A Versatile Ditopic Ligand System for Sensitizing the Luminescence of Bimetallic Lanthanide Bio-Imaging Probes

The homoditopic ligand 6,6’- [methylenebis(1-methyl-1H-benzimid- azole-5,2-diyl)]bis(4-{2-[2-(2-methoxy- ethoxy)ethoxy]ethoxy}pyridine-2-carboxylic acid) (H2LC2) has been tailored to self-assemble with lanthanide ions (LnIII), which results in the formation of neutral bimetallic helicates with the overall composition [Ln2(LC2)3] and also provides a versatile platform for further derivatization. The grafting of poly(oxyethylene) groups onto the pyridine units ensures water solubility, while maintaining sizeable thermodynamic stability and adequate antenna effects for the excitation of both visible- and NIR-emitting LnIII ions. The conditional stability constants (logb23) are close to 25 at physiological pH and under stoichiometric conditions. The ligand triplet state features adequate energy (0-phonon transition at ~21 900 cm-1) to sensitize the luminescence of EuIII (Q=21%) and TbIII (11%) in aerated water at pH 7.4. The emission of several other VIS- and NIR-emitting ions, such as SmIII (Q=0.38%) or YbIII (0.15%), for which in cellulo luminescence is evidenced for the first time, is also sensitized. The EuIII emission spectrum arises from a main species with pseudo-D3 symmetry and without coordinated water. The cell viability of several cancerous cell lines (MCF-7, HeLa, Jurkat and 5D10) is unaffected if incubated with up to 500 mm [Eu2(LC2)3] during 24 h. Bright EuIII emission is seen for incubation concentrations above 10 mm and after a 15-minute loading time; similar images are obtained with TbIII and SmIII. The helicates probably permeate into the cytoplasm of HeLa cells by endocytosis. The described luminescent helical stains are robust chemical species which remain undissociated in the cell medium and in presence of other complexing agents, such as edta, dtpa, citrate or l-ascorbate. Their derivatization, which would open the way to the sensing of targeted in cellulo phenomena, is currently under investigation.

Effect of the length of polyoxyethylene substituents on luminescent bimetallic lanthanide bioprobes

Jean-Claude Bünzli, Anne-Sophie Chauvin, Bo Song, Caroline Vandevyver

The new homoditopic ligand

H_2L^{C2'}

self-assembles with lanthanide ions (

Ln^{III}

) to yield neutral bimetallic helicates of overall composition

[Ln_2(L^{C2'})_3]

; it is fitted with two hexakis(oxyethylene) chains to test their effects on the thermodynamic, photophysical and biochemical properties of these complexes, with particular emphasis on their uptake by living cells. At physiological pH and under stoichiometric conditions, the conditional stability constants

\log \beta_{23}

are around 28 resulting in the speciation of the

Eu^{III}

helicate being > 92% for a total ligand concentration of 1 mM. The ligand triplet state features adequate energy (0- phonon transition at

\approx 21 800 cm^{-1}

) for sensitising the luminescence of

Eu^{III}

(Q = 19%) and

Tb^{III}

(Q = 10%) in aerated water at pH 7.4. The

Eu(^5D_0)

emission spectrum and lifetime (2.43 ms) are characteristic of a species with pseudo-

D_3

symmetry and without bound water in the inner coordination sphere. The viability of HeLa cancerous cells is unaffected when incubated with up to 500 microM

[Eu_2(L^{C2'})_3]

during 24 h. The

Eu^{III}

helicate permeates into the cytoplasm of these cells by endocytosis and remains essentially undissociated, despite a low intracellular concentration of

0.28 \mu M

. In addition, the leakage of the

Eu^{III}

helicate out of HeLa cells is very minimal over long periods of time. With respect to similar complexes with ligands bearing shorter tris(oxyethylene) chains, no substantial changes are observed, which opens the way for targeting experiments. This study also demonstrates that the

[Ln_2(L^{CX})_3]

helicates are fairly robust entities since their core is unaffected by the substitution in the pyridine 4-position.

2008

Luminescent Bimetallic Lanthanide Bioprobes for Cellular Imaging with Excitation in the Visible-Light Range

Jean-Claude Bünzli, Anne-Sophie Chauvin, Frédéric Gumy, Bo Song, Caroline Vandevyver

A series of homoditopic ligands H2LCX (X=4–6) has been designed to self-assemble with lanthanide ions (LnIII), resulting in neutral bimetallic helicates of overall composition [Ln2ACHTUNGTRENUNG(LCX)3] with the aim of testing the influence of substituents on the photophysical properties, particularly the excitation wavelength. The complex species are thermodynamically stable in water (logb23 in the range 26–28 at pH 7.4) and display a metal-ion environment with pseudo-D3 symmetry and devoid of coordinated water molecules. The emission of EuIII, TbIII, and YbIII is sensitised to various extents, depending on the properties of the ligand donor levels. The best helicate is [Eu2ACHTUNGTRENUNG(LC5)3] with excitation maxima at 350 and 365 nm and a quantum yield of 9%. The viability of cervix cancer HeLa cells is unaffected when incubated with up to 500 mm of the chelate during 24 h. The helicate permeates into the cells by endocytosis and locates into lysosomes, which co-localise with the endoplasmatic reticulum, as demonstrated by counterstaining experiments. The relatively long excitation wavelength allows easy recording of bright luminescent images on a confocal microscope (lexc=405 nm). The new lanthanide bioprobe remains undissociated in the cell medium, and is amenable to facile derivatisation. Examination of data for seven EuIII and TbIII bimetallic helicates point to shortcomings in the phenomenological rules of thumb between the energy gap D(3pp*–5DJ) and the sensitisation efficiency of the ligands.

2009