PET Imaging of Liposomal Glucocorticoids using 89Zr-oxine: Theranostic Applications in Inflammatory Arthritis

The encapsulation of Glucocorticoids (GCs) into long-circulating liposomes (LCLs) is a proven strategy to reduce the side effects of glucocorticoids and improve the treatment of inflammatory diseases, such as rheumatoid arthritis (RA). With the aim of supporting the development of GC-loaded LCLs, and potentially predict patient response to therapy clinically, we evaluated a direct PET imaging radiolabelling approach for preformed GC-LCLs in an animal model of human inflammatory arthritis. Methods: A preformed PEGylated liposomal methylprednisolone hemisuccinate (NSSL-MPS) nanomedicine was radiolabelled using [89Zr]Zr(oxinate)4 (89Zr-oxine), characterised and tracked in vivo using PET imaging in a K/BxN serum-transfer arthritis (STA) mouse model of inflammatory arthritis and non-inflamed controls. Histology and joint size measurements were used to confirm inflammation. The biodistribution of 89Zr-NSSL-MPS was compared to that of free 89Zr in the same model. A therapeutic study using NSSL-MPS using the same time points as the PET/CT imaging was carried out. Results: The radiolabelling efficiency of NSSL-MPS with [89Zr]Zr(oxinate)4 was 69 ± 8 %. PET/CT imaging of 89Zr-NSSL-MPS showed high uptake (3.6 ± 1.5 % ID; 17.4 ± 9.3 % ID/mL) at inflamed joints, with low activity present in non-inflamed joints (0.5 ± 0.1 % ID; 2.7 ± 1.1 % ID/mL). Importantly, a clear correlation between joint swelling and high 89Zr-NSSL-MPS uptake was observed, which was not observed with free 89Zr. STA mice receiving a therapeutic dose of NSSL-MPS showed a reduction in inflammation at the time points used for the PET/CT imaging compared with the control group. Conclusions: PET imaging was used for the first time to track a liposomal glucocorticoid, showing high uptake at visible and occult inflamed sites and a good correlation with the degree of inflammation. A subsequent therapeutic response matching imaging time points in the same model demonstrated the potential of this radiolabeling method as a theranostic tool for the prediction of therapeutic response - with NSSL-MPS and similar nanomedicines - in the treatment of inflammatory diseases


Materials and methods
All chemical reagents were purchased from commercial sources. Water (18.2 MΩ·cm) was obtained from an ELGA Purelab Option-Qsystem. UV titrations were carried out using a PerkinElmer Lambda 25 spectrometer, with samples in Brand 70 μL micro cuvettes. No-carrier-added 89Zr (produced at the BV Cyclotron, VU Amsterdam, NL) was purchased from PerkinElmer as [ 89 Zr]Zr(oxalate)4 in 1 M oxalic acid. Radioactivity in samples were measured using CRC-25R dose calibrator (Capintec). iTLC-SG and SA strips were purchased from Agilent, UK and scanned using the PerkinElmer Cyclone Plus Storage Phosphor Imager. Gamma counting was performed using a Wallac 1282 CompuGamma γ counter. The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an Institutional Review Board/Ethics Committee (IRB/EC) approved protocol. All animal studies were ethically reviewed and carried out in accordance with the Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals.

[ 89 Zr]Zr(oxinate)4 radiosynthesis
[ 89 Zr]Zr(oxalate)4 (5−75 MBq) was transferred to a 1.5 mL plastic vial, diluted to 100 µL with water (18.2 MΩ·cm) and gradually adjusted to pH 7.5−8 with 1 M sodium carbonate. The volume was then adjusted to 450 μL with Chelex®-treated water and 50 µL of a 10 mg/mL solution of 8hydroxyquinoline in chloroform was added. The mixture was vortexed for 5 min, a further 450 µL of chloroform were added and the mixture vortexed for 10 min. The organic phase was extracted into a conical glass vial and dried at 60 °C under a flow of nitrogen gas. The residue was dissolved in aqueous dimethyl sulfoxide (DMSO) for further use. Radiochemical yield was defined by the amount of radioactivity present in the dried organic extract divided by the starting amount of radioactivity.

Characterisation of the Zr-MPS complex
10 mg/mL solutions of methylprednisolone sodium hemisuccinate (MPS-Na) and non-radioactive ZrCl4 in MeOH were prepared. A 6 µL aliquot of ZrCl4 (60 µg, 0.26 µmol, 0.5 equivs.) was added to a 25 µL solution of MPS-Na (0.2 mg, 0.5 µmol, 2 equivs.) and the solution heated at 50 o C for 30 mins. The solution was applied to the ATR diamond on an ATR-IR system and the solvent removed via mild heating with a heat gun. This process was repeated until a thin film was visible on the ATR diamond.
The IR spectrum of the mixture was then taken. For an IR of MPS-Na, aliquots of the 10 mg/mL solution of MPS-Na was applied to the ATR diamond instead.

Dynamic light scattering (DLS) measurements of NSSL-MPS before and after radiolabelling
For size and polydispersity measurements:-After radiolabelling an aliquot of 89 Zr-NSSL-MPS was diluted 10x in 10% PBS in water and the size and polydispersity measured by DLS. For control measurements, an aliquot of NSSL-MPS was diluted in saline to match the same concentration of 89 Zr-NSSL-MPS after radiolabelling. An aliquot of this was then diluted 10x in 10% PBS in water and the size and polydispersity measured by DLS.
For zeta-potential measurements:-The same solutions used for the above size and polydispersity measurements were diluted a further 5x in 10% PBS in water and the zeta potential measured by DLS.

Biodistribution of free 89 Zr in RA mice
Biodistribution studies were carried out in accordance with British Home Office regulations governing animal experimentation. Rheumatoid arthritis was induced in female 9-week old C57Bl/6 mice (n = 2) as described in the main text, with visual inflammation scores assigned on each day post-serum injection and caliper measurements being performed on the wrists and ankles on Days 0, 2, 5, 7 & 9. At day 7 post-serum injection, all mice were anesthetised with isofluorane (1.5−2%) and [ 89 Zr]ZrCl4 (1.6-1.8 MBq, 120 μL) was injected i.v. into the mice. Mice were culled by cervical dislocation at 48 h p.i. whilst under anaesthesia, and the organs of interest were dissected, weighted and gamma-counted together with standard samples of the injected radiotracer to obtain percentages of the injected dose per mass values (%ID/g) for each organ/tissue ( Figure 6A). Each sample was weighed and counted with a γ counter (LKB compugamma), together with standards prepared from a sample of the injected [ 89 Zr]ZrCl4.