Characterization of MiRGD peptide nanocarrier for sFLT01 gene delivery to the retinal pigment epithelium cells
Somayeh Piroozmand1 *, Zahra-Soheila Soheili2 , Saman Hosseinkhani3 , Shahram Samiee4
- Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and
Biotechnology, Tehran, Iran.
- Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
Abstract: In development of drug delivery systems, some viral and non-viral carriers have been developed to improve gene delivery methods. Despite of many advantages, viral vectors face challenges such as limited DNA packaging capacity, immunogenicity, toxicity ...etc. Non-viral delivery systems with low toxicity and immunogenicity have been developed to overcome the aforesaid problems. One of the most important aspects in designing gene delivery vector is considering cell viability and the rate of toxicity. MiRGD peptide is composed of several functional motifs to overcome barriers in gene transduction pathway. In this study, physicochemical properties and toxicity of sFLT01, a novel fusion protein with antiangiogenic activity/ MiRGD peptide nanocarrier was examined in retinal pigment epithelium cells (RPE).
Methods: MiRGD peptide was purified by Ni-NTA affinity chromatography. The purity of the carrier was determined by 15% SDS-PAGE. Nanoparticles were prepared by optimization of sFLT01 plasmid DNA (pDNA) and MiRGD peptide nanocarrier at different N/P (nitrogen to phosphate) Ratios. The ability of MiRGD carrier to condense pDNA and stability of the nanoparticles were confirmed by gel retardation assay. Size and zeta potential were measured using DLS. Cell viability and toxicity was evaluated by MTT assay in RPE cells.
Results: MiRGD peptide effectively retarded and condensed pDNA into nanosize particles at higher N/P ratio which was stable in the presence of serum and effectively protected it from nucleases attack and prevent degradation. The surface charge and size of nanoparticles at higher N: P ratios remained positive and decreased, respectively. No significant toxicity was observed neither in the presence of free peptide nor on the exposure to MiRGD /pDNA nanoparticles which showed viability over 75-80 % after 48 h in contrast with polyethylene imine as positive control.
Conclusion: In order to deliver a desired gene to epithelial cell, a biodegradable peptide carrier with two repeats of 16 mer histone H1, which pack DNA in a particle with electrostatic binding forces, was used. According to DLS result, the surface charge and size of nanoparticles were reasonable and effective on the level of cellular uptake. The nontoxic properties observed in N/P ratios (4-16) nanoparticles, make it possible to use in cell transfection.
