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


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).


  1. Coyne, D.W. Ferumoxytol for treatment of iron deficiency anemia in patients with chronic kidney disease. Expert Opin. Pharmacother. 10, 2563–2568 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Auerbach, M., Strauss, W., Auerbach, S., Rineer, S. & Bahrain, H. Safety and efficacy of total dose infusion of 1,020 mg of ferumoxytol administered over 15 min. Am. J. Hematol. 88, 944–947 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Bashir, M.R., Bhatti, L., Marin, D. & Nelson, R.C. Emerging applications for ferumoxytol as a contrast agent in MRI. J. Magn. Reson. Imaging 41, 884–898 (2015).

    Article  PubMed  Google Scholar 

  4. Farrell, B.T. et al. Using iron oxide nanoparticles to diagnose CNS inflammatory diseases and PCNSL. Neurology 81, 256–263 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hamilton, B.E. et al. Comparative analysis of ferumoxytol and gadoteridol enhancement using T1- and T2-weighted MRI in neuroimaging. Am. J. Roentgenol. 197, 981–988 (2011).

    Article  Google Scholar 

  6. McCullough, B.J., Kolokythas, O., Maki, J.H. & Green, D.E. Ferumoxytol in clinical practice: implications for MRI. J. Magn. Reson. Imaging 37, 1476–1479 (2013).

    Article  PubMed  Google Scholar 

  7. Chen, F. et al. Chelator-free synthesis of a dual-modality PET/MRI agent. Angew. Chem. Int. Ed. Engl. 52, 13319–13323 (2013).

    Article  CAS  PubMed  Google Scholar 

  8. Shi, S. et al. Chelator-free radiolabeling of nanographene: breaking the stereotype of chelation. Angew. Chem. Int. Ed. Engl. 56, 2889–2892 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Freund, B. et al. A simple and widely applicable method to 59Fe-radiolabel monodisperse superparamagnetic iron oxide nanoparticles for in vivo quantification studies. ACS Nano 6, 7318–7325 (2012).

    Article  CAS  PubMed  Google Scholar 

  10. Israel, L.L. et al. Surface metal cation doping of maghemite nanoparticles: modulation of MRI relaxivity features and chelator-free 68 Ga-radiolabelling for dual MRI-PET imaging. Mater. Res. Express 2, 095009 (2015).

    Article  CAS  Google Scholar 

  11. Gaglia, J.L. et al. Noninvasive mapping of pancreatic inflammation in recent-onset type-1 diabetes patients. Proc. Natl. Acad. Sci. USA 112, 2139–2144 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yilmaz, A. et al. Imaging of myocardial infarction using ultrasmall superparamagnetic iron oxide nanoparticles: a human study using a multi-parametric cardiovascular magnetic resonance imaging approach. Eur. Heart J. 34, 462–475 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. Hasan, D.M. et al. Macrophage imaging within human cerebral aneurysms wall using ferumoxytol-enhanced MRI: a pilot study. Arterioscler. Thromb. Vasc. Biol. 32, 1032–1038 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Chalouhi, N., Jabbour, P. & Hasan, D. Inflammation, macrophages, and targeted imaging in intracranial aneurysms. World Neurosurg. 81, 206–208 (2014).

    Article  PubMed  Google Scholar 

  15. Vellinga, M.M. et al. Use of ultrasmall superparamagnetic particles of iron oxide (USPIO)-enhanced MRI to demonstrate diffuse inflammation in the normal-appearing white matter (NAWM) of multiple sclerosis (MS) patients: an exploratory study. J. Magn. Reson. Imaging 29, 774–779 (2009).

    Article  PubMed  Google Scholar 

  16. Maarouf, A. et al. Ultra-small superparamagnetic iron oxide enhancement is associated with higher loss of brain tissue structure in clinically isolated syndrome. Mult. Scler. 22, 1032–1039 (2016).

    Article  CAS  PubMed  Google Scholar 

  17. Usman, A. et al. Use of ultrasmall superparamagnetic iron oxide particles for imaging carotid atherosclerosis. Nanomedicine (Lond.) 10, 3077–3087 (2015).

