Viral nanoparticles decorated with novel EGFL7 ligands enable intravital imaging of tumor neovasculature.

Angiogenesis is a dynamic process fundamental to the development of solid tumors. Epidermal growth factor-like domain 7 (EGFL7) is a protein whose expression is restricted to endothelial cells undergoing active remodeling that has emerged as a key mediator of this process. EGFL7 expression is associated with poor outcome in several cancers, making it a promising target for imaging or therapeutic strategies. Here, EGFL7 is explored as a molecular target for active neovascularization. Using a combinatorial peptide screening approach, we describe the discovery and characterization of a novel high affinity EGFL7-binding peptide, E7p72, that specifically targets human endothelial cells. Viral nanoparticles decorated with E7p72 peptides specifically target tumor-associated neovasculature with high specificity as assessed by intravital imaging. This work highlights the value of EGFL7 as a target for angiogenic vessels and opens the door for novel targeted therapeutic approaches.

Discovery of novel integrin ligands from combinatorial libraries using a multiplex "beads on a bead" approach.

The development of screening approaches to identify novel affinity ligands has paved the way for a new generation of molecular targeted nanomedicines. Conventional methods typically bias the display of the target protein to ligands during the screening process. We have developed an unbiased multiplex "beads on a bead" strategy to isolate, characterize, and validate high affinity ligands from OBOC libraries. Novel non-RGD peptides that target α(v)ß(3) integrin were discovered that do not affect cancer or endothelial cell biology. The peptides identified here represent novel integrin-targeted agents that can be used to develop targeted nanomedicines without the risk of increased tumor invasion and metastasis.

A fast, reproducible and low-cost method for sequence deconvolution of 'on-bead' peptides via 'on-target' maldi-TOF/TOF mass spectrometry.

A novel approach to high-throughput sequence deconvolution of on-bead small peptides (MW < 2000 Da) using on-target MALDI-TOF/TOF instrumentation is presented. Short peptides of pentamer and octamer length, covalently attached to TentaGel polystyrene beads through a photolabile linker, were placed onto the MALDI target, apportioned with suitable matrix (2,5-dihydroxybenzoic acid) and then hit with the instrument laser (Nd : YAG, 355 nm). This induced easy and highly reproducible photochemical cleavage, desorption (MS mode) and fragmentation (MS/MS mode). Peptide fragments were identified with a mass accuracy of 0.1 Da of the expected values. This technique significantly accelerates the sequence determination of positive peptide hits obtained from random combinatorial libraries when screening against biological targets, paving the way for a rapid and efficient method to identify molecular imaging ligands specific to pathological targets in cancer and other diseases.

DNA cleavage and binding selectivity of a heterodinuclear Pt-Cu(3-Clip-Phen) complex.

The synthesis and nuclease activity of a new bifunctional heterodinuclear platinum-copper complex are reported. The design of this ditopic coordination compound is based on the specific mode of action of each component, namely, cisplatin and Cu(3-Clip-Phen), where 3-Clip-Phen 1-(1, 10-phenanthrolin-3-yloxy)-3-(1,10-phenanthrolin-8-yloxy)propan-2-amine. Cisplatin is not only able to direct the Cu(3-Clip-Phen) part to the GG or AG site, but also acts as a kinetically inert DNA anchor. The nuclease activity of this complex has been investigated on supercoiled DNA. The dinuclear compound is not only more active than Cu(3-Clip-Phen), but is also capable of inducing direct double-strand breaks. The sequence selectivity of the mononuclear platinum complex has been investigated by primer extension experiments, which reveal that its interaction with DNA occurs at the same sites as for cisplatin. The Taq polymerase recognizes the resulting DNA damage as different from that for unmodified cisplatin. The sequence-selective cleavage has been investigated by high-resolution gel electrophoresis on a 36-bp DNA fragment. Sequence-selective cleavages are observed in the close proximity of the platinum sites for the strand exhibiting the preferential platinum binding sites. The platinum moiety also coordinates to the other DNA strand, most likely leading only to mono guanine or adenine adducts.

Platinated copper(3-clip-phen) complexes as effective DNA-cleaving and cytotoxic agents.

The synthesis and biological activity of three heteronuclear platinum-copper complexes based on 3-Clip-Phen are reported. These rigid complexes have been designed to alter the intrinsic mechanism of action of both the platinum moiety and the Cu(3-Clip-Phen) unit. The platinum centers of two of these complexes are coordinated to a 3-Clip-Phen moiety, an ammine ligand and two chlorides, which are either cis or trans to each other. The third complex comprises two 3-Clip-Phen units and two chloride ligands bound in a trans fashion to the platinum ion. DNA-cleavage experiments show that the complexes are highly efficient nuclease agents. In addition, a markedly difference in their aptitude to perform direct double-strand cleavage is observed, which appears to be strongly related to the ability of the platinum unit to coordinate to DNA. Indeed, complex 6 is unable to coordinate to DNA, which is reflected by its incapability to carry out double-strand breaks. Nonetheless, this complex exhibits efficient DNA-cleavage activity, and its cytotoxicity is high for several cell lines. Complex 6 shows better antiproliferate activity than both cisplatin and Cu(3-Clip-Phen) toward most cancer cell lines. Furthermore, the cytotoxicity observed for 1 is for most cell lines close to that of cisplatin, or even better. Cu(3-Clip-Phen) induces very low cytotoxic effects, but a marked migratory activity. Complex 6 presents DNA-cleavage properties comparable to the one of Cu(3-Clip-Phen), but it does not show any migratory activity. Interestingly, both Cu(3-Clip-Phen) and 6 induces vacuolisation processes in the cell in contrast to complex 1 and cisplatin. Thus, the four complexes cisplatin tested, Cu(3-Clip-Phen), 1 and 6 stimulate different cellular responses.