Enhancing Cytosolic Internalization of [¹⁷⁷Lu]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, [¹⁷⁷Lu]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 [¹⁷⁷Lu]Lu–iPSMA–iGRP78 and 2.245 nM for [¹⁷⁷Lu]Lu–iPSMA. The dimeric radiopharmaceutical achieved 10.44 ± 2.43% cytosolic internalization and 4.81 ± 0.94% nuclear internalization, while the [¹⁷⁷Lu]Lu–iPSMA monomer showed 6.45 ± 0.60% cytosolic internalization and no uptake in the cell nucleus. In PC3 cells (PSMA–, CS-GRP78–), [¹⁷⁷Lu]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 [¹⁷⁷Lu]Lu–iPSMA (38.63 ± 6.13% of dead cells). In conclusion, conjugation of a GRP78 inhibitor to [¹⁷⁷Lu]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.