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.