Cell lines and animals
SPC-A1, a well-characterized human lung adenocarcinoma cell (HLAC) line, was obtained from Shanghai Cell and Biology Institute. Cells were maintained in RPMI 1640 medium (Gibco, USA) supplemented with 10% bovine serum (Gibco, USA), 2mmol/L L-glutamine and antibiotics (100U/mL penicillin and 100 μg/mL streptomycin) in a humidified atmosphere of 5% CO2 at 37. The 8-week-old male BALB/C nude and immunocompetent mice (body weight 20±2g) bought from Chinese Academy of Science, were kept in filter-topped cages with standard rodent chow and water available ad libitum, and a 12 hours light/dark cycle. Those nude mice were randomly allocated into different groups and there were 6 mice for each group. All animal protocols were approved by the Ethical Committee on Animal Experiments of the University of Fudan Animal Care Committee, Shanghai, China. All efforts were made to minimize suffering.
Human EGFR (NM_005228) gene targeted siRNA-EGFR (5′-GGAGCUGCCC AUGAGAAAUdTdT-3′/5′-AUUUCUCAUGGGCAGCUCCdTdT-3′) and non-specific siRNA-NEG (5′-GGAGCUGCCCAUGAGAAAUdTdT-3′/5′-AUUUCUCAUGGGCAG CUCCdTdT-3′), designed as previous description
, were chemically synthesized and annealed by GenePharma (Shanghai, China). Aqueous siRNA (20 μmol/L and 200 μmol/L) were subpackaged and stored at −80.
LPEI/siRNA complexes preparation and cell transfection in vitro
Commercial low molecular weight (22 kDa) Linear-PEI (jet PEITM), for in vitro DNA transfection, was bought from PolyPlus-transfection (Illkirch, France) and stored at −80. LPEI consisted of 7.5 mmol/L of NH4
+. LPEI/siRNA was made into a complex. In brief, 5 μl siRNA was dissolved in 100 μl of 10 mmol/L HEPES-150 mmol/L NaCl, pH 7.4, and incubated for 10 minutes. LPEI (2.5 μl) was dissolved in 100 μl of the same buffer and, after 10 minutes, was pipetted into the siRNA solution. This gave a net molar excess of ionizable nitrogen of LPEI to phosphate of siRNA (N/P) at a ratio of 5 as suggested for DNA by the manufacturer. Corresponding LPEI/siRNA complexes were constructed at N/P ratios of 3, 5 and 10. The total 200 μl mixture was vortexed and incubated at room temperature for 20 minutes to form a stable LPEI/siRNA-EGFR complex, and then dropped slowly into each well of 6-well plates. Serum-free DMEM was supplemented to a final volume of 2 mL, and 4 hours later replaced with RPMI 1640 medium.
Cells were harvested 24 hours after transfection. Total RNA was isolated from cells using TRIZOL Reagent (Invitrogen, USA), purified as recommended by the manufacturer, and then reverse-transcribed with M-MLV Reverse Transcriptase Kit (Promega, USA) following the manufacturer’s instructions. Real-time RT-PCR was performed using SYBR Green Real time PCR Master Mix (TOYOBO, Japan) and amplified with a Roter-Gene 3000 Amplification System (Corbett Research, Australia). Primer sequences used for EGFR (forward 5′-ACCGTGCCCTGATGGATGA-3′, reverse 5′-CCACGGTGGAATTGTT GCTG-3′) and β-actin (forward 5′-ATGACCCAGATCATGTTTGAGACC-3′, reverse 5′-GGAGGGCATACCCCTCGTAGA-3′) were designed and synthesized by Sangon Co. (Shanghai). All samples’ EGFR mRNA expression levels were normalized by β-actin amplification.
Flow cytometry assays were performed at 48 hours after transfection for the EGFR numbers scoring. After trypsinizing from plates, cells were washed twice with 1×PBS, then incubated with 20 μl of R-PE-conjugated mouse anti-human EGFR monoclonal antibody (BD Biosciences, USA) for 15 min in the dark at room temperature. Afterwards, cells received the same washing process and fixed in 0.5 mL of 4% paraformaldehyde. The EGFR numbers were analyzed on a Becton Dickinson FACScan flow cytometry with excitation and emission settings of 488 nm and 575 nm, respectively. To determine the change of EGFR numbers in SPC-A1 cells, we used positive cell percentage × mean intensity to evaluate the intensity of fluorescence.
