In vivo prostate tumor xenograft assay

PC-3 cells were grown to 60–70% confluent in T75 flasks. Next, the cells were collected and suspended in sterile normal saline. Cell suspension (3×10⁶ cells/100 µl) was subcutaneously injected into the right flank of 6 to 8-week-old male athymic nude mice (Harlan Laboratories, Inc.). All treatments (empty liposomes, SSL-IPA3, and SPRL-IPA3 (5 mg/kg) were started on day 3 from tumor implantation and were administered two times a week, by intraperitoneal (i.p.) injection. Tumor diameters were measured using digital calipers on days 7, 14, 19, and 21, and the tumor volume (mm³) was calculated by the modified ellipsoidal formula (tumor volume=½ [length × width²]). The average size of the tumors before treatment was 42 mm³. Mice were sacrificed on day 25 and tumors were dissected, weighed, and snap-frozen for further analysis. We included 12 mice in each group at the start of the experiment. However, one mouse from the empty liposome group, one from the SSL-IPA3 group and two mice from the SPRL-IPA3 group were later removed due to sickness.