Optimisation of the Synthesis and Cell Labelling Conditions for [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS: a Direct In Vitro Comparison in Cell Types with Distinct Therapeutic Applications

Abstract

Background There is a need to better characterise cell-based therapies in preclinical models to help facilitate their translation to humans. Long-term high-resolution tracking of the cells in vivo is often impossible due to unreliable methods. Radiolabelling of cells has the advantage of being able to reveal cellular kinetics in vivo over time. This study aimed to optimise the synthesis of the radiotracers [89Zr]Zr-oxine (8-hydroxyquinoline) and [89Zr]Zr-DFO-NCS (p-SCN-Bn-Deferoxamine) and to perform a direct comparison of the cell labelling efficiency using these radiotracers. Procedures Several parameters, such as buffers, pH, labelling time and temperature, were investigated to optimise the synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS in order to reach a radiochemical conversion (RCC) of >95 % without purification. Radio-instant thin-layer chromatography (iTLC) and radio high-performance liquid chromatography (radio-HPLC) were used to determine the RCC. Cells were labelled with [89Zr]Zr-oxine or [89Zr]Zr-DFO-NCS. The cellular retention of 89Zr and the labelling impact was determined by analysing the cellular functions, such as viability, proliferation, phagocytotic ability and phenotypic immunostaining. Results The optimised synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS resulted in straightforward protocols not requiring additional purification. [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS were synthesised with an average RCC of 98.4 % (n = 16) and 98.0 % (n = 13), respectively. Cell labelling efficiencies were 63.9 % (n = 35) and 70.2 % (n = 30), respectively. 89Zr labelling neither significantly affected the cell viability (cell viability loss was in the range of 1–8 % compared to its corresponding non-labelled cells, P value > 0.05) nor the cells’ proliferation rate. The phenotype of human decidual stromal cells (hDSC) and phagocytic function of rat bone-marrow-derived macrophages (rMac) was somewhat affected by radiolabelling. Conclusions Our study demonstrates that [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS are equally effective in cell labelling. However, [89Zr]Zr-oxine was superior to [89Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency.

Comparative in vivo biodistribution of cells labelled with [89Zr]Zr-(oxinate)4 or [89Zr]Zr-DFO-NCS using PET

“…Today, [ 89 Zr]Zr-(oxinate) 4 (also denoted as [ 89 Zr]Zr-oxine) is a well-evaluated radiotracer in preclinical studies and is currently in a first-in-human clinical study [ 9 , 11 – 15 ]. However, some limitations regarding radioactive leakage during the first 24 h should be further evaluated [ 10 , 16 , 17 ]. Our group have recently optimized the synthesis and cell labelling of both [ 89 Zr]Zr-(oxinate) 4 and [ 89 Zr]Zr-DFO-NCS [ 17 ].…”

“…However, some limitations regarding radioactive leakage during the first 24 h should be further evaluated [ 10 , 16 , 17 ]. Our group have recently optimized the synthesis and cell labelling of both [ 89 Zr]Zr-(oxinate) 4 and [ 89 Zr]Zr-DFO-NCS [ 17 ]. Both radiotracers were successfully synthesized with a radiochemical yield (RCY) of > 95% and used to label different cell types with high labelling efficiency.…”

Evaluation of different 89Zr-labeled synthons for direct labeling and tracking of white blood cells and stem cells in healthy athymic mice

“…Recently, Friberger et al reported a one-step clinically translatable method of synthesis of [ 89 Zr]Zr-oxine with a cell labeling efficiency of 61–68% with human decidual stromal cells (hDSCs), bone marrow-derived macrophages (rMac) and human peripheral blood mononuclear cells (hPBMCs). However, a 29–38% apparent efflux of 89 Zr from the labeled cells raised a further concern of radiotoxicity and non-specificity of the signal 57 .…”

“…Besides [ 89 Zr]Zr-oxine, the other reported method of cell labeling was covalent attachment of radiolabeled [ 89 Zr]Zr-DFO-Bn-NCS complex with the primary amines present on the cell surface proteins to form stable thiourea bonds, which has solved the efflux problem observed with [ 89 Zr]Zr-oxine 57 – 60 . The [ 89 Zr]Zr-DFO-Bn-NCS has been successfully used to radiolabel mouse melanoma cells, mouse dendritic cells and human mesenchymal stem cells with insignificant efflux of free 89 Zr from [ 89 Zr]Zr-DFO-Bn-NCS over time (7 days-post radiolabeling) 58 .…”

“…In this work, we have optimized and compared the radiolabeling yields of WBCs and SCs using three different ready-to-use labeling synthons [ 89 Zr]Zr-Hy 3 ADA 5 -NCS 66 , [ 89 Zr]Zr-Hy 3 ADA 5 -SA 66 and [ 89 Zr]Zr-DFO-Bn-NCS 57 – 60 (Fig. 1 ), and evaluated their applications in cell trafficking to better understand the biodistribution/pharmacokinetics of cell based therapies .…”