*3.6. Cell Internalization*

The HEP-G2 (human hepatocellular carcinoma), PC-3 (human prostate cancer), and fibroblasts cell lines, used for the internalization assays, were cultured in RPMI-1640 medium containing antibiotics (penicillin and streptomycin; 100 µg/mL) and fetal bovine serum at a concentration of 15% in an atmosphere of 5% carbon dioxide and 37 ◦C.

HEP-G2, PC-3, and fibroblast cells were harvested and diluted in PBS (pH 7.4). Each cell line (1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/tube) received two different treatments: (a) <sup>225</sup>Ac-rHDL (4 kBq/200 µL PBS at pH 7.4) (*n* = 3), and (b) <sup>225</sup>Ac-DOTA-benzene-p-SCN (4 kBq/200 µL PBS at pH 7.4) (*n* = 3). Cells were incubated with each treatment at 37 ◦C for 1 h. After the incubation time, tubes were measured in a well-type NaI(Tl) scintillation detector to determine the initial activity (100%). The tubes were centrifuged at 500× *g* for 10 min. The button was washed 2 times with PBS, pH 7.4. Then, a mixture of acetic acid/0.5 M NaCl was added, and the tubes were centrifuged again at 500× *g* for 10 min. The supernatant was removed, and activity of the button was measured, which corresponded to the percentage of activity internalized in the cells with regard to the initial activity.
