Wizard2 automatic γ counter

The lipophilicity of the [¹⁸F]-ATRi was acquired by adding 2.5 μCi to a mixture of 0.5 mL of 1-octanol and 0.5 mL of 25 mM phosphate buffered saline (pH 7.4) and mixed for 5 min. Then, the mixture was centrifuged at 15.000 rpm for 5 min. 100 μL samples were obtained from organic and aqueous layers, and the radioactivity of the samples were measured in a γ-counter WIZARD₂ automatic γ-counter (PerkinElmer, Boston, MA). The experiment was performed in triplicate, and the resulting logP_o/w was calculated as the mean ± SD.

Twenty-four hours prior to the in vitro studies, KB cells as well as A549 cells (1.0 × 10⁶ cells/well, >90 % viability for both lines) were seeded into 6-well plates containing folate-free RPMI 1640 medium (2.0 ml) and incubated to form subconfluent monolayers of the cells. Then, medium was removed from the wells, cells were washed with PBS (1 ml) and medium (900 µl) was added. After 30 min of incubation at 37 °C, the radiotracer (0.1 nmol, 100 µl) in PBS was added to the dedicated well (c = 100 nM). For blocking studies, cells were pre-incubated with an excess of folate (c = 10 µM) 5 min prior to the addition of radiotracer. After 30 min post incubation at 37 °C, the supernatant of each well was collected together with washing solution (ice-cold PBS, 2 × 1.0 ml). Then, cells were treated with 1 M sodium hydroxide solution (1.0 ml) for 10 min at room temperature. Finally, cell suspension was collected together with washing solution (ice-cold PBS, 2 × 1.0 ml). The radioactivity of all contents including initially added radiotracer (0.1 nmol, 100 µl) was measured using a WIZARD² automatic γ-counter from PerkinElmer. Receptor-specific uptake was calculated by cell-bound activity and expressed as a percentage of applied activity per million cells.

A binding assay was performed to determine the selectivity of the [¹⁸F]AlF-NODA-scVR1 for the VEGFR-1 receptor based on previously described protocols [16 (link)]. Commercially available HisPur™ Ni-NTA (Nickel-Nitrilotriacetic acid) magnetic beads (Thermo Fisher Scientific, Waltham, MA) were incubated with 1 μg of murine Fc-VEGFR-1 (BPS Biosciences, San Diego, CA) with an N-terminal His-tag at room temperature for 15 minutes in phosphate buffered saline with 1% bovine serum albumin for the tracer only and blocking cohorts. In the control group no VEGFR-1 was used and thus no receptor was bound to the surface of the magnetic beads. The beads were then washed and subsequently incubated for 5 minutes with approximately 1 ng of [¹⁸F]AlF-NODA-scVR1 in the control and tracer cohorts and 1 ng of [¹⁸F]AlF-NODA-scVR1 along with 5.0 μg of scVR1 in the blocking cohort. The beads were then washed by placing vials in a magnetic bead rack and collecting the supernatant. The bound tracer and unbound tracer collected in the washes was measured using a Wizard² automatic γ-counter (PerkinElmer, Waltham, MA).