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

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.

<p>A Radio-Nano-Platform for T1/T2 Dual-Mode PET-MR Imaging</p>

“…The radiochemical analysis ( Figure S3) demonstrates that under basic conditions (pH~8-9) 89 Zr 4+ ions react thermally with the FH core and result in high RCY and RCP. These results are also consistent with a previous study 27 and confirm the efficiency and robustness of the novel HIR technique. Therefore, cations with f-orbital electrons, more empty d-orbitals, larger radii, and higher positive charges (or higher oxidation state) result in a stronger electrostatic interaction and thus stronger bonding with the surface and a higher radiochemical yield (RCY).…”

“…The stability of the prepared radiolabeled FH SPION was fully characterized in our recent studies. 7,8,27 A complete picture for the stability of HIR-FH NPs includes three aspects. The first is FH's extremely high thermostability sourced from the good number of carboxyl groups on the CMD and presented by: i) the use of terminal sterilization at 121°C during manufacture; 28 ii) its storage for months at room temperature (20-25°C) as a concentrated (30 mg of Fe/mL) fluid for intravenous injection; and iii) minimal variation of NP sizes and MR relaxivities (r 1 & r 2 ) post HIR heating.…”

“…The first is FH's extremely high thermostability sourced from the good number of carboxyl groups on the CMD and presented by: i) the use of terminal sterilization at 121°C during manufacture; 28 ii) its storage for months at room temperature (20-25°C) as a concentrated (30 mg of Fe/mL) fluid for intravenous injection; and iii) minimal variation of NP sizes and MR relaxivities (r 1 & r 2 ) post HIR heating. 7,8,27 The second is the chemical stability of CMD-iron oxide interaction which permits the conversion of carboxyl groups to amino or azido/alkyne functional groups, followed by multifunctional modifications through amide formation 27 or click chemistry. 9 The third is their high radiochemical stability both in vitro analyzed by TLC of HIR-FH in references and in vivo without any noticeable bone uptake in PET/CT imaging both in mice 8,27 and monkey (unpublished results).…”

“…7,8,27 The second is the chemical stability of CMD-iron oxide interaction which permits the conversion of carboxyl groups to amino or azido/alkyne functional groups, followed by multifunctional modifications through amide formation 27 or click chemistry. 9 The third is their high radiochemical stability both in vitro analyzed by TLC of HIR-FH in references and in vivo without any noticeable bone uptake in PET/CT imaging both in mice 8,27 and monkey (unpublished results).…”

89Zr Labeled Fe3O4@TiO2 Nanoparticles: In Vitro Afffinities with Breast and Prostate Cancer Cells

“…Boros et al radiolabelled the FDA‐approved, carbohydrate coated commercial Fe 3 O 4 nanoparticles called ferumoxytol (Feraheme®) with a range of more commonly available radiometal isotopes, including 89 Zr and 64 Cu for PET and 111 In for SPECT . Particles were heated in an aqueous solution with the radiometal ions (80–120 °C) at a pH between 7–9 . However the nature of the chemical interaction between the metal isotopes and iron oxide nanoparticles remained unidentified in these reports.…”