Intracellular Protein Binding of Zr-89 Oxine Cell Labeling for PET Cell Tracking Studies

<b>Background/Objectives</b>: ⁸⁹Zr-oxine is an ex vivo cell labeling agent that enables cells to be tracked in vivo by positron emission tomography (PET) over a period of up to two weeks. To better understand where ⁸⁹Zr-oxine binds within cellular components, factors affecting labeling and intracellular distribution of ⁸⁹Zr were examined. <b>Methods</b>: Mouse primary T cells, natural killer cells, dendritic cells, and monocytes, and cell lines EL4 (mouse lymphoma), DC2.4 (mouse dendritic cell), Kit225K6 (human T cell leukemia) and MC38 (mouse colon adenocarcinoma) were labeled with ⁸⁹Zr-oxine or ¹¹¹In-oxine and protein binding within the cellular compartments, the labeling thresholds, and radioactivity retention were subsequently determined. <b>Results</b>: Cell incorporation of ⁸⁹Zr-oxine (27.8-71.8 kBq/10⁶ cells) positively correlated with cellular size and protein mass. Most (>97%) ⁸⁹Zr was protein-bound and primarily localized in the cytoplasm, membrane, and nuclear fractions (>81%) with distribution patterns varying by cell type. By contrast, ¹¹¹In-oxine showed lower protein-binding activity of approximately 59-65%, with 62-65% of ¹¹¹In localized in the cytoplasm. Autoradiography of electrophoresed subcellular fractionated cell samples indicated stable binding by ⁸⁹Zr-oxine to proteins in all subcellular fractions but unstable protein binding by ¹¹¹In. Saturation studies showed that ⁸⁹Zr-oxine labeling was saturable, and further labeling reduced cellular retention. Biodistribution of dendritic cells labeled with either ⁸⁹Zr-oxine or ¹¹¹In-oxine indicated greater retention of ⁸⁹Zr in the labeled cells in vivo than ¹¹¹In. <b>Conclusions</b>: ⁸⁹Zr-oxine stably binds many intracellular proteins and shows much higher and more stable protein binding than ¹¹¹In-oxine. Intracellular protein binding of ⁸⁹Zr accounts for the ability of ⁸⁹Zr-oxine labeling to successfully track cells in vivo long-term on PET.