Simple SummaryThere is a need to develop new theranostic approaches for malignant melanoma. Only 35% of patients with metastatic melanoma reach the milestone of 5-year survival, despite the success of new immunotherapy. We have developed a new class of peptides to target melanocortin-1 receptors (MC1Rs) that display elevated levels in human melanoma. In this study, we examined the melanoma targeting and biodistribution properties of two67Cu-tagged peptides in tumor-bearing mice. We found that one of the peptides, namely67Cu-NOTA-PEG2Nle-CycMSHhex, exhibited favorable melanoma targeting and biodistribution properties that underscored its potential as an MC1R-targeted therapeutic peptide for melanoma treatment in the future.AbstractThe aim of this study was to evaluate the effect of linker on tumor targeting and biodistribution of67Cu-NOTA-PEG2Nle-CycMSHhex{67Cu-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-polyethylene glycol-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and67Cu-NOTA-GGNle-CycMSHhex{67Cu-NOTA-GlyGlyNle-CycMSHhex} on melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhexand NOTA-GGNle-CycMSHhexwere synthesized and purified by HPLC. The biodistribution of67Cu-NOTA-PEG2Nle-CycMSHhexand67Cu-NOTA-GGNle-CycMSHhexwas determined in B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of67Cu-NOTA-PEG2Nle-CycMSHhexwas further examined in B16/F10 melanoma-bearing C57 mice.67Cu-NOTA-PEG2Nle-CycMSHhexexhibited higher tumor uptake than67Cu-NOTA-GGNle-CycMSHhexat 2, 4, and 24 h post-injection. The tumor uptake of67Cu-NOTA-PEG2Nle-CycMSHhexwas 27.97 ± 1.98, 24.10 ± 1.83, and 9.13 ± 1.66% ID/g at 2, 4, and 24 h post-injection, respectively. Normal organ uptake of67Cu-NOTA-PEG2Nle-CycMSHhexwas lower than 2.6% ID/g at 4 h post-injection, except for kidney uptake. The renal uptake of67Cu-NOTA-PEG2Nle-CycMSHhexwas 6.43 ± 1.31, 2.60 ± 0.79, and 0.90 ± 0.18% ID/g at 2, 4, and 24 h post-injection, respectively.67Cu-NOTA-PEG2Nle-CycMSHhexshowed high tumor to normal organ uptake ratios after 2 h post-injection. The B16/F10 melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT) using67Cu-NOTA-PEG2Nle-CycMSHhexas an imaging probe at 4 h post-injection. The favorable tumor targeting and biodistribution properties of67Cu-NOTA-PEG2Nle-CycMSHhexunderscored its potential as an MC1R-targeted therapeutic peptide for melanoma treatment.