Lipophilic ruthenium salen complexes: Incorporation into liposome bilayers and photoinduced release of nitric oxide

Article abstract of DOI:10.1039/c5dt02352a

A new lipophilic Ru salen complex with cholesterol groups can be efficiently incorporated into liposome bilayers, allowing the photoinduced release of nitric oxide (NO) and the membrane transport of NO to coexisting liposomes.

Full text of DOI:10.1039/c5dt02352a

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Lipophilic ruthenium salen complexes:
incorporation into liposome bilayers and
photoinduced release of nitric oxide†
Cite this: Dalton Trans., 2015, 44,
14200
Received 22nd June 2015,
Accepted 8th July 2015
Keita Nakanishi, Tomomi Koshiyama,* Soichi Iba and Masaaki Ohba*
DOI: 10.1039/c5dt02352a
www.rsc.org/dalton
A new lipophilic Ru salen complex with cholesterol groups can be reported, but would be useful especially for regulation of mem-
efficiently incorporated into liposome bilayers, allowing the photo- brane proteins such as cytochrome c oxidase, which is reversi-
induced release of nitric oxide (NO) and the membrane transport bly inhibited by NO.11 We therefore designed a new lipophilic
of NO to coexisting liposomes.
Ru nitrosyl complex, [Ru(L)Cl(NO)] (1, L = N,N′-ethylene-bis(4-
cholesteryl-hemisuccinate-salicylideneamine)), to fix the Ru
nitrosyl complex [RuII(salen)Cl(NO)]12–14 on the liposome
surface through specific hydrophobic interactions between
the cholesterol and phospholipid bilayers (Fig. 1).15,16 The
designed lipophilic Ru(salen) complex with cholesterol groups
is expected to be efficiently incorporated into liposome
bilayers, allowing light-controlled NO release.
Nitric oxide (NO) is known to act as a cellular signalling mole-
cule in mammals and to control physiological functions such
as vasodilation, smooth muscle relaxation, and platelet aggre-
gation inhibition.1,2 The development of NO delivery systems
is important not only for therapeutic applications but also to
provide tools to elucidate signalling mechanisms.3 Precise
spatiotemporal control of NO release is essential because of
the short half-life of NO (3–6 s). Because light is the best and
least invasive on/off trigger, various photoinduced NO donors
have been reported.4–7 In particular, metal complexes have
exhibited high potential as NO donors because of their high
stability under physiological conditions and excellent design-
ability. Some metal nitrosyl complexes, such as Cr, Mn, and
Ru nitrosyl, have been reported as light-responsive NO
precursors.5–7 However, their low water solubility prevents
further application in living systems.
To actualize a biocompatible NO delivery system, assem-
blies of photoactive metal nitrosyl complexes, using appropri-
ate materials such as polymers, nanoparticles, and vesicles,
have been constructed.8 A spherical vesicle, a liposome con-
sisting of a phospholipid bilayer, is an attractive platform for
targeted drug delivery because of its high biocompatibility,
long circulation time, and immunomodification.9 Ford et al.
reported the liposome encapsulation of a photochemical NO
precursor, trans-[Cr(cyclam)(ONO)2]+.10 However, the encapsu-
lation efficiency of the Cr complex was very low (ca. 1%) owing
to its limited specific interaction with liposomes. Light-con-
trolled NO release near a membrane surface has not been
Compound 1 was synthesized according to the method
described in the ESI,† and identified by elemental analysis
1
and H NMR. The UV-vis spectrum of 1 in chloroform (50 μM)
showed absorbance at 376 nm, which corresponded to the
σ–π* band (Fig. 2a).12,13 The IR spectrum of 1 exhibited a NO
stretch band at around 1833 cm−1 (Fig. 2b), which indicated
that the coordination geometry of the ruthenium centre in 1
was identical to that of the ‘unmodified complex’ [Ru(salen)-
Cl(NO)] in having a multiple bond between Ru(II) and nitroso-
nium (NO+).14 The NO releasing ability of 1 was determined by
measuring the time-dependent spectral changes of a chloro-
form solution of 1 (50 μM) under Xe irradiation (400–750 nm)
at 20 °C (Fig. 2a). The pale brown-coloured solution turned
green upon irradiation. New absorption peaks appeared
at around 396 nm and 648 nm after irradiation, and their
Department of Chemistry, Faculty of Science, Kyushu University, 6-10-1 Hakozaki,
Higashi-ku, Fukuoka, Japan. E-mail: ohba@chem.kyushu-univ.jp,
koshi@chem.kyushu-univ.jp; Fax: +81-92-642-2570; Tel: +81-92-642-2570
†Electronic supplementary information (ESI) available: Details of the synthesis
of lipophilic Ru complexes, physical measurements and additional experimental
data. See DOI: 10.1039/c5dt02352a
Fig. 1 Structure of 1 and schematic model for incorporation of 1 into
liposomes.
