Ruthenium polypyridyl peptide conjugates: Membrane permeable probes for cellular imaging

Article abstract of DOI:10.1039/b810403d

Two novel polyarginine labelled ruthenium polypyridyl dyes are reported, one conjugated to five, (Ru-Ahx-R₅), and one to eight arginine residues, (Ru-Ahx-R₈); both complexes exhibit long-lived, intense, and oxygen-sensitive luminesce

Full text of DOI:10.1039/b810403d

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Ruthenium polypyridyl peptide conjugates: membrane permeable probes
for cellular imagingw
Ute Neugebauer,a Yann Pellegrin,a Marc Devocelle,c Robert J. Forster,ab
William Signac,d Niamh Morand and Tia E. Keyes*ab
Received (in Cambridge, UK) 18th June 2008, Accepted 13th August 2008
First published as an Advance Article on the web 12th September 2008
DOI: 10.1039/b810403d
Two novel polyarginine labelled ruthenium polypyridyl dyes are
reported, one conjugated to five, (Ru–Ahx–R5), and one to eight
arginine residues, (Ru–Ahx–R8); both complexes exhibit long-lived,
intense, and oxygen-sensitive luminescence; (Ru–R8) is passively,
efficiently and very rapidly transported across the cell membrane
into the cytoplasm without requirement for its permeablisation.
ruthenium polypyridyl peptide conjugates which are
potentially useful targeted probes for luminescence and lumi-
nescence lifetime cellular imaging. It is important to mention
that a number of elegant examples of cell permeable inorganic
luminophores have been reported recently, based on Rh, Ir,
Re and f block elements,7 however this present report is, to our
knowledge, the first cell-permeable ruthenium–peptide
conjugate.
Luminescent dye molecules capable of passive cell delivery
may be used as molecular probes in cellular imaging, across
cell biology, molecular biology, microbiology, and flow cyto-
metry applications.
The two novel conjugates (Scheme 1) are based on the
parent ruthenium complex [Ru(bpy)2(pic)]2+, where pic is
2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline.8 Con-
jugation occurs through the terminal carboxy unit.9 The Rn
oligopeptides (n = 5 or 8) were obtained via Merrifield’s solid
phase peptide synthesis, according to the Fmoc/t-Bu strategy.
The detailed synthesis is described in ESI.w In this protocol,
the coupling efficiency of [Ru(bpy)2(pic)]2+ to the peptide
exceeded 85%.10 This is a significant improvement on more
typical conjugations of organic fluorophores which is ascribed
to the reactivity of the aryl acid pendant on the ruthenium
centre. The ruthenium dyes are functionalised unequivocally
through a single, reactive group, via reaction with nucleophilic
functions on peptides. They do not contain isomers or
competing functional groups which can lower the synthetic
yields of the labelling step and/or require their protection.11
The photophysical properties of the conjugates, Ru–Ahx–R5
and Ru–Ahx–R8 have been investigated in phosphate buffered
saline (PBS) at pH 6.7, which resembles the pH and ion
concentration found in living cells. The arginine modification is
relatively remote from the metal centre and is expected to have
very little influence on the electronic structure of the Ru-complex.
Consequently, the photophysical properties of Ru–Ahx–R5 and
Ru–Ahx–R8 are essentially indistinguishable from each other or
of the parent complex [Ru(bpy)2(picH)]2+.8 As for the parent
complex [Ru(bpy)2(picH)]2+, the electronic absorption spectra of
the two arginine derivatives show an absorption band at 460 nm
The most widely used probes in cellular imaging are
fluorescent and based on organic, typically polyaromatic,
chromophores. The short luminescence lifetimes of such species,
typically o10 ns, limits their environmental sensitivity, e.g.,
towards molecular oxygen and their application in fluorescence
lifetime imaging (FLIM).1 Ruthenium polypyridyl complexes
have unique photophysical properties which make them poten-
tially invaluable as probes for cellular imaging, including intense
polarised luminescence, large Stokes shifts, red emission wave-
lengths, and a good photostability. They are oxygen sensitive,
and do not exhibit the complication of dimer or excimer
formation observed for some O2-sensitive organic probes.2
However, there has been a longstanding barrier to their exploi-
tation in this context because conventional complexes do not
passively diffuse across the cell membrane.3 Therefore, cells
must be permeabilised by electroporation, detergent or treated
with some other transfection agent.4 Antibody conjugation has
been shown to permit external labelling of cells rather than
direct imaging.5 Very recently a ruthenium complex conjugated
to estradiol has been shown to be cell permeable, attributed to
the lipophilicity of the steroid pendant.6
Herein, we report on the preparation and characterization
of two novel intensely luminescent and oxygen-sensitive
a The Biomedical Diagnostics Institute, Dublin City University,
Dublin, 9, Ireland. E-mail: Tia.keyes@dcu.ie
b The School of Chemical Sciences, National Centre for Sensor
Research, Dublin City University, Dublin, 9, Ireland
c Centre for Synthesis and Chemical Biology, Department of
Pharmaceutical and Medicinal Chemistry, Royal College of Surgeons
in Ireland, 123 St. Stephen’s Green, Dublin, 2
d Royal College of Surgeons in Ireland, 123 St. Stephen’s Green,
Dublin, 2
w Electronic supplementary information (ESI) available: Experimen-
tal, synthesis and characterisation, video of cellular uptake, photo-
bleaching and temperature dependence of cellular uptake. See DOI:
10.1039/b810403d
Scheme 1 Structure of Ru-Ahx-Rn (n is 5 or 8). In the parent
,
complex, [Ru(bpy)2(picH2)]2+
imidazole ring is replaced by an aryl acid.