    Article  CAS  Google Scholar 

  18. Boros, E., Bowen, A.M., Josephson, L., Vasdev, N. & Holland, J.P. Chelate-free metal ion binding and heat-induced radiolabeling of iron oxide nanoparticles. Chem. Sci. 6, 225–236 (2015).

    Article  CAS  PubMed  Google Scholar 

  19. Normandin, M.D. et al. Heat-induced radiolabeling of nanoparticles for monocyte tracking by PET. Angew. Chem. Int. Ed. Engl. 54, 13002–13006 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sîrbulescu, R.F. et al. Mature B cells accelerate wound healing after acute and chronic diabetic skin lesions. Wound Repair Regen. 25, 774–791. (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Yuan, H. et al. Heat-induced-radiolabeling and click chemistry: a powerful combination for generating multifunctional nanomaterials. PLoS One 12, e0172722 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Balakrishnan, V.S. et al. Physicochemical properties of ferumoxytol, a new intravenous iron preparation. Eur. J. Clin. Invest. 39, 489–496 (2009).

    Article  CAS  PubMed  Google Scholar 

  23. Jahn, M.R. et al. A comparative study of the physicochemical properties of iron isomaltoside 1000 (Monofer), a new intravenous iron preparation and its clinical implications. Eur. J. Pharm. Biopharm. 78, 480–491 (2011).

    Article  CAS  PubMed  Google Scholar 

  24. Futterer, S., Andrusenko, I., Kolb, U., Hofmeister, W. & Langguth, P. Structural characterization of iron oxide/hydroxide nanoparticles in nine different parenteral drugs for the treatment of iron deficiency anaemia by electron diffraction (ED) and X-ray powder diffraction (XRPD). J. Pharm. Biomed. Anal. 86, 151–160 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Groman, E.V., Paul, K.G., Frigo, T.B., Bengele, H. & Lewis, J.M. Heat stable colloidal iron oxides coated with reduced carbohydrates and derivatives. US patent 6,599,498 (2003).

  26. Maruno, S. et al. Polysaccharide derivative/magnetic metal oxide composite. US patent 6,165,378 (2000).

  27. Maruno, S. & Hasegawa, M. Organic matter complex. US patent 5,204,457 (1993).

  28. Alcantara, D. et al. Fluorochrome-functionalized magnetic nanoparticles for high-sensitivity monitoring of the polymerase chain reaction by magnetic resonance. Angew. Chem. Int. Ed. Engl. 51, 6904–6907 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Cho, H. et al. Fluorochrome-functionalized nanoparticles for imaging DNA in biological systems. ACS Nano 7, 2032–2041 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Warner, C.L. et al. Manganese doping of magnetic iron oxide nanoparticles: tailoring surface reactivity for a regenerable heavy metal sorbent. Langmuir 28, 3931–3937 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. Chakravarty, R., Goel, S., Dash, A. & Cai, W. Radiolabeled inorganic nanoparticles for positron emission tomography imaging of cancer: an overview. Q. J. Nucl. Med. Mol. Imaging 61, 181–204 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Sun, X., Cai, W. & Chen, X. Positron emission tomography imaging using radiolabeled inorganic nanomaterials. Acc. Chem. Res. 48, 286–294 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Goel, S., Chen, F., Ehlerding, E.B. & Cai, W. Intrinsically radiolabeled nanoparticles: an emerging paradigm. Small 10, 3825–3830 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wunderbaldinger, P., Josephson, L., Bremer, C., Moore, A. & Weissleder, R. Detection of lymph node metastases by contrast-enhanced MRI in an experimental model. Magn. Reson. Med. 47, 292–297 (2002).

    Article  PubMed  Google Scholar 

  35. Thorek, D.L. et al. Non-invasive mapping of deep-tissue lymph nodes in live animals using a multimodal PET/MRI nanoparticle. Nat. Commun. 5, 3097 (2014).

