Controlled nitric oxide photo-release from nitro ruthenium complexes: The vasodilator response produced by UV light irradiation

Article abstract of DOI:10.1016/j.ica.2005.03.019

Preliminary pharmacological studies of various nitric oxide (NO) photo-releasing agents are reported based on the flash-photolysis studies of the nitro ruthenium complexes cis-[Ru^II(NO₂)L(bpy) ₂]⁺ (bpy = 2,2′-bipyridine and L = pyridine, 4-picoline and pyrazine) and [Ru^II(NO₂)(bpy)(terpy)] ⁺ (terpy = terpyridine) in physiological medium. The net photoreactions under these conditions are two primary photoproducts, in (I) there is Ru^II-NO₂ photoaquation, where the photoproducts are Ru^II-H₂O plus NO2- and (II) homolytic dissociation of NO from a coordinated nitrito to derive the Ru^II-OH₂ specie and NO. Based on photochemical processes, the nitro ruthenium complexes were incorporated in water in oil (W/O) microemulsion and used in the vasorelaxation induced experiment. Denuded rat aortas were contracted with KCl and nitro ruthenium complexes in microemulsion were added. Perfusion pressures were recorded while arteries were irradiated at 355 nm The time to reach maximum relaxation was longer for [Ru^II(NO₂)(bpy)(terpy)] ⁺ complex (ca. 50 min, n = 6) than for cis-[Ru(NO₂)L(bpy) ₂]⁺ with L = py and 4-pic complex (ca. 28 min, n = 6) and cis-[Ru(NO₂)(bpy)₂ (pz)]²⁺ complex (ca. 24 min, n = 5).

Full text of DOI:10.1016/j.ica.2005.03.019

Inorganica Chimica Acta 358 (2005) 2643–2650
www.elsevier.com/locate/ica
Controlled nitric oxide photo-release from nitro ruthenium
complexes: The vasodilator response produced by
UV light irradiation
a
Renata Galv a˜ o de Lima , Mar ´ı lia Gama Sauaia , Daniella Bonaventura ,
a
b
a,b
b
b
Antonio Cl a´ udio Tedesco , Renata Fonseca Vianna Lopez , Lusiane M. Bendhack ,
a,b,
*
Roberto Santana da Silva
a
Departamento de Qu ı´ mica da Faculdade de Filosofia Ci eˆ ncias e Letras de Ribeir a˜ o Preto, Universidade de S a˜ o Paulo, Av. dos Bandeirantes
3900, 14040-901 Ribeir a˜ o Preto, SP, Brazil
Faculdade de Ci eˆ ncias Farmac eˆ uticas de Ribeir a˜ o Preto, Universidade de S a˜ o Paulo, Av. do Caf e´ s/n, 14040-903 Ribeir a˜ o Preto, SP, Brazil
b
Received 24 September 2004; accepted 6 March 2005
Available online 6 April 2005
Abstract
Preliminary pharmacological studies of various nitric oxide (NO) photo-releasing agents are reported based on the flash-photol-
II
ysis studies of the nitro ruthenium complexes cis-[Ru (NO )L(bpy) ] (bpy = 2,2 -bipyridine and L = pyridine, 4-picoline and pyr-
+
0
2 2
II
+
azine) and [Ru (NO )(bpy)(terpy)] (terpy = terpyridine) in physiological medium. The net photoreactions under these conditions
2
II
are two primary photoproducts, in (I) there is Ru –NO
II
photoaquation, where the photoproducts are Ru –H
ꢀ
2
2
O plus NO2 and (II)
II
homolytic dissociation of NO from a coordinated nitrito to derive the Ru –OH
2
specie and NO. Based on photochemical processes,
the nitro ruthenium complexes were incorporated in water in oil (W/O) microemulsion and used in the vasorelaxation induced
experiment. Denuded rat aortas were contracted with KCl and nitro ruthenium complexes in microemulsion were added. Perfusion
II
pressures were recorded while arteries were irradiated at 355 nm The time to reach maximum relaxation was longer for [Ru
+
+
)(bpy)(terpy)] complex (ca. 50 min, n = 6) than for cis-[Ru(NO
(
NO
n = 6) and cis-[Ru(NO )(bpy) (pz)] complex (ca. 24 min, n = 5).
2
2
)L(bpy)
2
]
with L = py and 4-pic complex (ca. 28 min,
2
+
2
2
ꢀ
2005 Elsevier B.V. All rights reserved.
Keywords: Nitro ruthenium complexes; Nitric oxide; Nitrosyl complexes
1
. Introduction
ited to ca. 100 ms due to its high reactivity, the descrip-
tion and evaluation of its biochemical pathway in a
physiological medium became complex [3]. In this con-
text, compounds capable of releasing NO in a specific
biological target could provide a useful tool to study
the physiological actions of NO [4–8]. Besides this effect,
the design of new NO delivery agents could also have a
potential biomedical application, once it could act as a
pharmaceutical agent providing NO under demand to
a specific site. Therefore, it is very important to study
the chemical and physico-chemical properties of such
NO delivery agent in physiological medium.
The discovery that nitric oxide (NO) plays important
roles as a biological messenger in a wide range of phys-
iological processes has stimulated great interests in the
studies of its chemical and biochemical properties,
especially the investigation of NO-mediated events [1].
Considering that the chemistry of NO is the primary
determinant of its biological properties [2], which is lim-
*
Corresponding author. Tel.: +55166024428; fax: +55166331092.