the aryl amide pendant on the
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(e B16.9 Â 103 L molÀ1 cmÀ1) which is assigned to a metal-to-
ligand charge transfer (MLCT) transition. An intense
emission is observed at 607 nm with a quantum yield of 0.06,
which is slightly lower than that of the parent [Ru(bpy)2-
(picH)]2+ complex (F = 0.067), but still approximately 30%
higher than the quantum yield of the well known [Ru(bpy)3]2+
complex. The luminescence lies in the red, well away from
possible autofluorescence of biological material and the high
quantum yield permits easy detection, even at low dye concen-
tration. The presence of the Ahx-R8 moiety slightly reduces the
luminescence lifetime from 872 Æ 4 ns in the parent complex
(degassed aqueous solution) to 775 Æ 4 ns in Ru–Ahx–R8. This
long-lived luminescence may be well suited to explore some of the
longer lived, microsecond biodynamical processes, e.g.
membrane diffusion, protein rotation or folding. The lumines-
cence lifetime is, furthermore, oxygen sensitive. In air saturated
aqueous solution the lifetime of the excited state of Ru–Ahx–R8
decreases to 480 ns, this B50% decrease in lifetime indicates a
high sensitivity toward the concentration of dissolved oxygen.
To investigate cellular uptake of Ru–Ahx–R5, Ru–Ahx–R8
Fig. 2 (a) & (c) White-light differential-interference contrast image of
human blood platelets spread on a fibrinogen-coated glass slide
following 20 min incubation with 3 Â 10À5 M (a) Ru–Ahx–L5 and
(c) Ru–Ahx–L8. (b) & (d) The corresponding luminescence images
(lex = 458 nm, lem = 610 nm), on incubation with (c) Ru–Ahx–L5
and with (d) Ru–Ahx–L8.
and [Ru(bpy)2(picH)]2+
, we investigated myeloma cells
(Fig. 1) and human blood platelets, as examples of mamma-
lian cells (Fig. 2), using confocal laser scanning microscopy.
458 nm excitation was used, corresponding to the MLCT
absorbance of the metal complex and the emission images
were recorded at 610 nm. In a typical protocol, 3 mL of an
aqueous solution of the Ru-complex ([Ru(bpy)2(picH)]2+ or
Ru–Ahx–R8 (1.2 Â 10À3 M)) were added to 100 ml of the cell
suspension to yield a final dye concentration of 3.5 Â 10À5 M.
Fig. 1 compares the ability of the parent complex and the octa-
arginine derivative to transport across a myeloma cell
membrane. As expected, no luminescence was observed from
within the cells when they were incubated with [Ru(bpy)2-
(picH)]2+ (Fig. 1(c)), indicating the dye cannot penetrate the
cell membrane and enter the cell, even after extensive incuba-
tion. In contrast, Ru–Ahx–R8 transports passively through the
cell’s membrane and accumulates and preconcentrates inside
the cell. Transport is rapid and relatively irreversible.12 The
migration of the dye into the cell is complete within about
12 min yielding intense well contrasted cellular images as
shown in Fig. 1(a) and (b). The first 5 min of the passive
transmembrane transport of Ru–Ahx–R8 into myeloma cells is
shown in the accompanying video (see ESIw).13 During the
first 2–3 min, the dye concentrates in the cell membrane. From
here, the dye distributes into and throughout the cell. The
distribution of the dye inside the cell is not homogenous,
resulting in brighter and darker areas, according to the
different cellular compartments. After between 10 and
15 min the process is complete and no further changes to the
dye distribution within the cell are observed. In contrast,
[Ru(bpy)2(picH)]2+ could only be incorporated into the cell
through permeablisation of the cell membrane, in this case, on
exposure to detergent (Triton) as is shown in Fig. 1(d).14
The length of the polyarginine peptide (R5 and R8) is important
in dictating the membrane transport properties of the dye, as seen
in Fig. 2 for human blood platelets. Whereas platelets incubated
with Ru–Ahx–R8 show intense luminescence from their cyto-
plasm (Fig. 2(d)), the platelets incubated with Ru–Ahx–R5 do not
exhibit any luminescence. The cell penetrating ability of poly-
arginines is well known and generally thought to arise through
endocytosis. This ability is polyargenine chain length dependent
with R6 to R11 showing best endocytosis.15 Consistent with
endocytosis, we found the dye delivery in to the cell to be
temperature dependent.17 Thus, the inability of Ru–Ahx–R5 to
penetrate the cell membrane in this instance is consistent with
other peptide studies indicating the polyargenine is a useful carrier
peptide for cell penetration by metal complexes.16
A drawback of conventional organic chromophores for
laser scanning microscopy is their propensity to photobleach,
which limits their use over extended periods. Under imaging
conditions typically used for human blood platelets (see ESIw),
under 20 min continuous irradiation, Ru–Ahx–R8 retained
more than 50% of its initial luminescence intensity from
within a platelet. Such photostability is useful as it may allow
the dynamics of cellular processes to be studied at timescales
which are useful to the microscopist/biologist in obtaining
detailed or dynamic cellular information.
