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We reported that regioselectively addressable functionalized template (RAFT)-c(-RGDfK-)4 presenting four cyclic (Arg-Gly-Asp) (cRGD) peptides targets integrin αVβ3 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]