E-mail address: silva@usp.br (R.S. da Silva).
0
020-1693/$ - see front matter ꢀ 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2005.03.019

2644
R.G. de Lima et al. / Inorganica Chimica Acta 358 (2005) 2643–2650
Some metallonitrosyl complexes have been used as
2. Experimental
NO delivery agents [9–13]. The stability of the com-
plexes and the kinetics of NO release have been postu-
lated as dependent on the co-ligands and the metal ion.
Maybe the most studied system capable to release NO
under stimulation is the nitrosyl ruthenium species,
which is formally described as containing Ru –NO
bond [2]. Nitrosyl ruthenium complexes are attractive
because of their thermal stability, although under light
2.1. Apparatus
The ultraviolet–visible (UV–Vis) spectra were re-
corded on a Hitachi U-3501 and Genesys-2 apparatus
from Spectronic. The pH measurements were made
using a 430 pH meter from Corning. The photochemical
experiments were made using a laser flash-photolysis
apparatus consisting of a Continuum Q-switched
Nd-YAG laser (Continuum, Santa Clara, CA) with
excitation provided by the third harmonic at
k = 355 nm. The pulse length was 8 ns, the beam diame-
ter incident on the sample was 6 mm, and the repetition
rate was 10 Hz. The pulse energy was typically 15 mJ as
measured with a Field Master powermeter with an
L-30V head. NO release was measured with an ISO-
NOP NO meter from World Precision Instruments.
High Performance Liquid Chromatography (HPLC)
system consisted of a Shimadzu liquid chromatography
equipped with a LC-10AD pump, SPD-10AV UV–Vis
detector, and C-R6A integrator. A CLC-ODS column
(5 mm; 250_4.6 mm i.d.; Shimadzu) was used in all
experiments.
II
+
[
9–16] or electrochemical [17–20] stimulation they can
be NO delivery agents. Despite numerous investiga-
tions of nitrosyl ruthenium complexes, only few exam-
ples appear as stable in a physiological pH [21], which
has limited the use of those species as metal-based
drugs.
We have explored the potential of nitro ruthenium
complexes as an NO delivery agent in physiological
conditions. Some physical chemistry properties of cis-
II
+
[
methane [22]. Here, we present some photochemical
Ru (NO )(bpy) (py)] have been described in dichloro-
2 2
II
properties of cis-[Ru (NO )L(bpy) ] where bpy is
+
2
2
0
2,2 -bipyridine and L = pyridine, 4-picoline and pyra-
II
zine, and [Ru (NO )(bpy)(terpy)] with 2,2 :6 ,2 -terpy
+
0
0
00
2
is terpyridine (Scheme 1) in aqueous solution. The
observation that all nitro ruthenium complexes de-
scribed in this work are NO delivery agents directs us
to explore its vasodilator activity in an isolated rat
aorta. To avoid any side reactions in the cell using nitro
ruthenium complexes, we performed the pharmacologi-
cal assays with the complex inside of water in oil (W/O)
microemulsion, which was used as an efficient drug
delivery system. The aim of this work was to study the
effect of the nitro ruthenium complex as a pro-drug that
could release NO only when it was induced by light
irradiation.
2.2. Chemicals and reagents
0
RuCl ÆnH O, 2,2 -bipyridine (bpy), 4-picoline (4-
3
2
pic), pyridine (py), pyrazine (pz), and terpyridine (terpy)
were purchased as high purity reagents from Aldrich
Chemicals and were used as supplied. The recrystallized
II
+
complex salts cis-[Ru (bpy) (L)NO ](PF ) (L = py, 4-
2
6 3
II
+
pic) and [Ru (bpy)(terpy)NO ](PF ) were prepared as
6
3
previously described [23,24]. The complexes were char-
acterized by UV–Vis and infrared spectroscopy, and
then compared to the published results [23,24]. UV–
II
+
Vis spectra: k (nm) (loge) [Ru (bpy) (py)NO ](PF ) :
2
6 3
II
ꢀ1
3
34 (3.70), 300 (4.17) and FTIR m 1947 cm ; [Ru (b-
NO
+
2
py) (4-pic)NO ](PF ) : 332 (3.67), 300 (4.09) and FTIR
6 3
ꢀ
1
II
+
mNO 1944 cm
;
[Ru (bpy)(terpy)NO ](PF ) : 365
6 3
N
N
(3.87), 328 (4.05), 307 (4.24), 286 (4.30) and FTIR m
NO
1 II +
ꢀ
1949 cm . The cis-[Ru (bpy) (pz)NO ](PF ) was syn-
2 6 3
N
L
N
N
thesized by a similar procedure described for the synthe-
II
II
II
+
Ru
Ru
sis of cis-[Ru (bpy) (py)NO ](PF ) , but using pyrazine
2 6 3
N
N
NO2
as the attack ligand. Calc. for C H N OP F Ru: C,
24 20 7 3 18
NO2
3
1
0.44; H, 2.01; N, 10.53. Found: C, 30.08; H, 2.09; N,
II
N
N
0.23%. UV–Vis spectra: k (nm) (loge) [Ru (bpy) (pz)-
2
+
NO ](PF ) : 432 (3.34), 338 (3.74), 300 (4.26), and FTIR
6
3
ꢀ
1
L =
mNO 1950 cm
The cis-[Ru (NO )L(bpy) ]
.
