Delivery of plasmid DNA encoding Oct 4 with polyethylenimine-modified superparamagnetic iron oxide nanoparticles in HEK-293T cells

In order to improve the gene-delivering efficiency of polyethylenimine-modified superparamagnetic iron oxide nanoparticle (PEI-SPION) and elucidate the related mechanisms, the binding ratio and protection actions of PEI-SPION to plasmid DNA (pDNA), the physical characteristics of PEI-SPION/pDNA complex, and effects of exogenous factors (including magnetic field, inhibitors, and activator) on gene transfection in human embryonic kidney 293T cells (HEK-293T cells) were explored here. The results showed that the diameter of PEI-SPION was about 45 nm. When PEI-SPION and pDNA encoding Oct 4 (pDNA-Oct 4) were mixed at the mass ratio of 1: 1, the hydrodynamic diameter (299.10 ± 5.01 nm) of conjugate complex was significantly smaller than that of complex at the mass ratio of 1: 2 (469.23 ± 28.86 nm; P < 0.05) or 1: 4 (593.67 ± 20.81 nm; P < 0.01); but its zeta potential was significantly higher than that of these latter two treatment groups, respectively (23.57 ± 0.84 mV vs 18.07 ± 1.03 mV, 14.7 ± 0.44 mV; P < 0.01); if the proportion of PEI-SPION to pDNA-Oct 4 was further adjusted to 1: 8 or 1: 16, the zeta potential of conjugate became negative. DNA binding test also indicated that the combining ability of PEI-SPION to pDNA-Oct 4 was close to saturation state when the mass ratio was 1: 4. The PEI-SPION-mediated transfection efficiency would be significantly enhanced under the auxiliary action of external magnetic field (280 mTesla) for 20 min (P < 0.01). No significantly difference in transfection efficiency was observed among chlorpromazine (CPZ) treatment groups and control group (P > 0.05), and similar results were found for treatments of nystatin. Compared with control group, 2-deoxy-D-glucose (2-DG) and amiloride could significantly decrease the transfection efficiency (P < 0.01), and only 0.3 ± 0.58% cells were successfully transfected with pDNA-Oct 4 in 10 mM 2-DG treatment group. One hundred nanomolar phorbol-12-myristate-13-acetate (PMA) could enhance the cell transfection of pDNA-Oct 4, and the transfection efficiency was increased up to 83.44 ± 2.16%. Therefore, PEI-SPION could efficiently concentrate pDNA-Oct 4 and protect them from the digestion of DNase I. The uptake of PEI-SPION/pDNA-Oct 4 by HEK-293T cell was an energy-consuming process, and the main pathway for this process was macropinocytosis. One hundred nanomolar PMA and external magnetic field could improve the PEI-SPION-mediated gene transfection.