Evaluation of [68Ga]Ga-DOTA-AeK as a Potential Imaging Tool for PET Imaging of Cell Wall Synthesis in Bacterial Infections

The ability of bacteria to recycle exogenous amino acid-based peptides and amino sugars for peptidoglycan biosynthesis was extensively investigated using optical imaging. In particular, fluorescent AeK-NBD was effectively utilized to study the peptidoglycan recycling pathway in Gram-negative bacteria. Based on these promising results, we were inspired to develop the radioactive AeK conjugate [⁶⁸Ga]Ga-DOTA-AeK for the in vivo localization of bacterial infection using PET/CT. An easy-to-implement radiolabeling procedure for DOTA-AeK with [⁶⁸Ga]GaCI₃ followed by solid-phase purification was successfully established to obtain [⁶⁸Ga]Ga-DOTA-AeK with a radiochemical purity of ≥95%. [⁶⁸Ga]Ga-DOTA-AeK showed good stability over time with less protein binding under physiological conditions. The bacterial incorporation of [⁶⁸Ga]Ga-DOTA-AeK and its fluorescent Aek-NBD analog were investigated in live and heat-killed <i>Escherichia coli</i> (<i>E. coli</i>) and <i>Staphylococcus aureus</i> (<i>S. aureus</i>). Unfortunately, no conclusive in vitro intracellular uptake of [⁶⁸Ga]Ga-DOTA-AeK was observed for <i>E. coli</i> or <i>S. aureus</i> live and heat-killed bacterial strains (<i>p</i> > 0.05). In contrast, AeK-NBD showed significantly higher intracellular incorporation in live bacteria compared to the heat-killed control (<i>p</i> < 0.05). Preliminary biodistribution studies of [⁶⁸Ga]Ga-DOTA-AeK in a dual-model of chronic infection and inflammation revealed limited localization at the infection site with non-specific accumulation in response to inflammatory markers. Finally, our study demonstrates proof that the intracellular incorporation of AeK is necessary for successful bacteria-specific imaging using PET/CT. Therefore, Ga-68 was not a suitable radioisotope for tracing the bacterial uptake of AeK tripeptide, as it required chelation with a bulky metal chelator such as DOTA, which may have limited its active membrane transportation. An alternative for optimization is to explore diverse chemical structures of AeK that would allow for radiolabeling with ¹⁸F or ¹¹C.