II
+
with L = pyridine,
+
2
2
II
N
N
CH3
4-picoline and pyrazine, and [Ru (NO )(bpy)(terpy)]
2
N
N
were synthesized in situ by dissolving the nitrosyl specie
in a phosphate buffer solution, pH 7.4. The ionic
strength was adjusted with NaCl until 0.14 M in the
photochemical experiments. All preparations and mea-
surements were carried out under an argon atmosphere
py
4-pic
cis-[Ru(NO2)L(bpy)2]+
pz
[Ru(NO2)(bpy)(terpy)]+
Scheme 1.

R.G. de Lima et al. / Inorganica Chimica Acta 358 (2005) 2643–2650
2645
and protected from light. Doubly distilled H O was used
2
increments of 10 s. The calculated /NO values obtained
in the range studies were plotted versus t. These plots
were linear, with a negative slope, for the first 40% of
the reaction. The extrapolated spectroscopic quantum
for all experiments.
2
.3. HPLC mobile phase
yield at t = 0 was taken as /
for the photoaquation
NO
II
+
II
of NO from cis-[Ru (NO )L(bpy) ] and [Ru (NO )
Isocratic elution with (%) 20:80 ethanol:water con-
taining 0.1 M of phosphate buffer solution, pH 7.4,
2
2
2
+
(bpy)(terpy)] . Evaluation of /
at t = 0 eliminates
NO
ꢀ1
was used at a constant flow-rate of 1.0 ml min , at
room temperature. Samples for analysis were dissolved
in the mobile phase and 25 ll volumes were injected
possible complications resulting from secondary photo-
lysis of primary reaction product [30,31].
throughout the experiments and used to check the purity
II
of the complexes. Time retention for cis-[Ru (NO )L(b-
2.6. Preparation of nitro ruthenium complex formulation
2
+
py) ] with L = pyridine: 6.40 min; 4-picoline: 6.58 min;
Liquid water-in-oil (W/O) microemulsions were pre-
pared according to Wu et al. [32] and Gelfuso and Lopez
[33] by dissolving 64.25% (w/w) of olive oil and a 1:1
mixture of sorbitan monooleate (Span 80) and polyoxy-
ethylene 20 sorbitan monooleate (Tween 80) (32.50%
w/w) in an aqueous solution of nitro ruthenium com-
plex. The aqueous phase consisted of complexes (7.6 ·
2
II
pyrazine: 6.30 min and for [Ru (NO )(bpy)(terpy)] :
+
2
7
.46 min.
2.4. NO measurements with the ISO-NOP NO-meter and
the DUO-18 acquisition board
ꢀ
4
NO release was measured with an ISO-NOP NO me-
ter from World Precision Instruments. The sensitivity of
this apparatus ranges from 1 nM to 20 lM, with a 2 mm
sensor, which directly detects NO concentration by an
amperometric technique.
10 M) dissolved in propylene glycol/isotonic 0.1 M
phosphate buffer, pH 7.4 (1:1 v/v). To obtain a clear
isotropic system, all the ingredients were mixed by agi-
tating for 24 h. Microemulsions were stored at room
temperature and protected from light until used in the
experiments.
The calibration curve was constructed by dilutions of
nitric oxide stock solution. Nitric oxide gas was pur-
chased from Oxig eˆ nio do Brasil and passed through a
2.6.1. In vitro release studies of nitro ruthenium complex
The amount of nitro ruthenium complexes and their
release profiles was determined in vitro using a modified
Franz diffusion cell and a hydrophilic cellulose mem-
brane. The receptor medium was phosphate buffer solu-
tion, pH 7.4, at 37.0 ꢁC stirred at 300 rpm. The donor
compartment was filled with 1.5 g of microemulsion
ꢀ
1
0 M KOH solution to remove any trace of NO2
specie. Nitric oxide stock solution was prepared by
degassing aqueous phosphate buffer solution (pH 7.4)
followed by the introduction of nitric oxide as previ-
ously described [25]. The NO concentrations were calcu-
lated according to the reported molar fraction solubility
of NO (2.1 mM at 25 ꢁC) [26], which was confirmed by
II
+
II
containing cis-[Ru (NO )L(bpy) ] or [Ru (NO )(b-
2
2
2
+
ꢀ4
titration with KMnO as previously described [27].
py)(terpy)] complex at 7.6 · 10 M. The total avail-
able diffusion area of the cell was 1.3 cm . At regular
4
2
The 1 cm path length quartz cell containing the sample
solution was routinely thermostated at 25.0 ± 0.1 ꢁC. The
sample solution was stirred continuously during the flash-
photolysis experiment and the NO measurements were
made with the electrode positioned outside the light path
to avoid any photoelectric interference. The output of the
sensor was recorded with an IBM-PC computer linked to
a DUO-18 acquisition board from WPI.
intervals for up to 12 h, 1.5 ml of the receptor phase
was removed for the determination of the total drug
content by UV–Vis spectrophotometer, and replaced
by an equal volume of fresh receptor solution.
