Chemical reagents
Roswell park memorial institute 1640 (RPMI-1640) medium and fetal bovine serum (FBS) were purchased from Gibco. Penicillin-streptomycin and trypsin-EDTA were obtained from Bioidea. Cisplatin was purchased from Oncotic Pharma production GmbH, Germany. 25 kDa branched polyethylenimine was obtained from Sigma-Aldrich. 2′,7′-dichlorofluorescein diacetate (DCFDA) (ab113851) was purchased from Abcam. Propidium iodide (PI), (4H2O.FeCl2), (6H2O.FeCl3) and (NaOH) were obtained from Merck. Annexin V apoptosis detection kit was purchased from eBioscience, USA.
Synthesis and characterization of magnetic nanoparticles
The Fe3O4 MNPs synthesized by co-precipitation approach. Physical properties of nanoparticles and size-dependent parameters such as reaction temperature, pH of suspension, initial molar concentration were investigated (39). In this approach, nanoparticles synthesized from (4H2O.FeCl2), (6H2O.FeCl3) and (NaOH) with high purity and distilled water. The (4H2O.FeCl2) and (6H2O.FeCl3) solutions were mixed in the respective stoichiometry (i.e., ratio Fe (II):Fe (III) = 1:2) in presence of OH¯ deposited through the following reaction in equation (1).
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yAjTHGmBNwAjbGGGNOwAnYGGOMOQEnYGOMMeYEnICNMcaYE3ACNsYYY07ACdgYY4w5ASdgY4wx5gScgI0xxpgTcAI2xhhjTsAJ2BhjjDkBJ2BjjDHmcH7//h9YdXqY8+xBmgAAAABJRU5ErkJggg==)
Distilled water was initially deoxygenated by ultrasonic transducer and nitrogen gas. Then, iron salts II and III were added. The NaOH solution was also deoxygenated by the ultrasonic apparatus. The 12 ml of saline solution was added to 120 ml of NaOH solution under nitrogen atmosphere, and the solution was homogenized for 10 h at 10,000 rpm for 30 min. After that, the particles were washed three times with distilled water and once with acetone, and dried under vacuum conditions (40,41).
The binary complexes (PEI-CIS (PC)) and PEI-MNPs (PM)) were synthesized at 37°C for 1 h. for formation of ternary complexes (PEI-MNP/CIS (PM/C)) CIS bounded to PM binary complexes. Moreover, PEI, MNP and CIS bounded together at the same time and (PEI-MNP-CIS (PMC)) ternary complexes were synthesized at 37°C for 1 h.
The accuracy of MNPs and formation of binary and ternary complexes were confirmed by the Fourier transform infrared spectroscopy (FTIR) and dynamic light scattering analysis. FTIR spectra was done for dried sample of MNP, PM binary and PMC ternary complexes by FTIR spectrometer (Thermo scientific NICOLET IR100) in wave range of 4000 - 400 cm-1 with a resolution of 4 cm-1. In brief, the dried sample was placed on a silicon substrate transparent to infrared, and spectra were measured by transmittance method. Then, spectrums of synthesized products were plotted through Essential FTIR software. The size and surface charge distribution of PEI, MNP.PM and PMC ternary complexes in solutions and suspensions was determined using Dynamic light scattering (DLS Zeta Sizer, a nano-ZS model, UK). Briefly, 1 mg/ml solution of nanoparticles made in deionized-water and placed in the ultrasonic bath for 30 min. The sample was filtered with a 0.25 μm to remove larger and accumulated particles. Then, the particle size distribution and surface charge were analyzed by Zetasizer software (version 7.11).
Cell culture
The CIS-resistant human ovarian carcinoma A2780/CP and sensitive A2780 cells were obtained from National Cell Bank of Iran (NCBI). A2780/CP is a sub-line of A2780 that gained CIS resistance in the in vitro (27). The cells were allowed to grow in RPMI-1640 in the neutral PH (7.2–7.4) supplemented with 10% (v/v) heat-inactivated (50 °C, 30 min) fetal bovine serum (FBS) and 2 mM glutamine, 100 units/mL of penicillin and 100 mg/mL of streptomycin at 37 °C and 5% CO2 in a humidified incubator. The cells were trypsinized (0.025% trypsin, 0.02% EDTA) after they grown until 70–80% confluent. Prior to treatments, cells were allowed to reattach overnight.
Magnetic field exposure
We used permanent cobalt magnets to apply 20 millitesla (mT) homogenous SMF. The magnitude of this magnetic field was calculated by a Teslameter (13610.93, PHYWE, Gottingen, Germany) with a probe type of Hall Effect in respect to its cross-sectional area and thickness. We exposed 20 mT SMF to cells through placing magnets under bottom of cell culture plates during this study.
Cell treatments
The A2780/CP and A2780 cells were treated by CIS and PEI at concentrations of 5, 10, 25, 50 and 100 μg/ml, PC binary complex included concentrations of PEI (1, 2.5, 5 and 10 μg/ml) and CIS (2.5, 5, 7.5 μg/ml) and PM binary complex included concentrations of PEI (2.5, 5 and 10 μg/ml) and MNPs (1 μg/ml) in presence and absence of 20 mT SMF for 24 and 48 h. Moreover, cells were treated at mass ratio of PEI/CIS (0.4, 0.5, 1, 2 and 4 w/w) for 24 and 48 h. In addition, cells treated with PM/C and PMC ternary complexes at concentrations of 1 μg/ml PEI, 1 μg/ml MNPs and 2.5 μg/ml CIS in presence and absence of SMF for 48 h.
