The required materials were Iron (II) chloride tetrahydrate (99%), iron (III) chloride hexahydrate (98%), ammonia solution 25% GR for analysis, (Merck, Germany)(3-Dimethylaminopropyl)-3-ethylcarbodiimide (97%), Chitosan with molecular weight of 600,000–800,000 (Acros Organices) Monochloroacetic acid, synthetic Genistein (99%), NHS (N-Hydroxysuccinimide), DMSO (Dimethyl sulphoxide), MTT (Thiazolyl Blue Tetrazolium Bromide), Annexin V-FITC Apoptosis Detection Kit (Sigma Aldrich), FBS (Fetal bovine serum), Penstrep (Penicillin /Streptomycin), RPMI-1640, PI (Propidium Iodide Solution) (GIBCO). MOLT-4 cell lines were purchased from Pasteur Institute of Iran.
Synthesis of superparamagnetic Iron oxide nanoparticles
Magnetite nanoparticles were synthesized by coprecipitating iron (II) chloride and iron (III) chloride in alkaline solution. First 1.2 g of ferric chloride dissolved in 60 ml distilled water and ferrous solution prepared by dissolving 0.6 g of ferrous chloride in 30 ml of distilled water. After mixing of the solutions in a three neck flasks with magnetic stirring and purging the nitrogen gas for 10 min, 4 ml of 25% (w/w) NH3·H2O was added drop wise into the mixture solution and the pH of the solution was carefully monitored in order to reach 11.0. Change the color of the solution to dark black, showing formation of Fe3O4 and SPION precipitation. The black precipitate was separated from the solution with a neodymium magnet and rinsed several times with distilled water and ethanol and the pH value descended to 7.0. Resulted SPION dried in a vacuum oven in 70 °C for 4 h [23].
$$ {\mathrm{Fe}}^{2+}+\kern0.5em 2{\mathrm{Fe}}^{3+}+8\mathrm{OH}-\to {\mathrm{Fe}}_3{\mathrm{O}}_4\downarrow +4{\mathrm{H}}_2\mathrm{O} $$
Synthesis of carboxymethyl chitosan
For synthesis of O-carboxymethyl chitosan, 3 g chitosan was immersed in 25 ml of 50% wt NaOH solution to swell and alkalize for 24 h at room temperature. The alkalized chitosan was filtered using a G2 sintered funnel. The solution of 5 g of monochloroacetic acid in 25 ml of isopropanol added drop-wise on it for 30 min. After 4 h reaction at 60 °C temperature, the solvent was removed by filtering the mixture. The obtained product was dissolved in 100 ml of distilled water and 2.5 M HCl was used to adjust the pH to 7.0. Then this solution was filtered and carboxymethylated chitosan was precipitate by adding 400 ml of anhydrous ethanol. The final product was filtered, rinsed several times with ethanol and vacuum-dried at room temperature.
Preparation of Fe3O4 bound carboxymethylated chitosan
For preparation of carboxymethyl chitosan coated iron oxide nanoparticles, 28.25 μl of EDC (density 0.885 g/cm3) was added to 4 ml of phosphate buffer (0.2 mol/L Na2HPO4–NaH2PO4, pH 6.0), then 75 mg of Fe3O4 nanoparticles were added to the mixture and ultrasonicated for 10 min. After that 25 mg of carboxymethyl chitosan in 1 ml phosphate buffer was added to the obtained mixture and it was ultrasonicated for another 1 h. For distinction of the product from the mixture, neodymium magnet was used. Finally the product was washed with water and ethanol three times and vacuumed dried at room temperature.
Immobilizing genistein onto the CMC modified Fe3O4 nanoparticles
The next step was immobilization of genistein onto chitosan coated iron oxide nanoparticles. To reach this aim, genistein (2.5 mg) EDC (5.65 μl), NHS (6 mg) were dissolved in 5 ml of phosphate buffer (pH = 6.0, 2 mmol/L). Then 10 mg of Fe3O4-CMC nanoparticles was added. The suspension was then ultrasonicated for 10 min in 4 °C and shacked at room temperature for 24 h. Fe3O4-CMC-genistein was recovered by magnet from the reaction mixture. The precipitate was washed with 2 mM phosphate buffer (pH 6.0) and vacuumed dried at room temperature.
