Delivery of htsFLT01 to retinoblastoma cells by MiRGD peptide and Graphene Quantum Dots nanoparticles
Sina Goli garmestani1 , Zahra-Soheila Soheili 2 *, Saman Hosseinkhani 3 , Hamid Ahmadieh4 , Hamid Latifi-Navid 5 , Somayeh Piroozmand 2 , Naeimeh Bayatkhani 2 , Shahram Samiei 6
- 1- Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology,
Tehran 1497716316, Iran
- 1- Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran 1497716316, Iran
- 2- Department of Nanobiotechnology, Faculty of Biological Sciences,Tarbiat Modares University, Tehran, Iran 3- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
- 4- Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran
- 1- Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran 1497716316, Iran 2- Electrophysiology Research Center, Neuroscience Institute, Tehran University of Medical Sciences, Tehran 1416634793, Iran 3- School of Biological Sciences, Institute for Research in Fundamental Sciences (IPM), Tehran, Iran
- 7- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
Abstract: Retinoblastoma is the most common malignant intraocular tumor in children. The tumor is caused by mutations in the RB1 gene. Angiogenesis causes the tumor's growth and metastasis. So, inhibition of angiogenesis would be a promising solution to inhibit tumor invasion. We introduced the htsFLT01 chimeric construct to inhibit proangiogenic VEGF and PLGF molecules. The gene delivery is facilitated by the MIRGD peptide, which targets tumor cells, and the Graphene Quantum Dots )GQD( nanoparticle for its distinct properties in drug delivery.
Methods: Plasmids containing htsFLT01 prepared by maxi preparation method. The MIRGD peptide was expressed in E. coli, extracted using Ni-NTA column chromatography, and purified by dialysis. The purity was examined by SDS-PAGE. GQDs will be synthesized using the hydrothermal method and characterized by UV/Vis and FTIR analysis. For the next step, the MiRGD, plasmid, and GQDs complex will be assembled and analyzed by gel retardation, Dynamic light scattering (DLS), transmission electron microscopy, and Hemolysis. Finally, the complex is transfected into Y79 cells. Then, real-time PCR, apoptosis, flow cytometry, tube formation assay, and wound healing assays will be done.
Results: The quality of plasmids and peptides was confirmed by agarose gel electrophoresis and 15% Tris–Glycine SDS PAGE gel, respectively. UV/Vis Absorption spectroscopy will be used to reveal the synthesized GQDs peaks. FTIR will detect surface functional groups on GQDs with broad bands at 3000-3500 cm−1 to indicate amino and hydroxyl groups.
Conclusion: The htsFLT01 plasmid, MiRGD peptides, and GQD nanoparticles will be assembled and analyzed to study a novel nanocarrier complex for targeted gene delivery to retinoblastoma cells/tumors.
