4.9. Intracellular Uptake of Boron Compounds into SCC VII Cells and U87MG Delta EGFR Cells Evaluated with ICP-OES

SCC VII cells and U87MG delta EGFR cells were seeded on 10 cm dishes in cell culture mediums and incubated in 37 °C under 5% CO₂ incubator until 70~80% confluent. They were treated with boron compounds 1 (B1), 2 (B2), 4, or BPA in cell culture mediums for 4 h. The concentration of 10B in the medium was 0.35 or 0.7 ppm for B1 and B2, 0.48 ppm or 2.42 ppm for compound 4 and 20 ppm for BPA. A stock solution of ¹⁰B-para-boronophenylalanine (BPA) was used for all experiments. BPA was purchased from KatChem Ltd. (Prague, Czech Republic) and prepared by dissolving it in distributed water as a complex with 3% fructose for BPA. The ¹⁰B concentrations were measured by means of prompt gamma-ray spectrometry using a thermal neutron guide tube installed at the Kyoto University Research Reactor (KUR), and the value was about 1000 ± 4.55 ppm. To count the number of cells after treatment with the boron compounds, the cells (n = 1) were washed with PBS, detached by trypsin, and counted with a hemocytometer. For the measurement of boron uptake, the cells (n = 3) were washed with PBS (3 mL × 3) and digested with 60% HNO₃ aq (1 mL) at room temperature for 24 h, which were transferred to 15 mL centrifuge tubes with Milli-Q water (4 mL). These tubes were centrifuged at 3000 rpm and 4 °C for 10 min, and the resulting sample solutions were filtered. The concentration of boron atoms was determined by ICP-OES (ICPE-9000, Shimadzu, Kyoto, Japan). All reagents were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).