2.7. Pharmacological experimental protocols
2.7.1. Vessel preparation
Male Wistar rats (400–450 g) were killed by decapita-
2
.5. Quantum yield measurements
tion in accordance with the Ethical Animal Committee,
Ribeir a˜ o Preto Campus, University of S a˜ o Paulo, Bra-
zil. The thoracic aorta was quickly removed, dissected
free, and cut into 4 mm long rings. Since the response
to nitro ruthenium complexes does not require intact
endothelium, in the present study we investigated the
relaxation induced by this compound in endothelium-
denuded arteries. The endothelium was mechanically re-
moved and the aortic rings were placed between two
stainless-steel stirrups and connected to an isometric
force transducer (Letica Scientific Instruments) in order
Light intensities were determined by the classical
potassium ferrioxalate salt actinometry procedure after
each photolysis experiment [28,29]. The NO quantum
yield production was calculated based on NO concentra-
tions obtained by NO meter measurement. For most
II
runs, the initial concentration of cis-[Ru (NO )L
2
+
II
+
(
1
bpy) ] and [Ru (NO )(bpy)(terpy)] was ca. 1.0 ·
2
2
ꢀ
4
0
M. After the equilibration of the cell holder
temperature, photolysis was initiated by irradiating the
sample for a period of time ranging from 0 to 60 s with

2646
R.G. de Lima et al. / Inorganica Chimica Acta 358 (2005) 2643–2650
+
The [Ru(NO )L ] , which will be used to indicate the
to measure tension in the vessels. The rings were placed
in a 10 ml organ chamber containing Krebs solution
with the following composition (M · 10 ): NaCl 130,
2
5
II
+
cis-[Ru (NO )L(bpy) ] complex with L = pyridine,
2
2
3
II
4-picoline, pyrazine and [Ru (NO )(bpy)(terpy)] , was
+
2
KCl 4.7, KH PO 1.2, MgSO 1.2, NaHCO 14.9, glu-
cose 5.5, and CaCl 1.6. The solution was maintained
obtained based on the rate of conversion of nitrosyl to
2
4
4
3
3
nitro specie [22]. The [RuL (NO)] react very rapidly
+
2
5
at pH 7.4 gassed with 95% O and 5% CO at 37 ꢁC.
in solution with hydroxide ion to give deep yellow solu-
tion. The shape of the UV–Vis spectrum changed grad-
ually during the course of the reaction developing a new
2
2
The rings were initially stretched to a basal tension of
1
.5 g (previously determined by length–tension relation-
ship experiments), before allowing them to equilibrate
for 60 min in the bath, which was changed every 15–
3
+
band in visible region after dissolving [RuL (NO)] in
5
basic medium. This reaction is pH dependent once nitro-
syl ruthenium complex is regenerated in pH 1.0. One of
the spectral changes of the reaction of a nitrosyl ruthe-
nium complex with hydroxide ion can be observed in
Fig. 1.
20 min. Endothelial integrity was qualitatively assessed
by the degree of relaxation caused by acetylcholine
ꢀ
6
(
ACh, 1 · 10 M) in the presence of contractile tone in-
ꢀ
7
duced by phenylephrine (1 · 10 M) [34]. Since our
studies required endothelium-denuded aortas, the rings
were discarded if there was any degree of relaxation,
to avoid the possible influence of endothelial factors.
II
+
The cis-[Ru (NO )L(bpy) ] (L = py, 4-pic and pz)
2
2
II
and [Ru (NO )(bpy)(terpy)] complexes were character-
+
2
ized by UV–Vis spectrum (Table 1).
The flash-photolyses of cis-[Ru (NO )L(bpy) ] ,
II
+
2
2
0
2
.7.2. Light source
The radiation source for the photo-relaxation exper-
where bpy is 2,2 -bipyridine and L = py, 4-pic and pz,
II
and [Ru (NO )(bpy)(terpy)] were carried out with
+
2
iment was made using a UV lamp with peak intensity
at 355 nm. The UV lamp was placed next to the outer
wall of a jacketed glass incubation chamber. The dis-
tance from the lamp to the preparation during irradia-
tion was about 17 cm.
355 nm light irradiation in, pH 7.4, aqueous solution.
1.0
2
.7.3. Experimental protocols
Time-course for the relaxation induced by cis-[Ru (-
II
+
NO )L(bpy) ] (L = pyridine, 4-picoline and pirazine)
II
and [Ru (NO )(bpy)(terpy)] complexes.
2
2
+
0.5
2
ꢀ
4
The nitro ruthenium complexes (1 · 10 M) were
added to the organ chamber when a stable contraction
in response to 6 · 10 M KCl was achieved. Following
the addition of nitro ruthenium complexes, the 355 nm
light was irradiated for 50 min.
ꢀ
5
0.0
3
00
400
500
600
wavelength (nm)
3
. Results and discussion
Fig. 1. Spectral profile change with pH for the conversion of
+
II
Ru (bpy)(terpy)NO ] in [Ru (NO
+ 3+
II
[
2
)(bpy)(terpy)] . pH is 2.48, 4.3,
The production of NO using a coordination com-
5
.72, 7.16 and 10.29.
pound is of great interest, as one way to mimic many
NO dependent physiological processes in nature, and
of developing novel NO delivery systems. One of the
possibilities is to use light irradiation in the appropriate
wavelength on a solution containing metalonitrosyl
complexes leading to control release of NO. Unfortu-
nately, most nitrosyl ruthenium complexes, which could
act as NO delivery agent under light stimulation, have
been described mainly for the photolysis in acidic med-
ium [9,11,12]. Taking into account that a metal-based
drug should be stable in physiological pH and consider-
ing that a nitro compound has been described as a NO
delivery agent for some coordination compound [35–
Table 1
Electronic spectral data for nitro ruthenium complexes
a
Complex
k (nm)/loge
II
+
cis-[Ru (NO )(bpy) (py)]
2
2
2
2
2
2
240/4.20; 286/4.35
332/3.86; 416/3.57
II
cis-[Ru (NO
+
)(bpy)
)(bpy)
(4-pic)]
240/4.13; 286/4.37
342/3.74; 418/3.62
II
cis-[Ru (NO
+
(pz)]
246/4.15; 284/4.31
408/3.68
II
Ru (NO
+
[
2
)(bpy)(terpy)]
240/4.16; 274/4.26
2
82/4.34; 308/4.27
330/3.97; 420/3.69
48/3.72
3
9], we decided to evaluate the possibility to have nitro
4
ruthenium specie as a source of NO using 355 nm light
irradiation at pH 7.4.
a
All data were collected in phosphate buffer solution, pH 7.4.

