Cell-penetrating peptide MiRGD, a vehicle for efficient delivery of anti-angiogenesis htsFLT01 gene to Retinal Pigment Epithelium cells: physicochemical characterization and toxicity analysis
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: Gene therapy as a model of molecular medicine has gained considerable interest owing to its great
potential for the treatment of diseases like ocular neovascularization. Peptide-based nanocarriers
revealed great promise for gene delivery due to biocompatibility, specific targeting, high drug loading capacity, chemical diversity, low toxicity and immunogenicity. MiRGD peptide is a cell-penetrating peptide that composed of functional DNA condensing and endosome disruptive motifs
to overcome cellular barriers in the gene transduction pathway. We examined the toxicity and
physicochemical properties of novel fusion protein sFLT01 / MiRGD peptide nanocarrier in retinal
pigment epithelium cells (RPE).
Methods: sFLT01 gene was designed and constructed. The MiRGD peptide was expressed and purified by
Ni-NTA affinity chromatography. Nanoparticles were prepared at different N/P Ratios. The DNA
binding and stability were examined by gel retardation assay and serum stability. Size and zeta
potential analysis and activity of HIV gp41 motif of MiRGD peptide were determined by dynamic
light scattering (DLS) and hemolysis assay, respectively. Cell viability and toxicity of
nanoparticles were evaluated by MTT assay in RPE cells.
Results: Through increasing, N: P ratio, the DNA binding motif of MiRGD peptide effectively condensed
plasmid DNA(pDNA), neutralized its negative charges and stabilized it in the presence of serum
and effectively protected pDNA from degradation by the serum nucleases. At higher N: P ratios,
the size decreased and the surface charge of the nanoparticles remained positive. No significant
toxicity was observed neither in the presence of free peptide nor in the exposure to MiRGD /DNA
nanoparticles. HIV gp41 motif of peptide showed hemolytic activity in both acidic (pH 5.4) and
physiological conditions (pH 7.4). Furthermore, different N/P ratios represented more endosome
disruptive activity in physiological conditions than in acidic environments.
Conclusion: One of the most important issues considered for gene carriers is to protect the gene from
nuclease destruction. According to our results, it can be deduced that the peptide with DNA
binding motif condensed DNA and protected it from nucleases. HIV gp41 motifs prefer to have
endosomal escape activity under the milder conditions of the early endosome. Functional motifs
and nontoxic properties of MiRGD reduced the risk of degradation in transferring pathway.
