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.

Keywords: 89Zr; Cell labelling; Cell tracking; Deferoxamine; Imaging; Oxine; PET.

Fig. 1.

Molecular structures of ⁸⁹Zr-based PET radiotracers A [⁸⁹Zr]Zr-oxine and B [⁸⁹Zr]Zr-DFO-NCS, and the previously established SPECT radiotracer C [¹¹¹In]In-oxine.

Fig. 2.

Schematic illustration of the synthesis conditions and cell labelling mechanisms of (A) extracellular labelling using [⁸⁹Zr]Zr-DFO-NCS and (B) intracellular labelling using [⁸⁹Zr]Zr-oxine.

Fig. 3.

The radiochemical conversion (RCC) during synthesis over time for A [⁸⁹Zr]Zr-oxine with an oxine concentration of 7.7 mM at 65 °C and B [⁸⁹Zr]Zr-DFO-NCS at a concentration of 7.5 μM in room temperature. Both radiotracers reach an RCC > 98 % after 60 min incubation, and C pH-dependent RCC in the synthesis of [⁸⁹Zr]Zr-oxine. D Shelf-life of [⁸⁹Zr]Zr-DFO-NCS in pH 7.4 for optimal cell labelling efficiency (CLE). The maximum CLE was obtainable within 1-h post-synthesis. E Increased RCC of [⁸⁹Zr]Zr-DFO-NCS with increased DFO-NCS concentration over time.

Fig. 4.

Radiochemical conversion (RCC) >98 % with instant thin-layer chromatography (iTLC) for A [⁸⁹Zr]Zr-oxine and B [⁸⁹Zr]Zr-DFO-NCS synthesis. Radio-HPLC showing the C radioactive peak for [⁸⁹Zr]Zr-oxine and the D radioactive peak for [⁸⁹Zr]Zr-DFO-NCS.

Fig. 5.

Phenotyping by measuring antigen expression of hDSC pre- and post-labelling with [⁸⁹Zr]Zr-oxine (n = 3) or [⁸⁹Zr]Zr-DFO-NCS (n = 3). A flow panel for positive expression of CD29, CD44, CD73, CD90 and CD105 and a negative expression of CD14, CD34 and CD45 corresponds to hDSC phenotype. Radiolabelled cells showed a significant increase in expression of CD29, CD44, CD73 and CD105 compared to unlabelled cells (n = 3), which still corresponds to hDSC phenotype. Data were analysed as two parametric, paired-samples (pre- vs. post-labelling) with student T-test, P values set as <0.05*, < 0.01**. Bars represent mean and error bars standard deviation.

Fig. 6.

Phagocytic function measured with fluorescent dextran uptake in rat macrophages after labelling with A [⁸⁹Zr]Zr-oxine and B [⁸⁹Zr]Zr-DFO-NCS. The phagocytotic function shows a slight decrease for both compounds compared to unlabelled cells. [⁸⁹Zr]Zr-oxine labelled cells decrease with 11.2 % (3.6 SD) (n = 3, P = 0.048) and [⁸⁹Zr]Zr-DFO-NCS labelled cells decrease with 13.3 % (4.7 SD) (n = 3, P = 0.062). Bars represent mean and error bars standard deviation.