Supplementary Material

1) Supplementary Methods

Cell plating

For SNALP-mediated anti-miR-21/scrambled transfection, 24 hours before any experiment U87 cells were plated onto 6-well plates (1.5x105 cells/well, Western blot evaluation), 12-well plates (7x104 cells/well, miRNA and mRNA quantification), 24-well plates (3.5x104 cells/well, cell viability, caspase 3/7 activity and apoptotic cell death evaluation), 48-well plates (5x104 cells/well, flow cytometry) or 8-well plates (3x104 cells/well, confocal) at the indicated density, except otherwise stated.

Similarly, GL261 cells were plated onto 6-well plates (1.35x105 cells/well, Western blot evaluation), 12-well plates (6.5x104 cells/well, miRNA/mRNA quantification), 24-well plates (3.5x104 cells/well, cell viability and caspase 3/7 activity), 48-well plates (5x104 cells/well, flow cytometry) or 8-well plates (3x104 cells/well, confocal) at the indicated density, except otherwise stated. F98 cells were plated onto 8-well plates (confocal) at a density of 3x104 cells/well. Total RNA and protein were extracted 48 hours after cell exposure to liposomes, as described in Materials and Methods.

For experiments with sunitinib, 24 hours before any experiment U87 and GL261 cells were plated onto 24-well plates at a density of 3.5x104 cells/well (cell viability, caspase 3/7 activity and apoptotic cell death evaluation).

Confocal microscopy studies

Confocal microscopy images of cells were acquired in a point scanning confocal microscope Zeiss LSM 510 Meta (Zeiss, Germany), with a 40× EC Plan-Neofluar oil immersion objective, and diode (405 nm), argon (488 nm), DPSS (561 nm) and helium-neon (633 nm) lasers; cells were excited at 405 nm (Hoechst 33342), 488 nm (FAM) and 561 nm (rhodamine). Differential interference contrast (DIC) images were obtained using the helium-neon laser (633 nm). Digital images were acquired using the LSM 510 Meta software. All instrumental parameters pertaining to fluorescence detection and image analyses were held constant to allow sample comparison.

RNA extraction in cultured cells and cDNA synthesis

Total RNA from cultured cells was extracted using the miRCURY RNA extraction kit (Exiqon). Following RNA quantification, different transcription protocols were used depending on the type of RNA to be amplified by PCR.

For miRNA quantification, RNA conversion into cDNA was performed using the Universal cDNA synthesis Kit (Exiqon). For each sample, cDNA was produced from 25 ng of total RNA in an iQ5 thermocycler (Bio-Rad), by applying the following protocol: 60 min at 42°C and 5 min at 95°C. The cDNA was further diluted 1:60 with RNase-free water prior to quantification by qPCR.

For mRNA quantification, RNA conversion into cDNA was performed using the iScript™ cDNA synthesis Kit (Bio-Rad). For each sample, cDNA was produced from 1 µg of total RNA in an iQ5 thermocycler, by applying the following protocol: 5 min at 25°C, 30 min at 42°C and 5 min at 85°C. The cDNA was further diluted 1:3 with RNase-free water prior to quantification by qPCR.

QPCR quantification of mRNA expression

MRNA quantification in cultured cells was performed in an iQ5 thermocycler using 96-well microtitre plates and the iQ™ SYBR® Green Supermix Kit (Bio-Rad). The primers for the target genes (PDCD4, PTEN, survivin) and the reference gene (HPRT1) were pre-designed by Qiagen. A master mix was prepared for each primer set, containing a fixed volume of SYBR Green master mix, water and the appropriate amount of each primer to yield a final concentration of 150 nM. For each reaction, performed in duplicate, 10 μl of master mix were added to 2 μl of template cDNA. The reaction conditions consisted of enzyme activation and well-factor determination at 95°C for 1 min and 30 s followed by 40 cycles at 95°C for 10 s (denaturation), 30 s at 55°C (annealing) and 30 s at 72°C (elongation). The melting curve protocol consisted of 1 min heating at 55°C followed by eighty 10 s steps, with 0.5°C increases in temperature at each step. The percentage of mRNA knockdown was determined following the Pfaffl method for relative mRNA quantification in the presence of target and reference genes with different amplification efficiencies (47). The amplification efficiency for each target or reference gene was determined according to the formula: E=10(-1/S), where S is the slope of the standard curve obtained for each gene.

Protein extraction and quantification for electrophoretic separation

Total protein extracts were prepared from cultured U87 or GL261 cells, homogeneized at 4ºC in RIPA lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 50 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100) containing a protease inhibitor cocktail (Sigma), 2 mM DTT and 0.1 mM PMSF. The concentration of protein lysates was determined using the Bio-Rad Dc protein assay (Bio-Rad).

Evaluation of caspase 3/7 activity

Forty-eight hours after SNALP-mediated oligonucleotide transfection or immediately after exposure to sunitinib, cells were collected and lysed according to the manufacter’s instructions. Cell supernatant and caspase substrate were mixed and incubated in a black 96-well plate for 40 min (at RT), under gentle shaking. The production of the AMC fluorophore, produced as a result of caspase action on the substrate, was measured for a period of 6 to 8 hours, using a microplate reader (SpectraMax Plus 384, Molecular Devices) at excitation/emission of 354/442 nm. Results, presented as relative fluorescence units (RFU) to control untreated cells, were normalized for the number of cells in each condition.

