Figure 2.

Inhibition of c-Myb protein expression and increase of survival of neuroblastoma-bearing mice after treatment with c-myb antisense oligonucleotides (asODNs)-containing nanocarriers. (A) GI-LI-N neuroblastoma cells were treated with CpG-containing c-myb asODNs, either free (free CpG-myb-as) or encapsulated in untargeted [CCL(CpG-myb-as)] and targeted [Fab′-GD₂-CCL(CpG-myb-as)] nanocarriers, at a concentration of 100 μg/ml at the beginning of the experiment and 18 and 36 h later. Two hours after each addition, the cells were washed and transferred to CpG-myb-as-free fresh complete medium. The cells were harvested at 48 h (upper panel) or at the indicated time points (lower panels) and analysis of protein expression (c-Myb and c-Myc as control) was performed by immunoblotting. (B) Nude mice (n = 10 animals/group) were injected intravenously with 3.5 × 10⁶ HTLA-230 cells. Treatment with either as or scrambled (scr), CpG-containing ODNs, administered free and encapsulated in untargeted (CCL) and targeted (Fab′-GD₂-CCL) liposomes was started at 4 h after cell inoculation. Mice were treated 4 days a week, for 2 weeks, with 3 day rest between treatments. Each mouse received 50 μg ODN. Control mice (CTR) received HEPES-buffered saline. (C,D) Effects of either macrophages or natural killer (NK) cells depletion on anti-tumor activity mediated by treatment with liposomal formulations containing ODNs. Mice [nude, n = 8 animals/group (C) and SCID-bg, n = 10 animals/group (D)] were inoculated with HTLA-230 cells and then treated as already mentioned in Figure 1. In some treatment groups of (C), mice were injected with Clodronate-liposomes to deplete macrophages.