(Fig. 4c,d).
As DSASP polymer did not show any cytotoxicity, cell proliferation was reported in some weight ratio like 10 w/w compared to control. Additionally, by increasing the polymer mass ratio, except 100 w/w, no significant cytotoxicity was observed for any DSA8SP and DSA6SP magnetic complexes (Fig. 5a,b ). It is noticeable that the iron oxide nanoparticles, even after amphiphilic surface modification, sustained their low cytotoxicity in presence and absence of MFs (Fig. 5c ). The polyamines like spermine are fundamental factors in the cell proliferation cycle which naturally exist in cells [19, 46]. Indeed, although spermine’s increasing rate provides grounds to accelerate cell proliferation and restrict apoptosis by inhibiting cytochrome c release, its intracellular depletion leads cellular growth to be arrested [46].
Decrease in time association of nucleic acids with enzymatic conditions in cells and/or serum by the aim of accelerating DNA agglomeration to targeted cells and intensifying its rapid physical transportation, is demonstrated as another impressive factor in DNA protection which is studied through magnetofection [32]. Magnetofection through applying the external MF is considered as a non-invasive therapeutic approach, leading to cell viability without disrupting plasma membranes [23]. It is clearly shown that incubation with the application of a magnetic field in cell culture media remarkably reinforces cellular efficiency of complex for all DSASP–pDNA/MNPs, especially at weight ratio of 50 and 10 w/w in DSA6SP and also 50 and 20 in DSA8SP (Fig. 6) . However, DSA6SP–pDNA/Fe3O4nanoparticles show a preferable transfection efficacy presumably based on higher stearic acid residues which result in better condensation of complexes (Fig. 6b). DSA6SP with higher grafted rate of stearic acid (20%), gradually restricted more spermine conjugation with dextran chain which its amine content was evaluated at 0.26 µmol/mg as well as 0.35 µmol/mg and also 15% stearic acid in DSA8SP.
Spermine residues conjugated on dextran make it a more appropriate biodegradable polycations for gene delivery applications [47]. Moreover, based on our obtained results, the addition of both spermine and stearic acid conjugations along the dextran chain, affect the DNA-binding complex transfection efficiency and density, significantly. Hosseinkhani et al., considered stable interactions with various cationic polysaccharide derivatives and different grafts of oligoamines, only dextran-spermine polycations found active in transfection assay [47]. Additionally, the emphasis of grafted spermine moieties in transfection efficiency was represented properly by the study of Azzam et al., to point out the spermine residues high buffering capacity. Therefore, the more spermine incorporation in dextran-spermine compound, the more luciferase expression [14]. Furthermore, spermine could promote DNA condensation and simplify endo-lysosomal escape of oligonucleotide with two primary and two secondary amino groups respectively which results in an efficient transfection rate [19].
In conclusion, conjugation of fatty acids to polycations are utilized to protect the coated DNA-polymer complex from enzymes and ease the cellular uptake of amphiphilic complex into lipophilic plasma membrane based on hydrophobic surface [14]. Particle size in amphiphilic polymers, was directly affected by hydrophobic to hydrophilic chain ratios. So, the enhancement of stearic acid conjugation degree in amphiphilic structure simultaneously leads to increase in polymer hydrophobic properties, closer polymeric interactions in aqueous medium and assembly of smaller particles. Indeed, decreasing the size will subsequently increase the surface/volume ratio of particles. These phenomena lead more particles to communicate and destabilized the aqueous medium [22]. Therefore, through higher association of stearic acid with dextran in DSASP-pDNA/Fe3O4 complexes, the surface/volume ratio increases alongside decreasing complex size which consequent more hydrophobic interaction with plasma membrane. Considering DSA8SP with more amine content and also the efficient role of positive charges, the lower transfection efficacy was represented for this carrier compared to DSA6SP. Finally, the importance of hydrophilic content in transfection assay is explained based on the efficient effects on total size of complexes and enhancement of their cellular attachment due to plasma cell membranes.