Saleh Tayebeh, Bolhassani Azam, Shojaosadati Seyed Abbas, Hosseinkhani Saman
Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran.
Iran J Biotechnol. 2015 Mar;13(1):55-62. doi: 10.15171/ijb.1115.
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.
The aim of this study was to investigate the 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 .
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. 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.
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.
The results indicated that the efficient gene expression depends on both cell type and N/P ratio applied, . The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPGbased nanoparticles as a potent gene delivery system.
血浆膜和核膜对DNA质粒的低渗透性是这些治疗性分子发展的两个主要障碍。因此，基因治疗方法的成功取决于高效且安全的非病毒递送系统的开发。
本研究旨在研究使用细胞穿透肽递送系统递送编码HPV16 E7基因的质粒DNA，以实现细胞转染和蛋白质表达的最佳条件。为此，我们使用了一种阳离子肽递送系统MPG，它与核酸形成稳定的非共价复合物，用于递送作为模型抗原的pEGFP-E7。
大规模制备高纯度的编码HPV16 E7的DNA构建体（pEGFP-E7）。以不同的N/P（氮/磷）比制备MPG肽/DNA复合物，并研究纳米颗粒的物理化学特性和稳定性。进行肽介导的E7-GFP DNA转染，并在三种细胞类型中评估其表达。为了量化该递送系统的转染效率，收获转染的细胞并通过流式细胞术评估GFP阳性细胞。此外，通过蛋白质印迹分析证实E7-GFP表达。
基于MPG的纳米颗粒的细胞摄取显示与标准试剂PEI相当。以15:1的N/P比用基于MPG的纳米颗粒转染的COS-7细胞显示出最高的转染效率和基因表达。
结果表明，有效的基因表达取决于所应用的细胞类型和N/P比。通过蛋白质印迹和流式细胞术检测到的有效蛋白质表达支持基于MPG的纳米颗粒作为一种有效的基因递送系统的潜力。
