Materials and methods

Chemical r eagents

Dulbecco’s modified eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco. Penicillin-streptomycin and trypsin-EDTA were obtained from Bioidea. The 25 kDa branched polyethylenimine (PEI) and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich. FeCl3.6H2O, FeSO4.7H2O and NaOH were purchased from Merck. Dimethyl sulfoxide (DMSO) was obtained from Daejung Chem., Korea. Two prepared polymers of dextran-stearic acid-spermine polymers (DSASP) were obtained from Dr. Ebrahim Vasheghani-Farahani lab.

Synthesis of magnetic nanoparticles

Chemical co-precipitation method is defined as one of the Fe3O4 nanoparticles synthesized protocol [27]. In brief, FeCl3.6H2O and FeSO4.7H2O with a molar ratio of 2:1 were mixed under continuous stirring and nitrogen gas bubbling to inhibit oxidation. Subsequently, to reach the pH up to 10, NaOH (2.0 M) was slowly added into the mixture under continuous stirring. Moreover, the black precipitate as a result of NaOH addition, indicates the formation of Fe3O4 nanoparticles. Then, the mixture was vigorously stirred for 15 min and incubated at 70 °C for 30 min, after that it was cooled down at room temperature. Next, Fe3O4 nanoparticles were magnetically separated and frequently washed by deionized-water and ethanol solution to remove the impurities. Finally, MNPs were dried in an oven at 60 °C and collected by magnetic separation and stored under N2gas condition [28].

Plasmid purification

Plasmid DNA (pDNA) used in this study was pDB2 plasmid encoding luciferase, was propagated and isolated from DH5ɑ E. coli overnight. Qiagen Maxi kit-25 (Qiagen K.K., Tokyo, Japan) was applied for extraction and purification of pDNA. The concentration and purification of pDNA was measured using Nanodrop (Thermo, Wilmington, DE, USA) at an absorbance ratio of 260/280 nm ranging from 1.9 to 2.2.

Preparation of polymer-based magnetic complexes

The pDNA (1 μg/μl) and DSASP polymer at different mass ratios were diluted in deionized-water and incubated for 30 min at room temperature to from DSASP–pDNA complexes. Then, 1 μg/μl Fe3O4 magnetic nanoparticles (MNPs) were added to the mixture of DSASP–pDNA complexes and reincubated for extra 30 min.

Characterization of magnetic nanoparticles and complexes

The size and charge of MNPs and complexes were performed by dynamic light scattering (DLS). The size distribution and zeta potential were evaluated with a Zetasizer Nano-ZS instrument, Model ZEN3600 (Malvern Instruments, UK). Briefly, the dispersions of Fe3O4 were sonicated for 30 s in deionized-water and then incubated with DSASP–pDNA complex as previously mentioned. The particle size was measured in the scattering angle 173° at 25 °C. The zeta potential was measured using a universal zeta dip cell. DLS measurements were taken for DSASP–pDNA/Fe3O4 in the presence of increasing concentrations of DSASP. In addition, scanning electron microscopy (SEM) analysis was carried out to indicate the morphology and the size of both MNPs and complexes. Moreover, the magnetic properties of dried-SPIONs and DSASP–Fe3O4 were measured at a maximum applied field of 10 kOe using VSM 7300 vibrating sample magnetometer (VSM) (Lakeshore, USA). Furthermore, the chemical component and molecular vibrations of samples were analyzed by Fourier transform infrared spectroscopy (FTIR, Thermo Scientific Nicolet IR100, Madison, USA) using KBr pellet. Three scans per spectrum (400–4000 cm−1) at the resolution of 4 cm−1were measured for dried samples of Fe3O4, DSASP and DSASP–Fe3O4 mixed with KBr and made into pellets. The 6 mg/ml DSASP–Fe3O4was used in SEM, VSM and FTIR measurements.

Gel retardation assay

The gel retardation or electrophoretic mobility shift assay is utilized to evaluate the DNA interaction with complexes [29] and ensure about formation of ternary complexes of pDNA– DSASP–Fe3O4. DNA molecules migrate from the negative electrode towards the positive electrode. Moreover, addition of the small molecules such as DSASP to pDNA, the mobility will be restricted through the gel based on the emergence of electrostatic interaction between pDNA and polymer [30]. DNA condensation and mobility after binding with DSASP–Fe3O4complex was identified by gel retardation assay. Different weight-mixing ratios of DSASP–pDNA/Fe3O4 (1, 2.5, 5, 10, 25, 50 and 100) were prepared while the content of DNA to Fe3O4 was kept at 1 μg/μl and incubated at 37 °C for 15 min. After that, 8 μL of mixture suspension was analyzed by 1% agarose gel electrophoresis (90 V, 45 min). Naked–pDNA was utilized as a control group. The gel was stained in 0.5 mg/ml ethidium bromide for 30 min to visualize the localization of pDNA by UV illumination.

