Preparation of magnetite nanoparticles: Magnetite nanoparticles were prepared by co-precipitation method as previously mentioned ⁽⁹⁾. Briefly, a 50 ml solution containing 0.5 molar ferric iron chlorides (Merck Co.), and a 50 ml solution containing 0.25 molar ferrous iron chlorides (Merck Co.) were prepared. The solutions were then mixed and added to a 6 molar NaOH (Merck Co.) solution (pH of this medium was about 14) at room temperature. After stirring at the same temperature for 10 min, the obtained precipitate was washed off several times by distilled water while a pH of 7 was obtained. After each washing an ultrasonic bath was used to extract the ions through the precipitates. To dry the washed precipitate, a hot plate magnetic stirrer was used. This procedure was done at 70°C for 2 hr, so that a black powder was obtained.

The crystal structure of the powders was characterized by an X-ray diffractometer (Bruker, Advanced D8 model), using CuKα radiation (λ=1.5406 Å). Mean crystallite size of the nanoparticle was calculated by Scherrer's formula after applying the necessary corrections. Particle morphology of the sample has been investigated by a Transmission Electron Microscope (TEM), Philips CM12. Magnetization curves of the nanoparticles were obtained by a Vibrating Sample Magnetometer (VSM). To measure temperature increase, a mixture of one mg of the magnetite nanoparticles and 100 ml distilled water was prepared. Ten ml of the mixture was then placed at the center of a 2 turns RF coil and an AC current (f=400 kHz) was applied with an rms value of 400 A/m. A sensitive thermometer was used to measure the temperature increase.

Preparation of magnetite nanoparticles suspension: Magnetite nanoparticles (average particle size 10 nm) were dispersed in edible liquid paraffin according to previously published methods ⁽⁹⁾. Its solid concentration was 40% with final density of 1.13 g/cm ³ . This dark brown colored suspension had a viscosity of 130 Pa.s and shear stress yield of 20 Pa at room temperature. Prepared Fe₃O₄ ferro-fluid showed a very good stability up to 3 years.

Preparation of doxorubicin solution: Doxorubicin hydrochloride (Farmitalia, Italy) was dissolved in distilled water so that the final concentration was 20 µg/ml. This solution was used either as positive control or in combination with nanoparticles.

Cell culture and cell seeding: Human breast cancer cell line (MDA-MB-468) were cultured as monolayer in RPMI 1640 medium [Gibco, UK; each 500 ml of RPMI-1640 supplemented with 10% of Fetal Calf Serum (FCS, Gibco, UK), 5 ml of penicillin/ streptomycin (Sigma, USA; 50 IU/ml and 500 µg/ ml, respectively), 5 ml of sodium pyruvate (1 mM), NaHCO₃ (1 g) and 5 ml of l-glu-tamine (2 mM)]. Completed media was sterilized by 0.22 µ microbiological filters after preparation and kept at 4°C before using. Cells were kept in an incubator at 37°C, 5% CO₂ air humidified up to 15 subcultures. Cells were detached, using 0.25% trypsin and seeded (5×10⁴ cell/m1) in 15 ml tubes for biological evaluation.

Blank: Two ml of cell suspension (5×10⁴ cell/ml) in a 10 ml test tube was used as blank. An AC magnetic field (f=450 kHz and H=100 A/m) was applied on blank for 30 min. Then 20 µl of it was mixed with 20 µl of trypan blue (0.2% w/v) and the viability of cells was measured using a hemocytometer.

Four ml of cell suspension (5×10⁴ cell/ml) and 0.5 ml of edible paraffin oil (nanoparticle carrier) were mixed and divided into 2 equal portions in a 10 ml test tube. One test tube was left at room temperature and the second one was left in the magnetic field for 30 min. The magnetic field condition and the percent of viability were measured as mentioned for the blank treatment.

Four ml of cell suspension (5×10⁴ cell/ml) and 0.5 ml of doxorubicin (Farmitalia, Italy) solution (20 µg/ml) were mixed and the experiments were carried out as mentioned for the negative control.