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Fig. 3 UV-vis spectra of 1_Lipo ([Ru] = 11.2 μM) in 20 mM Tris/HCl
buffer (pH 7.4) and spectral changes associated with Xe irradiation
(400–750 nm) at 20 °C.
The average diameter of 1_Lipo was determined to be
141 nm by dynamic laser scattering analysis (Fig. S1†). The for-
mation of liposomes was also confirmed by the direct obser-
vation of a giant vesicle of 1_Lipo using confocal laser
scanning microscopy (Fig. S2†). The ruthenium and phos-
phorus concentrations of purified 1_Lipo were 33 μM and
0.94 mM, respectively. These values mean that the incorpor-
ation efficiency of 1 in 1_Lipo is 66%, and the molar ratio of 1
to DMPG is 1 : 28. The efficiency was significantly improved
compared with that of the previously reported liposomal
encapsulation of a NO precursor (about 1%).10 Moreover, the
unmodified complex [Ru(salen)Cl(NO)] was not incorporated
into liposomes. These results suggest that the Ru cores of 1
were located on the liposome surface with the support of the
lipophilic cholesterol groups.
The NO releasing ability of 1_Lipo under Xe irradiation was
evaluated by UV-vis spectroscopy (Fig. 3). As in the case of 1,
the absorbance of new absorption bands at 396 nm and
625 nm (LMCT band) gradually increased during Xe
irradiation over a period of 90 min. These spectral changes cor-
respond to the generation of the [RuIII(salen)Cl(H2O)] species
accompanying NO release. The particle size distribution of
1_Lipo hardly changed after irradiation (Fig. S1†); thus, aggre-
gation and disruption of liposomes did not occur during the
reaction. The amount of NO released from 1_Lipo upon
irradiation was confirmed using the fluorescent reagent 4,5-
diaminofluorescein (DAF-2). DAF-2 reacts with NO to yield a
compound with green fluorescence (DAF-2T) at 515 nm (exci-
tation λmax = 495 nm).18 The fluorescence spectra of the
mixture of 1_Lipo ([Ru] = 3.3 μM) and DAF-2 (10 μM) showed
strong fluorescence at 515 nm after Xe irradiation (Fig. 4). The
time profile of the fluorescence at 515 nm showed almost the
same curve as that of the absorbance at 396 nm in the UV-vis
spectra (Fig. S3†). This result indicates that the NO released
from 1_Lipo immediately diffused in the solution, and reacted
with DAF-2. The concentration of released NO after 60 min
irradiation was 2.3 μM, indicating that about 70% of 1 in
1_Lipo was converted to the photoproduct. The micromolar-
Fig. 2 (a) UV-vis spectra of 1 ([Ru] = 50 μM) in CHCl3 and spectral
changes associated with Xe irradiation (400–750 nm) at 20 °C, and (b) IR
spectra of 1 (black) and photoproduct (red).
absorbances increased with prolonged irradiation, with an iso-
sbestic point occurring at 331 nm. The broad band around
648 nm was assigned to a ligand-to-metal (RuIII) charge trans-
fer (LMCT) band of the Ru(III) salen complex.12,13 This spectral
change is consistent with the generation of the [RuIII(salen)Cl]
species accompanying NO release. After 120 s of Xe irradiation,
no significant further spectral changes were observed, and the
NO stretching band disappeared in the IR spectrum of the
photoproduct (Fig. 2b). These results indicate that modifi-
cation of the precursor complex [Ru(salen)Cl(NO)] with chole-
sterol groups did not inhibit the photoinduced NO releasing
ability, and 1 in CHCl3 immediately released all captured NO
upon Xe irradiation.