Serum stability assay

In the process of gene transfection, the usage of naked-DNA due to effects of nucleases and reticuloendothelial system cannot be possible. So, the necessity of appropriate vectors for protecting DNA play a crucial role against the enzymatic conditions. Moreover, to have an efficient gene delivery system especially in the in vitro, genetic materials’ carrier should protect DNA in front of serum enzyme during gene delivery, and also minimize the time association of pDNA with blood serum, which was investigated by DNaseI activity at weight ratio of 10 in the gel electrophoresis. Briefly, 6.5 μl of each complex was exposed to 0.75 μl of DNaseI (Promega, USA) in the final volume of 15 μL for 15 min at 37 °C. The reaction was inactivated followed by transferring complex into -20 °C. After the addition of 10% sodium dodecyl sulfate (SDS) to the solution, pDNA was released from complex and subsequently 8 µl of each sample comprising 3 μg DNA that was analyzed by 1% agarose gel electrophoresis (90 V, 45 min). The integrity of released-pDNA from the complex was compared with the naked-pDNA as control.

Cell Culture

The human embryonic kidney 293T cell line (HEK 293T) was obtained from Pasteur Institute of Iran (IPI). The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) 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 humidity incubator. The cells were then trypsinized (0.025% trypsin, 0.02% EDTA) after they were grown up to 70–80% confluent. Prior to treatments, cells were allowed to reattach overnight.

Cell viability assay

The viability of cells which are treated by nanoparticles were measured by MTT assay. This is a colorimetric assay based on ability of viable cells to reduce the water-soluble yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) by mitochondrial NAD(P)H-dependent oxidoreductase enzyme (succinate dehydrogenase) to purple insoluble formazan crystal. The HEK 293T cells were seeded into 96-well cell culture plates (SPL Life Sciences Co., Ltd. Korea) at the final concentration of 104 cells/well and incubated in 100 μL of DMEM containing 10% FBS and allowed to attach overnight. Then, cells were treated in the fresh supplemented DMEM with 10% FBS by DSASP–pDNA/Fe3O4 complexes including 1 μg/μL concentration of Fe3O4nanoparticles and pDNA, and different weight-ratio of DSASP (0.5, 1, 2.5, 5, 10, 20, 25, 50 and 100) in presence and absence of homogenous static magnetic field (SMF, 20 mT) for 30 min. After 24 and 48 h treatments, the media was removed and MTT solution (0.5 mg/ml in FBS-free DMEM) was added and incubated at 37 °C for extra 4 h in the dark. After that, the media was removed and 100 μL DMSO was added to each well. Finally, the absorbance of formazan intensity was quantified by a microplate reader (uQuant MQX200, BioTek, USA) at 570 nm.

Magnetofection

In this study, the gene delivery process, the magnetic ternary complexes were transferred to cells. The complexes comprised the pDNA and MNPs in the similar mass ratio of 1 μg. The extracted pDNA encoding the luciferase reporter gene that handles based on kinase activity, which converts luciferin to oxyluciferin and light using ATP. The ATP is converted to AMP and release the energy of the phosphate bonds in the form of light that was measured by luminometer [31]. The intensity of light indicates the activity of luciferase being equivalent to the amount of delivered nucleic acid to the cells. Briefly, 5 × 104 cells/well of HEK 293T cells were seeded in the 24 well cell culture plates and allowed to attach overnight. Then, 1 μg/μl pDNA was mixed with a DSASP solution at various mass ratios (0.5, 1, 2.5, 5, 10, 20, 25, 50 and 100) and incubated for 15 min. After that, the prepared-complexes gently were mixed with 1 μg/μl Fe3O4 nanoparticles and reincubated for extra 15 min. Each prepared complex’s DSASP–pDNA/Fe3O4 were diluted with FBS-free DMEM to a final volume of 300 μL and incubated at room temperature for extra 30 min. Next, polyplex solutions were added to cells in presence and absence of homogenous SMF (20 mT) at 37 °C for 30 min. After that, the media was replaced with DMEM containing 10% FBS, and cells were incubated for 48 h. Moreover, the 4.8 μg PEI at N:P ratio of 12 was utilized as control. Then, the media was completely removed and cell lysates were analyzed for luciferase activity that is followed by injection of assay buffer to a tube containing cell lysate and luminescence was evaluated using a luminometer (Berthold detection systems, GmbH, Germany).

Statistical Analysis

GraphPad Prism v5.07 (GraphPad Software Inc., San Diego, CA) was used for statistical analysis and data graphing. We applied one-way followed by post-hoc analysis using Newman–Keuls multiple comparison test to compare between independent variables. The error bars represented the mean ± standard deviation (mean ± SD) of at least three independent experiments (n = 3). P-values including *p < 0.05; **p < 0.001; ***p < 0.0001 were considered as a statistical significance difference.