Sample 1: Four ml of cell suspension (5×10⁴ cell/ml) and 0.5 ml of magnetite nanoparticles suspension were mixed and the experiments were repeated the same as for the negative control. To increase the contact of cells with nanoparticles test tubes were left on shaker either at room temperature or in the magnetic field.

Sample 2: Four ml of cell suspension (5×10⁴ cell/ml) and 0.25 ml of magnetite nanoparticles suspension plus 0.25 ml of doxorubicin were mixed and the experiments were repeated the same as mentioned for the sample 1.

Five µl of propidium iodide dye (PI; 1 mg/ ml in deionized water, Sigma) was added to 3 ml of all treated cell suspension (i.e. blank, negative control, positive control, samples 1 and 2) and the cell viability was evaluated. Each sample was analyzed in a PAS/Dako flow cytometer (Partec, Denmark) with the use of acquisition/analysis program Flo Max 2.4 (Partec). Cells were plotted according to forward scatter and side scatter profiles (a measure of size and granularity of an event, respectively) and gated to include cells only. Cells were located using these parameters and a live gate analysis was set around this population. Data were acquired from 10,000 cells (events) and the cells which showed high fluorescent in channel FL2 were regarded as dead (stained with PI).

Figure 1 shows XRD pattern of the co-precipitated magnetite nanoparticles. As can be seen all main peaks are related to a single-phase spinel structure. A mean crystallite size of about 5 nm has been obtained for the nanoparticles using Scherrer's Equation.

Figure 2 shows TEM photograph of the single-phase nanopowders and as can be seen there is a uniform distribution of particles with particle sizes between 3 and 10 nm, which is in agreement with the Scherrer's result.

Figure 3 shows room temperature hysteresis loop of the co-precipitated magnetite nanoparticles. As seen in Figure 3 the saturation magnetization of the nanoparticles is 32 emu/g, which is less than the saturation magnetization of bulk magnetite (92 emu/g) ⁽¹⁰⁾. This difference can be explained by core-shell model that explained elsewhere ⁽¹¹⁾. In this model, it is supposed that each particle consists of a core with ferrimagnetic order and a constant thickness nonmagnetic shell with spin glass phase. It is obvious that by decreasing the particle size the surface-to-volume ratio of a particle will increase, which leads to a reduction in magnetization of the particles ⁽⁸⁾. Temperature increase measurement due to a mixture of the nanoparticles and distilled water (10 mg/l) in presence of an AC magnetic field (f=400 kHz and H=400 A/m) show that a 15°C temperature increase is achievable after 30 min radiation. As the magnetic nanoparticles have super paramagnetic behavior, this can be due to relaxation losses ⁽⁶⁾.

Trypan blue exclusion assay: Trypan blue exclusion results are summarized in Table 1. Trypan blue exclusion assay showed that incubation of cells with a combination of doxorubicin and magnetite nanoparticles for 30 min in the magnetic field stained almost all the cells. Therefore, this treatment would be considered as a cytotoxic combination (Figure 4).

As seen in Figure 5 (A-C) more than 99% of cells were alive before any treatment. Therefore, these cells were used as control to setup the flow cytometer and other treatments were compared to this control. Thirty min incubation in magnetic field was not cytotoxic for cells (Figure 5D). In this study, edible paraffin oil was used as a carrier to make nanoparticle suspensions which had no effects on the cell viability either in RT (Figure 5E) or in the magnetic field (Figure 5F). Magnetite nanoparticles (Figure 5G) and doxorubicin (Figure 5H) alone or in combination (Figure 5I) at RT had no significant cytotoxic effects on cells. When cells were incubated with magnetite nanoparticles and magnetic field was applied for 30 min, 55.5% of cells died (Figure 5J). As seen in Figure 5K, 28% of cells which were incubated with doxorubicin at magnetic field were also dead; whereas dead cells reached up to 80% when incubated with both doxorubicin and magnetite nanoparticles together for 30 min in magnetic field (Figure 5L).

Considering the data presented in Table 1 and Figure 5, the following results can be concluded:

MDA-MB-468 cells were kept under magnetic field for 30 min and no significant reduction in the cell viability was seen.