R.G. de Lima et al. / Inorganica Chimica Acta 358 (2005) 2643–2650
2647
0
0
0
0
.16
.14
.12
.10
The electronic spectra after light irradiation of cis-
II
+
[
Ru (NO )(bpy) (4-pic)] complex could be observed
2 2
in Fig. 2 as example of the photochemical experiment
for a nitro ruthenium specie.
II
The absorption peaks of [Ru (NO )L ] observed in
+
2
5
4
25 nm region decrease in intensity, and a new peak ap-
pears at 478 nm region. The spectral changes exhibit
isosbetic points (316, 372 and 460 nm) during the course
of photolysis, which is consistent with the photoreaction
process. The photolysis of the nitro ruthenium com-
plexes was also carried out using a NO sensor, which
is an indubitable way to prove the NO release. The ob-
0.08
.06
0.04
.02
0.00
0
0
II
served quantum yield for the NO release from cis-[Ru
+
0
50
100
150
200
II
+
(
NO )L(bpy) ] and [Ru (NO )(bpy)(terpy)] com-
2 2 2
time (s)
plexes during photolysis at 355 nm was obtained by in
situ NO detection. The signal recorded by the NO sensor
increased quickly when the photolysis was initiated, then
decreased when the light was turned off owing to NO
consumption by various pathways [26] (Fig. 3). The
amplitude of the pulse is proportional to the concentra-
tion of free NO originated from the photolysis of cis-
Fig. 3. Chronoamperogram of NO released by flash-photolysis of
+
ꢀ5
II
2
using 355 nm light irradiation.
2 2
.0 · 10 M cis-[Ru (NO )(bpy) (4-pic)] at pH 7.4 buffer solution
Table 2
Quantum yield (/NO) values of nitro ruthenium complexes after flash-
photolysis at 355 nm
II
+
II
+
[
Ru (NO )L(bpy) ]
2
and [Ru (NO )(bpy)(terpy)]
2
2
ꢀ
/NO mol eistein
1a
Complexes
complexes, which was calculated based on the calibra-
tion curve. The NO concentration decreases after each
laser pulse, indicating that probably the photoproduct
absorbs part of the light. The calculated quantum yields
at 355 nm light irradiation are described in Table 2 for
the NO formation.
II
cis-[Ru (NO
+
2
2
2
)(bpy)
)(bpy)
)(bpy)
2
2
2
(py)]
0.007 ± 0.001
0.009 ± 0.001
0.037 ± 0.009
0.036 ± 0.009
II
cis-[Ru (NO
+
(4-pic)]
II
cis-[Ru (NO
+
(pz)]
II
Ru (NO
+
[
2
)(bpy)(terpy)]
a
All data were collected in phosphate buffer solution, pH 7.4.
The photochemical pathway was described mainly
based on the observations made on UV–Vis spectrum
and HPLC analysis. The resulting electronic spectrum
nium specie in physiological pH was made considering
II
the pKa of cis-[Ru (H O)L(bpy) ] and [Ru (H O)
2+
II
2
2
2
II
+
2+
for the photolysis of cis-[Ru (NO )L(bpy) ] and
2
(bpy)(terpy)] and by comparison to the synthesized
aqua ruthenium species [40,41]. By HPLC we also could
observe that the nitro ruthenium species exhibited only
one peak in aqueous solution, while after light irradia-
tion, at 355 nm, two peaks were observed for the studied
complexes as described for the photolysis of cis-[Ru
2
II
+
II
[
(
Ru (NO )(bpy)(terpy)] was attributed to cis-[Ru
2
2
+
II
2+
H O)L(bpy) ] and [Ru (H O)(bpy)(terpy)] , respec-
2 2 2
tively. The attribution of aqua ruthenium complex as a
product for the photochemical process of a nitro ruthe-
+
II
+
(NO )(bpy) (4-pic)] and [Ru (NO )(bpy)(terpy)] as
example for the photochemical studies of [Ru
2 2 2
0
0
0
0
0
.8
.6
.4
.2
.0
II
+
NO )L ] (Fig. 4).
The possibility to have nitrite release after light irra-
(
2 5
diation of a nitro ruthenium complex was also checked
by HPLC analysis in a similar procedure previously de-
scribed [42]. This technique allows the chromatographic
separation and online determination of the free nitrite
by comparison to the sodium nitrite HPLC analysis,
which appears at 4.30 min. We found no evidence for
the presence of free nitrite originated from light irradia-
II
+
II
tion at 355 nm of cis-[Ru (NO )L(bpy) ] and [Ru
2
2
+
NO )(bpy)(terpy)] complexes by HPLC, although it
(
2
has been related for some nitro ruthenium complexes
[35–39]. Taking these results into consideration, it is
2
00
300
400
500
600
ꢀ
possible to infer that limited amounts of NO2 could
wavelength (nm)
be produced during the photolysis of nitro ruthenium
complexes, which should be converted to NO by second-
ary photochemical reaction. On account for UV–Vis
II
Fig. 2. Electronic spectra in UV–Vis region of cis-[Ru (NO
2 2
)(bpy) (4-
+
ꢀ5
pic)] 2.0 · 10 M complex after flash-photolysis at 355 nm.

