Multimodal cell imaging by ruthenium polypyridyl labelled cell penetrating peptides

Article abstract of DOI:10.1039/b918611e

The capacity of ruthenium polypyridyl complexes as probes for combined confocal luminescence and resonance Raman imaging, enabled by their large Stokes shift, is demonstrated for a novel membrane sensitive Ru(ii) polypyridyl peptide. Confocal luminescence and resonance Raman imaging provides complementary information about the membrane phospholipid regions of the cell and the location of the dye within the cell.

Full text of DOI:10.1039/b918611e

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Multimodal cell imaging by ruthenium polypyridyl labelled cell
penetrating peptidesw
Lynda Cosgrave,a Marc Devocelle,b Robert J. Forsterb and Tia E. Keyes*a
Received (in Cambridge, UK) 9th September 2009, Accepted 6th November 2009
First published as an Advance Article on the web 19th November 2009
DOI: 10.1039/b918611e
The capacity of ruthenium polypyridyl complexes as probes for
combined confocal luminescence and resonance Raman imaging,
enabled by their large Stokes shift, is demonstrated for a novel
membrane sensitive Ru(II) polypyridyl peptide. Confocal
luminescence and resonance Raman imaging provides comple-
mentary information about the membrane phospholipid regions
of the cell and the location of the dye within the cell.
example, the sensitivity of the excited state lifetime and hence
the phosphorescent intensity to O2 is well known for many
ruthenium complexes.4 In addition, exploitation of ligands
such as dppz (dipyrido-[3,2-a:20,30-c]-phenazine) induces
sensitivity in these parameters to the aqueous environment.5,6
Examples of ruthenium polypyridyl complexes applied to
cell imaging are increasing,2 although a major barrier to this
application is that they do not generally cross the cell
membrane spontaneously. However, we recently described
how conjugation of a cell-penetrating polyarginine peptide
to a ruthenium polypyridyl complex allows ready passage of
the metal complex across the cell membrane.6,7
Advances in fluorescence microscopy, in particular scanning
confocal imaging technologies, have revolutionised the study
of live cells. Similar advances in Raman microscopy have
emerged in recent years, and scanning confocal Raman
microscopes are now commercially available, opening the
possibility of Raman imaging of cells. Label free Raman
mapping of cells has been the focus of a number of studies
as it holds the potential for mapping concentrations of key
biochemicals within the cell.
In this contribution we illustrate how a peptide linked
ruthenium complex can be exploited in multimodal imaging,
by demonstrating the application of
a novel environ-
mentally sensitive ruthenium polypyridyl peptide conjugate,
[Ru(dppz)2PIC-Arg8]10+, Scheme 1, as a membrane probe in
live SP2 myeloma cells; dppz is dipyrido-[3,2-a:20,30-c]-phenazine,
PIC is 2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline
and Arg is arginine. The complex was synthesized from
[Ru(dppz)2Cl2] and the parent ligand according to commonly
reported methods and the peptide modified at its N-terminus
with a 6-carbon spacer (6-amino-hexanoic acid) was prepared
by Merrifield’s solid phase peptide synthesis.11 The parent
complex and peptide conjugates were purified by HPLC and
structure was confirmed by NMR and MALDI-TOF mass
spectrometry.
The parent complex, [Ru(dppz)2PIC]2+, exhibits a metal-
to-ligand charge transfer (MLCT) absorbance centred at
approximately 450 nm, and in non-aqueous media, an intense
emission centred at 610 nm which is readily excited at 458 nm
by an argon ion laser.11
However, it is limited by the low sensitivity of conventional
Raman which requires long acquisition times; the resulting
dwell times of the laser on a single spot is potentially
damaging.1 In addition, in the complex matrix of a cell, it
inevitably suffers from background interference. However, if
as in fluorescence microscopy, exogenous labels can be
incorporated into the cell which can be excited under
resonance absorption conditions, the dye’s distribution and,
unique to Raman, its structure, can be probed with excellent
sensitivity. This may be particularly useful if that structure
exhibits environmental sensitivity.
