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.

Prostate-specific membrane antigen (PSMA) is a validated theranostic target with clinical relevance extending beyond prostate cancer to PSMA-expressing neovasculature in solid tumors. Despite the success of ¹⁷⁷Lu-PSMA therapy, a significant proportion of patients experience disease recurrence or partial responses, motivating the development of targeted alpha therapy (TAT) approaches. In this study, novel monomeric and dimeric ²²⁵Ac-labeled PSMA inhibitors, [²²⁵Ac]Ac-DOTA-iPSMA ([²²⁵Ac]Ac-D-iPSMA) and [²²⁵Ac]Ac-MACROPA-iPSMA ([²²⁵Ac]Ac-M-iPSMA), were designed through receptor-based computational screening, synthesized, radiolabeled, and evaluated in vitro and in vivo. Radiolabeling achieved high radiochemical purities (>96%), and saturation binding assays using lanthanum(III) surrogates confirmed cooperative nanomolar PSMA affinity. The binding affinities were Kd = 8.28 ± 0.51 nM (La-D-iPSMA) and Kd = 16.76 ± 2.51 nM (La-M-iPSMA). PSMA-specific uptake was validated in 4T1 breast cancer and HCT116 colorectal cancer cells. D-iPSMA induced superior late apoptosis (41.74% at 2 Gy), associated with reductions in GSK-3 α/β (Glycogen Synthase Kinase-3) and WNK1 (With No Lysine Kinase 1) phosphorylation, β-catenin downregulation, and dose-dependent DNA double-strand breaks (72.08% γ-H2AX [γ-phosphorylated histone]-positive cells at 6 Gy). [²²⁵Ac]Ac-M-iPSMA biodistribution in 4T1 tumor-bearing BALB/c mice revealed rapid blood clearance, a tumor-to-kidney dose ratio of 54:1, and a high tumor-absorbed dose. A combination of [¹⁷⁷Lu]Lu-D-iPSMA with [²²⁵Ac]Ac-M-iPSMA maximized reactive oxygen species generation. These findings support both conjugates as promising TAT candidates for PSMA-expressing tumors.