2648
R.G. de Lima et al. / Inorganica Chimica Acta 358 (2005) 2643–2650
+
(
a)
irradiation at 355 nm of cis-[Ru(NO )L ] can also pro-
2 5
III
duce {cis-[Ru (NO )L L ] }* at excited state, which
ꢀ +
2
4
can act as reducing agent, as described for similar sys-
III
tems [46]. The cis-[Ru (NO )L ] complex decom-
2+
2
5
poses in aqueous solution at pH 7.4 as observed by
UV–Vis spectrum after controlled potential electrolysis
at 0.9 V versus Ag/AgCl of an aqueous solution of cis-
II
+
[
Ru (NO )L ] complex.
2 5
The efficiency of the system was also evaluated in a
microemulsion system. Recent research work has fo-
cused on the use of microemulsion in several fields,
including drug delivery systems [47]. Microemulsion
could be used in order to solubilize drugs [48] and im-
prove systemic and topical drug availability [48,49]. In
this work, the principal target in the use of microemul-
sion is to minimize any side effect that could be caused
by the unreactive ruthenium compound, and also due
to the interest in creating nitric oxide (NO)-releasing
materials that can be used in a wide variety of biomed-
ical applications.
0
2
4
6
8
10
12
14
16
18
20
time (minute)
(b)
Owing to the solubility of nitro ruthenium complexes
in water, we assumed that the best type of microemul-
sion for this case is water/oil (W/O) system, where the ni-
tro ruthenium complex is solubilized in the internal
hydrophilic phase. The systematic approach used and
the presentation of the various physico-chemical and
biopharmaceutical aspects should facilitate the main
considerations involved in designing and characterizing
a specific self-dispersing NO release from a nitro ruthe-
nium complex in a microemulsion. Among these studies,
we evaluated the nitro ruthenium complex release from
the microemulsion using Franz diffusion cells [50].
Two compartment of Franz diffusion cells, equipped
with a synthetic membranes were employed for release
studies. The synthetic thin membrane separates the do-
nor compartment, filled with the nitro ruthenium com-
plexes-loaded microemulsion, from the receiver one,
filled with free aqueous phase. All experiments were per-
formed at 37.0 ꢁC and sampling was taken over 12 h,
with a sample being collected in each hour. The amount
of nitro ruthenium complexes released was determined
spectrophotometrically, in a way that we could observe
an electronic spectrum characteristic of a nitro ruthe-
nium complex described in this work. Results showed
0
2
4
6
8
10
12
14
16
18
20
22
time (minute)
II
Fig. 4. Chromatograms of (a) cis-[Ru (NO
+
(4-pic)] complex;
2
)(bpy)
2
II
+
(
2
b) [Ru (NO )(bpy)(terpy)] complex after flash-photolysis at 355 nm.
spectrum, NO measurement, HPLC analysis and by
comparison to the photochemistry of sodium nitrite
43–45], we could suggest the photochemical pathway
for the nitro ruthenium complexes as described in
Eqs. (1)–(5).
[
ꢁþ * ½RuL
hm
ðH
OÞꢁ2þ þ NO ꢀ
ð1Þ
½
RuðNO
2
ÞL
5
5
2
2
H2O
hm
ꢀ
Å
Å
ꢀ
NO2 * NO þ O
ð2Þ
ð3Þ
þ
hm
III
2þ
ꢀ
Å
½
RuðNO ÞL ꢁ * ½Ru L OHꢁ þ OH þ NO
2
5
5
H2O
II
+
that the systems formulated as [Ru (NO )(bpy)(terpy)]
2
+
and cis-[Ru(NO )L(bpy) ] complexes demonstrated
ꢁþ * f½Ru ðNO
hm
ÞL
Lꢁ gꢂ
þ
þ
ð4Þ
2
2
III
½
½
RuðNO
III
2
ÞL
5
2
4
that microemulsion systems provided a reservoir effect
for nitro ruthenium complexes in ca. 1 h (Fig. 5). After
this time, the nitro ruthenium specie was released from
the microemulsion into the receptor solution.
The W/O microemulsion containing the nitro ruthe-
nium complex was submitted to light irradiation at
355 nm to evaluate the NO release owing to the vasodi-
latation effect occasioned by free nitric oxide. In those
experiments, denuded rat aortic rings were previously
contracted with KCl 60 mM. The relaxation response
2þ
III
ꢂ
Ru L OHꢁ þ f½Ru ðNO ÞL Lꢁ g
5
2
4
þ
!
H
OÞꢁ2þ þ ½Ru ðNO
ꢁ2þ
The formation of cis-[Ru (OH)L ] specie, which
II
III
½Ru L
5
ðH
2
2
ÞL
5
ð5Þ
II
+
5
was observed by UV–Vis spectrum as well by HPLC
Figs. 2 and 4), could arise from the reduction of
(
cis-[Ru (OH)L ] originated from the homolytic cleav-
III
+
5
II
age of Ru –ONO during photolysis. We believe that the

R.G. de Lima et al. / Inorganica Chimica Acta 358 (2005) 2643–2650
2649
the hydroxo-ruthenium complex inside microemulsion
originating the nitro ruthenium specie. The recombina-
tion of NO to the fragment after photolysis was also ob-
served for some S-nitrosothiol specie, which was
accelerated due to the matrix effect [51]. In fact, we have
0
0
0
0
0
0
0
.020
.018
.016
.014
.012
.010
.008
2
+
observed that the reaction of [RuL (H O)] complex
5
2
with nitric oxide when carried out in physiological
+
solution originates [Ru(NO )L ] complex. Further
2
5
II
investigation of the kinetic behavior of cis-[Ru
2
+
II
2+
H O)L(bpy) ] and [Ru (H O)(bpy)(terpy)] with ni-
(
2 2 2
tric oxide in different pH is now in progress in our
laboratory.
