Figure 3.

Neuroblastoma-targeted nanoparticles entrapping siRNA specifically knockdown ALK. (A) Uptake and internalization of liposome-encapsulated FAM-labeled scrambled-siRNA (scr-siRNA-FAM) into GD₂-expressing neuroblastoma cells (SH-SY5Y). Cells were incubated at 37°C for 1 h, with either untargeted [CCL(scr-siRNA-FAM)] or Fab′ fragments GD₂-targeted coated cationic liposomes [Fab′-GD₂-CCL(scr-siRNA-FAM)]. After washing and cytospin centrifugation, cells were fixed and stained with a monoclonal antibody specific for the cellular adhesion molecule N-CAM (a-CD56) to reveal plasma membrane localization. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The cellular distribution of scr-siRNA-FAM (green), CD56 (red), nuclear DAPI staining (blue), and merged colors resulting from siRNA-liposome binding to the cell surface (orange) fluorescences was analyzed with a laser scanning spectral confocal microscope. Bars: 50 μm. (B–D) Tumor growth inhibition in vivo by ALK-siRNA encapsulated in Fab′-GD₂-CCL. SCID-bg mice (n = 8) were orthotopically injected with 1.5 × 10⁶ SH-SY5Y cells in the capsule of the left adrenal gland. Seven days after the tumor implantation, mice were treated, two-time a week for 3 weeks with ALK-siRNA, either free or encapsulated in GD2-targeted nanocarriers [Fab′-GD₂-CCL(ALK-siRNA)]. Another group of mice received a scrambled (scr) siRNA-loaded nanoparticles [Fab′-GD₂-CCL (scr-siRNA)] as control or HEPES-buffered saline (CTR). Tumor expansion over time measured by calipers (B) and survival times (D) were used for determination of the treatment efficacy. (C) The day after the last treatment (25 day), tumors from three mice per group were recovered for western blot analyses and ALK protein expression. *P < 0.05; **P < 0.01; ***P < 0.001, Fab′-GD₂-CCL(ALK-siRNA) vs. Fab′-GD₂-CCL(scr-siRNA).