Purification and [89Zr]Zr Radiolabelling of Bacteriophages for PET Tracking
Abstract
Purpose
Antimicrobial resistance is a growing global health threat, exacerbated by overuse of broad-spectrum antibiotics. Targeted strategies to detect and treat bacterial infections are needed. Bacteriophages (phages), viruses that infect specific bacteria, offer an alternative due to their selective bacterial targeting, minimizing disruption to host microbiota. Radiolabelling phages enables non-invasive investigation of their biodistribution and bacterial interactions in vivo. This study aims to develop a stable phage radiolabelling method using [89Zr]Zr-oxalate for PET tracking of bacterial infection.
Methods
Phage radiolabelling was performed using a bifunctional desferrioxamine (DFO) chelator containing an isothiocyanate group for covalent attachment to phage surface protein. Neutralized [89Zr]Zr-oxalate was chelated with DFO/phosphate-buffered saline pH 7.4 (PBS). Chelation efficiency was confirmed by radio-thin layer chromatography. Myoviridae phages LL12 and T4, both lytic toward Escherichia coli, were propagated in strain MG1655 and purified to remove host-derived protein. Purified phages were incubated with [89Zr]Zr-DFO for 1 hour at 37 °C. Unbound 89Zr was removed by precipitation with 6% polyethylene glycol 8000/0.75M NaCl, followed by centrifugation. The radiolabelled phage pellet was resuspended in PBS. Labelling efficiency was calculated as the ratio of final pellet activity to total initial activity. Plaque assays were used to compare infectivity of radiolabelled and unlabelled phages.
Results
[89Zr]Zr-DFO formation was efficient (96.24 ± 4.39%, n = 9). Labelling efficiency was 75.31 ± 15.15% (n = 7) for LL12 and 26.83% for T4 (n = 2). Radiolabelling did not adversely affect phage infectivity, with radiolabelled sample titers comparable to unlabelled controls within the tested activity ranges (LL12: ~1-3×10-4 Bq/PFU; T4: ~3×10-5 – 1.4×10-4 Bq/PFU).
Conclusion
DFO-based [89Zr]Zr enables efficient radiolabelling of phages for PET. Differences in labelling efficiency may reflect variation in surface-exposed proteins targeted by DFO conjugation. Future work will explore phages with diverse morphologies to assess radiolabelling variability and optimize a broadly applicable radiolabelling method.