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Heat-induced radiolabeling and fluorescence labeling of Feraheme nanoparticles for PET/SPECT imaging and flow cytometry

Nature Protocols volume 13, pages 392–412 (2018)Cite this article

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Abstract

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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Figure 1: Feraheme (FH) nanoparticles (NPs) are heat stable but acid labile.
Figure 2: Synthesis of HIR-FH and Cy5.5-FH nanoparticles.
Figure 3: Heat-induced radiolabeling of FH with 89Zr4+,64Cu2+, or 111In3+.
Figure 4: Radiochemical stability of HIR 89Zr-FH.
Figure 5: Monocyte internalization of Cy5.5-FH by dual-wavelength flow cytometry (Box 1 and Box 2).
Figure 6: Imaging of normal and abnormal monocyte trafficking with HIR 89Zr-FH (Box 3).

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Acknowledgements

This study was supported by grants NIH R01 EB017699, NIH T32EB013180, and NIH R01 MH100350.

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Authors and Affiliations

  1. Gordon Center for Medical Imaging, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA

    Hushan Yuan, Moses Q Wilks, Marc D Normandin, Georges El Fakhri, Charalambos Kaittanis & Lee Josephson

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Contributions

L.J. conceived of the HIR strategy and conducted synthetic and analytical experiments. H.Y. synthesized and analyzed all materials, and developed all analytical methods. M.Q.W., C.K., and M.D.N. performed in vivo imaging and flow cytometry studies. G.E.F. and L.J. analyzed data and wrote the manuscript.

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Correspondence to Lee Josephson.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Gating strategy for flow cytometry data.

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.

Supplementary information

Supplementary Figure 1

Gating strategy for flow cytometry data. (PDF 336 kb)

3D video

3D video of animal shown in Figure 6. (MPG 970 kb)

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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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