Luminescent lanthanide bimetallic triple-stranded helicates as potential cellular imaging probes

Article abstract of DOI:10.1039/b701482a

Water-soluble triple-stranded [Ln₂(L)₃] helicates have been successfully tested as imaging probes in human cervical adenocarcinoma cells (HeLa), the complex being not toxic and clearly staining their cytoplasm in a concentration-depe

Full text of DOI:10.1039/b701482a

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Luminescent lanthanide bimetallic triple-stranded helicates as potential
cellular imaging probes
Caroline D. B. Vandevyver,* Anne-Sophie Chauvin,* Steve Comby and Jean-Claude G. Bu¨nzli
Received (in Cambridge, UK) 31st January 2007, Accepted 26th March 2007
First published as an Advance Article on the web 5th April 2007
DOI: 10.1039/b701482a
Water-soluble triple-stranded [Ln2(L)3] helicates have been
successfully tested as imaging probes in human cervical
adenocarcinoma cells (HeLa), the complex being not toxic
and clearly staining their cytoplasm in a concentration-
dependent manner.
Lanthanide ions have provided a new incentive in the development
of luminescent molecular probes because of their peculiar proper-
ties including easily recognizable line-like emission spectra, long
excited-state lifetimes allowing the use of time-resolved measure-
ments, and large Stokes shifts upon ligand excitation.1 They are
now commonly used as luminescent responsive probes in
immunoassays2 and for the quantitative determination of analytes
present in biological fluids.3 Building on these applications, the
next step is to integrate them into luminescent stains for bio-
imaging purposes.4 To date, however, only a few labels are
available, presumably in view of the numerous requirements to be
fulfilled by lanthanide probes for time-resolved applications in vitro
and in vivo. The latter include solubility in aqueous media or in
solvents suitable for their conjugation to bio-specific probes,
thermodynamic stability, kinetic inertness, and marked sensitiza-
tion of the metal-centered luminescence. Recently, significant
advances in the spectral analysis of tissues have been reported.5–9
Despite many improvements still being needed to reach clinical
practicality, these investigations bring a proof of concept for the
use of lanthanide ions in in vivo and in vitro analyses.
In recent years, we have been tailoring homo- and hetero-
Scheme 1 Synthesis of the water-soluble ligand.
bimetallic helicates with the final purpose of designing bi-
functional lanthanide probes.10,11 Although not yet taken
was a cyclization in presence of iron to form the benzimidazole
advantage of, the intrinsic chirality of the helicates will be an
units. The ester functions were finally hydrolysed to provide the
asset in future developments of these probes. Herein, we report the
water-soluble H2L ligand.12 This receptor exists in two main forms
properties of the water-soluble and thermodynamically stable
in equilibrium at physiological pH, H2L and HL2, as determined
triple-stranded [Eu2(L)3] helicate in vitro, its interaction with the
by spectrophotometric titration.
human cervical adenocarcinoma cell line HeLa and its ability for
The bitopic ligand H2L self-assembles under stoichiometric
the imaging of these cells.
conditions with lanthanide perchlorate to yield 2 : 3 Ln : L edifices
The molecular design of the ligand relies on the ditopic
as the major species at pH 7.4 (in Tris-HCl buffer), as ascertained
hexadentate receptor H2L (see Scheme 1). To improve the
by spectrophotometric titration and mass spectrometry.13 The Ln
solubility of the neutral bimetallic helicates, polyoxyethylene arms
helicates appear to be thermodynamically quite stable with
acting as water-solubilizing groups were grafted in the para
conditional stability constants logb23 in the range 26–30
position of the pyridine ring. The substituent in 2 was introduced
(Ln 5 La, Eu, Lu).
