Enhanced Gene Delivery in Bacterial and Mammalian Cells Using PEGylated Calcium Doped Magnetic Nanograin

Abstract

Ehsan Hashemi,1,2 Hossein Mahdavi,3 Jafar Khezri,1 Farideh Razi,2,4 Mehdi Shamsara,1 Abbas Farmany5 1National Research Center for Transgenic Mouse & Animal Biotechnology Division, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran; 2Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran; 3School of Chemistry, College of Science, University of Tehran, Tehran, Iran; 4Diabetes Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran; 5Dental Implant Research Center, Hamadan University of Medical Sciences, Hamadan, IranCorrespondence: Abbas FarmanyDental Implant Research Center, Hamadan University of Medical Sciences, Hamadan, IranEmail [email protected]: Beyond viral carriers which have been widely used in gene delivery, non-viral carriers can further improve the delivery process. However, the high cytotoxicity and low efficiency impedes the clinical application of non-viral systems. Therefore, in this work, we fabricated polyethylene glycol (PEG) coated, calcium doped magnetic nanograin (PEG/Ca(II)/Fe3O4) as a genome expression enhancer.Methods: Monodisperse magnetic nanograins (MNGs) with tunable size were synthesized by a solvothermal method. The citrate anions on the spherical surface of MNGs capture Ca2+ ions by an ion exchange process, which was followed by surface capping with PEG. The synthesized PEG/Ca(II)/Fe3O4 was characterized using Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS) spectra, vibrating sample magnetometer (VSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). MTT test was utilized to assess the toxicity of PEG/Ca(II)/Fe3O4. Real time qPCR was applied for quantification of gene expression.Results: DLS spectra and TEM images confirmed a thin layer of PEG on the nanocarrier surface. Shifting the zeta potential in the biological pH window from −23.9 mV (for Fe3O4) to ≈ +11 mV (for PEG/Ca(II)/Fe3O4) confirms the MNGs surface protonation. Cytotoxicity results show that cell viability and proliferation were not hindered in a wide range of nanocarrier concentrations and different incubation times.Conclusion: PEGylated calcium doped magnetic nanograin enhanced PUC19 plasmid expression into E. Coli and GFP protein expression in HEK-293 T cells compared to control. A polymerase chain reaction of the NeoR test shows that the transformed plasmids are of high quality.Keywords: gene expression, calcium doped magnetic nanograins, cytotoxicity

Keywords

• gene expression
• calcium doped magnetic nanograins
• cytotoxicity