The magnetic fluids used were synthesized by the co-precipitation method of Fe2+ and Fe3+ ions in alkaline medium, subsequently oxidized by bubbling oxygen. The functionalized fluids of rhodium citrate associated with maghemite nanoparticles (MRC) and citrate-loaded maghemite nanoparticles (MC) were obtained by adsorption. MRC with 59.6 µM γ-Fe2O3 and 2.85 µM rhodium citrate, MC with 64.7 µM γ-Fe2O3 and 2.55 µM [citrate], and 2.513 mM [Rh2(C6H7O7)4(H2O)2 [rhodium (II) citrate] were synthesized at the Institute of Chemistry of the Goias Federal University (Goiania, Brazil). For maintenance of cell cultures, DMEM, RPMI and L15 mediums, fetal bovine serum (FBS) and 0.25% trypsin EDTA were used, and all were obtained from GIBCO Company-Life Technologies, Carlsbad, CA, USA, except Leibovitz’s L15 (Sigma-Aldrich, St. Louis, MO, USA). The MRC solution diluted in H2O had a mean hydrodynamic diameter of 119.5 nm with a polydispersity index (PDI) of 0.17, ζ-potential of − 35 mV, analyzed by Zetasizer Nano ZS machine (Malvern, Malvern, UK) using a technology called dynamic light scattering (DLS), and similar values were maintained after subsequent measurements at different time intervals. The antibodies used in this study were: anti-P-glycoprotein (monoclonal) produced in mice (Abcam, Cambridge, MA); anti-BrdU conjugated to Alexa Fluor 488 (Invitrogen Life Technologies, Carlsbad, CA, USA); HRP-conjugated rabbit anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA). The reagents used throughout the work were: MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide), DNAse (Sigma-Aldrich, St. Louis, MO, USA); bicinchoninic acid assay (BCA, Thermo Scientific, Rockford, IL); ATP (Invitrogen, San Diego, CA, USA). Inserts for invasion assay and basement membrane matrix (Matrigel™) were purchased from BD Biosciences, San Jose, CA, USA. For Western blot analyses, we used a protease inhibitor (Hoffman-La Roche, Basel, Switzerland). The development was made with Amersham ECL prime Western blotting detection reagent, and image acquisition and analyses were performed with an Image Quant LAS 4000 (GE Healthcare, Little Chalfont, UK); further image processing and analysis were done with the ImageJ program (NIH, USA). For analysis of cell migration, an Axiovert light microscope (5× magnification) was used and the images were captured with the AxioVision 100 software, both from Zeiss, Germany. The Spectramax M5 equipment and the data analyzed by the SoftMax Pro 5.2 program, both from Molecular Devices, LLC, USA, were used to read absorbances. The flow cytometer used was a FACS Calibur, BD Biosciences, Inc., San Jose, CA, and data analysis was performed on the Flow Jo v.5.2.7 program, Tree Star, Inc.
The human breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium and Leibovitz’s L15 medium without CO2, respectively, containing 1% (v/v) penicillin–streptomycin and 10% (v/v) heat-inactivated fetal bovine serum (FBS). Primary cultures of human non-tumor mesenchymal cells from dental pulp (HNTMC) were obtained from health volunteers under approval of the Human Ethics Committee of the University of Brasilia (UnB). HNTMC were cultured with DMEM, supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) penicillin–streptomycin. HNTMC isolation was performed according to the following protocol: Extracted dental pulp was placed directly to a sterile tube containing DMEM, supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) penicillin–streptomycin until it was transferred to a plate (6 well) in the biological safety cabinet. To keep the cells fixes and facilitate adhesion to the bottom of the plate, a sterile pipette folded in half was used as a hook. The dental pulp received culture medium DMEM and was maintained at 37 °C, 5% CO2 and 80% of humidity. A half of the culture medium was replaced twice a week, until the cells reach confluency (80–90%). Then, the adherent cells were dissociated with trypsin and were frozen as seed stock in the presence of a cryoprotective agent dimethylsulfoxide (DMSO).
In vitro cytotoxicity study
In order to understand how the cells respond to MRC nanoparticles, MDA-MB-231, MCF-7 and HNTMC cells were incubated for 24, 48 and 72 h with various concentrations of this compound. Furthermore, the tumor cells were also exposed to free rhodium citrate (RC) at the same concentrations as those in MRC. Viability of the cells after exposure to NPs and RC was evaluated by use of the MTT assay according to the manufacturer’s recommendations (Invitrogen Life Technologies, Carlsbad, CA, USA). Briefly, 1 × 104 cells/well were seeded in 96-well plates and exposed to the treatments at the concentrations of 50 μM, 100 μM, 200 μM (rhodium citrate). All experiments were repeated at least three times in triplicates and were preceded by kinetic tests to adjust the initial cells amount for each exposure time and to avoid the control group confluency at later time of exposition. Results were expressed as percent of viability (%V) according to the following formula (blank discounted): %V = absorbance (cells + medium + NPs) − absorbance (medium + NPs). After cell treatment, the medium was removed from each well and replaced with new medium containing MTT (15 μl of MTT solution at 5 mg/ml and 135 μl of culture medium) for two and a half hours at 37 °C in a humidified atmosphere with 5% CO2. The resulting formazan product was dissolved in 200 μl of dimethylsulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA). Afterward, the supernatants of each sample were transferred to a fresh 96-well plate (to avoid interference of nanoparticles that did not enter the cells and accumulate on the bottom of wells), and absorbance was measured by a spectrophotometer (SpectraMax M2, Molecular Devices, Sunnyvale, CA, USA) at the wavelength of 595 nm.