A particularly useful paradigm arises if a single label can be
used for both luminescence and resonance Raman imaging
of cells. Such multimodal imaging is impossible with the
fluorescent organic dyes commonly exploited for live cell
imaging since their small Stokes shift means fluorescence
overwhelms the weaker Raman response. However, as we
demonstrate here, ruthenium polypyridyl complexes which
typically exhibit up to 200 nm separation between their
absorbance and emission maxima can permit the collection
of intense interference free resonance Raman spectra at a
wavelength that also excites emission.3 Another key advantage
of Ru(II) complexes is the capacity to make their photo-
physical properties dependent on their environment. For
In degassed 9/1 acetonitrile–DMSO the complex exhibits a
luminescence lifetime of 0.68 ꢀ 0.017 ms. Following conjugation
of the polyarginine peptide to [Ru(dppz)2PIC]2+, no significant
11
change was observed in the absorption or emission lmax
However, the lifetime of the peptide conjugate in degassed
.
9/1 acetonitrile–DMSO increased to 0.78 ꢀ 0.022 ms. The
a National Bioimaging Platform, National Centre for Sensor Research,
The School of Chemical Sciences, Dublin City University, Dublin 9,
Ireland. E-mail: tia.keyes@dcu.ie
b 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, Ireland
w Electronic supplementary information (ESI) available: Detailed
synthesis and characterisation data; video. See DOI: 10.1039/b918611e
Scheme 1 Structure of [Ru(dppz)2PIC-Arg8]10+. In the parent com-
plex, the aryl amide on the imidazole ring is replaced with an aryl acid.
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Fig. 2 Confocal luminescence images of live SP2 myeloma cells at
37 1C after incubation for 48 h with (A) [Ru(dppz)2PIC-Arg8]10+ and
(B) [Ru(dppz)2PIC]2+. lex = 458 nm, lem = 620 nm.
pH 7.4. Both free dye and conjugated dye showed no emission
in the buffer alone but after addition to the vesicle suspension,
strong emission was observed from the vesicles.11 The capacity
of [Ru(dppz)2PIC]2+ and [Ru(dppz)2PIC-Arg8]10+ to function
as cellular membrane probes was then explored using confocal
fluorescence imaging using mouse SP2/0 myeloma cells.
Typically, 35 mM of dye or dye conjugate was added to the
cell culture medium and incubated with the cells for approxi-
mately 48 h. Although the complex shows some toxicity to the
cells after 48 h incubation (approx. 30% cell death) the cell
culture remains viable. The cells were collected and imaged on
a Zeiss Meta confocal microscope, live at 37 1C using an
incubator. Fig. 2 shows the confocal luminescence images for
Fig. 1 (i) The emission spectra of the free dye in an aerated
acetonitrile solution with sequential addition of deionised water.
(ii) The resonance Raman spectra of [Ru(dppz)2PIC-Arg8]10+
and [Ru(dppz)2PIC]2+ in PBS buffer and from within the cell
(from resonance Raman mapping) after excitation at 458 nm.
the myeloma cells stained with (A) [Ru(dppz)2PIC-Arg8]10+
(B) with the parent dye excited at 458 nm.10
,
enhanced luminescence of the conjugate was also reflected
in the quantum yields in aerated 9 : 1 acetonitrile–DMSO
which were 0.025 and 0.048 for [Ru(dppz)2PIC]2+ and
[Ru(dppz)2PIC-Arg8]10+ respectively.8 The origin of the
enhanced luminescence may arise from environmental protection
conferred by the large peptide chains on the dppz ligands.
Fig. 1(ii) shows the resonance Raman spectra of
[Ru(dppz)2PIC-Arg8]10+ and [Ru(dppz)2PIC]2+ in buffer
excited at 458 nm. Comparison with [Ru(dppz)3]2+ and
similar complexes9 confirms the majority of the modes can,
in each case, be assigned to the dppz ligand. This is consistent
with excitation resonant with a Ru(dp) to dppz p* MLCT
transition. The spectra are similar for dye and conjugate
except that bands at B1357 cmꢂ1 and B1390 cmꢂ1 are shifted
to higher energy (B20 cmꢂ1) for [Ru(dppz)2PIC-Arg8]10+. In
addition, a shoulder on the band at B1592 cmꢂ1 appears in
the spectrum of [Ru(dppz)2PIC-Arg8]10+ which may originate
from the peptide.
