Radiolabelling bacteria with ⁸⁹Zr-DBN for PET/MRI: comparing [⁸⁹Zr]Zr oxalate and [⁸⁹Zr]Zr phosphate

Our bodies host a diverse and complex community of bacteria, known as the microbiota, which play a crucial role in overall wellness. The gut microbiota perform essential functions, such as food fermentation, pathogen protection, and vitamin production. Dysbiosis, or the imbalance of these bacterial communities, can disrupt these processes, leading to diseases like cancer, respiratory infections, and neurological disorders. To enable non-invasive tracking of microbial distribution in vivo, this study investigates [⁸⁹Zr]Zr oxalate as a more efficient and cost-effective alternative to [⁸⁹Zr]Zr phosphate for radiolabelling diverse bacterial species for PET/MRI.

To test radiolabelling efficiency, the bifunctional chelator DBN, consisting of desferrioxamine (DFO) to chelate ⁸⁹Zr and a lysine-reactive group (isothiocyanate) for cell surface protein attachment, was used. The bifunctional chelator was mixed with either neutralized [⁸⁹Zr]Zr phosphate (titrated to pH 7 with 1 M K₂CO₃) in HEPES buffer or [⁸⁹Zr]Zr oxalate (titrated to pH 7 with 2 M Na₂CO₃) in phosphate buffered saline (PBS). The efficiency of isotope chelation was evaluated by radio thin layer chromatography (radio-TLC). The efficiency of bacterial conjugation was examined with commensal Lactobacillus crispatus ATCC33820 and probiotic Escherichia coli Nissle 1917. A t-test with unequal variances assessed statistical differences in chelation and bacterial labelling efficiencies.

[⁸⁹Zr]Zr oxalate showed significantly higher chelation efficiency to DBN (94.8 ± 5.5 %) compared to [⁸⁹Zr]Zr phosphate (66.6 ± 16.3 %; p < 0.05). Labelling efficiency for L. crispatus improved when [⁸⁹Zr]Zr oxalate was used either after one half-life had passed since production or within 1 half-life but diluted with water in a 1:1 ratio. Optimal labelling provided an average activity per live cell (colony-forming unit, CFU) between 0.003 and 0.12 Bq/CFU, with activities above 0.01 Bq/CFU causing significant bacterial death, while lower activities (below 0.006 Bq/CFU) effectively maintained bacterial viability. Comparable bacterial labelling efficiency results were obtained with Escherichia coli Nissle 1917.

This study establishes the first standardized protocol for radiolabelling viable bacteria using ⁸⁹Zr-DBN derived from [⁸⁹Zr]Zr oxalate. This method enables high chelation and labelling efficiency while preserving cell viability and is applicable to both L. crispatus (Gram-positive, facultative anaerobe) and E.coli (Gram-negative, aerobe), supporting its use in PET/MRI based bacterial imaging.