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Activatable Fluorescent Probes for Tumour‐Targeting Imaging in Live Mice

The direct and noninvasive in vivo visualisation of molecular processes such as ligand–receptor interaction, enzymatic activity, or gene expression, is the goal of molecular imaging. Optical imaging appears as a new complementary modality to the traditional nuclear or MRI (magnetic resonance imaging) techniques because of its low cost and fewer constraints. 4] Thus, the need for new optical probes with increased targeting and imaging capabilities arises. 6] Tung et al. introduced the concept of “smart” or activatable probes for imaging the activity of proteases over-expressed in mice tumours, such as MMP2 or cathepsin D. In these activatable probes, Cy5.5 dyes are grafted to peptide branching arms of a co-polymer, in such a way that they auto-quench initially. Whenever the proteases specifically cut the peptide sequence, the Cy5.5 fluorescence is recovered. In the present work, the concept of activatable probes is extended to the imaging of different molecular events, in the expectation of improving the image contrast in comparison to that obtained with classical targeting agents. The molecular structures we propose can be used for imaging enzymatic activity, specific targeting, and molecular processes triggered by ligand–receptor interaction. Moreover, their nonpolymeric nature can be very well controlled and characterised for pharmacology and medical applications. These activatable probes are built on a cyclodecapeptide template named RAFT (Regioselectively Addressable Functionalized Template), a new molecular vector for targeted drug delivery and molecular imaging of tumours and metastasis. This new class of molecules differs from other systems by the topological separation of two independent functional domains, which can be addressed in a spatially controlled manner: a cell targeting domain, and a therapeutic or imaging domain (Scheme 1). Clustered RGD-containing markers are used for cell recognition by the aVb3 integrin, [16–19] a vitronectin receptor over-expressed on the surface of endothelial cells of growing blood vessels, and therefore a cardinal feature of many malignant tumours. A multimeric presentation of the RGD motif is essential for integrin-mediated internalisation of the probe. 22–24] In the activatable probes we designed, the imaging function is composed of the cyanine 5 fluorescent dye (Cy5), a cleavable bond, and a quencher (Q). The cleavable bond is used to follow the cellular internalisation of the probe induced by binding to its receptor. Disulfide bridges are known to be reduced enzymatically by thioredoxines in the cytosol, and have been used to improve the efficiency of targeted drug delivery. The redox potential within the endosomal system could also be reducing. Therefore, -Cysteine-S S-Cysteine(abbreviated as S–S below) is chosen as a cleavable bond, to follow the internalisation of the fluorescent probe into the cells after its RGD-mediated binding on their surface. Demonstrating targeting can be crucial for screening and comparing the recognition efficiency of different biological markers. Moreover, demonstrating internalisation is also very important to estimate the ability of a molecule, such as the RAFT, to be used as a vector for targeted drug delivery. The self-quenching of the cyanine dyes allows the use of the Cy5-S S-Cy5 group as an activatable unit (Scheme 1). Alternatively, a diarylrhodamine derivative quencher (QSY21) can be used in place of one of the Cy5 units. Cy5 fluorescence is markedly inhibited in the Cy5-S S-QSY21 group (Table 1). This dynamic and static quenching in aqueous buffer can be accounted for by the hydrophobic nature and opposite charges of the molecules, which favour their stacking. In this context, the RAFT-(cRGD)4 Cy5-S S-Cy5 1, RAFT-(cRGD)4 Cy5-S SQSY21 2, and RAFT-(cRGD)4 QSY21-S S-Cy5 3 have been synthesised (Scheme 1). The peptide moiety of the molecule is prepared by a combination of solid and solution phase synthesis using a Fmoc/tBu strategy, followed by a chemoselective assembling of ligand moieties and template, as reported elsewhere. Npys-protected cysteine is chosen to graft the fluorescent unit to the RAFT. This allows a regioselective disulfide bond formation. Cy5 and QSY21, available in the activated ester form, are then attached successively to the macromolecule, providing, after RP-HPLC purification, the desired compounds in satisfying overall yields (13–18%). The activation of the fluorescent probes is demonstrated first in vitro. Figure 1A displays the absorption spectra of the RAFT-(cRGD)4 Cy5-S S-QSY21 2 before and 2 h after addition of 2-mercaptoethanol (2-MCE). The spectrum after cleavage matches that of the sum of Cy5 and QSY21, whereas for 2, the electronic interaction between the dye and quencher is evidenced by an intense absorption band at 600 nm. Similar results are obtained for 1: the 600 nm absorption band in this case is indicative of the presence of Cy5 dimers, whereas the 647 nm band is attributed to the dye monomer. Fluorescence measurements (Figure 1B) show the initial Cy5 fluorescence quenching and its 100% recovery upon the S S bond [a] Dr. J. Razkin, Dr. D. Boturyn, Prof. P. Dumy LEDSS, UMR CNRS 5616, 301 rue de la chimie, BP 53, 38041 Grenoble Cedex 9 (France) Fax: (+33)476-635-540 E-mail : Didier.Boturyn@ujf-grenoble.fr [b] Dr. I. Texier LETI/DTBS CEA Grenoble, 17 rue des martyrs, 38054 Grenoble Cedex 9 (France) Fax: (+33)438-785-787 E-mail : isabelle.texier-nogues@cea.fr [c] Dr. Z.-h. Jin, Prof. M. Favrot, Dr. J.-L. Coll INSERM U578, Institut Albert Bonniot, 38706 La Tronche (France) [d] Dr. V. Josserand ANIMAGE—CREATIS, B=timent CERMEP, 59 Boulevard Pinel, 69677 Bron Cedex (France) Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author.