Figure 1: SNALP internalization in U87 human GBM cells and mouse astrocytes, PTEN/PDCD4 expression, tumor cell proliferation and biodistribution of systemically-administered SNALP-formulated FAM-labeled oligonucleotides. For evaluation of SNALP internalization, (A, B) U87 cells and (C, D) mouse astrocytes were incubated with rhodamine-labeled CTX-coupled (CTX) or nontargeted (NT) liposomes encapsulating FAM-labeled anti-miR-21 oligonucleotides (for 4 hours at 37°C), at a final oligonucleotide concentration of 1 μM. Cells were then rinsed twice with PBS, stained with DNA-specific Hoechst 33342 (blue) and then observed by confocal microscopy. (E) PTEN and PDCD4 expression levels in U87 cells (corrected for individual α-tubulin signal intensity) 48 hours after cell incubation with CTX-coupled liposomes encapsulating anti-miR - 21 or scrambled oligonucleotides, as determined by Western blot. Results are presented as PTEN and PDCD4 expression levels relative to control. * p<0.05 to cells incubated with a similar amount of CTX-coupled liposomes encapsulating scrambled oligonucleotides. (F) For evaluation of tumor cell proliferation, U87 cells were incubated with CTX-coupled liposomes encapsulating anti-miR-21 or scrambled oligonucleotides for 4 hours, washed with PBS and further incubated for 24 hours with fresh medium. Cells were subsequently exposed to 15 μM of sunitinib for 24 hours, rinsed with PBS, after which cell viability was evaluated by the Alamar Blue assay. Scrambled/anti-miR-21 1 μM + S15: cells transfected with scrambled or anti-miR-21 oligonucleotides and further incubated with 15 μM sunitinib.* p<0.05 compared to cells incubated with SNALP-formulated scrambled oligonucleotides and further treated with 15 μM sunitinib. (G) Flow cytometry analysis (fluorescence intensity plots) of tumor and brain homogenates from animals injected intravenously with CTX-coupled and NT liposomes encapsulating FAM-labeled siRNAs or saline solution (PBS). # p<0.05 compared to animals injected with a similar amount of NT SNALP-formulated siRNAs. Adapted from Costa et al. [68].