Synthesis and characterization of Co-SPIONs. Co-SPIONs were synthesized in the thermostated glass reactor by Massart’s co-precipitation method (Massart, 1981) from the alkaline solutions of Co(II) and Fe(III) metal salts at 70 °C for 5 h. All reagents in synthesis procedure were at least of analytical grade and were used without any further purification, except for NaOH, which was purified by preparation of a saturated solution resulting in the crystallization of other sodium salts. CoCl2, Fe2(SO4)3 and citric acid were purchased from Aldrich Chemicals Inc. Ultrapure water was used throughout all experiments. For preparation of the working solutions 0.12 mol/L CoCl2, 0.06 mol/L Fe2(SO4)3, ...view middle of the document...
A small amount (40 µl) of Co-SPION solution was droped on freshly cleaved mica surface, spinning at 1000 rpm. Zeta potential of the particles was measured using a Brookhaven ZetaPALS zeta potential analyser (Brookhaven Instruments, USA).
Cell culture. Human pancreatic cancer cell line MiaPaCa2 and human ovarian cancer cell line A2780 were purchased from the European Collection of Cell Cultures (UK). MiaPaCa2 cells were cultured in Dulbecco’s Modified Eagle Medium. A2780 cells were cultured in Roswell Park Memorial Institute 1640 medium. Both growth media were supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin. All media and cell culture supplements were purchased from Biochrom AG (Germany). Cells were cultured at 37°C and 5% CO2 in a humidified incubator and subcultured twice a week.
Cell treatment with Co-SPIONs. Cells in a log phase were plated in surface-treated cell culture dishes at an appropriate density (3 x 104 cells/cm2 for MiaPaCa2 and 4 x 104 cells/cm2 for A2780) and cultured in a CO2 incubator for 24 hours. Afterwards, Co-SPIONs were added to cell growth medium.
For Co-SPIONs accumulation assay, cells were plated into 35 m Petri dish (containing 2 mL of growth medium), treated with Co-SPIONs until 0.48 µg/ml concentration and incubated for 24 hours. Untreated cells were used as controls. After treatment with Co-SPIONs cells were routinely rinsed 3 times with PBS and fixed with paraformaldehyde (4%) for 15 minutes. For iron content visualization in cells, Prussian Blue Cell Staining Reagent Pack (BioPAL) was used. Equal amounts of both reagents A (hydrochloric acid) and B (potassium ferrocyanide) were combined, added to fixed cells and left for 15 minutes. Staining solution was then aspirated and cells were rinsed 3 times with PBS. Cells were imaged using brightfield mode of Nikon Eclipse TE2000 EZ-C1 microscope (Japan).
For cell proliferation and viability analysis, cells were plated into 6-well plates, treated with 0.095, 0.48, 0.95 µg/ml of Co-SPIONs in 2 ml of growth medium per well and incubated for 24 and 48 hours. Untreated cells were used as controls. After treatment whole bulk of cells was collected by aspiring growth media and detaching rest of the cells via trypsinization. Accustain Kit (Digital Bio, Korea) was used to determine amount of total and non-viable cells according to manufacturer’s...
