Peptide synthesis, characterization and ⁶⁸Ga-radiolabeling of NOTA-conjugated ubiquicidin fragments for prospective infection imaging with PET/CT

Introduction: Human antimicrobial peptides are of interest for the development of positron emission tomography (PET) tracers as they exhibit desirable characteristics that make them good candidates for targeting vectors. Due to their natural role in the innate immune system they selectively bind to pathogenic bacteria and yeast, whilst remaining minimally immunogenic and cytotoxic to humans. Research into ubiquicidin (UBI)-based tracers has focused on (99m)Tc as a radionuclide, however, the use of bi-functional chelators such as 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), in combination with ⁶⁸Ga as a radionuclide, allows for a simple radiolabeling procedure which is preferable in a clinical setting using PET/CT.

Methods: The peptides fragments UBI29-41, UBI30-41 were synthesized by standard microwave Fmoc/tert-butyl (tBu)-solid phase synthetic protocols. Characterizations were performed using analytical HPLC and LC/MS. Both NOTA-conjugated peptides were exposed to (nat)Ga³⁺; their complexed form was quantified by direct LC/MS injection. This complexation was utilized to testify bacterial and mammalian cell binding potential of fluorophore-linked NOTA-UBI29-41/30-41. ⁶⁸Ga labeled NOTA-UBI fragments were also tested for competitive interaction to Staphylococcus aureus to proof the binding target. ⁶⁸Ga was eluted from SnO₂- and TiO₂-based ⁶⁸Ge/⁶⁸Ga generators using fractionated elution and anion exchanged-based post-procession. NOTA-peptide radiolabeling was carried out including optimization of buffer molarity, NOTA-peptide concentration(s), incubation temperature and -duration as well as considering various SPE purification cartridges.

Results: Pure UBI29-41, UBI30-41 and NOTA-UBI30-41 were successfully characterized. Both, NOTA-UBI fragments exhibited complexation rates to (nat)Ga³⁺)≥ 99%. The percentage binding was significantly higher to Staphylococcus aureus bacilli over Mt4 human leucocytes (P>0.05) for NOTA-UBI29-41[Lys(Abz)]<NOTA-UBI30-41[Lys(Abz)]. Significant lower binding was observed for both ⁶⁸Ga-labeled NOTA-UBI fragments (P >0.03) after pre-incubation with excess unlabeled NOTA-UBI. Reproducible ⁶⁸Ga radiolabeling ranged for 51-85% and 46-78% for NOTA-UBI29-41 and NOTA-UBI30-41, respectively.

Conclusion: Aside from successful peptide syntheses the first ever ⁶⁸Ga-radiolabeling method is reported for NOTA-UBI fragments. The NOTA-conjugation didn't compromise the selective and specific interaction with bacterial cells in vitro. Both tracers are warranting prospective imaging of infection with PET/CT.