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

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.

Luminescent lanthanides bioprobes emitting in the visible and/or near-infrared ranges

Steve Comby

This Ph.D. work deals with the development of lanthanide-based bioprobes for biomedical analyses and imaging. For this purpose, two different approaches have been followed, one focusing on visible-emitting complexes, while the second addresses the sensitization of NIR-emitting LnIII ions. The homoditopic ligands H2LC2 and H2LC3 have been tailored to self-assemble with lanthanide ions (LnIII) into neutral triple-stranded bimetallic helicates of overall composition [Ln2(L)3] and also to provide a versatile platform for further derivatization. The grafting of polyoxyethylene groups onto the pyridine (H2LC2) or the benzimidazole (H2LC3) units ensures water solubility, while maintaining large thermodynamic stability and adequate antenna effect for the excitation of LnIII ions. The conditional stability constants are large at physiological pH, with logβ23 reaching values in the range 22-25 for helicates with H2LC2 and H2LC3. The ligands triplet states feature adequate energy to efficiently sensitize the EuIII luminescence (QEu L = 21 and 11 % for L = H2LC2 and H2LC3, respectively) in aerated water at pH 7.4. Conversely, energy back transfer occurring in [Tb2(LC3)3] precludes a good sensitization of the TbIII luminescence, and only the TbIII helicate with H2LC2 displays sizeable quantum yield (QTbL = 11 %). Furthermore, it has been demonstrated that ligand H2LC2 sensitizes the emission of other visible- and NIR-emitting LnIII ions, such as SmIII or YbIII, for which in cellulo luminescence is evidenced, a rare observation. The EuIII emission spectra of both helicates arise from a main species with pseudo-D3 symmetry and without coordinated water. Cytotoxicity experiments reveal no significant effect of the helicates on the viability of several cancerous cell lines and cell imaging properties of the EuIII helicates are demonstrated for the HeLa cell line by luminescence microscopy. Bright EuIII emission is seen after relatively short time (15-30 min) and low helicate incubation concentrations (10 µM). Similar images were obtained for [Sm2(LC2)3] and [Tb2(LC2)3] using slightly higher loading concentration. The helicates stain the cytoplasm and the permeation mechanism is likely to be endocytosis. The described luminescent helical stains are robust chemical species which remain mostly undissociated in the cell medium and which resist to the presence of other complexing agents, as demonstrated in the case of H2LC2. All these properties make these helicates potential bioprobes for interacting with biological material and for cell imaging applications. As near-infrared (NIR) light, in addition to be less energetic than visible light, displays better penetrability into the human body, the development of bioprobes emitting in the near-infrared range is of prime importance for deeper tissue imaging, especially for early diagnosis of tumors. We therefore investigate the ability of new polydentate podands to sensitize the NIR luminescence of NdIII and YbIII ions. These ligands have been designed in a way to take advantage of the chelating effect of bidentate (Tsox, TsoxMe) or tridentate 8-hydroxyquinolinate (8-HQ) subunits (T2soxMe). The interaction between the ligands and LnIII ions in aqueous solution leads to the formation of stable 1:1 chelates (pLn in the range of 12-16), all the chromophoric 8-HQ units being coordinated to the metal center, exploiting the entropic effect generated by the anchor. At physiological pH, one major species is present for all the ligands regardless of the LnIII ion used. The low energy triplet states of the ligands are particularly well-suited for an efficient sensitization of the NdIII- or YbIII-centered luminescence (QNdL = 0.02-0.04 % and QYbL = 0.13-0.37 %) in buffered aqueous solution. ErIII NIR luminescence is also detected in water for the chelates with Tsox and TsoxMe. Lifetime determinations point in all cases to the absence of coordinated water molecules in the inner coordination sphere of the LnIII ion. It has also been demonstrated that methylation of the amide functions of Tsox removes the quenching mechanism induced by the proximate N-H groups and increases both the lifetimes and quantum yields of the TsoxMe chelates, with the YbIII complex being one of the most NIR luminescent lanthanide-based edifices described so far. Ligand TsoxMe thus appears to be the best candidate of the series regarding to the development of NIR probes for bioanalyses. Furthermore, other applications, for instance in telecommunications, are still conceivable since the incorporation of the Yb-TsoxMe chelate into sol-gel silica films has proven to be successful, the chelate withstanding the sol-gel process, while providing a slight enhancement of photophysical properties compared to the same chelate in aqueous solution.

