Ruthenium-decorated lipid vesicles: Light-induced release of [Ru(terpy)(bpy)(OH2)]2+ and thermal back coordination

Article abstract of DOI:10.1021/ja105025m

Electrostatic forces play an important role in the interaction between large transition metal complexes and lipid bilayers. In this work, a thioether-cholestanol hybrid ligand (4) was synthesized, which coordinates to ruthenium(II) via its sulfur atom and intercalates into lipid bilayers via its apolar tail. By mixing its ruthenium complex [Ru(terpy)(bpy)(4)]²⁺ (terpy = 2,2′;6′,2′′-terpyridine; bpy = 2,2′-bipyridine) with either the negatively charged lipid dimyristoylphosphatidylglycerol (DMPG) or with the zwitterionic lipid dimyristoylphosphatidylcholine (DMPC), large unilamellar vesicles decorated with ruthenium polypyridyl complexes are formed. Upon visible light irradiation the ruthenium-sulfur coordination bond is selectively broken, releasing the ruthenium fragment as the free aqua complex [Ru(terpy)(bpy)(OH ₂)]²⁺. The photochemical quantum yield under blue light irradiation (452 nm) is 0.0074(8) for DMPG vesicles and 0.0073(8) for DMPC vesicles (at 25 °C), which is not significantly different from similar homogeneous systems. Dynamic light scattering and cryo-TEM pictures show that the size and shape of the vesicles are not perturbed by light irradiation. Depending on the charge of the lipids, the cationic aqua complex either strongly interacts with the membrane (DMPG) or diffuses away from it (DMPC). Back coordination of [Ru(terpy)(bpy)(OH₂)]²⁺ to the thioether-decorated vesicles takes place only at DMPG bilayers with high ligand concentrations (25 mol %) and elevated temperatures (70 °C). During this process, partial vesicle fusion was also observed. We discuss the potential of such ruthenium-decorated vesicles in the context of light-controlled molecular motion and light-triggered drug delivery.

Full text of DOI:10.1021/ja105025m

Published on Web 12/16/2010
Ruthenium-Decorated Lipid Vesicles: Light-Induced Release
of [Ru(terpy)(bpy)(OH2)]2+ and Thermal Back Coordination
Sylvestre Bonnet,*,† Bart Limburg,† Johannes D. Meeldijk,‡
Robertus J. M. Klein Gebbink,§ and J. Antoinette Killian#
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden UniVersity, P.O. Box 9502,
2300 RA Leiden, The Netherlands, and Electron Microscopy Utrecht, Department of Biology,
Organic Chemistry & Catalysis, Debye Institute for Nanomaterial Science, and Biochemistry of
Membranes, BijVoet Center, Department of Chemistry, Faculty of Sciences, Utrecht UniVersity,
Padualaan 8, 3584 CH Utrecht, The Netherlands
Received June 9, 2010; E-mail: bonnet@chem.leidenuniv.nl
Abstract: Electrostatic forces play an important role in the interaction between large transition metal
complexes and lipid bilayers. In this work, a thioether-cholestanol hybrid ligand (4) was synthesized, which
coordinates to ruthenium(II) via its sulfur atom and intercalates into lipid bilayers via its apolar tail. By
mixing its ruthenium complex [Ru(terpy)(bpy)(4)]2+ (terpy ) 2,2′;6′,2′′-terpyridine; bpy ) 2,2′-bipyridine)
with either the negatively charged lipid dimyristoylphosphatidylglycerol (DMPG) or with the zwitterionic lipid
dimyristoylphosphatidylcholine (DMPC), large unilamellar vesicles decorated with ruthenium polypyridyl
complexes are formed. Upon visible light irradiation the ruthenium-sulfur coordination bond is selectively
broken, releasing the ruthenium fragment as the free aqua complex [Ru(terpy)(bpy)(OH2)]2+. The
photochemical quantum yield under blue light irradiation (452 nm) is 0.0074(8) for DMPG vesicles and
0.0073(8) for DMPC vesicles (at 25 °C), which is not significantly different from similar homogeneous
systems. Dynamic light scattering and cryo-TEM pictures show that the size and shape of the vesicles are
not perturbed by light irradiation. Depending on the charge of the lipids, the cationic aqua complex either
strongly interacts with the membrane (DMPG) or diffuses away from it (DMPC). Back coordination of
[Ru(terpy)(bpy)(OH2)]2+ to the thioether-decorated vesicles takes place only at DMPG bilayers with high
ligand concentrations (25 mol %) and elevated temperatures (70 °C). During this process, partial vesicle
fusion was also observed. We discuss the potential of such ruthenium-decorated vesicles in the context of
light-controlled molecular motion and light-triggered drug delivery.
mechanisms for the photobleaching of [Ru(bpy)3]2+, though, is
the photoejection of one of the bpy chelates.13,14 This process
Introduction
Over the years ruthenium(II) polypyridyl complexes have
become common tools in supramolecular chemistry1-3 and
photochemistry.4,5 They have also been used in biological
environments, either for photosensitizing,6 for probing the
structure of biological membranes,7,8 for sensing oxygen,9,10 or
as metal drugs.11,12 Compared to organic luminophores, [Ru-
(bpy)3]2+ (bpy ) 2,2′-bipyridine) shows peculiar photophysical
properties, namely, a triplet excited state with a long lifetime
and a good resistance toward photobleaching. One of the known
is caused by the thermal population, from the photogenerated
3MLCT state, of a metal-centered excited state 3MC with strong
dissociative character.15,16 When the ligand field around the
ruthenium center is decreased significantly as in [Ru(terpy)(phen)-
(L)]2+ (terpy ) 2,2′;6′,2′′-terpyridine; phen ) 1,10-phenanthro-
line; L is a sulfoxide, thioether, nitrile, or pyridine monodentate
ligand)17-21 or in the hindered complex [Ru(bpy)2(dmbpy)]2+
(dmbpy ) 6,6′-dimethyl-2,2′-bipyridine),22,23 ligand photoejec-
† Leiden University.
‡ Department of Biology, Utrecht University.
(7) Guo, X. Q.; Castellano, F. N.; Li, L.; Lakowicz, J. R. Biophys. Chem.
1998, 71, 51.
§ Organic Chemistry & Catalysis, Debye Institute for Nanomaterials
Science, Utrecht University.
(8) Svensson, F. R.; Li, M.; Norden, B.; Lincoln, P. J. Phys. Chem. B
2008, 112, 10969.
# Biochemistry of Membranes, Bijvoet Center, Department of Chemistry,
Utrecht University.
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(10) McNamara, K. P.; Rosenzweig, N.; Rosenzweig, Z. Mikrochim. Acta
1999, 131, 57.
(1) Campagna, S.; Puntoriero, F.; Nastasi, F.; Bergamini, G.; Balzani, V.
Top. Curr. Chem. 2007, 280, 117.
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ReV. 2006, 250, 1254.
(11) Novakova, O.; Kasparova, J.; Vrana, O.; Van Vliet, P. M.; Reedijk,
J.; Brabec, V. Biochemistry 1995, 34, 12369.
(3) Sauvage, J.-P.; Collin, J.-P.; Chambron, J.-C.; Guillerez, S.; Coudret,
C. Chem. ReV. 1994, 94, 993.
(12) Schatzschneider, U. Eur. J. Inorg. Chem. 2010, 1451.