A
composite of 1 and 1,2-ditetradecanoyl-sn-glycero-3-
phospho-(1′-rac-glycerol) (DMPG) liposome (1_Lipo) was pre-
pared by incorporation of 1 into a lipid bilayer of the liposome
by the Bangham method,17 in which a lipid film containing
DMPG, cholesterol, and 1 in a molar ratio of 20 : 4 : 1 was
hydrated with 20 mM Tris/HCl buffer (pH 7.4). The mixture
was purified using Sephadex G-25 to afford a pale yellow sus-
pension of 1_Lipo. The UV-vis spectrum of 1_Lipo showed a
characteristic absorbance at around 376 nm and a broad
absorption band from 400 to 800 nm (Fig. 3). These were
assigned to the σ–π* band of 1 and light scattering by the lipo-
some suspension, respectively.
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1_Lipo and DAF-2@Lipo showed a gradual increase in fluo-
rescence intensity resulting from the reaction between NO and
DAF-2 under Xe irradiation (Fig. 5b). Confocal laser scanning
microscopy images of the mixture of 1_Lipo and DAF-2@Lipo
before and after irradiation are shown in Fig. 5c. After
irradiation, green fluorescence was observed only in the
interior aqueous phase of DAF-2@Lipo, indicating the success-
ful membrane transport of NO from 1_Lipo to the coexisting
DAF-2@Lipo.
In conclusion, we have demonstrated the photoinduced
release of NO from liposomes incorporating the lipophilic Ru
salen complex 1. The efficient incorporation of 1 into liposome
bilayers was achieved as a result of the water insolubility of 1
and the high affinity of the cholesterol groups for phospho-
lipid bilayers. We consider our strategy to be a rational approach
for applying various water-insoluble photoinduced NO donors
in aqueous media. The composite of 1 and liposome gradually
released NO during Xe irradiation over a period of 90 min,
with relatively high efficiency. Furthermore, we succeeded in
achieving the membrane transport of NO to coexisting lipo-
somes. We have reported that the reactivity of metal complexes
on a liposome surface is influenced by the surrounding
environment of the reaction centre, including the position of
metal cores and the head groups of phospholipids.16 Thus,
further adjustment of the surroundings of lipophilic Ru nitro-
syl complexes could allow control of the rate and concentration
of released NO, leading to elucidation of the mechanisms of
biological effects such as vasorelaxant and anticancer effects.5
Fig. 4 Fluorescence spectra of the mixture of 1_Lipo ([Ru] = 3.3 μM) and
DAF-2 (10 μM) in 20 mM Tris/HCl buffer (pH 7.4) and spectral changes
associated with Xe irradiation (400–750 nm) at 20 °C (λex = 495 nm).
order concentration could be sufficient to control biological
activities such as anticancer activity.5
DAF-2-encapsulated liposome (DAF-2@Lipo) was then pre-
pared to examine transport of the NO from 1_Lipo to other
liposomes (Fig. 5). The fluorescence spectra of a mixture of
Acknowledgements
This work was supported by a Grant-in-Aid for Challenging
Exploratory Research (no. 26620049) from MEXT, Japan. T. K.
thanks MEXT for a Grant-in-Aid for Young Scientists (B) (no.
30467279), Kyushu University Interdisciplinary Program in
Education and Projects in Research Development. This work
was partly supported by the Nanotechnology Platform Program
(Molecule and Material Synthesis) of MEXT, Japan, and the
Center of Advanced Instrumental Analysis, Kyushu University.
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Fig. 5 (a) Schematic representation of NO transport from 1_Lipo to
DAF-2@Lipo, (b) fluorescence spectra of the mixture of 1_Lipo and
DAF-2@Lipo in 20 mM Tris/HCl buffer (pH 7.4) and spectral changes
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