Tayebeh Saleh1, Azam Bolhassani2, Seyed Abbas Shojaosadati3, Saman Hosseinkhani1. 1. Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. 2. Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran. 3. Biotechnology Group, Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, Iran.
Abstract
BACKGROUND: The poor permeability of the plasma and nuclear membranes to DNA plasmids are two major barriers for the development of these therapeutic molecules. Therefore, success in gene therapy approaches depends on the development of efficient and safe non-viral delivery systems. OBJECTIVES: The aim of this study was to investigate the in vitro delivery of plasmid DNA encoding HPV16 E7 gene using cell penetrating peptide delivery system to achieve the best conditions for cell transfection and protein expression. For this purpose, we have used a cationic peptide delivery system, MPG which forms stable non-covalent complexes with nucleic acids for delivery of pEGFP-E7 as a model antigen in vitro. MATERIALS AND METHODS: DNA construct encoding HPV16 E7 (pEGFP-E7) was prepared in large scale with high purity. MPG peptide/ DNA complexes were prepared at different N/P (nitrogen/phosphate) ratios and physicochemical characterization and stability of nanoparticles were investigated. In vitro peptide-mediated E7-GFP DNA transfection, and its expression was evaluated in three cell types. To quantify the transfection efficiency of this delivery system, transfected cells were harvested and assessed for GFP-positive cells by flow cytometry. Furthermore, E7-GFP expression was confirmed by western blot analysis. RESULTS: The cellular uptake of MPG based nanoparticles was shown to be comparable with standard reagent PEI. The COS-7 cells transfected by MPG-based nanoparticles at an N/P ratio of 15:1 showed the highest transfection efficiency and gene expression. CONCLUSIONS: The results indicated that the efficient gene expression depends on both cell type and N/P ratio applied, in vitro. The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPGbased nanoparticles as a potent gene delivery system.
BACKGROUND: The poor permeability of the plasma and nuclear membranes to DNA plasmids are two major barriers for the development of these therapeutic molecules. Therefore, success in gene therapy approaches depends on the development of efficient and safe non-viral delivery systems. OBJECTIVES: The aim of this study was to investigate the in vitro delivery of plasmid DNA encoding HPV16 E7 gene using cell penetrating peptide delivery system to achieve the best conditions for cell transfection and protein expression. For this purpose, we have used a cationic peptide delivery system, MPG which forms stable non-covalent complexes with nucleic acids for delivery of pEGFP-E7 as a model antigen in vitro. MATERIALS AND METHODS: DNA construct encoding HPV16 E7 (pEGFP-E7) was prepared in large scale with high purity. MPG peptide/ DNA complexes were prepared at different N/P (nitrogen/phosphate) ratios and physicochemical characterization and stability of nanoparticles were investigated. In vitro peptide-mediated E7-GFP DNA transfection, and its expression was evaluated in three cell types. To quantify the transfection efficiency of this delivery system, transfected cells were harvested and assessed for GFP-positive cells by flow cytometry. Furthermore, E7-GFP expression was confirmed by western blot analysis. RESULTS: The cellular uptake of MPG based nanoparticles was shown to be comparable with standard reagent PEI. The COS-7 cells transfected by MPG-based nanoparticles at an N/P ratio of 15:1 showed the highest transfection efficiency and gene expression. CONCLUSIONS: The results indicated that the efficient gene expression depends on both cell type and N/P ratio applied, in vitro. The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPGbased nanoparticles as a potent gene delivery system.
Authors: Klaus Kunath; Anke von Harpe; Dagmar Fischer; Holger Petersen; Ulrich Bickel; Karlheinz Voigt; Thomas Kissel Journal: J Control Release Date: 2003-04-14 Impact factor: 9.776
Authors: Flavia Rodrigues Oliveira Silva; Nelson Batista Lima; Maria Helena Bellini; Luiz Felipe Silva Teixeira; Eric Yiwei Du; Niloufar Jamshidi; Justin Gooding; Adam David Martin; Alexander Macmillan; Christopher Peter Marquis; Pall Thordarson Journal: Nanotheranostics Date: 2022-02-16