Characterization
Powder X-ray diffraction were recorded using a diffractometer (XRD-STOE-Stidy-mp) in 25 °C using CuKα radiation (λ = 1.54178 Å). Fourier transform infrared (FT-IR) spectroscopy of the samples was performed on a Bruker-Veator-22, FT-IR spectrophotometer for characterization the surface reaction of samples over the range of 400–4000 cm− 1.Morphology, mean particle size and size distribution was performed using Zeiss-EM10C, transmission electron microscopy (TEM), electron microscope operated at 100 KV DLS analysis. Magnetic characteristics were measured on a 7400 Lake shore vibrating sample magnetometer (VSM) at room temperature.
Cell culture
MOLT-4, MOLT17 and Jurket cell lines purchased from Pasteur Institute of Iran, centrifuged (130 g for 5 min) and suspended in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 mg/mL streptomycine, and 100 U/mL penicillin then cultured in 6-well micro plates (9.6 cm2) with concentration of 15 × 104 cells/ml and incubated in a humidified incubator by standard cell culture conditions (37 °C and 5% CO2).
Proliferation and MTT studies
In order to conduction the inhibiting effect of nano-conjugated Fe3O4-CMC-genistein in comparison with genistein and un-treated cells, briefly, a density of 15 × 104 /ml cells were seeded per each well of a six-well plate. Cells were treated with only genistein, Fe3O4-CMC nano-particles, and Fe3O4-CMC-genistein nano-conjugated with different doses from 20 and 40 μmol/L and incubated at 37 °C and 5% CO2. Doses 20 and 40 μmol/L selected in base of the IC50 obtained from MTT assay. After first 48 h, post treatment with 24 h’ interval were done for next remain 5 days. Hematocytometer was applied to count viable cells from three wells per dose. The appearance of healthy looking, rounded cells with intact cell membranes considered as viable cells versus dead or dying cells with ghostly, necrotic appearances and disrupted cell membranes. Tryphan-blue test was also performed.
Hematopoietic cancer cells were also treated with nano-conjugated Fe3O4-CMC-genistein as well as naked genistein in concentrations of 20, 40, 60, 80 and 100 μmol/L and then viable cells counted at 24, 48 and 72 h. Percent reduction of growth was calculated by dividing the number of treated cells with genistein or nano-conjugated Fe3O4-CMC-genistein to number of untreated cells.
MTT assay was also performed to detect the effect of Fe3O4-CMC-genistein nano-conjugate on proliferation of hematopoietic cancer cells as compare to naked genistein. Briefly, 20 μl MTT (5 mg/ml) was added up to each well of incubated cells with only genistein, Fe3O4-CMC nano-particles, and Fe3O4-CMC-genistein nano-conjugated, and then incubated for 4 h. Fe3O4-CMC nano-particles without genistein used as control the cytotoxic effect of nonao-particles. Then, the supernatants were removed and proceed by adding of 200 μl DMSO to dissolve formazen crystals. After 10 min incubation, the plate was read at 490 nm by micro plate reader versus to non-treated cells. Since, DMSO is solvent of genistein, and the final DMSO concentration n the medium was lesser than 0.1%, therefore, the cells treated with only DMSO also analyzed as control of cytotoxicity effect. Data of MTT assay was repeated 3 times for each cell line per dose.
Flow cytometry
Apoptosis particularly early apoptosis was also evaluated by flow cytometry analysis. For this purpose, hematopoietic cancer cells were cultured and treated with only genistein, synthesized nanoparticles Fe3O4-CMC without geneistein, and synthesized nano-conjugated Fe3O4-CMC-genistein for 48 h. Then, ice-cold PBS used to resuspend in 500 μL binding buffer and then incubated for 25 min more in dark. After that, solution of annexin-V and propidium iodide (PI) was added to the cells and then 400 μl binding buffer was added to each tube. FACS Calibur (BD; USA) instrument was used for analyzing.
Statistical analysis
The data were analyzed using Excel 2010, Graph Pad Prism 5.0 and SPSS softwares. Comparison of growth rate, apoptosis of cancer cell line between non-treated and treated, and nano-conjugated and naked genistein in different doses were performed by the non-parametric test followed by Mann-Whitney test and p-values < 0.05 were considered as statistically significant. Figure 1 shows a schematic illustration of synthesis methods and cell treatment.