(13) Durham, B.; Walsh, J. L.; Carter, C. L.; Meyer, T. J. Inorg. Chem.
1980, 19, 860.
(4) Graetzel, M. Acc. Chem. Res. 2009, 42, 1788.
(5) Concepcion, J. J.; Jurss, J. W.; Brennaman, M. K.; Hoertz, P. G.;
Patrocinio, A. O. T.; Murakami Iha, N. Y.; Templeton, J. L.; Meyer,
T. J. Acc. Chem. Res. 2009, 42, 1954.
(14) Van Houten, J.; Watts, R. J. J. Am. Chem. Soc. 1976, 98, 4853.
(15) Salassa, L.; Garino, C.; Salassa, G.; Gobetto, R.; Nervi, C. J. Am.
Chem. Soc. 2008, 130, 9590.
(6) Park, Y. T.; Noh, S. G. Int. J. Hydrogen Energy 1995, 20, 789.
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9
252 J. AM. CHEM. SOC. 2011, 133, 252–261
10.1021/ja105025m  2011 American Chemical Society

Ruthenium-Decorated Lipid Vesicles
A R T I C L E S
tion becomes the most efficient photophysical process, at the
cost of luminescence.
Thus, this study paves the way to the light-controlled hopping
of ruthenium complexes at a membrane-water interface.
In our quest toward the light-controlled motion of single
molecules24,25 we considered investigating the binding and
unbinding of ruthenium compounds to and from liposomes in
aqueous media. The bilayer of lipid vesicles offers a surface
on which metal complexes could eventually move. Meanwhile,
their “water solubility” allows for using bulk analytical tech-
niques such as UV-vis spectroscopy to probe the macroscopic
state of the system. In addition, vesicles are easy to deposit
onto mica or glass, where surface techniques can be used to
probe the motion at the single-molecule level.
Experimental Section
General. Acetone was dried on potassium carbonate and distilled
prior to use. KNO3 was used as a saturated aqueous solution, and
aqueous KPF6 was 40 g L-1. 1H (300.1/400.0 MHz) and 13C (75.5/
100.6 MHz) NMR spectra were recorded on a Varian INOVA 300
MHz or 400 MHz spectrometer; 31P{1H} (121.5 MHz) and 19F (376
MHz) NMR spectra were recorded on a Varian INOVA 400 MHz
spectrometer. Chemical shift values are reported in ppm (δ) relative
to Me4Si (1H and 13C NMR). MS measurements were carried out
on an Applied Biosystems Voyager DE-STR MALDI-TOF MS.
Elemental analyses were performed by H. Kolbe Microanalysis
Laboratories, Mu¨lheim, Germany. UV-vis absorption spectra were
taken on a Cary 5 spectrophotometer from Varian. For column
chromatography, Merck silica gel 60 (230-400 mesh) was used.
All standards reagents were purchased from Acros Organics and
Aldrich Chemical Co. Inc. and used as received. [Ru(terpy)-
(bpy)(OH2)](BF4)234 and [Ru(terpy)(bpy)(Cl)](Cl)34 were obtained
as described in the literature. Cholesterol, bromoacetylchloride,
sodium thiomethoxide, and acetic acid were from commercial
sources and used as received. 1,2-Dimyristoyl-sn-glycero-3-phos-
phoglycerol (DMPG) and 1,2-dimyristoyl-sn-glycero-3-phospho-
choline (DMPC) were obtained from Avanti Polar Lipids and stored
at -18 °C. A chloride-free buffer solution was prepared by mixing
KH2PO4 (313 mg, 2.3 mmol), K2HPO4 ·3H2O (593 mg, 2.6 mmol),
and K2SO4 (849 mg, 4.87 mmol) in a 500-mL volumetric flask
and dissolving with Milli-Q water (pH ) 7.03 at 23 °C). All
photosensitive solutions were protected from light by aluminum
foil.
To date, photosubstitution reactions with ruthenium poly-
pyridyl complexes have mostly been studied in coordinating
organic solvents, e.g., acetonitrile or pyridine, or in chlori-
nated solvents in the presence of chloride anions.13,17,18,23,26-31
However, several studies have shown that when irradiation
is performed in poorly coordinating solvents containing small
amounts of water, ruthenium aqua complexes can be formed
selectively.20,32,33 In this article, we consider ligand photo-
substitution reactions of [Ru(terpy)(bpy)(L)]2+ complexes at
the water-bilayer interface, i.e., in buffered aqueous solu-
tions. By covalent linkage of a monodentate ligand L (here
a thioether) to a membrane intercalator such as 3ꢀ,5R-
cholestanol and coordination of the sulfur ligand to ruthe-
nium, we obtained unilamellar vesicles decorated with
ruthenium polypyridyl complexes. Visible light irradiation
of the vesicles leads to the selective substitution of the
monodentate thioether ligand by a water molecule, thus
releasing [Ru(terpy)(bpy)(OH2)]2+. The efficiency of this
photochemical process at the membrane is not significantly
modified compared to comparable homogeneous systems,
with a measured photochemical quantum yield of 0.7% at
25 °C under blue light irradiation. With negatively charged
lipids, the released aqua complex remains in the vicinity of
the membrane through electrostatic interactions, which allows
for rebinding to the thioether-decorated vesicles upon heating.
Synthesis. Compound 2 was obtained by hydrogenation of
cholesterol. To a solution of 1 (7.73 g, 20 mmol) in tetrahydrofuran
(20 mL) were added palladium on charcoal (400 mg, 10%) and
acetic acid (1.7 mL). This mixture was transferred in a Parr
apparatus and subjected to hydrogenation at 60 °C under 5 bar of
H2 until consumption of H2 ceased (∼45 min). The cooled mixture
was filtered on Celite, the Celite was washed twice with THF (30
mL), the gathered organic phase evaporated to dryness, and the
crude product recrystallized from hot hexane (30 mL) down to 4
°C to give 5R-cholestan-3ꢀ-ol (compound 2, 5.90 g, 76%).
Compound 3. To a solution of 2 (5.90 g, 15.2 mmol) in dry
tetrahydrofuran (100 mL) was added bromoacetylchloride (4.5 mL,
53.1 mmol). The solution was heated to reflux for 2 h, the solvent
was evaporated on a rotary evaporator, and the minimum amount
of hot hexane was added to dissolve the crude product. After cooling
down to room temperature the hexane solution was left in the fridge
overnight to yield after filtration and drying bromoacetyl 5R-
cholestan-3ꢀ-oate as a pale green crystalline solid (compound 3,
(17) Hecker, C. R.; Fanwick, P. E.; McMillin, D. R. Inorg. Chem. 1991,
30, 659.
(18) Bonnet, S.; Collin, J.; Sauvage, J.; Schofield, E. Inorg. Chem. 2004,
43, 8346.
(19) Bonnet, S.; Collin, J.-P.; Gruber, N.; Sauvage, J.-P.; Schofield, E. R.
Dalton Trans. 2003, 4654.
(20) Bonnet, S.; Collin, J.; Sauvage, P. Inorg. Chem. 2006, 45, 4024.
(21) Bonnet, S.; Collin, J.-P.; Sauvage, J.-P. Inorg. Chem. 2007, 46, 10520.
(22) Laemmel, A.; Collin, J.; Sauvage, J. Eur. J. Inorg. Chem. 1999, 383.
