Preparation of Walterinnesia aegyptia venom
Walterinnesia aegyptia snakes were collected from the central region of Saudi Arabia. The snakes were kept in a serpentarium in the Zoology Department, College of Science, King Saud University. The snakes were warmed daily using a 100-watt lamp for nine hours, and water was always available. The snakes were fed purpose-bred mice every 10 to 14 days. The venom was milked from adult snakes, lyophilized and reconstituted in 1X phosphate-buffered saline (PBS) prior to use.
Combination of snake venom with silica nanoparticles
A total of 25 mg of mesoporous silica nanoparticles was added to a solution of 50 mg/ml venom in water. The suspension was stirred for 2 hour; the evaporation of water was prevented. The mesoporous silica nanoparticles loaded with venom were recovered using high-speed centrifugation and dried in a vacuum oven at 60°C.
Cell culture and reagents
Human MDA-MB-231 breast cancer cells were obtained from Dr. Douaa Sayed at Assiut University, Egypt and maintained in a culture medium consisting of MEM supplemented with 10% heat-inactivated fetal bovine serum (FBS, EuroClone, Life Science Division, Milan, Italy). The anti-proliferative effect of WEV and WEV+NP on MDA-MB-231 cells was determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) uptake method. The cells were plated at 1 × 106 cells/ml in 2 ml of culture medium in six-well Costar plates (Corning, Corning, NY). The cells were treated with different concentrations of WEV or WEV+NP for 1, 2, 6, 12, 24 or 48 h, and cytotoxicity was expressed as a relative percentage of the OD values measured in the control and WEV- and WEV+NP-treated cells. The experiments were repeated using human peripheral blood mononuclear cells (PBMCs). Morphological changes were observed after exposure to WEV and WEV+NP using a phase-contrast inverse microscope (Olympus, Japan).
Animals and lymphocyte isolation
Forty-five sexually mature 12-week-old male Swiss Webster (SW) mice weighing 25-30 g each were obtained from the Central Animal House of the Faculty of Pharmacy at King Saud University. All animal procedures were performed in accordance with the standards set forth in the Guidelines for the Care and Use of Experimental Animals by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the National Institutes of Health (NIH). The study protocol was approved by the Animal Ethics Committee of the Zoology Department, College of Science, King Saud University. All animals were allowed to acclimatize in metal cages inside a well-ventilated room for 2 weeks prior to the experiment. The animals were maintained under standard laboratory conditions (a temperature of 23°C, a relative humidity of 60-70% and a 12-hour light/dark cycle) and were fed a diet of standard commercial pellets and given water ad libitum. The animals were divided into 3 experimental groups (n = 15/group): group I was a control group that was subcutaneously injected with PBS, group II was subcutaneously injected with WEV (50 ng/ml for 12 hrs) and group III was subcutaneously injected with WEV+NP (20 ng/ml for 12 hrs). Lymphocytes were isolated from the animals' blood using Ficoll-Paque density gradients. The remaining red blood cells were osmotically lysed using ACK buffer. The cells were washed with phosphate-buffered saline (PBS), counted using the Trypan blue exclusion test, and cultured in R-10 culture medium (complete RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, and 50 μM 2-mercaptoethanol).
F-actin polymerization assay
The isolated blood lymphocytes were cultured for two hour in culture medium before the F-actin polymerization test. Intracellular F-actin polymerization was assessed as previously described . Briefly, cells were harvested and resuspended (4 × 106/ml) in HEPES-buffered RPMI 1640 at 37°C with or without CXCL12 (250 ng/ml). At the indicated times, cell suspensions (100 μl) were added to 400 μl of assay buffer containing 4 × 10-7 M FITC-labeled phalloidin, 0.5 mg/ml L-α-lysophosphatidylcholine (both from Sigma-Aldrich) and 4.5% formaldehyde in PBS. The fixed cells were analyzed using flow cytometry, and the mean fluorescence intensity (MFI) was determined for each sample. The percentage change in MFI was calculated for each sample at each time point using the following formula: (1-(MFI before the addition of CXCL12/MFI after the addition of CXCL12) × 100.
Whole-cell lysates were prepared from lymphocytes that were isolated from the control and WEV- and WEV+NP-treated mice in RIPA buffer (20 mM Tris-HCl, pH 7.5, 120 mM NaCl, 1.0% Triton X100, 0.1% SDS, 1% sodium deoxycholate, 10% glycerol, 1 mM EDTA and 1% protease inhibitor cocktail, Roche). Following centrifugation at 16,000 × g at 4°C for 15 min, the protein concentrations in the supernatants were determined using a protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of whole-cell protein (50 μg) were mixed with reducing sample buffer (0.92 M Tris-HCl, pH 8.8, 1.5% SDS, 4% glycerol, and 280 mM 2-ME) and separated using discontinuous SDS-PAGE. Proteins were transferred onto nitrocellulose membranes using a Bio-Rad Trans-Blot electrophoretic transfer device, and the membranes were blocked for 1 h at room temperature with 1% BSA or 5% skim milk dissolved in TBS (20 mM Tris-HCl, pH 7.4, and 150 mM NaCl) supplemented with 0.1% Tween 20. The membranes were then incubated in the same blocking buffer with anti-phospho-ERK, anti-phospho-AKT, anti-phospho-IκBα, anti-phospho-p38MAPK or anti-β-actin antibodies (1:1,000; Cell Signaling Technology, Beverly, MA). The blots were thoroughly rinsed and then incubated with an HRP-labeled species-matched secondary antibody for another 1 h. Protein bands were detected using enhanced chemiluminescence reagents (ECL, SuperSignal West Pico Chemiluminescent Substrate, Perbio, Bezons, France), and the ECL signals were recorded on Hyperfilm ECL. To quantify band intensities, the films were scanned, saved as TIFF files and analyzed using NIH ImageJ software.
CFSE proliferation assay
Isolated blood lymphocytes were harvested, washed twice in PBS and stained with 0.63 mM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) for 8 min at room temperature. Residual CFSE was removed by washing three times in PBS, and CFSE-labeled cells were seeded in 6-well plates, treated with or without a mitogen cocktail and grown for 4 days in cell culture medium. The CFSE fluorescence intensity was measured using FACS analysis.
Data are expressed as the mean ± standard error of the mean (SEM). Significant differences among groups were analyzed using a one-way analysis of variance (for more than two groups) followed by Tukey's post-test using SPSS software, version 17. Differences were considered statistically significant at P < 0.05. *P < 0.05, WEV-treated vs control; #P < 0.05, WEV+NP-treated vs. control; +P < 0.05, WEV+NP-treated vs. WEV-treated groups.