Wizard2

10 μL of [¹⁸F]AlF-PSMA-137 (1-2 MBq), 490 μL of phosphate-buffered saline (0.01 M, pH 7.4) and 500 μL of octanol was added into a tube. The mixture was vortexed for 3 min and centrifuged (3000 rpm, 5 min). Then, 5 samples (50 μL) from each phase were quantified using a γ-counter (Wizard II, Perkin Elmer Inc., Germany). The experiment was repeated 3 times. The value was expressed as logD7.4 (mean ± SD).
Stability in vivo and in vitro was studied by analyzing the radiochemical purity with radio-HPLC. For the in vitro stability, [¹⁸F]AlF-PSMA-137 was incubated in saline and 5% human serum albumin (HSA) for 1 h, 2 h and 4 h at 37°C. For in vivo stability assay, 37 MBq of [¹⁸F]AlF-PSMA-137 was injected into normal BALB/c mice, and the blood and urine were collected and treated at 5 min, 30 min and 60 min after injection.

The binding affinity of [¹²⁵I]IB-Mal-d-GDDDK-5F7 and [¹²⁵I]IB-Mal-d-GEEEK-5F7 for HER2 was determined using a saturation binding assay on the HER2-expressing BT474M1 cell line. Cells were seeded in 24-well plates at a density of 8 × 10⁴ cells/well and incubated at 37 °C for 24 h. On the next day, the cells were incubated in triplicate with increasing concentrations (0.1–100 nM; final volume 600 μL) of [¹²⁵I]IB-Mal-d-GDDDK-5F7 or [¹²⁵I]IB-Mal-d-GEEEK-5F7 for 2 h at 4 °C. A parallel assay was performed in which cells were coincubated with a 100-fold molar excess of trastuzumab, which competes with 5F7 for HER2 binding [17 (link)], to determine nonspecific binding at each concentration. The medium containing unbound activity was removed, and cells were washed twice with cold PBS and solubilized by treatment with 1 M NaOH (0.5 mL) at 37 °C for 10 min. Cell-associated activity was determined using a Perkin Elmer Wizard II (Shelton, CT, USA) dual-channel gamma counter. The dissociation constant (K_D) was determined by nonlinear regression using GraphPad Prism software (version 5.01). Triplicate samples were used for each concentration, and the entire experiment was repeated twice.