Enhancing Cytosolic Internalization of [(177)Lu]Lu-iPSMA in Prostate Cancer Cells: The Effect of Conjugating a GRP78 Inhibitor to the Radiotherapeutic Molecule.

Castration-resistant prostate cancer presents radiotherapeutic challenges, especially in optimizing the cytosolic internalization of therapeutic radiopharmaceuticals. This research aimed to design and evaluate in vitro, a new dimeric radiopharmaceutical, [(177)Lu]Lu-iPSMA-iGRP78, which combines PSMA and GRP78 inhibitors in a heterodimeric radioligand to improve the radionuclide internalization and cytotoxicity efficacy. Molecular docking showed that the dimer iPSMA-iGRP78 presents a higher affinity for GRP78 (CNN-docking score: -14.0 kcal·mol(-1), pKi: 10) and for PSMA (CNN-docking score: -17.0 kcal·mol(-1), pKi: 11.5) compared to the monomers iGRP78 (CNN-docking score: -11.0 kcal·mol(-1), pKi: 9.4) and iPSMA (CNN-docking score: -13.9 kcal·mol(-1), pKi: 10.2). The saturation binding assay using LNCaP cells (PSMA+, CS-GRP78+) showed an affinity (K(d)) of 1.883 nM for [(177)Lu]Lu-iPSMA-iGRP78 and 2.245 nM for [(177)Lu]Lu-iPSMA. The dimeric radiopharmaceutical achieved 10.44 ± 2.43% cytosolic internalization and 4.81 ± 0.94% nuclear internalization, while the [(177)Lu]Lu-iPSMA monomer showed 6.45 ± 0.60% cytosolic internalization and no uptake in the cell nucleus. In PC3 cells (PSMA-, CS-GRP78-), [(177)Lu]Lu-iPSMA-iGRP78 uptake was negligible, demonstrating specificity. Treatment with the dimeric radiopharmaceutical reduced cell viability (69.93 ± 4.85% of dead cells) significantly more than [(177)Lu]Lu-iPSMA (38.63 ± 6.13% of dead cells). In conclusion, conjugation of a GRP78 inhibitor to [(177)Lu]Lu-iPSMA improves the radionuclide internalization and cytotoxicity in prostate cancer cells, suggesting that the bispecific radiopharmaceutical is a promising strategy in prostate cancer treatment.