Discussion
Several methods are used for synthesis of superparamagnetic iron oxide
nanoparticles that are generally developed through two mechanisms
including thermal decomposition and co-precipitation. The latter is
represented to be more appropriate as it is considerably biocompatible
in the in vivo conditions. Iron oxide nanoparticles are coated with
biocompatible polymer for cellular transfection [27, 40]. Here, we
investigated the properties of DSA6SP and
DSA8SP–Fe3O4 for gene
delivery in the in vitro transfection systems.
In cells, spermine due to having a considerably cationic charge,
ameliorates DNA packaging into nucleus through neutralizing anionic
phosphate backbone charge which leads to DNA condensation. Spermine
could be applied as a valuable factor in gene delivery applications
[21]. Therefore, a decrease in the size of
DSASP–Fe3O4 complex at weigh rate above
10 nm tend to be attributed to higher spermine rate (Fig. 2c ).
On the other hand, different stearic acid grafted rates in DSASP
amphiphilic polymer of 6 and 8 tend to be another efficient factor in
polymer size rate that leads to more DNA condensation. Indeed, the
natural chemical structure of stearic acid enabled molecules to be more
flexible to move inward considering its free rotation of saturated
carbon atoms [21]. Moreover, the surface charge of dextran-stearic
acid polymers is often negative and close to zero due to the absence of
ionizing groups in the dextran chains and stearic acid molecules [15,
19, 22]. Furthermore, zetasizer measurement revealed that grafts of
spermine converted the zeta potential of
Fe3O4 nanoparticles from negative into
positive charges (Fig. 2d ). In aqueous environment, spermine
residues in DSASP–NPs are localized outside the micelle formation due
to its hydrophobicity property. Considering to cell membrane negatively
charged, the positively charged complexes facilitate its cellular uptake
[41].
Nanoparticles with critical diameter less than 25 nm have
superparamagnetic behaviors [42]. VSM magnetization curves indicated
superparamagnetic behavior with superior magnetic response for having
efficient magnetofection process in both unmodified
Fe3O4 nanoparticle and
DSASP–Fe3O4 complexes (Fig.
2c,d). Magnetic properties of nanoparticles strongly depends on the
size and shapes [42, 43], because their single magnetic domain below
critical diameter possess spherical structure which has parallel
direction with magnetic spins that would be a reliable confirmation on
SEM data and successful modification of the
Fe3O4 MNPs by DSASP. The magnetism
saturation of magnetic complexes is measured for 30.160
emu.g-1 relatively, being 35% lower than
Fe3O4 MNPs at 65.3
emu.g-1, reflecting the thick amphiphilic shells
covering Fe3O4 nanoparticles. This
superparamagnetism feature is significantly recommended in the in vivo
systems that prevents agglomeration of particles in the blood cycle
through the removal of MFs and vanish magnetization, subsequently
[42]. Results of FTIR spectra as shown in Fig. 3e ,
represent different interactions such as coordination between COO− and
Fe3+ (or Fe2+), hydrogen bonds, Van
der Waals force and electrostatic interactions which keep DSASP on the
surface of magnetite nanoparticles [38, 44].
The issue of stability in formation of polycations-based nanocomplexes
with negative phosphate backbone charge of DNA is considered as a
substantial requirement in gene delivery applications to achieve proper
transfection efficiency. Preparation of various weight ratio of
polycations to DNA is assumed as one of the most significant parameters
to achieve this process [41, 45]. So, a different mass ratio of
DSASP was mixed with similar volumes of pDNA and then results were
analyzed based on gel retardation assay. The pDNA band in mass ratio of
1 w/w illustrates amphiphilic polymer inability to have efficient
electrostatic interaction between amino groups and phosphate groups of
DNA to entrap plasmid. Whereas, the migration of DNA, above this mass
ratio was completely retarded, indicating DSASP enough cationic charge
to form sustained and strong complexation with negative charge of DNA.
The spermine residue conjugation on dextran molecules in DSASP polymer
is considered as a factor that neutralizes the negative surface charge
of DNA [45]. Moreover, the content of the spermine moieties on the
dextran chain in DSA8SP (0.35 µmol / mg) is higher than
the other formulation based on the lower conjugation of stearic acid to
polysaccharide which results in less space barrier for binding of the
primary amines. Additionally, in comparison with DSA8SP,
results show a more inconspicuous bond for gel retardation of
DSA6SP polymer in 1 w/w, which represents in an equal
mass ratio of polymers, DSA6SP was more effective in DNA
encapsulation (Fig. 4a,b ). Higher residues of non-cationic
fatty acids seem to be more effective in nucleic acid retardation
compared to amine positive charges. It means, the hydrophobicity of the
amphiphilic polymer, even in lower density, could neutralize a
considerable fraction of the DNA charges [21].
Enzymatic degradation discussed as a restriction factor, impressively
inhibits the process of gene transfection applications. Serum stability
results demonstrate that although free DNA was completely digested by
DNaseI due to existence of smear/fragmented band, both DSASPs
encapsulated DNA were efficiently protected pDNA under enzymatic
conditions attending to bright wells as an evidence for the presence of
DNA which was properly capsulated in amphiphilic biopolymer