Photochemical and pharmacological aspects of nitric oxide release from some nitrosyl ruthenium complexes entrapped in sol-gel and silicone matrices

Article abstract of DOI:10.1016/j.poly.2007.03.042

The entrapped [Ru(terpy)(L)NO](PF₆)₃, where terpy = 2,2′:6′,2″-terpyridine and L = 2,2′-bipyridine (bpy) and 3,4-diiminebenzoic acid (NH · NHq) complexes into sol-gel processed polysiloxane and silicone matrices, shows NO release characteristics when submitted to light irradiation at 355 and 532 nm, as judged by NO measurement using a NO-sensor electrode. The pharmacological properties of doped matrix showed vasodilator characteristics by visible light irradiation, which is of great interest because the target delivery system can avoid the occurrence of side effects possibly by the aquo ruthenium species. All matrices obtained showed to be amorphous materials. The scanning electron micrographs of the matrices showed irregularly shaped particles, with a broad size of 1000 μm for both matrices and homogeneous distribution.

Full text of DOI:10.1016/j.poly.2007.03.042

Polyhedron 26 (2007) 4620–4624
www.elsevier.com/locate/poly
Photochemical and pharmacological aspects of nitric oxide release
from some nitrosyl ruthenium complexes entrapped in sol–gel
and silicone matrices
a
a
b
c
Renata Galvao de Lima , Marilia Gama Sauaia , Camila Ferezin , Iuri Muniz Pepe ,
˜
d
b
d
´
´
Nadia Mamede Jose , Lusiane M. Bendhack , Zeˆnis Novais da Rocha ,
a,b,
*
Roberto Santana da Silva
a
ˆ
Departamento de Qu´ımica, Faculdade de Filosofia, Ciencias e Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Av. dos Bandeirantes 3900,
CEP 14040-901 Ribeira˜o Preto (SP), Brazil
Faculdade de Ciencias Farmaceuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Av. do Cafe s/n, CEP 14040-903 Ribeira˜o Preto (SP), Brazil
b
ˆ
ˆ
´
Optical Properties Laboratory, LaPO do Instituto de Fı´sica da Universidade Federal da Bahia, Campus Ondina, Salvador, Bahia, Brazil
c
d
Instituto de Quı´mica da Universidade Federal da Bahia, Campus Ondina, Salvador, Bahia, Brazil
Received 19 December 2006; accepted 27 March 2007
Available online 10 April 2007
Abstract
The entrapped [Ru(terpy)(L)NO](PF6)3, where terpy = 2,20:60,200-terpyridine and L = 2,20-bipyridine (bpy) and 3,4-diiminebenzoic
acid (NH Æ NHq) complexes into sol–gel processed polysiloxane and silicone matrices, shows NO release characteristics when submitted
to light irradiation at 355 and 532 nm, as judged by NO measurement using a NO-sensor electrode. The pharmacological properties of
doped matrix showed vasodilator characteristics by visible light irradiation, which is of great interest because the target delivery system
can avoid the occurrence of side effects possibly by the aquo ruthenium specie. All matrices obtained showed to be amorphous materials.
The scanning electron micrographs of the matrices showed irregularly shaped particles, with broad size of 1000 lm for both matrices and
homogeneous distribution.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Immobilized ruthenium complexes; Nitric oxide; Photo-release
1. Introduction
which are dependent on its site and source of production
[8,9]. A possible strategy would be to employ nitrosyl
ruthenium compounds that display relatively low thermal
reactivity but are photochemically active to release NO
when subjected to luminous stimulus [10–18]. Unfortu-
nately, many of these species present NO releasing ability
only at low pH, which make them unsuitable for clinical
therapy. The use of solid matrices with nitrosyl ruthenium
species capable of providing local and controlled NO deliv-
ery could overcome this kind of problem and yield an
important investigative tool to assess the effects of NO on
cells and tissues [19–21].
