Materials
Ferric sulfate heptahydrate (FeSO4·7H2O, 99% w/w), ferric chloride hexahydrate (FeCl3, 6H2O, 99% w/w), hydrochloric acid (HCl, 32% v/v), ammonia solution (NH3, 32% v/v), methanol (99.9% v/v), 3-aminopropyltriethoxysilane [NH2(CH2)3-Si-(OCH3)3, APTS], ethanol (99.9% v/v), methyl acrylate (99.5% v/v), methoxy-PEG and ethylenediamine (99% v/v), Eagle’s minimal essential medium (DEMEM), fetal bovine serum (FBS), and PenStrep were used in synthesis process and cell culture; all materials were purchased from Sigma-Aldrich (Germany).
Synthesis of IONPs and APTS-coated IONPs
IONPs were synthesized via the co-precipitation method which is explained in our previous study (Salimi et al. 2018). Briefly, 0.84 g of FeSO4 and 1.22 g of FeCl3 were dissolved in 20 ml deionized water followed by 30 min sonication. Then, 1 ml of 2 M HCl was slowly added with vigorous stirring in a nitrogen atmosphere. After 2 min, 4.6 ml ammonia was quickly added to the solution and stirring was continued for 1 min. The black precipitate of IONPs was washed five times with distilled water and ethanol through magnetic decantation. To IONPs coating with APTS, 150 ml ethanol was added to 25 ml of 5 g l−1 IONPs solution which was sonicated for 30 min; after 20 min of sonication, 300 µl of APTS was added to the mixture. Finally, the solution was stirred for 15 h at room temperature, and eventually, the resultant black precipitate was washed with ethanol three times (Khot et al. 2013).
Surface coating of IONPs with dendrimer and PEGylation
10 ml ethanol was added to the APTS@IONPs solution after 30 min sonication; subsequently, methyl acrylate/methanol solution (20%, v/v) was added (50 ml) at 0 °C during the sonication for 1 h followed by stirring for 48 h. Then, after washing the resultant solution with methanol, 15 ml ethylenediamine/methanol (50%, v/v) was added and the solution was sonicated for 3 h at 25 °C. This process was repeated to earn the fourth dendrimer generation (G4). The final solution was washed several times with methanol and water by magnetic decantation or centrifugation (Khodadust et al. 2013).
Eventually, mPEG molecules (molecular weight of 4000 Da) were conjugated to the surface of amino groups of dendrimers. The applied mPEG mass was three times more than the mass of iron. The mPEG was dissolved in the ethanol solution and added to the G4@IONPs solution followed by 18 h reflux.
Characterization
The morphology and size distribution of G4@IONPs were studied by transmission electron microscopy (TEM) and the hydrodynamic size and surface potential were measured through dynamic light scattering (DLS) and zeta potential, respectively. Magnetic properties of IONPs and G4@IONPs were measured by vibrating sample magnetometer (VSM) at 300 K under the magnetic field up to 15 kOe. Furthermore, the crystalline phase of IONPs was confirmed by X-ray diffraction (XRD, λ = 0.15406 nm) and G4 PAMAM bonds on the surface of IONPs was detected by Fourier-transform infrared spectroscopy (FTIR).
Cytotoxicity assay
MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay was used to determine the cytotoxicity of G4@IONPs in MCF7 and HDF1 cell lines. Cells were incubated in 96-well plates at a cell density of 4 × 103 cells per well and cultured in DMEM supplemented with 10% FBS and 1% PenStrep at 37 °C and 5% CO2 for 24 h. Subsequently, cells were washed twice with PBS and treated with serum-free culture media containing G4@IONPs in concentrations of 1500, 1000, 500, 100, 10, 1, and 0 (control) µg/ml. After incubation for 24 h, the culture media were removed, and 100 µl of serum-free medium and 10 µl of MTT solution were added to each well for 4 h. Finally, 100 µl of dimethyl sulfoxide (DMSO) was added; the absorbance of wells was measured using ELISA reader (Hiperion, microplate reader MPR4+) at 540 nm (Salimi et al. 2013).