(23) Mobian, P.; Kern, J.-M.; Sauvage, J.-P. Angew. Chem., Int. Ed. 2004,
43, 2392.
1
5.48 g, 71%). H NMR (400 MHz, δ in CDCl3): 4.75 (m, 1H,
CHOCO), 3.79 (s, 2H, CH2Br), 1.97 (dt, 1H, J ) 3.3, 12.4),
1.90-0.90 (m, 29H), 0.89 (d, 3H, J ) 6.5), 0.87 (d, 3H, J ) 6.6),
0.85 (d, 3H, J ) 6.6), 0.82 (s, 3H), 0.65 (s+m, 4H). 13C NMR
(101 MHz, δ in CDCl3): 166.92, 77.48, 77.16, 76.84, 76.13, 56.54,
56.41, 54.32, 44.76, 42.73, 40.11, 39.66, 36.80, 36.31, 35.94, 35.60,
35.58, 33.83, 32.10, 28.72, 28.38, 28.15, 27.35, 26.58, 24.34, 23.98,
22.96, 22.71, 21.36, 18.82, 12.37, 12.21. ESI MS exp (calcd):
531.26 (531.28, [M + Na]+), 1041.4 (1041.6, [2M + Na]+), 1551.4
(1551.9, [3M + Na]+). C,H,N expt 68.45/9.75/0.00; calcd 68.35/
9.69/0.0 for C29H49BrO2.
(24) Bonnet, S.; Collin, J.; Koizumi, M.; Mobian, P.; Sauvage, J. AdV.
Mater. 2006, 18, 1239.
(25) Bonnet, S.; Collin, J.-P. Chem. Soc. ReV. 2008, 37, 1207.
(26) Rillema, D. P.; Blanton, C. B.; Shaver, R. J.; Jackman, D. C.; Boldaji,
M.; Bundy, S.; Worl, L. A.; Meyer, T. J. Inorg. Chem. 1992, 31, 1600.
(27) Laemmel, A.; Collin, J.; Sauvage, J. C. R. Acad. Sci., Ser. IIc: Chim.
2000, 3, 43.
(28) Baranoff, E.; Collin, J.-P.; Furusho, Y.; Laemmel, A.-C.; Sauvage,
J.-P. Chem. Commun. 2000, 1935.
Compound 4. Compound 3 (1.02 g, 2.0 mmol) and sodium
thiomethoxide (289 mg, 4.12 mmol) were weighed in a round-
bottom flask and put under N2. Dry tetrahydrofuran (50 mL)was
cannulated under N2, and the suspension was heated to reflux for
2 h. THF was removed under vacuum, 100 mL of water was added,
and the product was extracted with Et2O (3 × 75 mL). The
(29) Collin, J.-P.; Laemmel, A.-C.; Sauvage, J.-P. New J. Chem. 2001, 25,
22.
(30) Collin, J.; Jouvenot, D.; Koizumi, M.; Sauvage, J. Inorg. Chem. 2005,
44, 4693.
(31) Collin, J.-P.; Jouvenot, D.; Koizumi, M.; Sauvage, J.-P. Inorg. Chim.
Acta 2007, 360, 923.
(32) Ossipov, D.; Gohil, S.; Chattopadhyaya, J. J. Am. Chem. Soc. 2002,
124, 13416.
(33) Schofield, E.; Collin, J.; Gruber, N.; Sauvage, J. Chem. Commun. 2003,
188.
(34) Takeuchi, K. J.; Thompson, M. S.; Pipes, D. W.; Meyer, T. J. Inorg.
Chem. 1984, 23, 1845.
9
J. AM. CHEM. SOC. VOL. 133, NO. 2, 2011 253

A R T I C L E S
Bonnet et al.
combined ether fractions were washed with water and brine, dried
with MgSO4, filtered, and evaporated to dryness. Column chroma-
tography on silica gel (200 mL) using pentane/dichloromethane
mixtures (8:3 to 1:1) afforded ligand 4 as a white solid (785 mg,
82%). 1H NMR (400 MHz, δ in CDCl3): 4.74 (m, 1H, 3〈), 3.14 (s,
2H, SCH2O), 2.20 (s, 3H, CH3S), 2.0-0.93 (m, 25H), 0.89 (d, 3H),
0.85 (dd, 6H), 0.82 (s+m, 4H). 13C NMR (100.6 MHz, δ in CDCl3):
169.95 (COO), 74.93 (CH〈O), 56.54, 56.41, 54.34, 44.80, 42.72,
40.11, 39.65, 36.86, 36.30, 36.15, 35.93, 35.60, 34.09, 32.12, 28.74,
28.38, 28.14, 27.58, 24.34, 23.98, 22.96, 22.70, 21.35, 18.81, 16.37,
12.38, 12.21. ESI MS exp (calc): 499.355 (499.359, [M + Na]+),
975.67 (975.72, [2M + Na]+), 1451.8 (1452.1, [3M + Na]+). C,H,N
expt 75.69/11.00/0.00; calc 75.57/10.99/0.0 for C30H52O2S.
microscope; the sample to irradiate was placed in a water bath at
25 °C to filter IR and UV radiations, and the reaction was followed
by UV-vis spectroscopy. For quantum yield determination the
continuous beam of a 1000 W xenon arc lamp from Lot was filtered
by a water filter of 15 cm diameter followed by an Andover
452FS10-50 interference filter from Lot Oriel (λex ) 452 nm).
Samples (3 mL) containing the vesicles (1.25 mM) functionalized
with 5 mol % of [5](PF6)2 were put in a closed, UV-vis quartz
cell (path length 1 cm) under an air atmosphere and stirred in a
water bath at 25 °C. Under these conditions, a light intensity of
6.4(3) × 10-8 einstein s-1 was measured using standard ferrioxalate
actinometry (see Supporting Information).35 The extinction coef-
ficients of [Ru(terpy)(bpy)(OH2)]2+ at the excitation wavelength
(452 nm) and in the presence of the ligand-functionalized vesicles
were determined by adding known amounts of the complex to
vesicle solutions containing 5 mol % of ligand 4 (values found:
ε452 ) 10800 cm M-1 for DMPG and 9750 cm M-1 for DMPC).
From the evolution of the UV-vis spectra of the vesicle-containing
solution (see Supporting Information), the variation of Ct, the
concentration in [5]2+, was determined as a function of irradiation
time t, and a linear regression of ln(Ct/C0) as a function of t gave
a pseudo-first-order rate constant of 2.34 × 10-3 s-1 for DPMG
and 2.27 × 10-3 s-1 for DPMC. The quantum yield for the
photosubstitution of ligand 4 by H2O at vesicles was calculated to
be 0.0074(8) for DMPG and 0.0073(8) for DMPC.
