Synthetic Cargo Internalization Receptor System for Nanoparticle Tracking of Individual Cell Populations by Fluorine...

Specific detection of target structures or cells lacking particular surface epitopes still poses a serious problem for all imaging modalities. Here, we demonstrate the capability of synthetic "cargo internalization receptors" (CIRs) for tracking of individual cell populations by ¹H/¹⁹F magnetic resonance imaging (MRI). To this end, a nanobody for green fluorescent protein (GFP) was used to engineer cell-surface-expressed CIRs which undergo rapid internalization after GFP binding. For ¹⁹F MR visibility, the GFP carrier was equipped with "contrast cargo", in that GFP was coupled to perfluorocarbon nanoemulsions (PFCs). To explore the suitability of different uptake mechanisms for this approach, CIRs were constructed by combination of the GFP nanobody and three different cytoplasmic tails that contained individual internalization motifs for endocytosis of the contrast cargo (CIR1-3). Exposure of CIR⁺ cells to GFP-PFCs resulted in highly specific binding and internalization as confirmed by fluorescence microscopy as well as flow cytometry and enabled visualization by ¹H/¹⁹F MRI. In particular, expression of CIR2/3 resulted in substantial incorporation of ¹⁹F cargo and readily enabled in vivo visualization of GFP-PFC recruitment to transplanted CIR⁺ cells by ¹H/¹⁹F MRI in mice. Competition experiments with blood immune cells revealed that CIR⁺ cells are predominantly loaded with GFP-PFCs even in the presence of cells with strong phagocytotic capacity. Importantly, binding and internalization of GFP-PFCs did not result in the activation of signaling cascades and therefore does not alter cell physiology. Overall, this approach represents a versatile in vivo imaging platform for tracking of individual cell populations by making use of cell-type-specific CIR⁺ mice.