct regions, for which the
assignments were made on the basis of similar mononuclear
species.39,41 The bands at 634 nm (log ε = 4.52 M−1 cm−1) and
576 nm (log ε = 4.39 M−1 cm−1) were assigned as due to the
Q-band described for phthalocyanines. The shoulder at 318 nm
(log ε = 4.89 M−1 cm−1) was attributed to the metal ligand
charge transfer (MLCT) from ruthenium(II) to the bipyridine
dπ(RuII) → π*(bpy) and nitrosyl dπ(RuII) → π*(NO) ligands,
respectively. A band ascribed to the intraligand transition was
detected at 290 nm (log ε = 5.17 M−1 cm−1). Compared with
The produced NO amount decreased with increasing pH
(Table 1), which was attributed to the interconversion nitrosyl-
nitro species due to the attack of the hydroxide ion, which
should diminish the nitrosyl character of the nitrogen oxide
ligand. A very low NO quantity was also observed for light
irradiation at 660 nm, probably because the photochemical
mechanism for this process involves a photoinduced electron
transfer from the phthalocyanine site to the nitrosyl moiety.
Direct singlet oxygen production was measured in aceto-
nitrile solution and determined by time-resolved phosphor-
escence studies at 1270 nm by using an Nd-YAG laser λex
operating at 355 nm (inset, Fig. 3).44 The assays were also con-
ducted by photooxidation using 1,3-diphenylisobenzofuran
(DPBF) as a substrate. The singlet oxygen measurement was
performed via an indirect method monitoring DPBF consump-
tion by UV-Vis spectroscopy.45 The sample with (I) and DPBF
was irradiated with a laser (Colibri, Quantum TechR), λirr
660 nm. The decrease in DPBF concentration was followed at
415 nm (Fig. 3). The measured singlet oxygen quantum yield
was 0.35 when the sample was excited at 355 nm, which is in
the range of some phthalocyanine complexes,46,47 and 0.80
when the excitation was carried out at 660 nm. There was no
reduction in DBPF absorbance when the sample was irradiated
at 377 nm.
Photocytotoxicity assays using (I) were performed against
B16F10 cells. For this purpose, a stock solution of the com-
pound was stored in DMSO. The cells were seeded in a 96-well
plate for 4 hours prior to the treatment. Then, the cellular
Fig. 2 Absorption spectra of (I) (4.0 × 10−5 M) in DMSO (solid line) and
[Ru(pc)(pz)2] (1.84 × 10−5 M) in chloroform (dashed line).
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Fig. 5 Time course of NO release from (I) (2.0 × 10−6 M) in aqueous/
DMSO solution under reduction with ascorbic acid.
Fig. 3 Absorption spectra of (I) in the presence of DPBF after different
times of irradiation (30 s) with a 660 nm laser. Inset: exponential decay
of singlet oxygen emission after excitation at 355 nm with an Nd-YAG
laser.
light irradiation at 660 nm, while no effect was observed in the
dark.
The reduction of complex (I) with concomitant generation
of NO can easily be assessed in biological medium, as verified
by carrying out reduction with ascorbic acid (Fig. 5). Because it
is known that NO donors can be used as cancer chemothera-
peutic agents23,38,43,48,49 depending on their concentration, we
pursued the hypothesis that the NO released by photolysis of
(I) could act as an anticancer agent, which could explain the
cytotoxicity observed in Fig. 4.
Fig. 4 Cell viability vs. concentration for (I) against the B16F10 cell line.
The cells were treated with (I) under the indicated concentration. The
number of viable cells was assessed by MTT 4 hours after light exposure.
Results are presented as a percentage compared with the control and
Conclusions
The effect of (I) on cell viability seems to be due to a synergism
between NO and singlet oxygen. The potential application of a
system like (I) in clinical therapy against cancer should be con-
sidered an upgrade to normal photodynamic therapy. The use
of low intensity laser and the consequent marked cell viability
decrease in the B16F10 cell line described in this work is a
novelty and has never been observed by photobiological assays
using a photosensitizer. A possible biological mechanism
related to this cytotoxicity could be due to inhibition of mito-
chondrial respiration by nitric oxide increasing the bioavail-
ability of oxygen to produce ROS.50 The details of NO
and singlet oxygen generation and the relationship to the
biological applications of this complex are currently under
investigation.
the values are the mean
carried out in duplicate.
SEM of three independent experiments
medium was removed, and different concentrations of (I)
were added. Cells were exposed to the compounds for four
hours, followed by exposure to a light irradiation dose (λirr
660 nm; D = 5 J cm−2). The response to the light treatment was
measured 4 hours after irradiation. The same experimental
conditions were also employed in a control assay carried out
without light stimulus. Cell viability was measured by means
of the MTT assay (Fig. 4).43
These preliminary results showed that in the presence of (I)
at 4.0 × 10−6 M, the cell viability decreased to 21.78 0.29%
under light irradiation compared to controls (no compound)
while in the dark and at the same concentration of compound
(I) there was little cytotoxicity (91.82 0.97% viability com-
pared to the control). A similar experiment performed with
[Ru(pc)]36 demonstrated that the cell viability was reduced to
80.00 0.69% of the control (no compound) under 5 J cm−2
Acknowledgements
The authors thank FAPESP, CNPq, CAPES, National Science
Foundation and NAP photochem for financial support.
4024 | Dalton Trans., 2014, 43, 4021–4025
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