EPFL2008

Luminescent Lanthanide Helicates Self-Assembled from Ditopic Ligands Bearing Phosphonic Acid or Phosphoester Units

Matthias Baud, Jean-Claude Bünzli, Anne-Sophie Chauvin, Steve Comby, Cyntia De Piano, Céline Duhot

A series of hexadentate ditopic receptors incorporating benzimidazole moieties have been designed, which are fitted with phosphonic acid or phosphoethylester coordinating units. In addition, poly(oxyethylene) pendants have been introduced on the benzimidazole backbone of two ligands to increase water solubility. The ligands self-assemble with lanthanide ions under stoichiometric conditions, yielding triple-stranded homobimetallic helicates, as ascertained by mass spectrometry and UV-visible titrations. The helicates display large thermodynamic stability, for example, log β23 ≈ 21-24 for all the EuIII complexes. Photophysical measurements reveal sensitization of the metal-centered luminescence in the europium and terbium complexes, which is modulated by the nature of the ligand. Hydration numbers determined by the lifetime method are essentially zero. The Eu(5D0) lifetimes are long and reach values up to 3.2 ms, while quantum yields as high as 25% are obtained in water at pH 7.4. Back transfer limits the sensitization efficiency for TbIII luminescence, and both lifetimes and quantum yields are much smaller. The properties of the helicates are discussed with respect to those self-assembled from ligands bearing carboxylate coordinating units.

2009

Stable 8-hydroxyquinolinate-based podates as efficient sensitizers of lanthanide near-infrared luminescence

Jean-Claude Bünzli, Anne-Sophie Chauvin, Steve Comby

New polydentate ligands (e.g., Tsox and TsoxMe) have been synthesized to take advantage of the chelating effect of bidentate 8-hydroxyquinolinate subunits connected to a N,N,N¢,N¢-tetraaminopropyl-1,2-ethylenediamine framework and with the aim of sensitizing the NIR luminescence of NdIII and YbIII ions. Ten pKa’s have been determined and the interaction between the ligands and LnIII ions in dilute aqueous solution has been probed both by potentiometric and spectrophotometric titrations. These studies have been mostly performed with the EuIII ion, which is in the middle of the lanthanide series, and extended to other ions (LaIII, ErIII, LuIIII). Stable complexes with LnIII ions are formed (pLn in the range of 14-16), the four chromophoric units being coordinated to the metal center, exploiting the entropic effect generated by the anchor. The monometallic complexes [Ln(H2L)]3- exist as the major species at physiological pH regardless of the lanthanide used. Lifetime determinations of the Nd(4F3/2)and Yb(2F5/2) excited levels in both H2O and D2O at buffered pH point to the absence of water molecules bound in the inner coordination sphere of the LnIII. Photophysical properties of the free ligands and of their lanthanide complexes have been investigated in buffered aqueous solutions both at room temperature and 77 K. The lowenergy triplet state makes energy transfers from the ligand to the metal ions possible; this leads to a sizable sensitization of the NdIII- or YbIII-centered luminescence (QNd L ) 0.02% and QYb L ) 0.18%) for Tsox chelates. Methylation of the amide functions removes the quenching mechanism induced by the proximate N-H vibrations and increases both the lifetimes and quantum yields of the TsoxMe chelates (QNd L ) 0.04% and QYb L ) 0.37%). In fact, TsoxMe yields one of the most luminescent YbIII compounds known in water, and this ligand appears to be suitable for the development of NIR probes for bioanalyses.