Development of a Radiogallium-Labeled Heterodivalent Imaging Probe Targeting Negative Charges and Integrin on the Surface of Cancer Cell Membranes

Radiopharmaceuticals targeting tumor-specific environments are powerful tools for cancer diagnosis and treatment. We previously demonstrated the considerable high tumor uptake of the cationic amphiphilic peptide, ⁶⁷Ga-NOTA-KV6, in vivo. However, because this radioligand shows a relatively rapid clearance from the tumor over time, further structural optimization is necessary. In this study, to enhance tumor accumulation and retention, we synthesized and evaluated a heterobivalent radiogallium-labeled radiotracer, [⁶⁷Ga]Ga-DOTA-KV6-Mal-c(RGDyK) ([⁶⁷Ga]<b>6a</b>), fusing the KV6 peptide targeting negatively charged sites on the cancer cell membrane and cyclic RGD peptide targeting integrin α_vβ₃ on the cancer cell membrane. Cellular uptake study revealed high accumulation of [⁶⁷Ga]<b>6a</b> in integrin α_vβ₃-expressing U-87MG cancer cells, but uptake was significantly inhibited in the presence of an excess of the cyclic RGD peptide, c(RGDyK) (<b>1</b>). Peptide <b>6a</b> exhibited integrin α_vβ₃-binding affinity comparable to those of RGD peptides <b>1</b> and DOTA-Mal-c(RGDyK) (<b>8</b>). In vivo biodistribution studies of U-87MG tumor-bearing mice revealed that [⁶⁷Ga]<b>6a</b> exhibited better accumulation and retention in tumor tissues than [⁶⁷Ga]Ga-DOTA-KV6-Mal-Et ([⁶⁷Ga]<b>6b</b>; without the RGD peptide motif) and [⁶⁷Ga]Ga-DOTA-Mal-c(RGDyK) ([⁶⁷Ga]<b>9</b>; without the KV6 peptide motif). Single-photon emission computed tomography analysis also revealed high signals of [⁶⁷Ga]<b>6a</b> in tumor tissues, which were significantly blocked in the presence of excess peptide <b>1</b>. Although reducing radiotracer accumulation in nontumor tissues, such as the kidneys, remains a challenge, our developed approach exhibits potential to enhance the selectivity and retention of radiopharmaceuticals in tumor tissues.