Ultracentrifugation. The UV-vis spectrum of a freshly prepared
vesicle sample was first measured between 250 and 800 nm. A
1.0-mL sample was ultracentrifuged at 25 °C and 100 krpm (RCF
35 500 g) for 1 h. Next, 0.70 mL of the supernatant was pipetted
out, and its UV-vis spectrum was measured. The lipid content of
the supernatant was measured by a Rouser assay36 after Bligh and
Dyer extraction.37 The Bligh and Dyer method was used to extract
the phospholipids from the aqueous phase as follows. To each
sample (0.4 mL) were added methanol (1.2 mL) and chloroform
(0.5 mL), and the mixture was homogenized. It was then extracted
3 times by addition of chloroform (0.5 mL), mixing, centrifugation
at 3000 rpm (RCF 1620g) for 3 min, and removal of the chloroform
phase. The combined organic fractions were evaporated under a
flow of N2 for 1 h, and dried under vacuum for 30 min. The Rouser
assay consists of a spectrophotometric titration of the phosphate
concentration using a molybdate salt. Each extracted sample was
decomposed with HClO4 (0.3 mL, 70-72%) at 180 °C for 1 h.
After cooling of the samples to room temperature, water (1 mL)
was added, followed by ammonium heptamolybdate (0.4 mL, 1.25%
w/v) and ascorbic acid (0.4 mL, 5% w/v), and the samples were
cooked 5 min in boiling water, using marbles as stoppers to prevent
evaporation. The absorbance of the solution at 797 nm was
measured and compared to a calibration curve obtained using 6
samples containing 0, 10, 20, 30, 40, 50 nmol of phosphate and
prepared in exactly the same conditions. Typical linear regression
coefficients R2 ) 0.9997 were found, and the amount of lipids found
in the supernatant after ultracentrifugation was calculated accordingly.
[5](PF6)2. [Ru(terpy)(bpy)(Cl)](Cl) (150 mg, 0.27 mmol) and
ligand 4 (138 mg, 0.29 mmol) were weighed in a round-bottom
flask and put under N2. Dry, degassed acetone was added (20 mL),
and an acetone solution of AgBF4 (113 mg, 0.58 mmol) was
cannulated under N2. The reaction mixture was heated at reflux
overnight (16 h), cooled to room temperature, and filtered over
Celite, and acetone was removed under vacuum. The crude product
was purified by chromatography on silica gel (200 mL) using an
acetone/water/KNO3sat mixture (100:10:1). The bright orange frac-
tion was collected, 50 mL of aqueous KPF6 was added, acetone
was removed on a rotary evaporator, and the precipitate was filtered
on a glass filter, washed thoroughly with water and Et2O, and dried
under vacuum. Yield: 94 mg of compound 4 as a bright orange
1
solid (50%). H NMR (400 MHz, δ in acetone-d6): 10.1 (dd, 1H,
A2), 8.94 (d, 3H), 8.74 (d, 2H), 8.71 (d, 1H), 8.52 (m, 2H), 8.20
(m, 3H), 8.01 (m, 3H), 7.53 (m, 3H), 7.31 (m, 1H), 4.45 (m, 1H,
CHO), 3.00 (s, 2H, SCH2O), 1.62 (s, 3H, CH3S), 2.02-0.82 (m,
39H), 0.78 (s, 3H), 0.67 (s+m, 4H). 13C NMR (100.6 MHz, δ in
acetone-d6): 166.9, 159.1, 158.4, 157.74, 157.66, 154.7, 153.3,
150.9, 140.0, 139.3, 138.2, 129.5, 128.7, 128.3, 126.1, 125.7, 125.4,
124.9 (18 Carom), 76.5 (CH2O), 57.28, 57.15, 55.01, 45.24, 43.35,
40.84, 40.23, 37.24, 36.92, 36.89, 36.58, 36.25, 36.08, 34.52, 32.69,
29.22, 28.89, 28.67, 27.95, 24.83, 24.51, 23.06, 22.82, 21.89, 19.07,
15.58, 12.46, 12.41 (SMe + 27 Calkyl). 19F NMR (376.3 MHz, δ in
acetone-d6): -72.9 (d, JF-P ) 707.8 Hz). UV-vis: λmax in nm (ε
in cm M-1): 454 (7760), 328 (15900), 332 (15900). ESI MS exp
(calc): 483.709 (483.719 for C57H75N5O3RuS, [M - 2PF6]2+).
C,H,N expt 52.59/5.69/5.32; calcd 52.54/5.69/5.57 for C55H71F12-
N5O2P2RuS.
Vesicle Preparation. Aliquots of phospholipids (0.005 mmol)
and ligand 4 or complex [5](PF6)2 (1-25 mol %, see Table 1) were
mixed from chloroform stock solutions and dried under a flow of
nitrogen for a few hours. They were subsequently placed under
vacuum to remove traces of chloroform. Then the lipid mixtures
were hydrated in a chloride-free buffer containing 10 mM of
phosphates and 40 mM of K2SO4 (total ionic strength 50 mM), at
pH ) 7.0. The final concentration of the lipids was 2.5 mM. The
lipid suspensions were freeze-thawed 10 times (from liquid N2
temperature to +50 °C) and then extruded 10 times (at +50 °C)
through 200 nm polycarbonate filters. The vesicle-containing
samples were conserved in the dark at 4 °C and used within 5 days.
Dynamic Light Scattering. Vesicle size was determined by
dynamic light scattering in a Zetasizer (Malvern Instruments Ltd.,
U.K.), operated at a wavelength of 633 nm.
Cryo-transmission Electron Microscopy. A 3-µL aliquot of
sample solution was pipetted onto a glow discharged Quantifoil
R2/2 copper grid 200 mesh in the environmental chamber of a
Vitrobot with a RH of 100% at 30 °C. The sample was blotted
once during 2 s and plunged into liquid ethane. The grid was
transferred to a Gatan cryoholder Model 626. The transmission
electron microscope used was a Philips Tecnai12 equipped with a
Biotwin-lens and a W filament, operated at 120 kV acceleration
voltage. Images were captured with a SIS Megaview II CCD-camera
and processed with iTEM software.
Results
Ligand Synthesis and Coordination to Ruthenium. The three-
step synthesis of ligand 4 is shown in Scheme 1: hydrogenation
of cholesterol was achieved first, followed by esterification with
bromoacetylchloride, and nucleophilic substitution of the bro-
mide by a methanethiolate group. Coordination of 4 to
ruthenium was realized by reacting it with [Ru(terpy)(bpy)-
(Cl)](Cl) in the presence of 2 equiv of AgBF4 in acetone,
followed by column chromatography (see Scheme 2). Anion
(35) Calvert, J. G.; Pitts, J. N., Chemical actinometer for the determination
of ultraviolet light intensities. In Photochemistry; Wiley and Sons:
New York, 1967; pp 780.
(36) Rouser, G.; Fleische, S.; Yamamoto, A. Lipids 1970, 5, 494.
(37) Bligh, E. G.; Dyer, W. J. Can. J. Biochem. Physiol. 1959, 37, 911.
