Abstract

Gallium-68 labeled peptide radiotracers have attracted significant attention in positron emission tomography (PET) imaging of various diseases. While 68 Ga-DOTA radiolabeling protocols are well-established, two classes of peptides, disulfide-directed multicyclic peptides (DDMPs) and amphiphilic antimicrobial peptides (AAMPs), presented challenges in achieving satisfactory radiolabeling efficiency using existing methods. This study therefore aimed to establish optimized 68 Ga-DOTA radiolabeling protocols specifically tailored to these peptides, in order to achieve high radiochemical purity (RCP) and radiochemical yield (RCY) suitable for clinical translation. By systematically varying key labeling conditions and purification methods such as pH, 68 GaCl 3 concentration, reaction time, purification cartridge and eluent types, and sterile filtration, two new radiolabeling protocols were developed for DDMP- and AAMP-based radiotracers. Detailed optimization identified an optimal pH of 3.6 combined with the use of concentrated 68 GaCl 3 for efficient radiolabeling of DDMPs, achieving RCY > 60% and RCP > 95%. For AAMPs, extended reaction time, hydrophilic–lipophilic balanced (HLB) cartridges, and ethanol/HCl elution improved RCY to >70% with RCP > 90%. These results demonstrate that precise pH control and concentrated 68 GaCl 3 are essential for high labeling efficiency of DDMPs, whereas optimized purification methods are key for AAMPs. Importantly, this work provides efficient radiolabeling strategies for these two clinically promising classes of peptides and offers insights for optimizing labeling protocols for other peptides. The established methods will support subsequent Good Manufacturing Practice (GMP)-compliant manufacturing and facilitate the clinical translation of high-value radiopharmaceuticals with favorable in vivo performance.