Formation of MNPs and DSASP–pDNA/MNPs
The formation of ternary nanocomplexes is fundamental in transferring
nucleic acids comprising magnetic cores with polymeric shells that are
conjugated to pDNA. In the delivery systems, the size of nanocarriers
have significant roles that were obtained from DLS about 62 nm for the
synthesized Fe3O4 and 100–300 nm for
the SPIONs formulated in DSASP–pDNA complex at weight ratios of 10–100
w/w (Fig. 2a,c) . The zeta potential of the superparamagnetic
iron oxide nanoparticles was -27 mv (Fig. 2b) . The stronger
negative surface charge distribution of nanoparticles can facilitate the
electrostatic interaction with positive charge particles. Additionally,
results demonstrated that increasing the positive charge and decreasing
the particle size as a result of rise in the mass ratio of DSASP (0.5,
1, 2,5, 5, 10, 25, 50, 100) at the a concentration of
Fe3O4/pDNA (1 mg/ml), can efficiently
affect the electrostatic interactions with pDNA and lead to more
condensed molecules (Fig. 2c,d) . Successful synthesis of
DSA6SP–pDNA/Fe3O4nanoparticles sustained due to the positive zeta potential of +35 mv
value at weight rate of 50 w/w. In addition, DSASP 6 and 8 have a
relative difference in amine contents as a result of divers’ conjugation
of stearic acid, in which the amount of 20% and 15% were evaluated for
their hydrophobic part, respectively.
Boosting the graph rate of stearic acid can decrease the micelles
particle size. There is a harmony with this phenomenon that the ratio of
hydrophobic to hydrophilic chain length, remarkably impresses the
particle size in linear amphiphilic polymers (Fig. 2c) . So,
smaller particle size with higher surface/volume ratios will have higher
zeta potential, which could efficiently surround negative charge of
nucleic acids (Fig. 2d) . Negative surface charge of
DSASP–pDNA/Fe3O4 complexes at w/w ratio
below 10, were gradually transferred into positive potential as result
of an increase in w/w rate of polymer. Furthermore, in this study the
negative surface charge almost up to 5 w/w demonstrated insufficient
formation of complexation between polymer and DNA though electrostatic
repulsion which indicated complex inability to condense nucleic acids
negatively charged phosphate backbone properly (Fig. 2d) [12,
32].
As shown in Fig. 3a , the SEM image of MNPs declares a
homogeneously spherical shape for Fe3O4particles with diameter around 12-18 nm, which has proper agreement with
the result of DLS assay. Obtained SEM results also represent roughly
spherical form for agglomeration of
DSASP–Fe3O4 nanoparticles with
desirable dispersibility and no significant size difference in
comparison with Fe3O4 MNP (Fig.
3b ). Therefore, due to optimum size of delivery vectors from 50 to 100
nm [12], the MNPs derived from
DSASP–pDNA/Fe3O4 seems to be efficient
in gene delivery. Size comparison between SEM imaging and zetasizer
measurement is attributed to dried and hydrated state of samples,
respectively. It means that the DLS information represents the
hydrodynamic size of nanoparticles being naturally larger than their
actual size [33].
Moreover, VSM was used to investigate the magnetic behavior of
nanoparticles and the formation of
DSASP–Fe3O4 complexes. The maximum size
for superparamagnetic behavior is generally taken to be almost 30 nm
[34]. The hysteresis loops of bare
Fe3O4 and coated MNPs of
DSASP–Fe3O4 was shown in Fig.
3c,d . Reduction in hysteresis magnetization from plateau level to zero
was remarkably shown at room temperature (~298 K) for
both curves through removal of MFs confirming their superparamagnetic
property. In addition, the magnetic saturation of MNPs complexes is
estimated to be 30.160 emμ·g–1, being lower than bulk
Fe3O4 at 65.3
emμ·g–1. The lower rate of magnetic saturation of
DSASP–Fe3O4 compared to
Fe3O4 could be attributed to the
attachment of DSASP to nanoparticles. Indeed, cationic surface spines of
polymer as modification, affect the core spin of
Fe3O4 from along the MFs that decrease
the complex saturation magnetization values [35].
The IR spectrum being obtained from the FTIR spectrometer is located in
the mid-IR region (666-4000 cm-1). Considering the
mid-IR region, many functional groups’ transition energy due to their
alternation in vibrational energy state could be estimated to determine
whether specific functional groups exist in the structure of molecules
[36]. The FTIR spectrum shows striking information on the chemical
structure of Fe3O4 nanoparticle and
DSASP–Fe3O4 complexes in the region of
400-4000 cm-1 (Fig. 3e ). The first gradual
absorption pick at 3413.89 cm-1 originated from strong
stretching vibrations of hydrogen band with hydroxyl groups (OH) that
were absorbed by the sample from the medium. The evaluated peak at 1617
cm−1 is attributed to the O–H bending [37]. In
addition, the second absorption peak at 567.06 cm-1 is
assigned to Fe-O bond in Fe3O4nanoparticles which indicates the synthesization of nanoparticles.