Hemolysis assay
Blood samples from healthy male BALB/c mice were collected in heparin-coated tubes. The red blood cells (RBCs) were obtained by centrifuging the blood samples at 1500 rpm for 5 min and removing the upper plasma. The RBCs were washed three times with sterile isotonic 0.9% NaCl solution, and the purified RBCs were suspended in sterile isotonic 0.9% NaCl. The RBC suspension (300 µl) was mixed with 1 ml of 1000, 500, 250, 100, 50, and 10 µg/ml of G4@IONPs. All samples were incubated at 37 °C for 2 h and then centrifuged for 2 min at 4000 rpm. Distilled water and isotonic 0.9% NaCl were applied as positive and negative controls, respectively. The supernatant absorbance was measured at 540 nm using ELISA reader (Hiperion, microplate reader MPR4+) (Li et al. 2015). The hemolysis percentage was calculated using the following equation:
$${\text{\% Hemolysis}} = \frac{{{\text{OD}}_{\text{sample}} - {\text{OD}}_{\text{negativecontrol}} }}{{{\text{OD}}_{\text{positivecontrol}} - {\text{OD}}_{\text{negativecontrol}} }} \times 100.$$
(1)
Stability of G4@IONPs
The stability of G4@IONPs was investigated by recording the change in turbidity in 50% FBS. 150 µl of G4@IONPs suspension (100 µg/ml) was added to 150 µl of FBS in 96-well plates and incubated for different times up to 72 h at 37 °C. After that, the absorbance of samples was measured at 405 nm. A solution of 5% glucose was employed as a negative control (Li et al. 2015).
Temperature–time curves
The radiofrequency (RF) absorption of G4@IONPs was determined by establishing the AMF-specific absorption rate (SAR), which is defined as the amount of induced heat per unit mass of MNPs per unit of time (\(\frac{\Delta T}{\Delta t}\)) (Wolinsky and Grinstaff 2008; Xia et al. 2009). A magnetic hyperthermia research system (LABA, HT-1000W, NATSYCO) with a frequency range of 100–400 kHz was used. An Eppendorf microtube containing G4@IONPs solution (200 µl) was inserted inside the water-cooled magnetic induction copper coil (6 cm in diameter). The temperature rise was measured with a digital thermometer and plotted against time (temperature–time curve) at frequencies of 200 and 300 kHz with a field intensity of 12 kA/m. The SAR values were calculated using the following equation:
$${\text{SAR}} = \left( {\frac{1}{{m_{\text{Fe}} }}} \right)C\left[ {\frac{\Delta T}{\Delta t}} \right],$$
(2)
where mFe is the mass of iron in the sample, C is the specific heat capacity of the sample, and \(\left[ {\frac{\Delta T}{\Delta t}} \right]\) is the initial slope of the temperature–time curve (Natividad et al. 2008, 2009). The net temperature change was yield by the following equation:
$$\Delta {\text{T}} = {\text{T}}_{\text{n}} - {\text{T}}_{0} ,$$
(3)
where T0 and Tn are initial temperature and temperature at the interval, respectively. The SAR was estimated from the initial and steepest part of the slope of the time–temperature curve. We determined the appropriate interval for calculating the slope by analyzing the plot of incremental temperature change and selecting the region with a constant first derivative of the heating rate. The temperature change was calculated over every interval (i.e., Tn − Tn−1), and the results were plotted versus heating time (t) (Bordelon et al. 2011).
Simulation of heat generation and transfer
To verify the measurement results, simulation of heat generation due to magnetic hyperthermia was performed using COMSOL Multiphysics. The microtube, MNPs solution, and surrounding atmosphere were implemented to assess the heat transfer in a time-transient manner. Different heat transfer mechanisms were included in the simulations, e.g., heat transfer in fluids for the solution and surrounding air, and heat transfer in solids for the tube. Based on certain values of SAR for 1 ml of G4@IONPs suspension, heat generation and heat transfer rates were modeled as functions of time.