Cell viability

At defined time points, 10% (v/v) resazurin dye in complete DMEM medium was added to each well and cells were incubated at 37°C until the development of a pink coloration. Two-hundred microliters of supernatant were collected from each well, transferred to clear 96-well plates and the absorbance at 570 (reduced form) and 600 nm (oxidized form) was measured in a microplate reader (SpectraMax Plus 384, Molecular Devices). Cell viability was calculated as percentage of control cells using the equation: (A570 − A600) of treated cells × 100 / (A570 − A600) of control cells.

Histological evaluation of brain sections (cresyl violet staining)

Frozen brain sections (20 μm-thick) were allowed to thaw at room temperature, until they were completely dried. The subsequent steps of the protocol were performed in a fume hood with individual protection (mask, goggles and gloves). Sections were emerged in cresyl violet solution for 10 min, washed twice in acetate buffer (80% acetic acid, 20% sodium acetate), washed twice with absolute ethanol and then emerged in xylene for 5 min. Excess solution was removed between washes. Sections were mounted with two drops of Eukitt mounting medium (Sigma) and a glass cover was placed on top for protection. Slides were maintained at RT (protected from light) until observation under the microscope.

Homogenization of tissue samples for fluorescence detection by flow cytometry

Four hours after intravenous injection of SNALP-formulated FAM-labeled siRNAs (2.5 mg/kg), animals were sacrificed by intracardiac perfusion with 30 mL of ice-cold PBS and brain, lungs, liver and kidneys were harvested into polystyrene tubes containing PBS supplemented with 2% FBS. The brains were further dissected to separate tumors from healthy brain tissue. Tissue processing was performed on ice and protected from light (whenever possible) in order to minimize the loss of viable cells and protect the fluorescent probe.

Healthy brain and tumors were homogenized using a mortar and pestle (approximately 50 movements were executed, until pieces of tissue were no longer differentiated); liver, kidneys and lungs were disrupted into smaller pieces using a scalpel, and further incubated in 1 mL of filtered lysis buffer (PBS, 2% FBS, 0.6% BSA, 1 mg/mL colagenase IV) for 30 min (lungs) or 1 hour (liver, kidneys). Vortex was performed every 15 min and tissue homogenates were then strained through 40 μm strainers (BD Biosciences), in order to obtain a uniform cell suspension and then transferred to 50 mL polystyrene tubes and washed twice with ice-cold PBS. Erythrocyte lysis was performed in lung homogenates by incubation with 500 μL of ACK lysing buffer (Lonza, USA) for 8 min at 37ºC. Cells were centrifuged at 2000 rpm for 5 (brain, tumor) or 8 (lungs, liver, kidneys) min (at 4ºC), the supernatant was removed and the cells were resuspended in 1 (brain, tumor, lung, kidney) or 2 (liver) mL of PBS supplemented with 2% FBS. From this cell suspension, 1x106 cells were transferred to 1.5 mL polystyrene tubes for detection of fluorescence (triplicates per condition were performed), whereas the remaining cells were processed for cryopreservation. Cells were centrifuged at 5000 rpm for 5 min (at 4ºC), the supernatant was removed and cells were fixed with 150 μL of Cytofix/Cytoperm™ solution (BD Biosciences) for 20 min at 4ºC. Cells were then washed twice with Perm/Wash™ solution (BD Biosciences), centrifuged at 5000 rpm for 5 min (at 4ºC) and further incubated with 50 μL of anti-FAM antibody (Sigma, clone FL-D6, 1:100), prepared in Perm/Wash™ solution, for 30 min (at 4ºC). After incubation with a primary anti-FAM antibody, cells were washed twice with Perm/Wash™ solution (BD Biosciences) and further incubated with 50 μL of an Alexa488-conjugated anti-mouse secondary antibody (1:200; Molecular Probes, Life technologies, USA), prepared in Perm/Wash™ solution, for 30 min (at 4ºC). After being washed twice with Perm/Wash™ solution, samples were ressuspended in 500 μL of PBS supplemented with 2% FBS and immediately analyzed in a FACS Calibur flow cytometer.

2) Supplementary Data

See Supplementary Table S1 in attachment.

Supplementary Table S1. Oligonucleotide, siRNA and primer sequences.

3) Supplementary Results

See Supplementary Figures 1, 2, 3, 4 and 5 in attachment.

Supplementary Figure 1. Association of SNALPs with mouse primary astrocyte cells.

Supplementary Figure 2. Internalization of SNALPs in GL261 mouse and F98 rat glioma cells.

Supplementary Figure 3. Tumor cell internalization of SNALPs three months after their preparation.

Supplementary Figure 4. Association and internalization of liposomes encapsulating anti-survivin siRNAs in survivin-expressing U87 glioma cells, and effect on survivin mRNA expression.

Supplementary Figure 5. Effect of sunitinib on the viability of GL261 glioma cells.

References for supplementary material

46. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29(9):e45.