Modulation of adiponectin receptors AdipoR1 and AdipoR2 by phage display-derived peptides in in vitro and in vivo models.

Type 2 diabetes (T2D) is often linked to metabolic syndrome, which assembles various risk factors related to obesity. Plasma levels of adiponectin are decreased in T2D and obese subjects. Aiming to develop a peptide able to bind adiponectin receptors and modulate their signalling pathways, a 12-amino acid sequence homologous in AdipoR1/R2 has been targeted by phage display with a linear 12-mer peptide library. The selected peptide P17 recognises AdipoR1/R2 expressed by skeletal muscle, liver and pancreatic islets. In HepaRG and C2C12 cells, P17 induced the activation of AMPK (AMPKα-pT172) and the expression of succinate dehydrogenase and glucokinase; no cytotoxic effects were observed on HepaRG cells. In db/db mice, P17 promoted body weight and glycaemia stabilisation, decreased plasma triglycerides to the range of healthy mice and increased adiponectin (in high fat-fed mice) and insulin (in chow-fed mice) levels. It restored to the range of healthy mice the tissue levels and subcellular distribution of AdipoR1/R2, AMPKα-pT172 and PPARα-pS12. In liver, P17 reduced steatosis and apoptosis. The docking of P17 to AdipoR is reminiscent of the binding mechanism of adiponectin. To conclude, we have developed an AdipoR1/AdipoR2-targeted peptide that modulates adiponectin signalling pathways and has therapeutic relevance for T2D and obesity associated pathologies.

Development of a peptide-functionalized imaging nanoprobe for the targeting of (FXYD2)γa as a highly specific biomarker of pancreatic beta cells.

Diabetes is characterized by a progressive decline of the pancreatic beta cell mass (BCM), which is responsible for insufficient insulin secretion and hyperglycaemia. There are currently no reliable methods to measure non-invasively the BCM in diabetic patients. Our work describes a phage display-derived peptide (P88) that is highly specific to (FXYD2)γa expressed by human beta cells and is proposed as a molecular vector for the development of functionalized imaging probes. P88 does not bind to the exocrine pancreas and is able to detect down to ~156 human pancreatic islets/mm(3) in vitro after conjugation to ultra-small particles of iron oxide (USPIO), as proven by the R2 measured on MR images. For in vivo evaluation, MRI studies were carried out on nude mice bearing Capan-2 tumours that also express (FXYD2)γa. A strong negative contrast was obtained subsequent to the injection of USPIO-P88, but not in negative controls. On human histological sections, USPIO-P88 seems to be specific to pancreatic beta cells, but not to duodenum, stomach or kidney tissues. USPIO-P88 thus represents a novel and promising tool for monitoring pancreatic BCM in diabetic patients. The quantitative correlation between BCM and R2 remains to be demonstrated in vivo, but the T2 mapping and the black pixel estimation after USPIO-P88 injection could provide important information for the future pancreatic BCM evaluation by MRI.

In vitro and in vivo characterization of several functionalized ultrasmall particles of iron oxide, vectorized against amyloid plaques and potentially able to cross the blood-brain barrier: toward earlier diagnosis of Alzheimer's disease by molecular imaging.

Alzheimer's disease (AD) is a neurodegenerative disorder most often diagnosed 10 years after its onset and development. It is characterized by the accumulation of amyloid-ß peptide (ABP) into amyloid plaques between nerve cells, which produces a massive local neurodegeneration. Molecular magnetic resonance imaging allows diagnosis of AD by showing ABP accumulation in the brain. The ultrasmall particles of iron oxide (USPIO) derivatives proposed in the present work were functionalized with peptides that present an affinity for ABP, independently of its state of aggregation. Their nanomolar Kd * confirms the high affinity of our vectorized contrast agents (VCA) for ABP and therefore their high labeling potential, specificity and sensitivity. Their lack of toxicity has been demonstrated, both by in vitro studies using the MTT method on several cell types, and by in vivo investigations with assessment of renal and hepatic biomarkers and by histopathology evaluation. The results of biodistribution studies corroborated by MRI demonstrate that USPIO-PHO (USPIO coupled to peptide C-IPLPFYN-C) are able to cross the blood-brain barrier without any facilitating strategy, and accumulates in the brain 90 min after its injection in NMRI mice. None of the USPIO derivatives were found in any organs one week after administration. To conclude, USPIO-PHO seems to have a genuine potential for labeling amyloid plaques in the brain; it has a nanomolar binding affinity, no toxic effects, and its elimination half-life is about 3 h. Further tests will be made on transgenic mice, aimed at confirming the potential of early AD diagnosis using our VCA.

Carboxy-silane coated iron oxide nanoparticles: a convenient platform for cellular and small animal imaging.

This study reports the synthesis of stabilized ultrasmall iron oxide nanoparticles (USPIO) as bimodal probes for magnetic resonance and optical imaging. These nanosystems are based on small iron oxide cores surrounded by a thin polysiloxane shell exhibiting carboxylic acid functions. Thanks to these functions, hybrid particles were obtained by conjugating a fluorophore to the superparamagnetic contrastophore. Such modification allowed us to directly follow these USPIO in cellulo, which provided interesting information about their internalization pathway and cellular distribution upon mitosis. Finally, the efficiency of these systems as probes for bimodal imaging was emphasized by the observation of their in vivo behavior in mice using magnetic resonance and optical imaging.