Histochemistry analysis
For Prussian blue staining, used to detect the presence of iron, MCF7 cells were incubated in a medium containing G4@IONPs (500 µg/ml) for 2 h. Subsequently, cells were fixed with 4% formalin at room temperature for 20 min and washed with PBS, followed by the incubation with 10% potassium ferrocyanide in 10% HCl (50%, v/v) for 20 min (Samanta et al. 2008). MCF7 cells after 2 h incubation with 500, 250, 100, 50, and 0 (control) μg/ml G4@IONPs were trypsinized and collected by centrifugation. The collected cells were lysed by 2 ml 65% nitric acid. The amount of the nanoparticles cell uptake was quantified using inductively coupled plasma mass spectrometry (ICP-MS) (Varian Inc, Palo Alto, CA) and the resulting concentration was divided by counting the cell numbers.
Magnetic hyperthermia in breast cancer and normal cells
At 24 h after seeding 4 × 105 cells (MCF7 and HDF1) in a 35 mm dish, cells were incubated with medium with/without the G4@IONPs in a concentration of 500 μg/ml for 2 h at 37 °C. For hyperthermia treatment, these cells were put in the magnetic coil for 120 min (12 kA/m and 300 kHz), and control cells were left in the incubator at 37 °C. Immediately afterward, the viability of cells was assessed by MTT assay.
Apoptosis assay
After hyperthermia condition, identifying of apoptotic cells was determined using in situ cell death detection kit (Roche, Mannheim, Germany) terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Assay performed according to the manufacturer’s protocol. Briefly, MCF7 cells were fixed by 4% paraformaldehyde for 10 min, permeabilized with 0.2% Triton X-100 for 2 min on ice, and incubated with a mixture of TUNEL reaction. Cells treated with 5% ethanol for positive apoptosis control. For negative apoptosis control, cells were induced only with label solution. Evaluation performed by an inverted fluorescent microscope.
Relaxivity measurements of G4@IONPs
Relaxivity is a measure of the ability of a contrast agent to enhance the relaxation of adjacent hydrogen spins, which can improve contrast in MRI images (Barick et al. 2014). To assess the relaxivity of G4@IONPs, samples were prepared at different concentrations of 1, 2, 4, 8, 10, 12, 16, and 20 µg/ml and placed in a plastic container. Longitudinal and transverse relaxation times (T1 and T2) were measured by 3 T MRI scanner (Trio Tim/SIEMENS, Munich, Germany). To measure T1, 6 spin echoes (SE) images were acquired with an echo time (TE) of 12 ms and repetition times (TR) of 3000, 2000, 1000, 500, 250, and 100 ms. To measure T2, 32 SE images were obtained with TR of 3000 ms and TE of 12–384 ms. The signal intensity (SI) of each concentration was determined via RadiAnt Dicom viewer software and calculated by Eqs. 4 and 5:
$${\text{SI}} = S_{0} \left( {1 - {\text{e}}^{{\frac{{ - {\text{TR}}}}{{T_{1} }}}} } \right)$$
(4)
$${\text{SI}} = S_{0} {\text{e}}^{{\frac{{ - {\text{TE}}}}{{{\text{T}}_{2} }}}} ,$$
(5)
where R1 and R2 curves were obtained via logarithmic fitting to SI versus TR and TE curves, respectively, using the OriginPro 2016 software. Eventually, the relaxivity values (r1 and r2) were estimated using a linear fit to R1 and R2 versus G4@IONPs concentration curves, respectively. All sequences were acquired using a 280 × 280 mm2 field of view (FOV), a resolution of 256 × 230 pixels, and slice thickness of 7 mm.
To evaluate the in vivo capability of G4@IONPs, an MRI study was performed on animal models. BALB/c mice were intravenous injected with 0.2 ml G4@IONPs at Fe concentration of 1 mg/ml. The MRI experiments were performed using a 3 T MRI scanner (MAGNETOM Prisma/SIEMENS, Munich, Germany) with a magnetic field intensity of 3 T. The following parameters were adopted for obtaining in vivo MR images and signal intensity analysis in vivo: FOV 6 × 6 cm, matrix size = 256 × 125, slice thickness 4 mm, TEs 41.6, 71, 113.6 ms, and TR 2000 ms.
Statistical analysis
All data were expressed as mean ± SD and one-way ANOVA was used for statistical analysis. P < 0.05 was considered statistically significant.