    Article  PubMed  CAS  Google Scholar 

  36. Keliher, E.J. et al. 89Zr-labeled dextran nanoparticles allow in vivo macrophage imaging. Bioconjug. Chem. 22, 2383–2389 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Weissleder, R. et al. Superparamagnetic iron oxide: pharmacokinetics and toxicity. Am. J. Roentgenol. 152, 167–173 (1989).

    Article  CAS  Google Scholar 

  38. Wong, R.M., Gilbert, D.A., Liu, K. & Louie, A.Y. Rapid size-controlled synthesis of dextran-coated, 64Cu-doped iron oxide nanoparticles. ACS Nano 6, 3461–3467 (2012).

    Article  CAS  PubMed  Google Scholar 

  39. Liu, K.X. et al. Controllable preparation of ternary superparamagnetic nanoparticles dual-doped with Mn and Zn elements. J. Nanosci. Nanotechnol. 12, 8437–8442 (2012).

    Article  CAS  PubMed  Google Scholar 

  40. Chen, S., Alcantara, D. & Josephson, L. A magnetofluorescent nanoparticle for ex vivo cell labeling by covalently linking the drugs protamine and Feraheme. J. Nanosci. Nanotechnol. 11, 3058–3064 (2011).

    Article  CAS  PubMed  Google Scholar 

  41. Lu, M., Cohen, M.H., Rieves, D. & Pazdur, R. FDA report: ferumoxytol for intravenous iron therapy in adult patients with chronic kidney disease. Am. J. Hematol. 85, 315–319 (2010).

    CAS  PubMed  Google Scholar 

  42. Wang, C. et al. Comparative risk of anaphylactic reactions associated with intravenous iron products. JAMA 314, 2062–2068 (2015).

    Article  CAS  PubMed  Google Scholar 

  43. Ning, P., Zucker, E.J., Wong, P. & Vasanawala, S.S. Hemodynamic safety and efficacy of ferumoxytol as an intravenous contrast agents in pediatric patients and young adults. Magn. Reson. Imaging 34, 152–158 (2016).

    Article  CAS  PubMed  Google Scholar 

  44. Finn, J.P. et al. Cardiovascular MRI with ferumoxytol. Clin. Radiol. 71, 796–806 (2016).

    Article  CAS  PubMed  Google Scholar 

  45. Gahart, B.L., Nazareno, A.R. & Ortega, M.Q. Intravenous Medications: A Handbook for Nurses and Health Professionals 3rd edn 555–556 (Elsevier, 2016).

  46. Lacroute, P. & Levoy, M. Fast volume rendering using a shear-warp factorization of the viewing transformation. Computer Graphics Proceedings, Annual Conference Series, 451. (1994).

  47. Bar-Shalom, R. et al. SPECT/CT using 67Ga and 111In-labeled leukocyte scintigraphy for diagnosis of infection. J. Nucl. Med. 47, 587–594 (2006).

    PubMed  Google Scholar 

  48. Lou, L. et al. 99mTc-WBC scintigraphy with SPECT/CT in the evaluation of arterial graft infection. Nucl. Med. Commun. 31, 411–416 (2010).

    Article  PubMed  Google Scholar 

  49. Schnell, M.A., Hardy, C., Hawley, M., Propert, K.J. & Wilson, J.M. Effect of blood collection technique in mice on clinical pathology parameters. Hum. Gen. Ther. 13, 155–161 (2002).

    Article  CAS  Google Scholar 

  50. Harisinghani, M., Ross, R.W., Guimaraes, A.R. & Weissleder, R. Utility of a new bolus-injectable nanoparticle for clinical cancer staging. Neoplasia 9, 1160–1165 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Turkbey, B. et al. A phase I dosing study of ferumoxytol for MR lymphography at 3 T in patients with prostate cancer. Am. J. Roentgenol. 205, 64–69 (2015).

    Article  Google Scholar 

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

Author information

Authors and Affiliations



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.

Corresponding author

Correspondence to Lee Josephson.

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Competing interests

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).

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