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

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. Supplementary Information The online version contains supplementary material available at 10.1007/s11307-021-01622-z.

irradiated with 25 µA protons in a cyclotron for 1-3 hrs. Proton energy was degraded to a nominal 12. 6 MeV to minimize co-production of long-lived 88 Zr and 88 Y. Separation of 89 Zr from 89 Y was performed with a separation module (Taddeo PRF, Comecer) using a home-made hydroxamate column as described previously [1,2]. Prior to separation, the column was

Optimization of [ 89 Zr]Zr-oxine synthesis
To exclude the need for chloroform, different non-toxic buffers and solvents were investigated, avoiding the need for separation and evaporation. To accelerate the reaction, the concentration was increased by decreasing the volume and the temperature was increased (not exceeding the melting point of oxine at 75°C). To investigate the optimal incubation and pH conditions, a pH range from 5.0 to 10.0 was tested, following escalating incubation times of 10, 20, 30, 40, 50 and 60 minutes. The final protocol was obtained: oxine (Honeywell) was dissolved in 99% ethanol (Sigma-Aldrich) to a concentration of 204 mM and incubated on a heat shaker (Eppendorf Thermomixer C) at 65°C for approximately 5 minutes. . The RCC was also confirmed by radio high-performance liquid chromatography (radio-HPLC) (as described below). RCC was consistently over 95%, therefore no further separation was needed.

[ 89 Zr]Zr-DFO-NCS synthesis
To achieve a stable RCC of >95% without the need for purification, we optimised several conditions such as synthesis buffer, reaction time, DFO-NCS concentration, and temperature as below. The RCC in all tests was determined by iTLC using a mobile phase of 50 mM DTPA (Sigma-Aldrich) pH 4). [ 89 Zr]Zr-DFO-NCS remained at the origin (Rf ~ 0) while unbound 89 Zr was chelated by DTPA, travelling with the solvent front (Rf ~ 0.9). The RCC was also confirmed by radio-HPLC (as described below).

Radio-HPLC analysis of [ 89 Zr]Zr-oxine and [ 89 Zr]Zr-DFO-NCS
To confirm the RCC, analysis using radio high-performance liquid chromatography (radio-HPLC) was performed. An HPLC system (UFLC Shimadzu), equipped with a radio detector

Human decidual stromal cells
The human decidual stromal cells (hDSCs) were isolated, prepared and cultured as previously described [3,4]. Briefly, hDSCs were isolated from human placenta, obtained through a caesarean section of healthy donors after informed consent, according to legislation by the cytometry, hence confirming that the cells were of maternal origin and of mesenchymal lineage [4,5].

Rat bone marrow-derived macrophages
In accordance with the ethics approval (9328-2019 and N138/14), rat bone marrow-derived macrophages (rMac) were collected as previously described [6]. Briefly, the femurs were surgically removed and the bones were cut at both ends to expose the bone marrow (BM) cells.
The BM cells were then thoroughly flushed with Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich) using a syringe and needle. To disaggregate potential cell clusters, the cells were carefully resuspended and passed through a cell strainer (40 μm, BD Biosciences). The collected cell mixture was then suspended in complete DMEM containing 20% fetal bovine serum (Sigma-Aldrich), penicillin-streptomycin (Sigma-Aldrich), Lglutamine (Sigma-Aldrich), 2-mercaptoethanol (Gibco, Life Technologies) and sodium pyruvate (Thermo Fisher) and was then stimulated with 20 ng/mL rat macrophage colonystimulating factor M-CSF (400-28, PeproTech). Cells were then cultured in T175 culture flasks for 8 days in a humid incubation chamber at 37°C and with 5% CO2. Mature macrophages were resuspended in PBS and characterised using flow cytometric analysis (BD Biosciences or Merck Guava H12), as previously described [6]. Cell proliferation was determined using dimethyl sulfoxide with 5 mM carboxyfluorescein succinimidyl ester (CFSE) (BioLegend), measured by flow cytometry and analysed using the Calibur software (FlowJo v10, BD Biosciences).

Human peripheral blood mononuclear cells
Human peripheral blood mononuclear cells (hPBMC) were isolated from buffy coats under ethics approval Dnr 2006/229-31/1 as previously described [7]. In short, blood cells were