Cell migration test
To assess the migration/invasion capacity of MDA-MB-231, the cells were treated with MRC and plated (1 × 106) in 25-cm2 flasks for 24 h. Treatment with 200 μM nanoparticles was performed in the culture flasks for 24 h. Cell viability was evaluated by means of trypan blue assay. The cells were trypsinized, resuspended (2 × 104) in L15 medium without fetal bovine serum (FBS) and added to the top of inserts (Transwell for 24-well plates, BD Biosciences) with 8-μm pores coated with a thin layer (30 μl) of Matrigel™ (BD Biosciences) diluted 5× in frozen fetal bovine serum-free L15 medium. The plate was left for 1 h at 37 °C for solidification of the Matrigel layer. In the wells of the plate, below the inserts, the L15 medium was supplemented with 10% SFB to attract a migration of the cells through the pores. After 48 h, the inserts were removed from the plate and the cells that adhered to the top of the Transwell (which did not migrate) were removed with a cotton swab. Cells remaining in the lower part of the insert were fixed (3.7% paraformaldehyde, 15 min) and stained with 0.1% crystal violet for 5 min. All stained cells were photographed (5× magnification), and an invasiveness rate was given by automated counting of cells processed with the ImageJ software.
The effect of MRC nanoparticles on DNA synthesis
For analysis of DNA replication and cell cycle, 5 × 105 MDA-MB-231 and MCF-7 cells were plated in 6-well plates (10 cm2) in appropriate medium. After adherence, medium was exchanged for another containing 10 μM bromodeoxyuridine (BrdU) and incubated for 2 h at 37 °C for incorporation of BrdU into the DNA. As a negative control, one group did not receive BrdU. Subsequently, the cells received new medium, and in the experimental group, nanoparticles containing 200 μM MRC were added, and the treatment lasted for 24 h. The group which did not receive the treatment was considered the zero point and was collected immediately after incorporation of BrdU, and processed until the overnight fixation step at 4 °C. The cells were trypsinized and centrifuged at 1500 rpm for 5 min. The medium was discarded, and MDA-MB-231 cells were washed with ice-cold phosphate-buffered saline (PBS) and fixed with 70% ice-cold ethanol for 30 min. The fixative solution was removed, and DNAse (0.3 mg/ml) was added for 10 to 15 min for denaturation of the DNA at 37 °C. After discarding the solution containing DNAse, the pellet was resuspended in blocking solution (30 min) containing 50 μl of Tween 20 and 1% serum albumin (BSA)/PBS. Samples were incubated for 1 h with primary anti-BrdU antibody (1:100), and after 45 min with FITC-conjugated secondary antibody (1:10,000) in a solution containing 5 μl of RNAse (10 mg/ml). Subsequently, the pellet was resuspended in 1 ml with 5 μg/ml propidium iodide (PI) for 10 min. The cells (10,000 of each group in triplicate) were analyzed on a flow cytometer.
For western blot, MDA-MB-231, MCF-7 and HNTMC cells (1x106) were grown in flasks. Fresh medium with 100 µM MRC was added to the flasks, which were incubated for 6 h at 37 °C. Cells were rinsed three times in ice-cold PBS, and protein extraction was performed with lysis buffer (50 mM Tris pH 7,4; 150 mM NaCl; 5 mM ethylenediamine tetraacetic acid (EDTA); and 1% Triton X100 and protease inhibitor cocktail). Lysates were centrifuged (10 min, 4 °C). Protein concentrations were determined by the bicinchoninic acid assay. Protein total lysates (20 µg) were separated by 4–12% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS)—0.1% Tween, followed by incubation with primary (1:1000 anti-P-glycoprotein) and secondary (goat anti-mouse HRP—1:5000) antibodies. Development was performed by ECL chemiluminescence kit. The experiment was repeated three times.
The quantitative data were submitted to the ‘t’ test for unpaired samples (between two groups) or simple variance (ANOVA) (between three or more groups) followed by the Bonferroni post-test. Statistical analyses were conducted using the GraphPad Prism software, and the results were expressed as mean ± SEM. Values of p < 0.05 were considered statistically significant.