Irradiation and Quantum Yield Measurement. White light
irradiations were performed using the 150 W halogen lamp of a
9
254 J. AM. CHEM. SOC. VOL. 133, NO. 2, 2011

Ruthenium-Decorated Lipid Vesicles
A R T I C L E S
Scheme 1. Synthesis of Ligand 4
Scheme 2. Synthesis of Complex [5](PF6)2 and Notation of the Protons
Table 1. Composition of Samples A-F and Characterization by
Dynamic Light Scattering
exchange using KPF6 in excess yielded the water-insoluble
orange complex [5](PF6)2, which was characterized by 1H, 19F,
and 13C NMR, mass spectrometry (ESI-MS), elemental analysis,
and UV-vis spectroscopy. Coordination of the sulfur atom of
4 to the ruthenium atom in complex [5](PF6)2 is characterized
by an absorption maximum at 454 nm in acetone,38,39 as well
as by a low-field shifted doublet for the A2 proton on the
,
sample
lipida
additiveb c
Zaverage (nm)
polydispersity index
A
B
B′
DMPG
DMPG
140.9 ( 6.1 0.038 ( 0.043
156.3 ( 4.6 0.113 ( 0.059
4
DMPG 4 + [Ru-OH2]2+ 156.3 ( 4.6 0.113 ( 0.059
C
DMPG [5]2+
160.1 ( 3.7 0.156 ( 0.030
167.8 ( 3.8 0.114 ( 0.034
169.9 ( 7.2 0.070 ( 0.034
177.8 ( 1.8 0.097 ( 0.032
C irradiated DMPG [5]2+ + hν
1
D
E
E′
DMPC
DMPC
bipyridine in H NMR spectroscopy (10.1 ppm in acetone-d6,
see Scheme 2 for notations).40
4
DMPC 4 + [Ru-OH2]2+ 177.8 ( 1.8 0.097 ( 0.032
DMPC [5]2+
155.1 ( 2.0 0.093 ( 0.033
155.7 ( 3.4 0.109 ( 0.024
Vesicle Preparation and Characterization. Large unilamellar
vesicles (LUVs) including 1 mol % of either complex [5](PF6)2
or ligand 4 were prepared as described (see Experimental
Section). Eight types of samples were prepared as indicated in
Table 1. Although [5](PF6)2 is not water-soluble, upon incor-
poration into the vesicles transparent yellow suspensions were
obtained for sample C and F (i.e., those containing 1 mol % of
[5](PF6)2). The UV-vis spectrum of the vesicle solutions
F
F irradiated DMPC [5]2+ + hν
a Lipid concentration is 2.5 mM. b Unless otherwise noted, additives
are introduced with a concentration of 1 mol % compared to the lipids.
c [Ru-OH2]2+ stands for [Ru(terpy)(bpy)(OH2)](BF4)2 and is added after
preparation of the vesicles.
TEM) was performed by vitrification above Tm on samples A-F
(see Figure 2).
3
showed the characteristic weak MLCT absorption bands of
[5]2+ in the visible region (absorption maximum at 450 nm)
With anionic lipids (DMPG, samples A-C, see Figure 2) a
relatively low concentration of vesicles was observed on the
negatively charged support, probably due to repulsive electro-
static interactions. Samples of type A and B contained only
unilamellar vesicles with a size compatible with DLS measure-
ments (∼150 nm). Sample C also contained unilamellar vesicles,
but another type of structure was observed as well, which
appeared as dark lines, ovoids, or discs with a uniform contrast
(see black arrow in Figure 2c). Finally some of these unilamellar
vesicles appeared faceted or as open bilayer fragments. On
average, the diameter of the particles is roughly ∼150 nm, which
is consistent with DLS measurements. The bilayer thickness
was 7 ( 1 nm, which corresponds to the expected value for a
single bilayer. Pixel size of the images was in the range of 1
nm, which limited the accuracy of the measurements.
When neutral DMPC lipids were used (samples D-F) a much
higher number of vesicles was observed (see Figure 2d-f), as
and several more intense bands in the UV region (see Figure
1).
The size distribution of the vesicles was measured by dynamic
light scattering (see Table 1). A narrow distribution centered
between 140 and 180 nm was obtained, which is consistent with
the nominal size of the filter holes used in the preparation (φ
200 nm). In order to obtain more insight into the morphology
of the vesicles, cryo-transmission electron microscopy (cryo-
(38) Root, M. J.; Deutsch, E. Inorg. Chem. 1985, 24, 1464.
(39) Bonnet, S.; Collin, J.; Gruber, N.; Sauvage, J.; Schofield, E. Dalton
Trans. 2003, 4654.
(40) The chemical shift of the A2 proton is very sensitive to the nature of
the monodentate ligand coordinated to ruthenium. For example, in
D2O the A2 proton of [Ru(terpy)(bpy)(Cl)]+ appears at 9.93 ppm, that
of [Ru(terpy)(bpy)(OH2)]2+ at 9.55 ppm, and that of [Ru(terpy)(bpy)(2-
thiomethoxyethanol)]2+ at 9.80 ppm (unpublished results).
9
J. AM. CHEM. SOC. VOL. 133, NO. 2, 2011 255

A R T I C L E S
Bonnet et al.
Figure 1. UV-vis spectra of [5](PF6)2 (0.025 mM) in acetone, DMPG vesicles, and DMPC vesicles.
Figure 2. Cryo-TEM pictures of samples A-F. In each picture the scale bar is 200 nm. For sample C the black arrow shows the uniform disk mentioned
in the text.
there was no repulsive interaction with the negatively charged
carbon support. Although the vesicles were mostly unfaceted
and unilamellar, a significant number of multilamellar vesicles
were also present. Contrary to DMPG samples no uniform discs
were observed. The size of the vesicles was consistent with the
DLS measurements (∼50-200 nm), and the bilayer thickness
was 7 ( 1 nm.
Irradiation Experiments. When the ruthenium-functionalized
vesicles (samples C and F) are irradiated with white light at 25
°C, a gradual color change from yellow to red is observed. At
any point of the experiment if the irradiation is stopped the
spectrum of the solution also stops evolving, which shows the
thermal inertness of the ruthenium complex in the dark. Figure
3a shows the evolution of the UV-vis spectrum of sample C
as a function of irradiation time. Clear isosbestic points are
observed at 458, 386, 332, 311, 297, and 288 nm, showing that
a single photoreaction is taking place. The initial absorption
band at 457 nm gradually vanishes to give rise to a new species
characterized by an absorption band at 492 nm. The absorption
spectrum in the end of the photoreaction is very close to that
of sample B′, in which [Ru(terpy)(bpy)(OH2)]2+ was added to
vesicles functionalized with ligand 4 (see Figure 3b). It is also
close to that of [Ru(terpy)(bpy)(OH2)]2+ in water; we attribute
the small differences between vesicle-containing and vesicle-
free samples to the interaction between the aqua complex and
the membrane. Similar results were obtained with DMPC
vesicles: the UV-vis spectrum of sample F after irradiation
was found nearly identical to that of sample E′ (data not shown).
As shown in the insert of Figure 3a, a pseudo-first-order
kinetics is observed for the photoreaction using white light
(ln(Ct/C0) ) -kt, where C0 and Ct are the concentration of [5]2+
before irradiation and after an irradiation time t, respectively).
The quantum yield of the process was measured at 25 °C using
monochromatic light set at the wavelength of the isosbestic point
(λex ) 452 nm). In the conditions of the experiment (see
Experimental Section), the quantum yield for the photosubsti-
tution of 4 by an aqua ligand at the ruthenium center was found
to be 0.0074(8) for DMPG and 0.0073(8) for DMPC vesicles.
9
256 J. AM. CHEM. SOC. VOL. 133, NO. 2, 2011

Ruthenium-Decorated Lipid Vesicles
A R T I C L E S
Table 2. Absorption Maximum of the Solution before
Ultracentrifugation and Color of the Lipid Pellets after
Centrifugation for Samples A-Fa
λmax of solution before
ultracentrifugation (nm)
Color of pellet after
ultracentrifugation
sample
A, B
B′
C
C irradiated
D, E
E′
F
n.o.