Nitric oxide (NO) is an endogenously produced free rad-
ical in mammals that is responsible for mediating a wide
variety of important physiological processes including neu-
rotransmission, platelet and tumor cell adhesion, immune
response, and vasodilation [1–7]. The several biological
functions of nitric oxide make NO-releasing agents poten-
tial therapeutic candidates for a range of disease states,
*
Corresponding author. Address: Faculdade de Cieˆncias Farmaceˆuticas
´
de Ribeirao Preto, Universidade de Sao Paulo, Av. do Cafe s/n, CEP
˜
˜
14040-903 Ribeirao Preto (SP), Brazil. Tel.: +55 16 36024428; fax: +55 16
˜
36331092.
With the aim of stabilizing nitrosyl ruthenium com-
pounds that could act as NO-releasing agents in a control-
E-mail address: silva@usp.br (R.S. da Silva).
0277-5387/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2007.03.042

R.G. de Lima et al. / Polyhedron 26 (2007) 4620–4624
4621
lable manner, as well as limiting NO exposure to selected
sites within the body, we have entrapped [Ru(terpy)(L)-
NO](PF6)3 complexes (where terpy = 2,20:60,200-terpyridine
and L = 2,20-bipyridine (bpy) and 3,4-diiminebenzoic acid
(NH Æ NHq) into sol–gel processed polysiloxane and sili-
cone matrices. Recently some physico-chemical properties
of nitrosyl complexes entrapped in a silicate sol–gel matrix
have been described [19,20]. Although the authors have
suggested its use in therapeutic applications due to the con-
trolled NO release, there is no experimental indication that
kind of system will work in biological experiments.
the 0.5 cm3 of acetonitrile, and five drops of di-n-butyl-
tin–dilaurate complex, 5% in hexane (Gelest) as catalyst.
The final mixture was stirred for 5 min. The wet gels were
air-dried at room temperature for 24 h.
2.4. Apparatus
Ultraviolet-visible (UV–Vis) spectra were recorded on a
Hitachi U-3501 and a Hewlett Packard HP8452A diode
array spectrophotometer. Infrared (IR) spectra were
´
recorded on either a Protege 460 series or a MB Bomem
In this study, NO measurements were carried out and
we have found that [Ru(terpy)(L)NO](PF6)3 complexes
can also promote NO-release from the sol–gel and silicone
membranes by ultraviolet and visible light irradiation.
Some pharmacological properties of the entrapped [Ru(ter-
py)(NH Æ NHq)NO](PF6)3 complexes have also been
described based on the photolysis at 532 nm showing the
versatility of these materials for therapeutic applications.
102 FTIR spectrometer. Scanning electron microscopy
was performed using a LEO 440 microscope equipped with
an Oxford EDS detector. The pH measurements were
made using a 430 pH meter from Corning. NO release
was measured with an ISO-NOP NO meter from Word
Precision Instruments. Continuous photolysis with mono-
chromatic irradiations at 355 and 436 nm was carried out
using a 150 W Xenon lamp in a model 6253, Oriel Univer-
sal Arc Lamp Source. The irradiation wavelength was
selected with an Oriel interference filter, with 10 nm band
path, for photolysis at the appropriate wavelengths. The
progress of the photoreactions was monitored
spectrophotometrically.
2. Experimental
2.1. Chemicals and reagents
RuCl3 Æ nH2O, terpyridine (terpy) and bipyridine (bpy)
were purchased as high purity reagents from Aldrich
Chemicals and were used as supplied. Doubly distilled
water was used for all experiments. All preparations and
measurements were carried out under an argon atmosphere
and protected from light.
2.5. Nitric oxide release measurement
The experiment was performed using a photo-reactor
(0.18 m3) containing 250 W fluorescent lamps (Philips),
which were used as a visible light source. The 450–
800 nm wavelength range was achieved using a filter. A
total of 0.200 g of membrane containing incorporated
nitrosyl ruthenium complexes were added to a quartz
UV–Vis reservoir containing 10 mL of 0.01 M phosphate
buffer solution pH 7.4. This reservoir was connected by a
polyethylene tube (20 cm; 1 mm diameter) to another
chamber containing 10 mL of phosphate buffer solution
where the NO-sensor (amino-700 from Innovative Instru-
ments, Inc) was adapted. Argon flux was bubbling in both
reservoirs during the experiment.
