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 . 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 . 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 .
Nanoparticles with critical diameter less than 25 nm have superparamagnetic behaviors . 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 . 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 . 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 .
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 (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 .
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 . Magnetofection through applying the external MF is considered as a non-invasive therapeutic approach, leading to cell viability without disrupting plasma membranes . 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/Fe3O4 nanoparticles 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 . 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 . 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 . 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 .
The 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 . 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 .