492
454
492
n.o.
492
454
492
colorlessb
red
yellow
red
colorlessb
colorlessb
yellow
colorlessb
F irradiated
a n.o.
)
not observed. b The colorless pellets were not fully
transparent and slightly diffused light.
(see below). The size of the particles ranged between 100 and
200 nm. For sample F (DMPC, Figure 4 right), a mixture of
unilamellar, multilamellar, and vesicular vesicles was found after
irradiation, with sizes of 50-200 nm. DLS measurement of the
vesicles led to the same conclusion (see Table 1): visible light
irradiation neither alters the size, nor the shape of the ruthenium-
functionalized vesicles C and F, and leads to a state that is
similar to sample B′ and E′ (see Discussion).
Ultracentrifugation Experiments. Samples C and F were
subjected to ultracentrifugation, before and after irradiation. The
absorbance of the supernatant was quantitatively measured at
the absorption maximum of the ruthenium complex (454 nm
before irradiation and 492 nm after, see Table 2) and compared
to the absorbance before centrifugation (see Table 3).
With both DMPG and DMPC vesicles, the pellets obtained
by ultracentrifugation before irradiation are yellow, and the
absorbance of the supernatant is low, thus showing attachment
of the ruthenium complex to the lipid vesicles. After irradiation
the situation is more contrasted (see Tables 2 and 3): for anionic
DMPG vesicles red pellets are observed, and according to the
UV-vis spectrum of the supernatant only ∼15% of the initial
absorbance is retained. Thus, ∼85% of the photochemically
produced [Ru(terpy)(bpy)(OH2)]2+ complexes are contained in
the pellet. On the contrary, for neutral DMPC vesicles the lipid
pellets obtained after ultracentrifugation are colorless, and the
absorbance at 492 nm is identical before and after centrifugation.
Thus, in this case the released [Ru(terpy)(bpy)(OH2)]2+ is
essentially noninteracting with the lipid bilayer.
Figure 3. (a) Evolution of the UV-vis spectrum of sample C upon white
light irradiation at 25 °C in a pH ) 7.0, chloride-free phosphate buffer (t
) 0, 1, 2, 3, 4, 5, 10, and 15 min). Insert shows the evolution of ln(Ct/C0)
as a function of irradiation time t, where Ct and C0 stand for the concentration
of [5]2+ at time t and t ) 0, respectively. (b) Superimposed UV-vis spectra
of sample C after irradiation (black), sample B′ (orange), and a reference
sample of [Ru(terpy)(bpy)(OH2)](BF4)2 in water (red).
These values are of the same order of magnitude as the ones
published for nitrile ligands,17,18 which highlights that the
membrane environment does not significantly modify the
photosubstitution properties of the ruthenium complex.
For samples C and F, cryo-electron microscopy shows that
the morphology of the ruthenium-functionalized vesicles after
visible light irradiation is very similar to that before irradiation
(see Figure 4). For sample C (DMPG, Figure 4 left), unfaceted
unilamellar vesicles were observed, as well as uniform discs
For sample B′ and E′, after ultracentrifugation the pellets were
found red and colorless, respectively (see Table 2), and the
supernatant showed similarly low (16%) and high (75%)
Figure 4. Cryo-TEM pictures of sample C (left) and F (right) after visible light irradiation. For sample C, black arrows show the uniform discs also seen
before irradiation (see Figure 2C).
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J. AM. CHEM. SOC. VOL. 133, NO. 2, 2011 257

A R T I C L E S
Bonnet et al.
Table 3. Percentages of Immobilization of Ruthenium at DMPG
and DMPC Vesicles before and after Irradiation
absorbance at λmax
before
after
remaining Ru in
remaining lipid in
sample
centrifugation centrifugation supernatant (%)c supernatant (%)d
A′ a
0.292
0.282
0.314
0.239
0.273
0.350
0.258
0.226
0.050
0.045
0.044
0.059
0.283
0.262
0.277
0.126
17
16
14
<0.8
<2.1
<1.8
<1.4
<0.3
<0.2
<0.1
<0.1
B′
C irradiated
C
25
D′ a
103b
75
E′
F irradiated
F
107b
56
a Obtained by adding 1 mol % of [Ru(terpy)(bpy)(OH2)](BF4)2 to
sample A (A′) or D (D′). b With DMPC samples (D′, E′, F, and F
irradiated) light scattering was found to be large, which significantly
increased the absorbance of the baseline and hence the errors during
application of the Beer-Lambert law. Estimated absolute errors on these
values are 10-20%. c According to UV-vis spectroscopy. d According
to Rouser assays.
ruthenium content, which is comparable to sample C and F after
irradiation (see Table 3). In all cases, a Rouser assay showed
that the supernatant did not contain significant amounts of lipids,
as the phosphate content was lower than 2% (see Table 3).
Back-Coordination. After irradiation, samples C and F were
heated at 60 °C for several hours, and up to 80 °C overnight
(16 h). The same heat treatment was applied to sample B′ and
E′. In all cases, the UV-vis spectrum after heating showed a
3
broadening of the MLCT band in the visible but no lowering
of the wavelength of the absorption maximum (see Figure 5a).
Partial precipitation was observed for neutral DMPC vesicles.
As we suspected that the kinetics of the coordination reaction
would be dependent on the concentration of the ligand, we
prepared samples B′′ (DMPG) and sample E′′ (DMPC) by
mixing 25 mol % of ligand 4 with the appropriate lipid,
preparing the vesicles, and finally (as for B′ and E′) adding 1
mol % of complex [Ru(terpy)(bpy)(OH2)]2+. Upon heating to
70 °C for several hours, DMPC vesicles (sample E′′) were not
stable and precipitated without appreciable color change. For
anionic DMPG vesicles, however, a gradual color change from
red to yellow was observed after a few hours. The reaction was
followed by UV-vis spectroscopy, which showed that the
absorption band at 492 nm gradually disappeared and that a
new band appeared around 450 nm, showing the slow formation
of a sulfur-bonded ruthenium complex (see Figure 5b). The band
at 492 nm did not completely vanish, however, and eight
supplementary hours of heating at 70 °C did not improve the
situation. Deconvolution of the final spectrum concludes to 63%
of sulfur-bound ruthenium complex (see Figure S4 in Supporting
Information). We suspect some degradation processes to occur
upon heating for a long time (see Discussion). Cryo-TEM
pictures were taken from sample B′′ after heating (see Figure
6). Unilamellar vesicles and flat discs were still present, which
looked similar to the pictures obtained before heating for sample
B′ (1 mol % of L). However, some much larger vesicles and
flat discs were also observed, with a diameter of up to 500 nm.
This is indicative of partial fusion of the lipid bilayers.
Figure 5. Evolution of the UV-vis spectrum of (a) sample B′ (1 mol %
of ligand 4) after being heated to 80 °C for 16 h and (b) sample B′′ (25
mol % of ligand 4) after being heated to 70 °C for 14 h.
which back binding to the membrane took place. In order to
clearly establish the nature of these discs, we tilted the sample
holder in the electron microscope. In all cases, the uniform discs
and dark lines turn into ovoids, whereas unilamellar vesicles
remain spherical (see Figure 7). Reversely, ovoids can be turned
into dark lines upon increasing tilt angles (data not shown).