2.2. Synthesis of ruthenium complexes
The recrystallized complex salt [RuCl(bpy)(terpy)]Cl,
[Ru(bpy)(terpy)(NO)](PF6)3, [RuCl(NH Æ NHq)(terpy)]Cl
and [Ru(NH Æ NHq)(terpy)NO](PF6)3 were prepared as
previously published [22–24].
2.3. Preparation of the membranes
The hybrid composite was synthesized by preparing a
mixture of 24 mL tetraethoxysilane (Aldrich, Milwaukee,
WI, USA) and 19 mL of bidistilled water. The reagents
were carefully added and three drops of concentrated
HCl were added in order to catalyze the reaction. The
nitrosyl ruthenium complexes (ca. 0.001 g) were added with
constant stirring into 4.5 mL of hydrolyzed. The solution
was stirred for 5 min at 60 ꢁC and aliquots (2.0 mL) of
the mixture were then transferred to a Petri dish (58 mm
diameter) to complete the reaction. The molds remained
opened for 3 days until they no longer exhibited weight loss
due to evaporation of residual water and solvent.
The silicone resin was prepared by mixing poly-
dimethylsiloxane (PDMS Dow Corning) (2.000 g) with tet-
raethoxysilane (TEOS, Fluka) (0.500 g) in 2.5 cm3 of
isopropylic alcohol. To this mixture we add the nitrosyl
ruthenium compound (0.001 g), previously dissolved in
The reservoir containing the membrane was submitted
to light irradiation and the NO was detected by measuring
the current.
2.6. Vessel preparation
Male Wistar rats (180–200 g) were killed by decapitation
and the thoracic aorta was quickly removed and dissected
free and cut into rings 4 mm long. The endothelium was
mechanically removed by gently rolling the lumen of the
vessel on a thin wire. The aortic rings were placed between
two stainless-steel stirrups, and connected to an isometric
force transducer (50630-45, Harward Bioscience, South
Natick, MA, USA) and the other was connected to a fixed
support in the chamber in order to record the tension on a
Harward Bioscience Oscillograph polygraph. The rings
were placed in a 10 mL organ chamber containing Krebs

4622
R.G. de Lima et al. / Polyhedron 26 (2007) 4620–4624
solution with the following composition (mmol/L): NaCl
130, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, NaHCO3 14.9,
Glucose 5.5, CaCl2 1.6. The solution was maintained at
pH 7.4 gassed with 95% O2 and 5% CO2, at 37 ꢁC. The
rings were initially stretched to a basal tension of 1.5 g,
before allowing to equilibrate for 60 min in the bath fluid
that was changed every 15–20 min. Endothelial integrity
was qualitatively assessed by the degree of relaxation
caused by acetylcholine (ACh,1 lmol/L) in the presence
of contractile tone induced by phenylephrine (0.1 lmol/
L). Because our studies require endothelium-denuded aor-
tas, the rings were discharged if there was any degree of
relaxation to ACh, in order to avoid possible influence of
endothelial factors. The tissues were washed, pre-con-
tracted with the EC50 of phenylephrine (0.1 lmol/L). On
top of the contraction, the chamber was loaded with
0.134 g 0.004 (n = 7) of the membrane containing
entrapped [Ru(NH Æ NHq)(terpy)NO]3+, which was added
in a dialysis bag to avoid any adverse physiological effect
due the organic reagents. Similar experiment was run with
the control (membrane without ruthenium complex
ꢀ0.129 0.004 (n = 7)). After addition of the membrane,
the light was activated and time-course for relaxation
induction was evaluated. Relaxation was calculated as a
percentage of the maximal contraction induced by
phenylephrine.
Spectral changes accompanying this reaction for [Ru(bpy)
(terpy)NO]3+ are shown in Fig. 1. The product was charac-
terized as [Ru(NO2)(L)(terpy)]+ (nitro form) based mainly
on the similarity of the analogous ruthenium complexes
[24]. The spectral profiles show that, above pH 3.00 for
[Ru(bpy)(terpy)NO]3+ and pH 6.00 for [Ru(NH Æ NHq)
terpy)NO]3+, the nitro form exists in significant amounts
at equilibrium. Low pH is somehow crucial for both nitrosyl
ruthenium species generate NO release by light irradiation.
