Targeting of Formyl Peptide Receptor 2 for i<i>n vivo</i> imaging of acute vascular inflammation

Abstract

Inflammatory conditions are associated with a variety of diseases and can significantly contribute to their pathophysiology. Neutrophils are recognised as key players in driving vascular inflammation and promoting inflammation resolution. As a result, neutrophils, and specifically their surface formyl peptide receptors (FPRs), are attractive targets for non-invasive visualization of inflammatory disease states and studying mechanistic details of the process. <b>Methods</b>: A small-molecule Formyl Peptide Receptor 2 (FPR2/ALX)-targeted compound was combined with two rhodamine-derived fluorescent tags to form firstly, a targeted probe (Rho-pip-C1) and secondly a targeted, pH-responsive probe (Rho-NH-C1) for <i>in vivo</i> applications. We tested internalization, toxicity and functional interactions with neutrophils <i>in vitro</i> for both compounds, as well as the fluorescence switching response of Rho-NH-C1 to neutrophil activation. Finally, <i>in vivo</i> imaging (fluorescent intravital microscopy [IVM]) and therapeutic efficacy studies were performed in an inflammatory mouse model. <b>Results</b>: <i>In vitro</i> studies showed that the compounds bound to human neutrophils via FPR2/ALX without causing internalization at relevant concentrations. Additionally, the compounds did not cause toxicity or affect neutrophil functional responses (e.g. chemotaxis or transmigration). <i>In vivo</i> studies using IVM showed Rho-pip-C1 bound to activated neutrophils in a model of vascular inflammation. The pH-sensitive ("switchable") version termed Rho-NH-C1 validated these findings, showing fluorescent activity only in inflammatory conditions. <b>Conclusions</b>: These results indicate a viable design of fluorescent probes that have the ability to detect inflammatory events by targeting activated neutrophils.