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.