In this regard, novel strategies to preserve the properties of
[Ru(terpy)(L)NO]3+ should be developed for biomedical
applications. We found that silicone and sol–gel matrices
could allow stabilizing the nitrosyl species as well be a con-
venient target delivery system for NO release.
The physico-chemical properties of [Ru(terpy)(L)NO]3+
seems to be unaffected by the entrapping process with sol–
gel or silicone membranes as judged by the UV–Vis and
FTIR analysis. The solid sate absorbance spectra for sol–
gel and silicone samples doped with nitrosyl ruthenium
complexes show some similarities to those obtained in
aqueous solution. Fig. 2 shows the electronic spectrum of
[Ru(bpy)(terpy)NO]3+, as an example of a [Ru(ter-
Light irradiation for the pharmacological assays was
performed using a photo-reactor. The reactor consists of
a 0.5 m3 box inside which aluminum was placed to increase
light reflection. Three 250 W fluorescent lamps (Philips)
were used as a visible light source with 450–800 nm wave-
length range achieved with a filter (7.05 cm2 area) posi-
tioned in front of each channel on the chamber.
2.7. Statistical analysis
Relaxant responses to the NO donor were measured
from the plateau of the phenylephrine contraction and
were expressed as percent reversal of the phenylephrine
pre-contraction. Data are expressed as mean SEM. In
each set of experiments,n indicates the number of rats stud-
ied. We considered the maximal relaxing effect for com-
pounds when the concentration used reached the
baseline. Statistical significance was tested by one-way
ANOVA (Bonferroni’s Multiple Comparison Test) and
values of p > 0.05 were considered to be significant.
Fig. 1. Spectral profile change with pH for the conversion of [Ru-
(NH Æ NHq)(terpy)NO]3+ in [Ru(NH Æ NHq)(terpy)(NO2)]+. Concentra-
tion of the complex = 2.6 · 10ꢀ5 M. pH 2.02, 2.40, 3.20, 3.58, 6.30, 7.40.
3. Results and discussion
The nitrosyl ruthenium complexes, [Ru(terpy)(L)NO]3+
(L = bpy; NH Æ NHq), used in the present study have been
proposed as a NO donor agents in aqueous acidic solution
by external stimulation [24].
These species react very rapidly in aqueous medium with
hydroxide ion to give deep yellow solutions (Eq. (1)).
Fig. 2. Electronic spectrum of [Ru(bpy)(terpy)NO]3+ in solution pH 2.03
(solid line) and in silicone matrix (dashed line).
Keq
3þ
½RuL5ðNOÞꢁ þ 2OHꢀ ꢀ ½RuL5ðNO2Þꢁþ þ H2O
ð1Þ

R.G. de Lima et al. / Polyhedron 26 (2007) 4620–4624
4623
py)LNO]3+ species in solid state (dashed line) and in aque-
ous solution (solid line) performed at room temperature. A
series of three bands were observed in both spectra in the
region of 250–300 nm attributed to intra-ligand transition.
However, the electronic spectrum in solution shows a
shoulder at 360 nm, attributed to a metal ligand charge
transfer band [24], which was not observed in the solid
state, probably due the medium effect.
Scanning electron microscopy (SEM) of the studied
membranes in this study was applied to determine the dis-
tribution pattern and size of particles in the solid matrices.
All matrices obtained were found to be amorphous materi-
als (Figure S1: Supporting Material). The SEM micro-
graphs revealed irregularly shaped particles with broad
size of 1000 lm for both membranes as well as a homoge-
nous distribution. The samples of [Ru(bpy)(terpy)-
NO](PF6)3 complex doped in sol–gel and silicone
matrices formed three-dimensional networks while the
samples of [Ru(NH Æ NHq)(terpy)NO](PF6)3 complex
showed edges and conchoidal fractures, as well as globular
particle shapes with low-dimensional structures.
Lꢀ1 HCl and also in phosphate buffer solution at pH
7.40 for 30 min. The electronic spectra of these dispel ali-
quots showed insignificant absorption, which was consid-
ered as a qualitative indicator of the efficiency of the
entrapment of ruthenium nitrosyl complexes in sol–gel
and silicone matrices.
