Jesus Razkin

Noninvasive Optical Imaging of Ovarian Metastases Using Cy5-labeled RAFT-c(-RGDfK-)4

Our group has developed a new molecular tool based on the use of a regioselectively addressable, functionalized template (RAFT) scaffold, where four cyclic (Arg-Gly-Asp) (cRGD) peptide motifs were grafted. The aim of this study was to determine whether RAFT-c(-RGDfK-)4 combined with optical imaging could allow noninvasive detection of deep ovarian metastases. Human ovarian adenocarcinoma IGROV1 cells expressing low levels of integrin αvβ3 (the main receptor for the cRGD peptide) were used for in vitro and in vivo assays in combination with Cy5-labeled RAFT-c(-RGDfK-)4, cRGD, or RAFT-c(-RβADfK-)4. In vivo fluorescence imaging was performed on subcutaneous (SC) tumors and intraperitoneal IGROV1 metastases in nude mice. The accumulation of RGD-Cy5 conjugates in cultured cells or in tumor tissues was examined using confocal laser scanning microscopy. RAFT-c(-RGDfK-)4 exhibited stronger staining in vitro, enhanced tumor-to-background ratio for SC tumors, and allowed early detection of 1- to 5-mm large intraabdominal nodules using noninvasive optical imaging. Histological study revealed that RAFT-c(-RGDfK-)4 accumulated into tumor neovasculature but also into tumor cells. Our data demonstrate that a Cy5-labeled RAFT-c(-RGDfK-)4 is an efficient optical probe for early and noninvasive tumor detection.

Synthetic Peptide Templates for Molecular Recognition: Recent Advances and Applications

The creation of molecular systems that can mimic some of the properties of natural macromolecules is one of the major endeavors in contemporary protein chemistry. However, the construction of artificial proteins with predetermined structure and function is difficult on account of complex folding pathways. The use of topological peptide templates has been suggested to induce and stabilize defined secondary and tertiary structures. This is because the recent advances in the chemistry of coupling reagents, protecting groups, and solid‐phase synthesis have made the chemical synthesis of peptides with conformationally controlled and complex structures feasible. Besides their use as structure‐inducing devices, these peptide templates can also be utilized to construct novel structures with tailor‐made functions. Herein, we present recent advances in the field of peptide‐template‐based approaches with particular emphasis on the demonstrated utility of this approach in molecular recognition, along with related applications.

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.

In vivo noninvasive optical imaging of receptor-mediated RGD internalization using self-quenched Cy5-labeled RAFT-c(-RGDfK-)(4).

We reported that regioselectively addressable functionalized template (RAFT)-c(-RGDfK-)4 presenting four cyclic (Arg-Gly-Asp) (cRGD) peptides targets integrin aVb3 with an improved specificity compared with monomeric cRGD. In this study, we improved this vector by creating a “stealth” molecule in which a fluorescence quencher (Q) is linked to Cy5 via a disulfide bond (-SS-). RAFT-c(-RGDfK-)4-Cy5-SS-Q fluorescence is quenched unless activated by reduction during internalization. RAFT-c(-RGDfK-)4-Cy5-SS-Q fluorescence was negligible when compared with the control but totally recovered after cleavage of the disulfide bridge. Confocal microscopy showed that only the intracellular Cy5 signal could be detected using RAFT-c(-RGDfK-)4-Cy5-SS-Q, confirming that uncleaved extracellular molecules are not visible. Whole-body imaging of mice bearing subcutaneous tumors injected intravenously with RAFT-c(-RGDfK-)4-Cy5-SS-Q showed a very significant enhancement of the fluorescent contrast in tumors compared with the unquenched molecule. Histology of the tumor confirmed the intracellular accumulation of Cy5. These results demonstrate that the presence of a labile disulfide bridge between the targeting vector and a drug mimetic is an efficient way to deliver a dye, or a drug, intracellularly. In addition, this quenched RAFT-c(-RGDfK-)4-Cy5-SS-Q probe is a very powerful vector for imaging tumor masses and investigating in vivo RGD-mediated internalization.

Luminescent probes for optical in vivo imaging

Going along with instrumental development for small animal fluorescence in vivo imaging, we are developing molecular fluorescent probes, especially for tumor targeting. Several criteria have to be taken into account for the optimization of the luminescent label. It should be adapted to the in vivo imaging optical conditions : red-shifted absorption and emission, limited overlap between absorption and emission for a good signal filtering, optimized luminescence quantum yield, limited photo-bleaching. Moreover, the whole probe should fulfill the biological requirements for in vivo labeling : adapted blood-time circulation, biological conditions compatibility, low toxicity. We here demonstrate the ability of the imaging fluorescence set-up developed in LETI to image the bio-distribution of molecular probes on short times after injection. Targeting with Cy5 labeled holo-transferrin of subcutaneous TS/Apc (angiogenic murine breast carcinoma model) or IGROV1 (human ovarian cancer) tumors was achieved. Differences in the kinetics of the protein uptake by the tumors were evidenced. IGROV1 internal metastatic nodes implanted in the peritoneal cavity could be detected in nude mice. However, targeted metastatic nodes in lung cancer could only be imaged after dissection of the mouse. These results validate our fluorescence imaging set-up and the use of Cy5 as a luminescent label. New fluorescent probes based on this dye and a molecular delivery template (the RAFT molecule) can thus be envisioned.

045 Optical imaging of lung cancer using near-infrared fluorescence

Introduction We report here the in vitro and in vivo characteristics of a new molecular conjugate vector for targeting and imaging of tumors. Its core is a cyclo-decapeptide plateform named RAFT, onto which two spatially independent functional domains can be cova-lently and stereo-specifically linked : a cell targeting domain for tumor targeting and a labeling domain able to carry two drugs and/or labeling agent. In order to prove the interest of this carrier, we used a well known cRGD cyclopeptide, a ligand for the α v β 3 integrin. Methods Using competition assays and confocal microscopy in vitro , as well as in vivo tumor formation and near-infrared optical imaging in mice, we analyzed the characteristics of the RAFT(cRGD)4 vector. Results We demonstrate that this vector presenting 4 cRGD motifs very efficiently prevents (α v β 3 -mediated cell adhesion to vit-ronectin. Furthermore, it is actively endocytosed because of the multivalent cRGD presentation, a major advantage for drug delivery. In vivo experiments in nude mice reveal that repeated intratu-moral injections of low doses of RAFT(cRGD)4 reduce tumor growth by reducing the number of tumor blood vessels. Furthermore, RAFT(cRGD)4 significantly improves the targeting specifi-city of subcutaneous tumor masses as well as disseminated metastasis after intravenous injection. Conclusion Thus, RAFT (cRGD)4 is specific, internalized, perfectly controlled and can carry multiple biological functions on a single, spatially defined backbone, making of it a powerful and versatile synthetic vector for drug delivery, molecular imaging or both. This versatility is a major advantage since we can load it with multiple functions. This was demonstrated by the addition of a cytotoxic KL peptide on the cargo domain of the RAFT. RAFT-RGD-KL shows a very promissing specific antitumoral activity when injected intravenously in tumors bearing mice. Finally, recent developments in the optical System allowed us to follow these optical probes using a new 3D optical tomographic system in lung metastasis. This allowed us to detect lung metastasis in 3D for the first time to our knowledge using optical NIR probes.