• Delivery of htsFLT01 to Y79 Cells by MiRGD peptide and Graphene Quantum Dots Nanoparticles
  • Sina Goli Garmestani,1,* Zahra-Soheila Soheili,2 Saman Hosseinkhani,3 Hamid Ahmadieh,4 Hamid Latifi-Navid,5 Naeimeh Bayatkhani,6
    1. Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
    2. Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
    3. Department of Nanobiotechnology, Faculty of Biological Sciences,Tarbiat Modares University, Tehran, Iran 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
    5. Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
    6. Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran


  • Introduction: Retinoblastoma is a common intraocular malignancy that affects children. Vascular endothelial growth factor (VEGF) is a potent proangiogenic factor highly expressed in retinoblastoma. Inhibiting angiogenesis has shown promise in killing retinoblastoma cells, making anti-angiogenic therapy a potential new treatment strategy. htsFLT01 is a fusion protein that can neutralize mouse and human VEGF and PlGF. MiRGD peptide contains various motifs, can penetrate cancerous tissue, and bind to overexpressed receptors in tumor cells. Graphene quantum dots have great potential in bio-imaging applications due to their biocompatibility, low cytotoxicity, and tunable fluorescence properties.
  • Methods: The htsFLT01 plasmids were cloned in XL10 bacteria and extracted using an anion exchange affinity column per the Favorgen Maxi preparation kit protocol. BL21 bacteria containing the MiRGD gene were cultured in a 2xyt medium and induced with IPTG at a final concentration of 0.5 mM. The bacterial lysate was then transferred to a Ni-NTA chromatography column with an increasing imidazole and decreasing urea gradient. After observing the peptide bands on a 15% SDS-PAGE gel, the purified peptide was desalted by dialysis. Graphene quantum dots were produced using the hydrothermal method with citric acid and urea. Their absorption and emission wavelengths were examined using Bio Tek cytation, and their functional surface groups were analyzed using FTIR. In the next step, 1.0 μg plasmid was mixed with different amounts of the purified peptide at different nitrogen-to-phosphate ratios and incubated at room temperature for 40 minutes. Various concentrations of GQDs were then mixed with the prepared pDNA/MiRGD complexes, and this step was performed on ice. These mixtures were incubated at 4 °C with controlled agitation overnight. The formation of the complexes was preliminarily investigated using a gel retardation assay. Finally, DLS was conducted to determine the size and charge of GQDs, MiRGD, and the complexes. The human RB cell line Y79 was maintained in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin and was placed in a 37°C, 5% CO2 incubator.
  • Results: The agarose gel electrophoresis results showed that the plasmid has good quality. The presence of the peptide band was confirmed using a 15% Tris-Glycine SDS-PAGE. The GQDs Cytation exhibited absorption at 330 nm and emission at 440 nm wavelengths. Gel retardation assays using acrylamide-based gels demonstrated stable dual and ternary complexes. Agarose-based gel retardation assays, followed by ethidium bromide staining, confirmed the binding of pDNA to the complexes. Dynamic Light Scattering determined the charges of GQDs, MiRGDs, and the complexes as -23, +6, and +11, respectively.
  • Conclusion: We will conduct additional molecular investigations after identifying the optimal dose and timing for treating cells with the nano complex using the MTT assay. These will include apoptosis flow cytometry and real-time PCR to assess the final impact of the targeted gene delivery by the nano complex on inhibiting the retinoblastoma cell line by reducing angiogenesis.
  • Keywords: Retinoblastoma – gene delivery – peptide – nanoparticles