Feraheme (FH) nanoparticles (NPs) have been used extensively for treatment of iron anemia (due to their slow release of ionic iron in acidic environments). In addition, injected FH NPs are internalized by monocytes and function as MRI biomarkers for the pathological accumulation of monocytes in disease. We have recently expanded these applications by radiolabeling FH NPs for positron emission tomography (PET) or single-photon emission computed tomography (SPECT) imaging using a heat-induced radiolabeling (HIR) strategy. Imaging FH NPs using PET/SPECT has important advantages over MRI due to lower iron doses and improved quantitation of tissue NP concentrations. HIR of FH NPs leaves the physical and biological properties of the NPs unchanged and allows researchers to build on the extensive knowledge obtained about the pharmacokinetic and safety aspects of FH NPs. In this protocol, we present the step-by-step procedures for heat (120 °C)-induced bonding of three widely employed radiocations (89Zr4+ or 64Cu2+ for PET, and 111In3+ for SPECT) to FH NPs using a chelateless radiocation surface adsorption (RSA) approach. In addition, we describe the conversion of FH carboxyl groups into amines and their reaction with an N-hydroxysuccinimide (NHS) of a Cy5.5 fluorophore. This yields Cy5.5-FH, a fluorescent FH that enables the cells internalizing Cy5.5-FH to be examined using flow cytometry. Finally, we describe procedures for in vivo and ex vivo uptake of Cy5.5-FH by monocytes and for in vivo microPET/CT imaging of HIR-FH NPs. Synthesis of HIR-FH requires experience with working with radioactive cations and can be completed within <4 h. Synthesis of Cy5.5-FH NPs takes ∼17 h.
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This study was supported by grants NIH R01 EB017699, NIH T32EB013180, and NIH R01 MH100350.
The authors declare no competing financial interests.
Integrated supplementary information
Representative gating strategy for excluding doublets (a), and debris (b) in flow cytometry analysis of 1×10^6 events. Singlets are defined on the Forward-Scatter(height) vs. Forward-Scatter(area) dot plot (a). Singlets comprise 84.35% of total events in this data set. Small debris are excluded on a Side-Scatter(Area) vs. Forward-Scatter(Area) dot plot (b). Debris comprise 78.75% of all events in this data set. The data that was shown previously and that was used for analysis was those events that were non-debris singlets. In this data set, the non-debris singlet populations comprised 16.52% of all events.
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Yuan, H., Wilks, M., Normandin, M. et al. Heat-induced radiolabeling and fluorescence labeling of Feraheme nanoparticles for PET/SPECT imaging and flow cytometry. Nat Protoc 13, 392–412 (2018). https://doi.org/10.1038/nprot.2017.133
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