For specificity evaluation, non-specific siRNA-NEG was complexed by LPEI at corresponding N/P ratios and thereafter transfered into SPC-A1 cells. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) complexed siRNA-EGFR transfecting group was set as a positive control. The non-treatment group was taken as a blank control. EGFR mRNA or protein expression levels were normalized by setting the amount of blank control at 1.0.
Size and zeta-potential measurements
Dynamic light scattering (DLS) was employed to determine hydrodynamic diameters of the complexes, and laser Doppler anemometry (LDA) was utilized to measure their zeta potentials. LPEI complexed siRNA nanoplex was prepared as above. Plasmid DNA (p ShNEG) provided by pSilencerTM 2.1-U6 neo kit (Ambion, Inc., Austin, TX, USA) was complexed with LPEI at the same N/P ratio of 5 as the manufacturer’s instructions. When the LPEI/siRNA and LPEI/DNA complexes were formed, their mean particle size and zeta-potential distribution were measured using a Zeta potential/particle Sizer NicompTM 380 ZLS (PSS Nicomp particle size system, USA). Position and attenuator were optimized by the device. Measurements were conducted in quadruplicates.
LPEI/siRNA complexes transfection in vivo by intraperitoneal (i.p.)injection
SPC-A1 cell suspension was prepared and inoculated subcutaneously into flanks of nude mice (1×107 cells /100 μl per site), (n = 6). As grown up tumors were removed and divided into uniform masses of 1 mm × 1 mm, and implanted subcutaneously into the right flank of the untreated mice. When tumors reached 6 mm × 6 mm, the SPC-A1-xenografted mice model was successfully established and i.p. treatment started.
For i.p. injection, the in vivo-jet PEITM transfection reagent (optimized cationic linear PEI-based transfection reagent for in vivo experiments), also from Polyplus-transfection (Illkirch, France), was used, which provided as a ready-to-use solution at 150 mM nitrogen concentration and less than 0.1EU/mL endotoxin. SiRNA-EGFR (3 μl, 200 μmol/L) and LPEI (0.75μl) were dissolved in 250μl of 5% glucose solution (GS), respectively. The latter was pipetted into the siRNA solution 10 minutes later. After vortexing and incubating at room temperature for 20 minutes, stable LPEI/siRNA-EGFR complexes under N/P ratio of 5 were formed in 5% GS at a final volume of 500μl. Afterwards, they were intraperitoneally administrated to each mouse in 1h. At the same time, a corresponding dose of LPEI complexed non-specific siRNA-NEG or 5% GS were given to the other two groups as controls.
In the experiment of discontinued administration, i.p.injection was started (day 0) when tumors had reached 6 mm × 6 mm, and repeated 2~3 times per week until the 22nd day, 24 h after the last injection. Tumor growth was measured every 3 days using a digital caliper by an observer blinded to treatment allocation. Tumor volume was calculated (0.52× longest diameter × shortest diameter2)
. At the end of the experiment, tumors were harvested, weighed, and examined for EGFR expression, proliferation, and apoptosis evaluations. No deaths of nude mice was observed during the experiment process.
Western blot analysis
EGFR expression in tumor samples was detected by western blot. Tissue was homogenized, centrifuged, and supernatants collected. Equivalent amounts of extracted protein (50 mg) were mixed with sample buffer containing 5% 2-mercaptoethanol, boiled, cooled, and loaded in each lane of a 7.5% polyacrylamide gel. Electrophoresis was performed at a constant voltage of 80V and, subsequently, proteins were transferred to a PVDF membrane. The membrane was blocked overnight with 3% gelatin in Tris-buffered saline (TBS). Subsequently, membranes were incubated with mouse anti-EGFR primary antibody (1:1000, BD Biosciences, USA) at 4°C overnight, and after washing twice in TBST, with peroxidase-conjugated goat anti-mouse IgG (Santa Cruz biotechnology, Inc., Santa Cruz, CA, USA) at room temperature for 1 hour. GAPDH was used as an internal control. Protein blots were visualized with chemiluminescence reagent ECL(Amersham, Freiburg, Germany). Membranes were washed thrice and then exposed to X-ray film and bands were quantified by scanning densitometry.