Changing the peak location of Fe–O from 567cm-1 to
583cm-1 showed relative interaction of stearic acid
with SPIONs. Although the intensity of this peak replacement was
expected to be stronger, decline in
Fe3O4 absorption peak spectrum,
demonstrated its sufficient entrapment by biopolymer. Moreover, decline
in COO– vibration from 1709.29 to 1699.78 could be considered as
another evidence for stearic acid interactions. Furthermore, both
binding groups of C-N at wavelength peak of 1650-1620
cm-1 and noticeable decline in absorption peak of 580
to 631 cm-1 in Fe3O4could be related to dextran-spermine entrapment [18, 38, 39]. So,
results showed a successful synthesis of MNPs grafted to polymers.
Estimating the protection capability of
nanocomplexes
Protection capability for both 6 and 8 DSASP polymers in different
complexes charge and mass ratios at 1, 2.5, 5, 10, 20, 25, 50, 100 w/w
were prepared in 1% agarose gel electrophoresis. As shown inFig. 4a,b , for both of DSA6SP and
DSA8SP at mass ratio above 1 w/w, with increasing
concentration, no migration was observed for complexes since they
remained in the well. Turning to 1 w/w for both polymer weak
interactions was shown between DNA and DSASP and the positive charge of
complexes could not completely prevent the plasmid from migration toward
the positive electrode. In addition, at this mass ratio compared to
polymer 8, it seemed that DSA6SP had better
electrostatic interaction with DNA due to the appearance of a more
inconspicuous bond on agarose gel. Therefore, these differences
indicated that in equal proportions of these polymers, the
DSA6SP ability in pDNA capsulation was greater than
DSA8SP polymer. The instability of pDNA in presence of
DNaseI was incubated with
pDNA-DSASP–Fe3O4 ternary complexes. As
seen in Fig. 4c,d , the fragment smear at the bottom of agarose
gel indicated that naked DNA is degraded within 30 min while duplex and
ternary complexes with pDNA were completely protected from enzymatic
degradation. Furthermore, the plasmid bands after the addition of SDS to
incubated complexes with DNaseI indicates the pDNA complexes with DSASP
or DSASP–Fe3O4 particles remained
intact whereas naked pDNA was almost digested.
The effects DSASP on cell viability in presence of
SMF
The cell viability based on the efficacy of MNPs with
DS6ASP and DS8ASP was performed through
MTT assay at different mass ratios in presence and absence of SMF. As
shown in Fig. 5 , the MFs do not show any excess cytotoxicity in
both nanocomplexes compared to the absence of SMF. Moreover, increasing
the mass ratios of DS8ASP complexes, toxicity did not
surge significantly (P≥0/05) except 100 w/w in presence of SMF, and also
in some mass ratios such as 10 w/w increase in cell proliferation was
estimated compared to control. In addition, in all treatments of
DS6ASP polymer except the 100 w/w, by increasing the
mass ratios, no toxicity was significantly observed (P≥0/05(. In the
following treatments, the concentration of DSASP and also MNPs were
prepared at 10 w/w and incubated in presence and absence of SMF. The
results indicated no significant cytotoxicity in presence and absence of
SMF compared to control after 48 h.
Transfection efficiency of
DSA6SP and DSA8SP polyamines in presence
SMF
We transfected nucleic acids based on the magnetofection process, the
DSA8SP–pDNA/MNP and DSA6SP–pDNA/MNP
ternary complexes were transferred to cells using an external SMF 20 mT.
The ratio of polymer to DNA was determined due to different mass ratios
including 10, 20, 25, 50 and 100 w/w. Moreover, the amount of
nanoparticles in the complex was optimized based on the weight ratio of
pDNA. In different mass ratios of magnetic DSA8SP, an
increase in luciferase activity was recorded compared to treated cell
with pDNA alone. The transfection rate of the PEI as a positive control
did not increase significantly compared to most mass ratios of
DSA8SP–pDNA/MNP complex (P>0.05). In
addition, the SMF enhanced the transfection efficiency compared to
control, especially in the mass ratios of 10 and 50 w/w
(P<0.05) (Fig. 6a ). The magnetic
DSA6SP polymer with different mass ratios surged the
activity of luciferase significantly and also the SMF affected the
transfection efficacy of polymer specifically at the mass ratio of 20
and 50 w/w (P<0.05). Moreover, transfection rates of magnetic
nanocomplexes were higher than PEI polymer (P<0.05)
(Fig. 6b ). As seen in Fig. 6c, comparison of both
complexes illustrated appropriate cellular internalization as a result
of a composed hydrophobic core of stearic acid, but based on a slight
difference in the amount of fatty acids in polymers, the transfection
efficiency of DSA6SP was higher than
DSA8SP polymer.