Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA038

Poster Communications

Imaging of human neutrophil granulocytes by active targeting of perfluorocarbon-nanoemulsion and non-invasive 19F MRI

P. Bouvain1, V. Flocke1, B. Steckel1, S. Temme1, U. Flögel1

1. molecular cardiology, Heinrich-Heine Universität Düsseldorf, Düsseldorf, Germany.

Magnetic resonance imaging (MRI) is one of the most important clinical imaging modalities since it provides excellent contrast between soft tissues and is free of radiation. However, conventional 1H MRI is unable to visualize distinct cell-types. To overcome this, contrast agents based on iron oxide or gadolinium have been applied for cell tracking. Recently, 19F-based contrast agents (Perfluorocarbon nanoemulsions = PFCs) emerged for non-invasive imaging of monocytes and macrophages at inflammatory hot spots. 19F is nearly absent from biological tissue, therefore accumulation of 19F-loaded cells results in unequivocal ‘hot spots' without any background. Since neutrophil granulocytes are the first cells that infiltrate the infarcted myocardium and play a pivotal role for the subsequent healing process, we developed a targeting approach for the specific targeting of PFCs to human neutrophil granulocytes and their visualization by 19F MRI. For targeting of neutrophils granulocytes, we utilized the neutrophil specific peptide (NG2), as well as non-specific control peptide (NGC)1. First, we tested the binding of NG2/NGC to neutrophils and found that the NG2, but not NGC bound to neutrophils and that neither of the peptides bound to monocytes or lymphocytes. In addition, we found that NG2 does not bind to neutrophils from mouse, rat or pig. Interestingly, we found a stronger binding of NG2 to neutrophils from patients with ST-elevated myocardial infarction (STEMI) (MFI: 4,27±1,85; n=8) compared to healthy donors (MFI: 1,71±0,59; n=6). Stimulation of healthy neutrophils with LPS also increased the binding of NG2. For 19F-MRI, we coupled the NG2/NGC peptides to fluorescently labelled PFCs to generate NG2-/NGC-PFC. Upon incubation with NG2- or NGC-PFCs 19F-MRI revealed a strong 19F signal in healthy neutrophils (AUC: 1.3±0.5; n=6) compared to NGC-PFC (AUC: 0.2±0.1, n=9). Again, we observed a stronger 19F signal in neutrophils from patients after myocardial infarction (AUC: 2.8±1.1; n=5). These results were confirmed by flow cytometry which revealed that NG2-PFCs but not NGC-PFCs were specifically taken up by neutrophils. Monocytes showed little and lymphocytes no uptake of NG2-PFCs. Internalization of NG2-PFCs was verified by confocal microscopy. Here, we could demonstrate that NG2 specifically binds to human granulocytes and that NG2-PFCs can be utilized to visualize neutrophils by 19F-MRI and flow-cytometry. Importantly, STEMI patients showed the increased binding of the NG2 and stronger labelling with PFCs. Our targeting approach holds the potential to specifically visualize the infiltration of human neutrophil granulocytes into inflamed tissue via non-invasive 19F MRI.

Where applicable, experiments conform with Society ethical requirements