Immunohistochemisty and terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labelling (TUNEL) assay
Tumors were fixed in 4% paraformaldehyde and embedded in paraffin. IHC detection of EGFR and proliferating cell nuclear antigen (PCNA) were performed in 3μm histological sections. Briefly, sections were immersed in xylene, 95% alcohol, and 80% alcohol for 10 min, respectively, and washed with PBS (pH 7.4) for three times after each immersion. After protein denature, using microwave and non-specific biding blocking with normal goat serum for 20 min at RT, sections were incubated with primary antibodies against EGFR or PCNA (both diluted to 1:100, Maixin-Bio, China) overnight at 4°C. Sections were washed with PBS and incubated with goat anti-mouse secondary antibody (Cell Signaling) at a 1:100 dilution for 20 min at 37°C. Sections were again washed with PBS and incubated for 20 min with SABC. After again being washed with PBS, sections were incubated with 3, 3-di-aminobenzidine (DAB) for 3~5 min, and the reaction stopped by washing in PBS. Microscopically, brown particles appeared within cells, indicating positive expression of the protein molecules assessed. Five consecutive high-power fields (×200) were examined in each of specimen under a light microscope (Carl Zeiss, Germany) in a blinded fashion. The proliferation index was calculated according to the following formula: number of PCNA positive cells/ total cell count × 100%.
LPEI/siRNA complexes inducing cell apoptosis was assessed by measuring DNA strand breaks using an in situ cell death detection kit (Roche Applied Science, Indianapolis, IN, USA) based on TUNEL staining. Tissue slides were fixed in 4% paraformaldehyde for 10 minutes, followed by washing in phosphate buffered saline (PBS) and blocking in 3% H2O2 methanol for 10 min. After permeabilisation in a solution containing 0.1% Triton X-100 and 0.1% sodium citrate, tissues were labelled with 25 ml of TUNEL reaction mixture containing a 1:2 dilution of enzyme for 2 hours at 37°C in a humidified chamber. Signals were then converted into HRP using antifluorescein antibody and visualized by 3,3′diaminobenzidine coloration (Roche Applied Science, Indianapolis, Indiana, USA) as recommended by the manufacturer. Tissues were counterstained with methylgreen and TUNEL-positive cells were counted in five randomly selected × 200 high-power fields under microscopy. The apoptosis index was calculated according to the following formula: number of apoptotic cells/total number of nucleated cells × 100%.
Blood biochemical analysis
At the endpoint of the above experiment, all blood samples were collected for toxicity testing. By retro-orbital puncture, blood was collected into heparinized tubes. Within 1 h, samples were centrifuged at 5000 × g for 10 min. The plasma was taken for biochemical parameters analysis, carried out with an automatic analyzer (HITACHI 7020, JAPAN). Liver enzymes, including aspartate aminotransferase (AST), and alanine aminotransferase (ALT), were measured. Urea and creatinine (Cr) were also measured for renal function evaluation.
SiRNA-EGFR or non-specific siRNA-NEG (0.6 nmol), both complexed with LPEI in 5% glucose solution, were administrated by i.p. injection to 8-week-old male BALB/C nude or immunocompetent mouse, and 500 μl of 5% glucose solution was given as a negative control. After 2 days, the injection was repeated at 6 h before the end of the experiment. As a positive control based on a previous study, 40 μg of lipopolysaccharide (LPS, Sigma, St. Louis, MO) in 500 μl of phosphate-buffered saline (PBS) was intraperitoneally administrated to immunocompetent mouse and the experiment terminated after 2 h. Mice blood was taken by retro-orbital puncture and allowed to clot overnight at 4°C. Samples were centrifuged at 5000 × g for 10 min, and the supernatants collected. Serum concentrations of TNF-α and IFN-γ were determined by enzyme-linked immunosorbent assay (ELISA) kits according to manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). The amount of cytokine was determined on 100 μl of × 5 diluted serum, loaded in duplicate.
Data were presented as the means ± standard deviation. Statistical differences among multiple groups were calculated by one-way analysis of variance (ANOVA). If a ANOVA was statistically significant, an unpaired two-tailed Student’s t-test was used for between-group comparisons. P values of less than 0.05 were considered statistically significant.