Michael Claron

Reduction of renal uptake of 111In-DOTA-labeled and A700-labeled RAFT-RGD during integrin αvβ3 targeting using single photon emission computed tomography and optical imaging

Integrin αvβ3 expression is upregulated during tumor growth and invasion in newly formed endothelial cells in tumor neovasculature and in some tumor cells. A tetrameric RGD‐based peptide, regioselectively addressable functionalized template‐(cyclo‐[RGDfK])4 (RAFT‐RGD), specifically targets integrin αvβ3 in vitro and in vivo. When labeled with indium‐111, the RAFT‐RGD is partially reabsorbed and trapped in the kidneys, limiting its use for further internal targeted radiotherapy and imaging investigations. We studied the effect of Gelofusine on RAFT‐RGD renal retention in tumor‐bearing mice. Mice were imaged using single photon emission computed tomography and optical imaging 1 and 24 h following tracer injection. Distribution of RAFT‐RGD was further investigated by tissue removal and direct counting of the tracer. Kidney sections were analyzed by confocal microscopy. Gelofusine significantly induced a >50% reduction of the renal reabsorption of 111In‐DOTA‐RAFT‐RGD and A700‐RAFT‐RGD, without affecting tumor uptake. Injection of Gelofusine significantly reduced the renal retention of labeled RAFT‐RGD, while increasing the tumor over healthy tissue ratio. These results will lead to the development of future therapeutic approaches. (Cancer Sci 2012; 103: 1105–1110)

Redox‐Driven Host–Guest Interactions Allow the Controlled Release of Captured Cells on RGD‐Functionalized Surfaces

A quartz crystal microbalance technique with dissipation monitoring and a complementary optical microscopy technique were used for monitoring the capture and release of specific cells on a surface displaying a bifunctional molecular device, composed of a molecular scaffold endowed with the cell recognition property of an RGD ligand and a β‐CD/Fc redox‐switchable system.

PET imaging and biodistribution analysis of the effects of succinylated gelatin combined with l-lysine on renal uptake and retention of 64Cu-cyclam-RAFT-c(-RGDfK-)4 in vivo

(64)Cu-cyclam-RAFT-c(-RGDfK-)4, an αVβ3 integrin-targeting tetrameric cyclic RGD peptide probe, is a potential theranostic compound for positron emission tomography (PET) of tumor angiogenesis and for internal radiotherapy owing to the multiple decay modes of (64)Cu. Since kidneys are dose-limiting organs in internal radiotherapy, we aimed to reduce the renal accumulation of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 by co-injection with Gelofusine (GF), a succinylated gelatin solution, and/or L-lysine (Lys), and to explore, for the first time, the related mechanisms using the noninvasive and quantitative PET imaging technology. Biodistribution assays, dynamic and static PET scans, and metabolism studies with radio-thin-layer chromatography (radio-TLC) were performed in healthy or αVβ3-positive tumor-bearing mice. In the results, co-injection with GF markedly reduced the renal uptake and slightly increased the tumor uptake of (64)Cu-cyclam-RAFT-c(-RGDfK-)4. L-Lysine alone had no effect on the probe biodistribution, but the combined use of Lys and GF tended to enhance the effect of GF. Dynamic PET and metabolite analysis by radio-TLC highly revealed that GF blocks the renal reabsorption of (64)Cu-cyclam-RAFT-c(-RGDfK-)4, but does not interfere with its metabolism and excretion. In conclusion, administration of GF and Lys is a useful strategy for kidney protection in (64)Cu-cyclam-RAFT-c(-RGDfK-)4-based internal radiotherapy.

Micro–Positron Emission Tomography/Contrast-Enhanced Computed Tomography Imaging of Orthotopic Pancreatic Tumor–Bearing Mice Using the αvβ3 Integrin Tracer 64Cu-Labeled Cyclam-RAFT-c(-RGDfK-)4

The purpose of this study was to develop a clinically relevant orthotopic xenotransplantation model of pancreatic cancer and to perform a preclinical evaluation of a new positron emission tomography (PET) imaging probe, 64Cu-labeled cyclam-RAFT-c(-RGDfK-)4 peptide (64Cu-RAFT-RGD), using this model. Varying degrees of αvβ3 integrin expression in several human pancreatic cancer cell lines were examined by flow cytometry and Western blotting. The cell line BxPC-3, which is stably transfected with a red fluorescence protein (RFP), was used for surgical orthotopic implantation. Orthotopic xenograft was established in the pancreas of recipient nude mice. An in vivo probe biodistribution and receptor blocking study, preclinical PET imaging coregistered with contrast-enhanced computed tomography (CECT) comparing 64Cu-RAFT-RGD and 18F-fluoro-2-deoxy-D-glucose (18F-FDG) accumulation in tumor, postimaging autoradiography, and histologic and immunohistochemical examinations were done. Biodistribution evaluation with a blocking study confirmed that efficient binding of probe to tumor is highly αvβ3 integrin specific. 64Cu-RAFT-RGD PET combined with CECT provided for precise and easy detection of cancer lesions. Autoradiography, histologic, and immunohistochemical examinations confirmed the accumulation of 64Cu-RAFT-RGD in tumor versus nontumor tissues. In comparative PET studies, 64Cu-RAFT-RGD accumulation provided better tumor contrast to background than 18F-FDG. Our results suggest that 64Cu-RAFT-RGD PET imaging is potentially applicable for the diagnosis of αvβ3 integrin–expressing pancreatic tumors.

Biomolecular Assemblies Combining Two Orthogonal Copper‐Mediated Ligations in a One‐Pot Reaction

The access to multifunctional biomolecular compounds involves multistep reactions usually with a complicated protection scheme and lengthy separation processes. The development of a strategy combining several orthogonal ligations is highly desirable. Herein, we introduce a new method that involves two orthogonal copper-mediated ligations of azide with alkyne, and amine with thioacid. We established compatible conditions to carry out molecular assemblies of three different chemical components in a single one-pot reaction. The effectiveness of the method was demonstrated in the synthesis of biomolecular compounds that are known to target tumor tissue. The simple reaction conditions suggest that this strategy of combining several orthogonal ligations could have wide potential for the chemical synthesis of complex macromolecules.

Development of a selective cell capture and release assay: impact of clustered RGD ligands

There is a growing interest in isolating tumor cells from biological samples. Considering that circulating tumor cells can be rare in blood, it appears challenging to capture these cells onto a surface with high selectivity and sensitivity. In the present paper, we describe the design of functionalized surfaces aimed at selectively capturing tumor cells by using an RGD peptide ligand with either a tetrameric or a monomeric presentation. β-Cyclodextrin-coated self-assembled monolayers were used as platforms for the binding of RGD ligands endowed with a redox ferrocene cluster. The dissociation of the inclusion complex on the surface accounts for the release of the captured cells upon the electrochemical oxidation of ferrocene. For this purpose, we determined suitable RGD densities for both monovalent and tetravalent ligand presentations. The results indicate that the clustered RGD architecture efficiently improves selective cell capture at a very low RGD surface density (∼10 RGD per μm2) compared to the monovalent presentation (∼1000 RGD per μm2).