via a Mitsunobu reaction starting from the ethyl diester of
Upon ligand excitation at 325 nm, the emission spectrum of the
dipicolinic acid 1, which was further converted into a monoester
neutral [Eu2(L)3] helicate displays the usual metal-centered
product 3, followed by Phillips coupling to give 4. The next step
3
luminescence (Fig. 1, bottom). The energy of the pp* 0-phonon
transition is around 22 100 cm21, leading to efficient sensitization
´
Laboratory of Lanthanide Supramolecular Chemistry, Ecole
of the EuIII luminescence, with an overall quantum yield Q 5 18 ¡
2% under physiological conditions, as determined in two different
Polytechnique Fe´de´rale de Lausanne (EPFL), BCH 1405, 1015
Lausanne, Switzerland. E-mail: caroline.vandevyver@epfl.ch; anne-
sophie.chauvin@epfl.ch; Fax: 41 21 693 9815; Tel: 41 21 693 9824
ways, using
a comparison method with Cs3[Eu(dpa)3] as
1716 | Chem. Commun., 2007, 1716–1718
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Fig. 2 Cells were incubated in presence of different concentrations of the
helicate in RPMI-1640 for 24 h. The images were taken using a Zeiss LSM
500 META confocal microscope (Objective: Plan-Apochromat, 63/1.30
oil; lexc 5 405 nm (Ar laser), LP 505 filter). Scale bar: 17 mm.
relatively highly luminous laser source is used. A solution of the
helicate in water at pH 7.4 gives exactly the same emission
spectrum whatever the excitation wavelength is, 325 or 405 nm.
The complex [Eu2(L)3] clearly permeates into HeLa cells and
stains their cytoplasm in a concentration dependent manner
(Fig. 2); the higher the concentration, the brighter the images.
However, the apparent limiting concentration to get the brightest
images is 250 mM; at higher concentration no increase in the image
brightness is observed. In addition, some diffuse auto-fluorescence
of the cells was observed at 0 mM which in principle could be
eliminated by time-gating microscopy (not presently available in
our laboratories).
Fig. 1 Corrected emission spectra of [Eu2(L)3] in HeLa cells loaded with
the helicate 500 mM (after washing with PBS buffer, top), in RPMI-1640
(middle), and 15 mM in water at pH 7.4 (Tris-HCl, bottom); lex 5 325 nm.
standard14 and an absolute method with an integration sphere.15
The Eu(5D0) lifetime is long (t 5 2.4 ¡ 0.1 ms) and reflects the
absence of water molecules in the inner coordination sphere.
Working in time-resolved mode avoids problems associated with
auto-fluorescence and Rayleigh scattering so that these photo-
physical properties, long lifetime and sizeable quantum yield, will
allow sensitive detection in biological medium. A more detailed
analysis of the ligand-field splitting reflected in the transitions to
A key question is whether the cells remain viable and healthy
over the period of examination. Therefore the influence of the EuIII
helicate on cell proliferation and viability was examined by the
WST-1 assay18,19 at time intervals in the range of 30 min to 24 h.
As shown in Fig. 3, no significant difference can be observed
between the proliferation of the cells in absence or presence of the
helicate up to 500 mM. This observation was confirmed by the
viability of the cells, after 24 h of incubation with [Eu2(L)3]: 100%
for 0 mM, 89 ¡ 3% for 125 mM, 101 ¡ 1% for 250 mM and 108 ¡
2% for 500 mM.
7
the various FJ levels clearly points to the overall coordination
arrangement around the metal ions being derived from D3
7
symmetry. In particular, the crystal field splitting of the F1 level
corresponds to an A2 (273 cm21) + split E (453, 479 cm21) scheme;
the latter 26 cm21 splitting is consistent with a relatively weak
deviation from the ternary symmetry.
