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INTERNATIONAL CONGRESS OF

CancerGenetics

• Investigation of drug delivery of Aflibercept to retinoblastoma cancer cells by MiRGD peptide and Graphene Quantum Dots nano-carrier

• Naeimeh Bayatkhani,^1,* Saman Hosseinkhani,² Zahra-Soheila Soheili,³ Somayeh Piroozmand,⁴ Hamid Latifi-Navid,⁵ Sina Goli Garmestani,⁶
  1. Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
  2. Department of Nanobiotechnology, Faculty of Biological Sciences,Tarbiat Modares University, Tehran, Iran
  3. Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
  4. Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, 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 known as the most common intraocular malignant tumor in childhood and approximately 8000 new cases are diagnosed worldwide each year. Mutations in the retinoblastoma gene (RB1), located on chromosome 13q14.2, are responsible for this condition. Although Retinoblastoma is known as a curable cancer, untreated one can be fatal and even with treatment, advanced tumors can limit globe salvage. Chemotherapy is an effective treatment for retinoblastoma but many affected children suffer from undesirable side effects. Therefore, there is a necessity for designing a new drug delivery system. A novel nano-carrier with MiRGD peptides and graphene quantum dots (GQDs) has been developed due to the structural differences between cancerous and normal cells. The iRGD motif penetrates cancerous tissues, while the other motifs deliver both hydrophobic and hydrophilic drugs. The non-toxic GQDs facilitate biological tracking and enhance drug binding to peptides. Consequently, this nano-carrier seems to be suitable for delivering Aflibercept, an anti-VEGF drug, to prevent the activation of angiogenesis. Therefore, these novel nanoparticles could potentially play a significant role in reducing tumor size and invasion.
• Methods: To purify the MiRGD peptide, Ecoli BL21 was cultured in a 2XYT medium containing kanamycin antibiotic and IPTG as inducer of protein expression. After using a lysis buffer, the MiRGD peptide was purified by Ni-NTA column chromatography under a urea-imidazole gradient. Then the impurities were removed by washing buffers. The purity of the peptide was examined by SDS-PAGE. Purified peptide was desalted by dialysis against PBS buffer. Graphene quantum dots (GQDs) were synthesized by dissolving citric acid and urea in water using a hydrothermal method. The solution was then autoclaved, and ethanol was added. After centrifugation, the solution was dried and redispersed in deionized water. The UV/Vis and fluorescence spectra of the synthesized GQDs and Aflibercept were examined using a Cytation reader. Dynamic Light Scattering (DLS) was performed to determine the ζ-potential of GQDs, the MiRGD peptide, and Aflibercept. Fourier-transform infrared spectroscopy (FTIR) was conducted to identify the bands related to the surface functional groups present on the GQDs. After assembling the complexes of the drug, GQDs, and varying concentrations of MiRGD, the ζ-potential and UV/Vis spectrum of the complexes were examined.
• Results: The MiRGD peptide band was observed on a 15% Tris-glycine SDS-PAGE gel, with a molecular weight of approximately 9.6 kD. The UV/Vis spectrum of the MiRGD peptide, investigated at various wavelengths, demonstrated a peak at 207 nm. Likewise, the UV/Vis spectrum of the synthesized graphene quantum dots (GQDs) showed two peaks at 199 nm and 338 nm. Similarly, Aflibercept's UV/Vis absorption spectroscopy displayed a peak at 216.5 nm, while the fluorescence spectrum of the drug illustrated a peak at 420 nm. The fluorescence spectrum of the synthesized GQDs was analyzed at different excitation wavelengths, with the maximum emission observed at 440 nm. The ζ-potential measurements of GQDs, Aflibercept, and the peptide were found to be −23.3 mV, +4.70 mV, and +6.57 mV, respectively. FTIR spectroscopy of GQDs demonstrated an absorption band in the range of 3000-3500 cm−1, indicating the presence of amino and hydroxyl groups on the surface of the GQDs. The bands at 1700 cm−1 correspond to the vibrational absorption of C=O, and the band at 1400 cm−1 is related to the bending vibrations of C=C. The UV/Vis spectrum of the complexes revealed peaks between 200 and 220 nm. The ζ-potential of the complexes ranged from 10 to 12 mV.
• Conclusion: In conclusion, finding a new targeted drug delivery system for the treatment of retinoblastoma is essential. A novel nano-carrier containing MiRGD peptide and graphene quantum dots (GQDs) has been developed for this purpose. The goal of this study is to investigate the effect of this nano-carrier on retinoblastoma. So far, the MiRGD peptide and GQDs have been prepared and characterized. Aflibercept has also been characterized as an anti-angiogenesis drug which is going to be used in the complex. The assembly and characterization of the complex, which includes MiRGD peptide, the drug, and GQDs, have been completed. The next step will involve investigating the effects of these complexes on a retinoblastoma cell line.
• Keywords: Retinoblastoma, Drug Delivery, Aflibercept, Peptide, GQDs