Thus, these structures clearly correspond to flat discs that appear
differently depending on the observation angle.
Discussion
As recently shown by Hunter et al.,41 binding and unbinding
of transition metal complexes to and from membrane-embedded
ligands is an interesting biomimetic approach toward the binding
and unbinding of biomolecules at membrane proteins. Usually,
metal-steroid conjugates are considered as protein targeting
tools because steroids are protein substrates,42-47 but we are
(41) Dijkstra, H.; Hutchinson, J.; Hunter, C.; Qin, H.; Tomas, S.; Webb,
S.; Williams, N. Chem.sEur. J. 2007, 13, 7215.
(42) Jackson, A.; Davis, J.; Pither, R.; Rodger, A. Inorg. Chem. 2001, 40,
3964.
(43) Buil, M. L.; Esteruelas, M. A.; Garces, K.; Onate, E. Organometallics
2009, 28, 5691.
Nature of the Flat Discs. The uniform discs observed by cryo-
TEM for DMPG vesicles including 1 mol % of complex [5]2+
(sample C) were not present either if the charge of the ruthenium
head was removed (sample B or A) or if lipids were used that
do not have a net charge (sample D, E or F). In addition, when
sample C was irradiated, these structures were conserved, and
they were also observed after heating sample B′′ at 70 °C, upon
(44) Jaouen, G.; Vessieres, A.; Butler, I. Acc. Chem. Res. 1993, 26, 361.
(45) Lo, K. K.-W.; Lee, T. K.-M.; Lau, J. S.-Y.; Poon, W.-L.; Cheng, S.-
H. Inorg. Chem. 2008, 47, 200.
(46) Lo, K. K.-W.; Tsang, K. H.-K.; Sze, K.-S.; Chung, C.-K.; Lee, T. K.-
M.; Zhang, K. Y.; Hui, W.-K.; Li, C.-K.; Lau, J. S.-Y.; Ng, D. C.-M.;
Zhu, N. Coord. Chem. ReV. 2007, 251, 2292.
(47) Schobert, R.; Bernhardt, G.; Biersack, B.; Bollwein, S.; Fallahi, M.;
Grotemeier, A.; Hammond, G. L. Chem. Med. Chem. 2007, 2, 333.
9
258 J. AM. CHEM. SOC. VOL. 133, NO. 2, 2011

Ruthenium-Decorated Lipid Vesicles
A R T I C L E S
Figure 6. Cryo-TEM pictures of sample B′′ after heat treatment at 70 °C for 22 h (B′′ ) DMPG including 25 mol % of ligand 4, to which 1 mol % of
[Ru(terpy)(bpy)(OH2)]2+ was added).
Figure 7. Cryo-TEM pictures of sample C after irradiation at two different tilt angles of the sample holder: ∠ ) 0° (left) and 30° (right). White arrows,
spherical liposomes; black arrows, dark lines (left) turning to ovoids (right) upon tilting; dashed arrows, uniform discs. Similar pictures are observed before
irradiation.
using cholesterol derivatives for their ability to insert into
biological membranes.48 By covalently binding a 5R-cholestan-
3ꢀ-ol fragment to a monodentate thioether ligand and subse-
quently coordinating 4 to ruthenium, we aimed at decorating
unilamellar vesicles with ruthenium polypyridyl complexes.
UV-vis spectra of the functionalized DMPG and DMPC
vesicles were comparable to the spectrum of [5]2+ in acetone.
In addition, the yellow color due to the presence of the sulfur-
bonded ruthenium complex significantly diminished upon spin-
ning down the lipid vesicles. Thus, we can conclude that the
ruthenium complexes were attached to DMPG and DMPC
membrane via (1) direct coordination of the sulfur atom to
ruthenium and (2) supramolecular insertion of the cholestanol
moiety into the lipid bilayer.
Interestingly, a small (25%) to medium (56%) fraction of
S-bound ruthenium complex was also found in the supernatant
before irradiation for samples C and F, respectively. As a Rouser
assay of the supernatant excludes small unilamellar vesicles that
would not be spun down by centrifugation, such minor fraction
might be caused by (1) partial hydrolysis of the ester bond in
complex [5]2+, which would liberate the partially water-soluble
complex [Ru(terpy)(bpy)(S(Me)CH2COOH)]2+, or (2) exchange
of the counteranion of complex [5]2+. Indeed, the solubility of
complex [5]2+ in water highly depends on its counteranion.
During the purification of compound [5]2+ by chromatography,
for example, evaporation of acetone from the acetone/water/
KNO3 fractions ([NO3]- ≈ 0.032 M) does not lead to precipita-
tion of the orange complex, unless large amounts of saturated
aqueous KPF6 solution are added. Thus, compound [5]2+ in the
nitrate form, i.e., [5](NO3)2, is partly soluble in aqueous solution
despite its long apolar tail, whereas [5](PF6)2 is not. Thus, the
lipophilic hexafluorophosphate anions of the complex initially
introduced in the vesicle-containing samples might gradually
be exchanged by the more hydrophilic hydrogenophosphates
or sulfates anions present in the buffer, thus increasing the
solubility of complex [5]2+ in aqueous solution, which might
explain the amount of complex still present in the supernatant
after centrifugation.
The photochemistry of [Ru(terpy)(N-N)(Y)]2+, where N-N
is a bidentate diimine ligand and Y is a monodentate ligand,
has been studied thoroughly in (wet) organic solvents.17,20,27,32,33,39
However, to our knowledge it is the first time that the selective
photosubstitution of Y by an aqua ligand is reported in purely
aqueous solution. The presence of clear isosbestic points during
irradiation shows that a single photoreaction is taking place.
The first-order kinetics also corresponds to previous work, where
it was shown that upon ligand photoexpulsion coordination of
a solvent molecule is taking place.18 All the experiments carried
out in the present work (UV-vis spectroscopy, TEM, centrifu-
gation) converge to a great similarity between samples C after
irradiation and sample B′ on the one hand, and between sample
F after irradiation and sample E′ on the other hand (see Table
1). Thus, we conclude that the following reaction is taking place
at the membrane:
(48) d’Hardemare, A. D.; Torelli, S.; Serratrice, G.; Pierre, J. L. Biometals
2006, 19, 349.
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J. AM. CHEM. SOC. VOL. 133, NO. 2, 2011 259

A R T I C L E S
Bonnet et al.
hν
{[Ru(terpy)(bpy)(4)2+]}@LUV
8 [Ru(terpy)(bpy)(OH2)]2+
+ {4}@LUV
pH ) 7.0
water, r.t..
As the cholestanol fragment of 4 is inserted in the membrane,
the above reaction should lead to the detachment of the
ruthenium complex from the bilayer. As depicted in Figure 8,
however, depending on the charge of the lipids, two different
situations were found. First, with anionic lipids (DMPG)
ultracentrifugation after irradiation leaves a small fraction
(∼15%) of the initial absorbance of [Ru(terpy)(bpy)(OH2)]2+
in the supernatant, which accounts for a strong electrostatic
interactions between the “free”, positively charged ruthenium
complex and the negatively charged membrane. On the contrary,
with neutral lipids (DMPC) ultracentrifugation after irradiation
leaves a large fraction (75-100%) of the initial absorbance of
[Ru(terpy)(bpy)(OH2)]2+ in the supernatant, which accounts for
a weak interaction between the ruthenium complex and the
membrane. As no bias was introduced for incorporation of the
complexes in the outer and inner monolayer, one would expect
close to 50% of the liberated complexes in the supernatant after
centrifugation, if the ruthenium complexes situated inside the
vesicles were not able to cross the membrane. Considering the
large size of the vesicles, finding significantly more than 50%
is an indication that DMPC membranes might be leaky toward
[Ru(terpy)(bpy)(OH2)]2+. Two factors might explain such
permeability of the membrane toward [Ru(terpy)(bpy)(OH2)]2+.