The human cervical adenocarcinoma cell line HeLa was
cultured in RPMI-1640 medium supplemented with 10% foetal
calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-
essential amino-acids, and 1% 2-[4-(2-hydroxyethyl)-1-piperazi-
ne]ethanesulfonic acid monosodium salt (HEPES buffer).16
HeLa cells at 90% confluency were loaded with the helicate
(500 mM in RPMI-1640 culture medium) for 6 h, washed with PBS
(phosphate buffer saline) ten times, and then harvested with
trypsin. The mixture was centrifuged (3000 tr min21, 5 min), and
the pellet was re-suspended in PBS (0.8 mL). The emission
spectrum of the EuIII helicate localized in cells displays an overall
shape very similar to the emission spectrum in aqueous solution at
pH 7.4 (Fig. 1, top). In particular, the intensity ratios of the various
transitions are the same (e.g. I(7F2)/I(7F1) 5 0.98 as compared to
1.04), and the lifetime remains long (1.7 ¡ 0.3 ms), thus indicating
that the EuIII helicate is un-dissociated inside the cells. The only
noticeable difference is an increase in the splitting of the E(7F1)
sub-level to 42 cm21, probably reflecting some distortion of the
helical structure due to second-sphere interactions.17
Although live cell imaging is a rapidly emerging field, fixed cells
are still often used in diagnostic tests. The usefulness of the EuIII
helicate for visualizing them is demonstrated in Fig. 4. The cells
were fixed with glutaraldehyde (0.4%, 5 min) or using ice-cold
methanol (220 uC, 15 min), resulting in a much higher
luminescence than found in living cells.
When the complex is loaded at 4 uC in living cells, we clearly
observe its presence in the cytoplasm with luminosity close to that
The cells grown on glass bottomed cell culture dishes were
incubated in cell culture medium with the complex at different
concentrations. Cells were examined with a confocal microscope
24 h after complex loading, under 405-nm excitation (argon laser);
this was the shortest wavelength available and excitation of the
incorporated helicate at this wavelength is made possible because
the absorption spectrum extends up to this spectral range and a
Fig. 3 WST-1 proliferation test of HeLa cells in absence or presence of
different concentrations of [Eu2(L)3]. Each point represents the average
over three nominally identical measurements.
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3 D. Parker and J. A. G. Williams, in The lanthanides and their
interrelations with biosystems, ed. A. Sigel and H. Sigel, Marcel Dekker
Inc., New York, 2003, Metal Ions in Biological Systems, Vol. 40, Ch. 7.
4 S. Pandya, J. H. Yu and D. Parker, Dalton Trans., 2006, 2757; R. Pal
and D. Parker, Chem. Commun., 2007, 474.
5 J. C. Frias, G. Bobba, M. J. Cann, C. J. Hutchison and D. Parker, Org.
Biomol. Chem., 2003, 1, 905.
6 H. C. Manning, T. Goebel, R. C. Thompson, R. R. Price, H. Lee and
D. J. Bornhop, Bioconjugate Chem., 2004, 15, 1488.
7 Y. Bretonniere, M. J. Cann, D. Parker and R. Slater, Org. Biomol.
Chem., 2004, 2, 1624.
8 B. Tang, H. Huang, K. Xu, L. Tong, G. Yang, X. Liu and L. An,
Chem. Commun., 2006, 3609.
Fig. 4 The cells were incubated in presence of 500 mM of the helicate in
RPMI-1640 for 24 h. The images were taken as for Fig. 2. Scale bar:
17 mm.
9 J. H. Yu, D. Parker, R. Pal, R. A. Poole and M. J. Cann, J. Am. Chem.
Soc., 2006, 2294.
10 M. Elhabiri, R. Scopelliti, J.-C. G. Bu¨nzli and C. Piguet, J. Am. Chem.
Soc., 1999, 121, 10747.
11 N. Andre´, T. B. Jensen, R. Scopelliti, D. Imbert, M. Elhabiri,
G. Hopfgartner, C. Piguet and J.-C. G. Bu¨nzli, Inorg. Chem., 2004,
43, 515.
measured at 37 uC. These observations indicate that the helicate is
unlikely to enter the cells by non-caveolae and non-clathrin
endocytosis and therefore an active uptake is more probable.
