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Abstract Background The cRGD peptide is a promising probe for early non-invasive detection of tumors. This study aimed to demonstrate how RAFT-c(-RGDfK-)4, a molecule allowing a tetrameric presentation of cRGD, improved cRGD-targeting potential using in vivo models of αVβ3-positive or negative tumors. Results We chose the human embryonic kidney cells HEK293(β3) (high levels of αVβ3) or HEK293(β1) (αVβ3-negative but expressing αV and β1) engrafted subcutaneously (s.c.) in mice. Non-invasive in vivo optical imaging demonstrated that as compared to its monomeric cRGD analogue, Cy5-RAFT-c(-RGDfK-)4 injected intravenously had higher uptake, prolonged retention and markedly enhanced contrast in HEK293(β3) than in the HEK293(β1) tumors. Blocking studies further demonstrated the targeting specificity and competitive binding ability of the tetramer. Conclusion In conclusion, we demonstrated that Cy5-RAFT-c(-RGDfK-)4 was indeed binding to the αVβ3 receptor and with an improved activity as compared to its monomeric analog, confirming the interest of using multivalent ligands. Intravenous injection of Cy5-RAFT-c(-RGDfK-)4 in this novel pair of HEK293(β3) and HEK293(β1) tumors, provided tumor/skin ratio above 15. Such an important contrast plus the opportunity to use the HEK293(β1) negative control cell line are major assets for the community of researchers working on the design and amelioration of RGD-targeted vectors or on RGD-antagonists.

The modular synthesis and biological characterization of template assembled cyclopeptides as a multimeric system for targeting and endocytosis of cells expressing alphavbetta(sub 3) integrin are reported. The alphavbetta(sub 3) integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis.

Background: The cRGD peptide is a promising probe for early non-invasive detection of tumors. This study aimed to demonstrate how RAFT-c(-RGDfK-)4, a molecule allowing a tetrameric presentation of cRGD, improved cRGD-targeting potential using in vivo models of αVβ3-positive or negative tumors. Results: We chose the human embryonic kidney cells HEK293(β3) (high levels of αVβ3) or HEK293(β1) (αVβ3-negative but expressing αV and β1) engrafted subcutaneously (s.c.) in mice. Non-invasive in vivo optical imaging demonstrated that as compared to its monomeric cRGD analogue, Cy5-RAFT-c(-RGDfK-)4 injected intravenously had higher uptake, prolonged retention and markedly enhanced contrast in HEK293(β3) than in the HEK293(β1) tumors. Blocking studies further demonstrated the targeting specificity and competitive binding ability of the tetramer. Conclusion: In conclusion, we demonstrated that Cy5-RAFT-c(-RGDfK-)4 was indeed binding to the αVβ3 receptor and with an improved activity as compared to its monomeric analog, confirming the interest of using multivalent ligands. Intravenous injection of Cy5-RAFT-c(-RGDfK-)4 in this novel pair of HEK293(β3) and HEK293(β1) tumors, provided tumor/skin ratio above 15. Such an important contrast plus the opportunity to use the HEK293(β1) negative control cell line are major assets for the community of researchers working on the design and amelioration of RGD-targeted vectors or on RGD-antagonists. [ABSTRACT FROM AUTHOR]

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]

Background The herpes simplex virus type 1 (HSV-1) VP22 protein has the property to mediate intercellular trafficking of heterologous proteins fused to its C- or N-terminus. We have previously shown improved delivery and enhanced therapeutic effect in vitro and in vivo with a P27-VP22 fusion protein. In this report, we were interested in studying the spread and biological activity of VP22 fused to the P53 tumor suppressor. Methods Expression of the VP22-P53 and P53-VP22 fusion proteins was shown by Western blot and intercellular spreading was monitored by immunofluorescence on transiently transfected cells. In vitro antiproliferative activity of wild-type (wt) P53 and P53-VP22 was assessed by proliferation assays and transactivating ability was studied by a reporter gene test and a gel-shift assay. Antitumor activity was also tested in vivo by intratumoral injections of naked DNA in a model of subcutaneous tumors implanted in nude mice. Results Our results show that the C-terminal fusion or the N-terminal P53-VP22 fusion proteins are not able to spread as efficiently as VP22. Moreover, we demonstrate that VP22-P53 does not possess any transactivating ability. P53-VP22 has an antiproliferative activity, but this activity is not superior to the one of P53 alone, in vitro or in vivo. Conclusions Our study indicates that a gene transfer strategy using VP22 cannot be considered as a universal system to improve the delivery of any protein. Copyright © 2005 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The receptor for the iC3b fragment of complement, CR3, is involved in monocytes/macrophages and neutrophils phagocytosis. CR3 is known to interact with the low affinity receptor for Ig (CD16) and previous studies have suggested that this cooperation modulates CR3 functions. Herein we have studied the effect of CD16 on the ability of human monocytes CR3 to bind to iC3b. We show that iC3b binding to CR3 is inhibited by several reagents that are known to dissociate the CD16/CR3 complex. In addition, treatment of monocytes with soluble CD16 inhibited iC3b binding to CR3. Together, these data indicate that iC3b binding to monocyte CR3 is up-regulated by an interaction between membrane CD16 and CR3. The implication of CD16 in CR3 binding to iC3b was also analyzed after monocyte differentiation into dendritic cells (DC). Differentiation of monocytes into DC abrogates the cooperation between CD16 and CR3, due to a loss of CD16/CR3 interaction. In accordance, this phenomenon is associated with a lack of iC3b binding to DC. As a consequence, deposition of iC3b on apoptotic cells does not modify their phagocytosis by DC. In conclusion, we demonstrate a cooperation between CD16 and CR3 that favors iC3b binding to CR3 but is lost on DC. [ABSTRACT FROM AUTHOR]

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]