Cell viability assay
The cell viability of A2780/CP and A2780 cells were measured by tetrazolium-based colorimetric assay (MTT assay). Briefly, cells (104 cells/ well) were seeded into 96-well culture plate (SPL Life Sciences Co., Ltd. Korea) and incubated in a total volume of 100 μL supplemented RPMI at 37 °C and 5% CO2 in a humidified incubator. Cells were initially allowed to attach overnight. Following the treatments, 100 μL FBS-free RPMI containing of 0.5 mg/mL MTT was added to each well and kept at 37 °C for 4 h in the dark. Then, formazan was dissolved by 100 μL/well DMSO. The relative number of living cells in each group was measured by microplate reader (uQuant MQX200, BioTek, USA) at 570 nm. Cell viability results were shown as percentage compared to control cells. Sensitivity of selected cell types evaluated by the half-maximal inhibitory concentration (IC50).
Quantitation of intracellular ROS accumulation
Intracellular ROS levels under normal and stress conditions were detected using 2′,7′–dichlorofuorescein diacetate (DCFDA) assay kit. The A2780/CP and A2780 cells were treated in 1 µg/ml of PEI and 2.5 µg/ml of CIS as well as PM/C and PMC complexes (at concentrations of 1 µg/ml of PEI, 1 µg/ml of MNP and 2.5 µg/ml of CIS) compared to untreated cells in the presence and absence of SMF for 48 h, which were prepared in supplemented RPMI media with 10% FBS in the 6-well cell culture plate (SPL Life Sciences Co., Ltd. Korea). After treatments, cells were prepared immediately as recommended by the manufacturer. Briefly, the cells were washed with PBS. Then the samples were suspended in the conical test tube with 20 µM DCFDA in the 1X buffer and incubated at 37 °C in dark for 30–45 min. Measurement of ROS production was monitored immediately by FACScalibur Becton-Dickinson flow cytometry (Franklin Lakes, NJ). The DCFDA flow cytometric data were analyzed by flowjo software (version 7.6.1) (27,42,43).
Cell cycle analysis
The A2780/CP and A2780 cells were treated to 1 µg/ml of PEI and 2.5 µg/ml of CIS as well as PM/C and PMC complexes (at concentrations of 1 µg/ml of PEI, 1 µg/ml of MNP and 2.5 µg/ml of CIS) in presence and absence of SMF for 48 h, which were prepared in supplemented RPMI media with 10% FBS in the 6-well cell culture plate (SPL Life Sciences Co., Ltd. Korea) and incubated at 37 °C and 5% CO2 in a humidified incubator. After that, cells were trypsinized and collected through centrifugation at 400g for 5 min. Then, cells were resuspend in 0.5 ml of PBS and fixed by adding 4.5 ml of 70% (v/v) cold ethanol, and centrifuged at 400g for 5 min. After that, cells were washed in 5 ml PBS, centrifuged at 400g for 5 min and incubate at room temperature for 5 min, and recentrifuged at 400g for 5 min, respectively. Then, supernatant was removed and resuspended in 1 ml of DNA staining solution. The prepared cells were incubated for at least 30 min at room temperature in the dark. The obtained cell suspension was analyzed by FACScalibur Becton-Dickinson flow cytometry (Franklin Lakes, NJ). Data were collected from at least 104 cells. The flow cytometric data were analyzed by flowjo software (version 7.6.1) (44).
Detection of cell apoptosis
Apoptosis was detected using annexin V-FITC/PI staining. The A2780/CP and A2780 cells were treated in 1 µg/ml of PEI and 2.5 µg/ml of CIS as well as PM/C and PMC complexes (at concentrations of 1 µg/ml of PEI, 1 µg/ml of MNPs and 2.5 µg/ml of CIS) in presence and absence of SMF for 48 h, which were prepared in supplemented RPMI media with 10% FBS in the 6-well cell culture plate (SPL Life Sciences Co., Ltd. Korea) and incubated at 37°C and 5% CO2 in a humidified incubator. After that, cells were collected and labeled by annexin V/PI in 1X binding buffer for 15 min. Then, the apoptotic and necrotic cells were evaluated by FACScalibur Becton-Dickinson flow cytometry (Franklin Lakes, NJ). The flow cytometric data were analyzed by flowjo software (version 7.6.1). Total number of apoptotic and necrotic cells were defined as sum of Annexin V+/PI-and Annexin V+/PI+ populations including Q1 = Necrosis; Q2 = Late Apoptosis; Q3 = Early Apoptosis; Q4 = Live Cells (45).
Statistical analysis
GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, USA) was used for data graphing and estimating the values of inhibitory concentration 50% (IC50) for any types of cytotoxicity. All experiments were performed with three independent repetitions. Data were showed as mean ± standard deviation (SD) and were analyzed by factorial analysis of variance (ANOVA) followed by Tukey’s post hoc tests. Differences were assessed to be significant p-value was less than 0.05.