0
2
4
6
8
10
12
time (hour)
Fig. 5. Plot of absorbance at 448 nm vs. time during the liberation
II
experiment of [Ru (NO
+
)(bpy)(terpy)] load in microemulsion to
4. Conclusion
2
receptor solution.
Considering that all of these nitro ruthenium com-
plexes continuously release NO upon initiation of the
reaction, which was observed in an aqueous physiologi-
cal solution as well in a microemulsion, it is possibile for
us to infer that the photo-relaxation occurs due to the
NO photo-release. The report of the NO-releasing mate-
rial that utilizes light as an external on/off trigger per-
mits a precise control of the rate of NO released. This
control may allow the use of this gel, topically, which
can be a source of administration of NO in cancer
therapy.
0
1
2
3
4
5
6
7
8
9
0
0
0
0
0
0
0
0
0
1
1
00
10
Acknowledgment
0
5
10
15
20
25
30
35
40
45
50
Time (min)
This work was supported by grants from FAPESP,
CNPq and Pronex.
Fig. 6. Time-course for nitro ruthenium complexes-induced relaxa-
tion. Denuded thoracic aortic rings pre-contracted with 60 mM KCl
II
+
and 100 lM cis-[Ru (NO
2
)(bpy)
2
(py)] complex (ꢃ, n = 6), cis-
II
Ru (NO
+
(pz)] complex (,, n = 5) and [Ru (NO
II
[
2
)(bpy)
2
2
)(bpy)
+
terpy)] complex (n, n = 6) were added. Data are means ± SEM of
(
n experiments performed on preparations obtained from different
animals.
References
[
[
1] L.J. Ignarro, Nitric Oxide Biology and Pathobiology, first ed.,
Academic Press, San Diego, CA, 2000, p. 41.
2] J.A. McCleverty, Chem. Rev. 104 (2004) 403.
was recorded while arteries were irradiated. The rapid
decrease of tension was obtained when the Krebs solu-
[3] S. Herold, W.H. Koppenol, Chimia 55 (10) (2001) 870.
4] R.F. Furchgott, Biosci. Rep. 19 (4) (1999) 235.
[5] D.M. Djuric, M.T. Nesic, I.Z. Andjelkovic, Mediat. Inflamm. 5
1) (1996) 69.
6] S. Lorens, J. Jordan, E.J. Nava, Physiol. Biochem. 58 (3) (2002)
79.
[7] D. Levy, D.W. Zochodne, Eur. J. Neurosci. 10 (5) (1998) 1846.
[
II
tion containing cis-[Ru (NO )L(bpy) ] complexes in-
+
2
2
(
side a microemulsion was irradiated at 355 nm (Fig.
II
[
6
). Similar results were also observed when the [Ru
+
1
(
NO )(bpy)(terpy)] complex in a microemulsion was
2
[
8] S. Gulbenkian, R. Uddman, L. Edvinsson, Peptides 22 (6) (2001)
95.
9] V. Togniolo, R.S. da Silva, A.C. Tedesco, Inorg. Chim. Acta 316
1–2) (2001) 7.
used as vasodilator agent (Fig. 6). The relaxation was
completely terminated when a microemulsion without
the nitro ruthenium complex was submitted to the same
light irradiation.
9
[
(
[
[
[
10] M.G. Sauaia, F.S. Oliveira, A.C. Tedesco, R.S. da Silva, Inorg.
Chim. Acta 355 (2003) 191.
The time to reach maximum relaxation was longer for
+
II
11] F.D. Oliveira, V. Togniolo, T.T. Pupo, A.C. Tedesco, R.S. da
Silva, Inorg. Chem. Commun. 7 (2) (2004) 160.
[
than for cis-[Ru (NO )L(bpy) ] with L = py and
Ru (NO )(bpy)(terpy)] complex (ca. 50 min, n = 6)
2
II
+
2
2
12] M.G. Sauaia, R.G. de Lima, A.C. Tedesco, R.S. da Silva, J. Am.
Chem. Soc. 125 (48) (2003) 14718.
II
4
-pic complex (ca. 28 min, n = 6) and cis-[Ru (NO )
2
+
(bpy) (pz)] complex (ca. 24 min, n = 5). This difference
2
may be an indication that nitric oxide can combine with
[13] Y.X. Wang, P. Legzdins, J.S. Poon, C.Y. Pang, J. Cardiovasc.
Pharmacol. 35 (2000) 73.

2650
R.G. de Lima et al. / Inorganica Chimica Acta 358 (2005) 2643–2650
[
14] R.M. Carlos, A.A. Ferro, H.A.S. Silva, M.G. Gomes, S.S.S.
Borges, P.C. Ford, E. Tfouni, D.W. Franco, Inorg. Chim. Acta
[33] G.M. Gelfuso, R.F.V. Lopez, Braz. J. Pharm. Sci. 39 (2003)
326.
3
57 (5) (2004) 1381.