Following incubation with [Ru(dppz)2PIC]2+ (B), only the
outer membrane of the cells exhibits emission, Fig. 2(B). In
contrast, following incubation with [Ru(dppz)2PIC-Arg8]10+
,
the outer and internal membranes associated with the
sub-cellular structures emit strongly. Counter-staining indi-
cates that these are the nucleus and cell organelles. In addition,
on the cytoplasmic side of the outer cellular membrane, small
features can be seen which are thought to be endosomal
vesicles that occur when the dye is internalised via endocytosis.
The emission spectrum of the internalised dye, collected on the
Zeiss Meta, corresponded to the 3MLCT emission of the
ruthenium complex. A z-stack was also performed to investi-
gate the lipid structures and dye distribution throughout the
cell at 0.2 mm increments over 20 mm; this is presented as a
video in supplementary material.w11 Similar results are
observed on shorter incubation times but the most intense
images require overnight incubation.
The dppz ligands confer ‘molecular light-switch’ properties
on the ruthenium complex wherein the emission is quenched in
an aqueous environment due to hydrogen bonding to the
phenazine nitrogens. This effect is illustrated in Fig. 1(i) where-
in emission is seen to steadily decrease with addition of
increasing volumes of water to a 100% acetonitrile solution
of [Ru(dppz)2PIC]2+. Complete extinction of emission occurs
at approximately 18% v/v water. Overall, the photophysical
properties of the conjugated complex mirror those of the
parent, which exhibits the same aqueous light switch property.
The ability of a lipid bilayer to switch the luminescence of
these complexes on was confirmed by applying the complex
to a suspension of large unilamellar vesicles formed from
dipalmitoylphosphatidylglycerol (dppg) in PBS buffer at
Although the emission images indicate that the distribution
of the parent and conjugate are different, as only dye associated
with membrane emits fluorescence imaging could not be
used to assess the true distribution of the dye in the cell or
to determine conclusively whether [Ru(dppz)2PIC]2+ had
crossed the membrane. Resonance Raman mapping, however,
does not rely on the dye emission and was employed for the
first time here as a complementary technique to the same cell
medium to assess the location of the dye and conjugate. Fig. 3
shows the white light image and resonance Raman map for the
SP2 cells incubated with the dye or the dye conjugate as
described above for the confocal luminescence imaging; the
excitation line was 458 nm. The resonance Raman image was
generated by analysing the intensity of the 1598, 1564 and
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spectroscopy could be used to validate these results and
showed the dye distribution within the cell, confirming the
parent complex accumulates in the outer cell membrane but
does not penetrate the cell. Overall, the Ru(II) complexes are
highly versatile molecular probes for cell imaging and we
believe their use in multimodal imaging as described here
represents a powerful new paradigm in cell imaging.
This material is based upon work supported by Science
Foundation Ireland under Grant No. [05/IN.1/B30] and The
Higher Education Authority under PRTLI IV.
IRCSET are gratefully acknowledged for postgraduate
scholarship funding.
Prof. Richard O’Kennedy, DCU, is sincerely thanked for
providing cell culture.
Fig. 3 Resonance Raman intensity map of a live myeloma cell after
incubation with [Ru(dppz)2PIC-Arg8]10+ (A1) and with the free dye
[Ru(dppz)2PIC]2+ (B1) after excitation at 458 nm. A and B represent
the corresponding white light images of the live cells.
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examples of the resonance Raman spectra used to construct
the map.
The plot intensity represents the intensity of these
vibrational modes and therefore the relative dye concentration
around the cell. Fig. 3 clearly reveals that whereas the parent
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11 See supplementary material.
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