Fig. 1 Luminescence images (lex = 458 nm, lem = 610 nm) of
myeloma cell incubated with Ru–Ahx–R8 (3.5 Â 10À5 M) after (a)
3 min and (b) after 5 min. (c) Myeloma cell incubated with the parent
complex [Ru(bpy)2picH]2+ (3.5 Â 10À5 M) for 26 min and (d) for
5 min after permeablizing the cell with Triton (1% v/v).
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Overall, Ru-labelled cell-permeable peptides are likely to be an
important tool in cellular imaging for probing cellular events
and the cellular environment.
This material is based upon work supported by the Science
Foundation Ireland under the Biomedical Diagnostics Insti-
tute (Award No. 05/CE3/B754) and SFI investigator
programme (Award No. 05/IN.1/B30). The authors express
their profound gratitude to Prof. Richard O Kennedy and
Dr Marie LeBeurre for supplying the myeloma cell culture.
Fig. 3 (a) Fluorescence intensity image of myeloma cells after
incubation for 15 min with Ru–Ahx–R8, 3.5 Â 10À5 M in aqueous
PBS buffer. (b) False colour fluorescence lifetime image of the same
cell (fast FLIM).
Notes and references
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A key advantage of ruthenium polypyridyl complexes over
many conventional fluorescent imaging dyes are their long lived
excited states. This property renders such complexes far more
sensitive to their environment, e.g., dissolved oxygen concen-
tration, pH, dielectric constant and potential. Significantly, the
fluorescence lifetime is independent of luminophore concentra-
tion, the optical path of the microscope, the local excitation
light intensity, as well as the luminescence detection efficiency.
This makes luminescence lifetime an ideal parameter to measure
in biological systems where the exact concentration of dye after
cellular uptake is difficult to determine and replicate accurately.
As described, the luminescence of Ru–Ahx–R8 exhibits signifi-
cant oxygen dependence. Fig. 3(a) shows a scanning confocal
luminescence image of myeloma cells incubated with
Ru–Ahx–R8, Fig. 3(b) shows the false colour luminescence
lifetime image of the same cells. The lifetime of the residual
dye in the external buffered solution is monoexponential,
however, the lifetimes of selected compartments within the cells
are typically biexponential. The false colour image reflects the
average lifetime of the probe.
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10 In a typical protocol, 50 mmol of resin, produced an average weight
of 7 mg of the desired product after purification, with excellent
reproducibility.
As can be seen from the false colour coding, the average
lifetime of the dye varies across the various compartments of
the cells. The dye residing in the membrane of the cell, for
example, exhibits the shortest lifetime. This is in agreement
with the anticipated higher solubility of O2 in the membrane17
and demonstrates the potential of these dyes for cellular
oxygen sensing.
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reduces slightly with time however, the decrease is small (o ca.
30% over 2 h) and even after 2 h the cells remain strongly
luminescent.
In conclusion, we have demonstrated a high yield synthetic
strategy for production of peptide labelled ruthenium lumino-
phores. We demonstrate that an octa-arginine labelled
ruthenium complex Ru–Ahx–R8, is an oxygen sensitive
luminophore that transports rapidly and passively across the
cell membrane to preconcentrate inside the cell. This beha-
viour was demonstrated for myeloma and for human blood
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lived and intense, and has appropriate absorption and emis-
sion characteristics to suit most conventional confocal laser
systems. Its long lifetime, makes it quantitatively sensitive to
oxygen concentration and the counter ligands can be readily
altered to target the environmental sensitivity of the probe.
For example, permitting sensitivity to pH, water content and
the rigidity of the microenvironment. Labelling of peptides
assembled by solid-phase synthesis can be more broadly
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13 See video—ESIw.
14 The cytotoxicity of the dye was investigated using Trypan Blue and
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(42 h) there was evidence for cytotoxicity. This is currently under
further investigation.
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