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Published on: March 8, 2012
Evaluation of Cell Penetrating Peptide Delivery System on HPV16E7 Expression in Three Types of Cell Line
Tayebeh Saleh1, Azam Bolhassani2, Seyed Abbas Shojaosadati3
1Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
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.