Due the spectroscopic and photochemical characteristics
of [Ru(NH Æ NHq)(terpy)NO]3+ complex in aqueous solu-
tion [24] as well encapsulated in a solid matrix, some phar-
macological assays was performed. In a vasodilator test,
denuded rat aortic rings were previously contracted with
phenylephrine and vasorelaxation was verified in the pres-
ence of doped matrix. In this experiment, we used the sol–
gel matrix (ca. 0.200 g), which was kept in a cellulose dialysis
tubing to avoid any other side reaction. The addition of the
membrane without entrapped compound in the absence of
light did not produce vasodilation response. However, as
shown in Fig. 4, in the presence of visible light irradiation
and after depletion of endogenous photoactivable NO
stores, NO release caused relaxation of denuded pre-con-
tracted aorta with maximum within about 100 s. In pre-con-
tracted aortic rings, pre-incubation with oxyhaemoglobin
10 lmol/L for 30 min, a known NO scavenger [25], com-
pletely abolished the relaxation induced by the matrix indi-
cating that NO is released in the extracellular medium.
Vasodilation using sol–gel or silicone as target delivery
system shows similar vasorelaxation effects observed for
[Ru(NH Æ NHq)(terpy)NO]3+ in physiological solution [7].
The entrapped [Ru(terpy)LNO]3+ complex on the matri-
ces was submitted to light irradiation at 355 nm, while the
[Ru(NH Æ NHq)(terpy)NO]3+ complex was also irradiated
at 532 nm. NO monitoring of these photochemical pro-
cesses in aqueous solution, as observed for [Ru(NH Æ NHq)-
(terpy)NO]3+ as an example of nitrosyl ruthenium complex
entrapped in silicone matrix, showed NO release (Fig. 3)
although in the absence of light no current increase was
recorded. The amount of available NO per volume of the
matrices can be controlled by the amount of [Ru(bpy)(ter-
py)LNO]3+. It implies that tissue exposure to such materials
could be convenient because it provides different dosages of
NO. After exhaustive photolysis, the UV–Vis spectrum of
the entrapped [Ru(terpy)LNO]3+ showed [Ru(terpy)-
L(H2O)]2+ characteristics (Figure S2: Supporting Material).
The stability of the solid matrices to entrap the nitrosyl
compounds was tested by rinsing the samples in 0.1 mol
The entrapped ruthenium complexes induced Emax
:
29.9 3.6% for sol–gel matrix. It seems to be less effective
when compared to the [Ru(NH Æ NHq)(terpy)NO]3+ in
physiological solution, which is possibly explained by the
fact that activation of guanylyl-cyclase is nitric oxide con-
centration dependent. In addition, the membrane releases
NO extracellularly and some NO amount can be lost out-
side the cells. In this way, less NO could be available inside
the cell to promote the guanylyl-cyclase activation and
Fig. 4. Time-course for [Ru(NH Æ NHq)(terpy)NO]3+-induced relaxation.
Denuded aortic rings were pre-contracted with 100 nmol Lꢀ1 phenyleph-
rine and [Ru(NH Æ NHq)(terpy)NO]3+ doped in sol–gel matrix (s, n = 7)
and control (sol–gel matrix) (d, n = 7) were added. Data are mean-
Fig. 3. Time course of NO release from [Ru(NH Æ NHq)(terpy)NO]3+
complex in silicone matrix under 532 nm light irradiation. Mass
membrane = 0.200 g.
s
SEM of n experiments performed on preparations obtained from
different animals.

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In the present study, we showed for the first time that the
NO released from entrapped [Ru(NH Æ NHq)(terpy)-
NO]3+by light irradiation induces rat aorta relaxation, as
demonstrated by its vasodilatory effect. This NO-release
ability might represent an effective mechanism for some
therapeutic applications because NO has been shown to pre-
vent biofilm formation. Studies are being conducted at our
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vent, for example, bacterial adhesion, which may be useful
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Appendix A. Supplementary material
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.poly.2007.
03.042.
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