Nano-carrier based targeted delivery of aflibercept to retinoblastoma cells
Nano-carrier based targeted delivery of aflibercept to retinoblastoma cells
Naeimeh Bayatkhani,1,*Zahra-Soheila Soheili,2Saman Hosseinkhani,3Hamid Latifi-Navid,4Somayeh Piroozmand,5Sina Goli Garmestani,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 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 the most common intraocular malignancy in children, with an incidence of approximately 1 in 17,000 live births worldwide. Mutations in the retinoblastoma gene (RB1), located on chromosome 13q14.2, are responsible for this condition. It is a curable cancer if diagnosed in a timely manner. However, the mortality rate remains high in developing countries, and advanced tumors can limit the possibility of globe salvage. Although chemotherapy is an effective treatment for retinoblastoma, many affected children suffer from undesirable side effects. Therefore, there is a pressing need to design a new drug delivery system that is more efficient and has fewer side effects. In recent years, a novel nano-carrier containing MiRGD peptides and graphene quantum dots (GQDs) has been developed, based on the structural differences between cancerous and normal cells. Since αv integrins are overexpressed in tumor cells, the iRGD motif in the peptide facilitates deep penetration into cancerous tissues by binding to them. Other motifs enhance the delivery of both hydrophobic and hydrophilic drugs. The non-toxic GQDs assist with non-invasive biological tracking and improve drug binding to the peptides. Consequently, this nano-carrier is deemed suitable for delivering Aflibercept, an anti-VEGF drug, to prevent the activation of angiogenesis.
Methods: In order to extract the MiRGD peptide, E. coli BL21 containing the expression vector (pET28a) was cultured in 2xYT medium supplemented with kanamycin and IPTG as an inducer of protein expression. Then, the bacterial cells were harvested by centrifugation, and the cell pellets were resuspended in lysis buffer. A Ni-NTA column chromatography was employed to purify the MiRGD peptide, and impurities were removed using buffers with a urea-imidazole gradient. Following the examination of the peptide's purity by SDS-PAGE, the 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 investigated.
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, examined at various wavelengths, revealed a peak at 207 nm. Similarly, the UV/Vis spectrum of the synthesized graphene quantum dots (GQDs) showed two peaks at 199 nm and 338 nm. The fluorescence spectrum of the synthesized GQDs was analyzed at different excitation wavelengths, with the maximum emission observed at 440 nm. Aflibercept's UV/Vis absorption spectroscopy, conducted at various wavelengths, displayed a peak at 216.5 nm, while the fluorescence spectrum of the drug revealed a peak at 420 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 illustrated peaks between 200 and 220 nm. The ζ-potential of the complexes ranged from 10 to 12 mV.
Conclusion: In conclusion, there is a pressing need for a new targeted drug delivery system for the treatment of retinoblastoma. A novel nano-carrier containing MiRGD peptide and graphene quantum dots (GQDs) has been developed for this purpose. This study aims to investigate the effect of this nano-carrier on retinoblastoma. So far, the MiRGD peptide and GQDs have been prepared and characterized. Additionally, aflibercept, an anti-angiogenesis drug, has also been characterized and will be incorporated into the complex. The assembly and characterization of the complex, which includes MiRGD, the drug, and GQDs, have been completed. The next step will involve investigating the effects of these complexes on a retinoblastoma cell line.