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