Either the DMPC vesicles have holes because the temperature
during ultracentrifugation (25 °C) is close to their phase
transition temperature (24 °C), in which case the leakiness is
due to the coexistence, at that temperature, of domains that are
in the fluid and in the gel phase.49 Alternatively, [Ru(terpy)(b-
py)(OH2)]2+ might be lipophilic enough to cross the membrane
by partial solubilitization, which has been recently suggested
for related polypyridyl ruthenium(II) complexes.50,51
Quantum yield measurements show that photocleavage of the
ruthenium complexes from the vesicles is as efficient as
comparable homogeneous systems,17,18 and according to DLS
and cryo-TEM analysis it does not change the size or shape of
the vesicles themselves. By contrast, thermal coordination of
[Ru(terpy)(bpy)(OH2)]2+ to the thioether-decorated vesicles is
a slow process, and heating to 70 °C for prolonged times is
required to observe partial coordination. DMPC vesicles are not
stable under such hard conditions and aggregate into larger
particles that ultimately precipitate. For DMPG vesicles repul-
sive electrostatic interactions between the vesicles prevent
precipitation of the vesicles, which allows for the observation
of back-coordination by UV-vis spectroscopy. Thus upon
heating the following reaction takes place:
Figure 8. Model for the irradiation of [5](PF6)2 at membranes: (a) with
anionic DMPG lipids and (b) with neutral DMPC lipids.
Figure 9. Model for the formation of the “pancakes” observed by cryo-
TEM: (a) double bilayers and (b) single bilayers (bicelles).
(≈35%, see deconvolution Figure S4) of ruthenium complexes
cannot coordinate to the thioether ligands imbedded in the
membrane. The interpretation of this is still unclear; it is not
possible to fully exclude degradation processes such as lipid
hydrolysis, hydrolysis of the ester linker in ligand 4, or slow
oxidation of the sulfur ligands to sulfoxides or sulfones.
Flat discs made of lipids might either be bilamellar bilayers,
i.e., flattened vesicles, or bicelles, i.e., unilamellar lipid discs
(see Figure 9). As electrostatic interactions are necessary to
observe these discs, we initially considered the first hypothesis,
where flattening of the vesicles would be caused by attractive
electrostatic interactions between the ruthenium heads situated
in the inner monolayer, and the negative lipids facing them at
the opposite side of the inner monolayer (Figure 9a). Similar
electrostatic interactions are commonly invoked to explain how
divalent Ca2+ or Mg2+ cations stabilize negatively charged lipid
bilayers on negatively charged glass or mica surfaces.52
However, the thickness of the discs was measured under an
angle where they appear as black lines. We found only 7 ( 1
nm, which corresponds to single discoidal bilayers, thus bicelles
(see Figure 9b). Bicelles were typically described when a
combination of very short lipids (typically in C6) and long lipids
(C12-C18) was used, and usually the short lipids concentrate at
the rim of the bicelle.53-55 Our system is deprived of such short
lipids, but [5]2+ being composed of a polar head and an apolar
2+
(
)(
)
Ru terpy bpy OH
(
[
) ]
2
heating
8 {Ru(terpy)(bpy)(4)2+
}
@DMPG
{ }@DMPG
4
+
pH ) 7.0
However, cryo-TEM also shows that after heating aggregation
of the LUVs have occurred, as much larger vesicles and bicelles
are observed. In addition, the remaining shoulder at 492 nm
after heating (see Figure 5b) indicates that a minor fraction
(49) Noordam, P. C.; Killian, A.; Elferink, R. F. M. O.; Degier, J. Chem.
Phys. Lipids 1982, 31, 191.
(52) Richter, R.; Brisson, A. Biophys. J. 2005, 88, 3422.
(53) Diller, A.; Loudet, C.; Aussenac, F.; Raffard, G.; Fournier, S.; Laguerre,
M.; Grelard, A.; Opella, S. J.; Marassi, F. M.; Dufourc, E. J. Biochimie
2009, 91, 744.
(50) Schatzschneider, U.; Niesel, J.; Ott, I.; Gust, R.; Alborzinia, H.; Wolfl,
S. Chem. Med. Chem. 2008, 3, 1104.
(51) Zava, O.; Zakeeruddin, S. M.; Danelon, C.; Vogel, H.; Gratzel, M.;
Dyson, P. J. ChemBioChem 2009, 10, 1796.
(54) Katsaras, J.; Harroun, T.; Pencer, J.; Nieh, M. Naturwissenschaften
2005, 92, 355.
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Ruthenium-Decorated Lipid Vesicles
A R T I C L E S
cholestanol moiety might behave as a surfactant, which are
known to sometimes lead to the formation of bicelles and open
vesicles.56 Very recently, a similar effect has been reported with
sulfate-functionalized cholesterols in DMPC/DHPC lipid bi-
layers.57
therapy,59-61 light activation62-64 and light-triggered delivery65-67
of metal drugs represent fast-developing research fields. Our
system combines a photocleavable metal complex and lipid
bilayers as biocompatible carriers.68-72 It represents thus a
potentially new alternative to introduce drugs in the body, where
the drug is attached to the membrane itself by a photocleavable
coordination bond, instead of being encapsulated into it.73,74
Conclusion
Acknowledgment. The Dutch Organization for Scientific
Research (NWO-CW/Vernieuwingsimpuls) is kindly acknowledged
for a Veni grant to S.B.
The present work represents our first attempt of probing the
(photo)coordination properties of ruthenium complexes in the
biomimetic environment of a lipid bilayer. By synthesizing a
cholestanol-thioether hybrid that coordinates to ruthenium via
its sulfur atom, we were able to decorate large unilamellar
vesicles with ruthenium polypyridyl complexes. When using
negatively charged lipids to build up the membrane (DMPG),
both electrostatic interactions and insertion of the apolar tail of
the ligand into the bilayer seem to play a role in the
metal-membrane interaction. After visible light irradiation and
cleavage of the Ru-S bond, the free aqua complex [Ru(terpy)-
(bpy)(OH2)]2+ still interacts with the membrane through elec-
trostatic forces, which allows for back-coordination of the
complex to the vesicles, though at high ligand concentrations
and elevated temperatures. Increasing the rate of thermal binding
to the membrane-embedded ligands is a prerequisite before fast
hopping of metal complexes at the surface of a vesicle can be
obtained. Ultimately, organization of the ligands at the mem-
brane might allow for light-controlled motion of single mol-
ecules at the membrane-water interface.
Supporting Information Available: Evolution of the absor-
bance of vesicle solutions during quantum yield measurement,
actinometry data, and deconvolution of the spectrum after
heating (shown in Figure 5b). This material is available free of
charge via the Internet at http://pubs.acs.org.
JA105025M
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