[34] R.F. Furchgott, J.V. Zawadzki, Nature 288 (1980) 373.
[35] I.V. Lorkovic, K.M. Miranda, B. Lee, S. Bernhard, J.R. Schoo-
nover, P.C. Ford, J. Am. Chem. Soc. 120 (1998) 11674.
[36] M.A. DeLeo, P.C. Ford, Coord. Chem. Rev. 208 (2000) 47.
[37] K.S. Suslick, R.A. Watson, Inorg. Chem. 30 (5) (1991)
912.
[
[
15] A.K. Patra, P.K. Mascharak, Inorg. Chem. 42 (23) (2003) 7363.
16] C.F. Works, C.J. Jocher, G.D. Bart, X.H. Bu, P.C. Ford, Inorg.
Chem. 41 (14) (2002) 3728.
[
[
[
[
[
[
[
[
[
[
17] B. Serli, E. Zangrando, T. Gianfeffara, L. Yellowlees, E. Alessio,
Coord. Chem. Rev. 245 (1–2) (2003) 73.
18] V.R. de Souza, A.M.D. Ferreira, H.E. Toma, J. Chem. Soc.,
Dalton Trans 3 (2003) 458.
[38] M. Yamaji, Y. Hama, Y. Miyazaki, M. Hoshino, Inorg. Chem.
31 (1992) 932.
19] A.K.M. Holanda, D.L. Pontes, I.C.N. Di o´ genes, I.S. Moreira,
L.G.F. Lopes, Transition Metal Chem. 29 (4) (2004) 430.
20] J.C. Toledo, H.A.S. Silva, M. Scarpellini, V. Mori, A.J. Camargo,
M. Bertotti, D.W. Franco, Eur. J. Inorg. Chem. 9 (2004) 1879.
21] D. Bonaventura, F.S. Oliveira, V. Togniolo, A.C. Tedesco, R.S.
da Silva, L.M. Bendhack, Nitric Oxide 10 (2004) 83.
[39] R.M Carlos, D.R. Cardoso, E.E. Castellano, R.Z. Osti, A.J.
Camargo, L.G. Macedo, D.W. Franco, J. Am. Chem. Soc. 126
(2004) 2546.
[40] B.A. Moyer, T.J. Meyer, Inorg. Chem. 20 (1981) 436.
[41] K.J. Takeuchi, M.S. Thompson, D.W. Pipes, T.J. Meyer, Inorg.
Chem. 23 (1984) 1845.
22] B. Durham, J.L. Walsh, C.L. Carter, T.J. Meyer, Inorg. Chem.
[42] V. Jedlickov a´ , Z. Paluch, S.J. Alusik, Chromatography B 780
(2002) 193.
1
9 (1986) 860.
23] M.G. Sauaia, R.S. da Silva, Transition Metal Chem. 28 (3) (2002)
54.
[43] K.J. Reszka, P. Bilski, C.F. Chignell, J. Am. Chem. Soc. 118
(1996) 8719.
2
24] W.R. Murphy, K. Takeuchi, M.H. Barley, T.J. Meyer, Inorg.
Chem. 25 (1986) 1041.
[44] J. Mack, J.R. Bolton, J. Photochem. Photobiol. A: Chem. 128
(1999) 1.
25] D.A. Wink, J.F. Darbyshire, R.W. Nims, J.E. Saavedra, P.C.
Ford, Chem. Res. Toxicol. 6 (1993) 23.
[45] A. Dejam, P. Kleinbongard, T. Rassaf, S. Hamada, P. Gharini,
J. Rodriguez, M. Feelisch, M. Kelm, Free Radical Biol. Med.
35 (12) (2003) 1551.
26] S. Kudo, J.L. Bourassa, S.E. Boggs, Y. Sato, P.C. Ford, Anal.
Biochem. 247 (1997) 193.
[46] X.-Y. Wang, A.D. Guerzo, R.H. Schmehl, J. Photochem.
Photobiol. C 5 (2004) 55.
[
[
[
27] V. Mori, M. Bertotti, Analyst 125 (9) (2000) 1629.
28] W.D. Bowman, J.N. Demas, J. Phys. Chem. 80 (21) (1976) 2434.
29] J.N. Demas, W.D. Bowman, E.F. Zalewski, R.A. Velapoldi, J.
Phys. Chem. 85 (1981) 2766.
[47] U. Schmalfub, R. Neubert, J. Wohlrab, J. Control. Release 46
(1997) 279.
[48] P.P. Constantinides, J.P. Scarlat, Int. J. Pharm. 158 (1997) 57.
[49] D.W. Osborne, A.I. Ward, K.J. Oneill, J. Pharm. Pharmacol. 43
(6) (1991) 451.
[
[
[
30] R.P. Waine, Principles and Applications of Photochemistry,
Oxford Science Publication, New York, 1991, p. 8.
31] D.A. Chaisson, R.E. Hintze, D.H. Stuemer, J.D. Petersen, D.P.
McDonald, P.C. Ford, J. Am. Chem. Soc. 94 (1972) 6665.
32] H. Wu, C. Ramachandran, N.D. Weiner, B.J. Roessler, Int. J.
Pharm. 220 (2001) 63.
[50] E.B. Souto, S.A. Wissing, C.M. Barbosa, R.H. Muller, Int. J.
Pharm. 278 (2004) 71.
[51] S.M. Shishido, A.B. Seabra, W. Loh, M.G. de Oliveira, Bioma-
terials 24 (2003) 3543.