However, the exact mechanism by which the cells take up the
chelate has yet to be unraveled.
12 Anal. Calcd for C43H50N6O12?2H2O: C, 58.76; H, 6.19; N, 9.56. Found:
C, 58.48; H, 6.71; N, 9.64%.
Similar experiments are being conducted with the TbIII (Q 5 11.4
¡ 0.3%, t 5 0.65 ¡ 0.05 ms) and SmIII (Q 5 0.38 ¡ 0.06%,
t 5 30.4 ¡ 0.4 ms) helicates and we are also testing the influence of
grafting the polyether arm on the benzimidazole unit. In summary,
a new family of strongly luminescent and highly stable probes has
been developed. The EuIII helicate is taken up by HeLa cells and is
localized in the cytoplasm. The luminescent stain can be used in
both live and fixed cells imaging with excitation wavelengths up to
405 nm despite its main absorption peaking at 325 nm. Work is in
progress to fully study the interaction process with cells, to find
better experimental conditions for the imaging, and to take
advantage of the long EuIII luminescence lifetime in time-resolved
microscopy.
13 [Eu2(L3)]?2MeCN: calc. for [C129H144N18O36Eu2?2MeCN]2+ 5 1434.942,
found 1434.916.
14 A.-S. Chauvin, F. Gumy, D. Imbert and J.-C. G. Bu¨nzli, Spectrosc.
Lett., 2004, 37, 517; erratum: 2007, 40, 193.
15 J. C. de Mello, H. F. Wittmann and R. H. Friend, Adv. Mater., 1997, 9,
230.
16 The HeLa (ATCC CCL-2) cell cultures were maintained at 37 uC under
5% CO2 and 95% air atmosphere. The growth medium (from Gibco1
Cell Culture, Invitrogen, Basel, Switzerland) was changed every other
day until the time of use of the cells. Cell density and viability, defined as
the ratio of the number of viable cells over the total number of cells, of
the cultures were determined by trypan blue staining and a Neubauer
improved hemacytometer (Blau Brand, Wertheim, Germany).
17 C. Piguet, J.-C. G. Bu¨nzli, G. Bernardinelli, G. Hopfgartner and A.
F. Williams, J. Am. Chem. Soc., 1993, 115, 8197.
18 M. Ishiyama, M. Shiga, K. Sasamoto, M. Mizoguchi and P. G. He,
Chem. Pharm. Bull., 1993, 41, 1118; M. Ishiyama, K. Sasamoto,
M. Shiga, Y. Ohkura, K. Ueno, K. Nishiyama and I. Taniguchi,
Analyst, 1995, 120, 113.
19 Cells were seeded in a 96-well tissue culture microplate at a
concentration of 7.5 6 104 cells/well in 100 mL culture medium and
incubated overnight at 37 uC and 5% CO2. The complex was dissolved
in fresh RPMI medium at 37 uC, at a concentration of 500 mM; after
removal from the medium, 100 mL/well were added, then 10 mL of
WST-1 reagent. The plate was shaken for 1 min at 450 rpm and further
incubated at 37 uC and 5% CO2. Absorbance was measured at 450 nm
with an ELISA reader (Spectra MAX 340, Molecular Devices,
Sunnyvale, CA, USA). The results are expressed as an average over
three nominally identical measurements.
This research is supported through grants from the Swiss
National Science Foundation. We thank Mr Fre´de´ric Gumy
(luminescence) and Mr Fre´de´ric Thomas (ligand synthesis) for
their invaluable assistance.
Notes and references
1 J.-C. G. Bu¨nzli, Acc. Chem. Res., 2006, 39, 53; J.-C. G. Bu¨nzli and
C. Piguet, Chem. Soc. Rev., 2005, 34, 1048.
2 I. Hemmila¨ and V. M. Mukkala, Crit. Rev. Clin. Lab. Sci., 2001, 38,
441.
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This journal is ß The Royal Society of Chemistry 2007