Mice bearing the orthotopic model were treated starting from day 21 after NB cell implant; mice with the pseudo-metastatic model received the first treatment 4h after NB cell injection. These therapeutic schedules were designed to test the effects of our targeted formulations against both established and pseudo-metastatic preclinical models of human NB, as described [16,19]. Animals were treated i.v. once a week for 3 weeks with untargeted (SL[DXR]) or peptide-targeted SL[DXR] (5mg/kg). Scrambled peptide-functionalized liposomes were used as a control, and in every experiment a group of control mice received HEPES-buffered saline. Survival times were used as the main criterion for determining treatment efficacy. In the orthotopic model, time-dependent anti-tumor activity was also evaluated by bioluminescence imaging (BLI) and X-ray analyses. For this purpose, the GI-LI-N cell line was infected with a retrovirus expressing the firefly luciferase gene, as previously reported [17]; luciferase activity of retrovirally-transduced cells was visualized in vivo by BLI (IVIS Caliper Life Sciences, Hopkinton, MA) after a 10min incubation with 150μg/mL of d-luciferin (Caliper Life Sciences), as described [17]. X-ray analysis was superimposed to the luminescence for a better visualization of the tumors.
