Retinoblastoma is a common intraocular malignancy that occurs during childhood. VEGF is the most potent and specific proangiogenic factor secreted by almost all solid tumor cells. Studies have shown that VEGF is highly expressed in retinoblastoma. Inhibition of angiogenesis has been shown to kill retinoblastoma cells, suggesting that anti-angiogenic therapy may be a promising new treatment strategy against a specific vulnerability of the cancer. htsFLT۰۱ is a fusion protein combining vascular endothelial growth factor receptor-۱ domain with IgG۱ Fc. This protein can neutralize both mouse and human VEGF and PlGF. MiRGD
peptide consists of histone H۱, glycoprotein ۴۱, nuclear localization signal of simian virus ۴۰, and iRGD motifs. iRGD motif provides deep penetration into cancerous tissue by binding to αv integrin, overexpressed receptor in tumor cells. Graphene quantum dots have shown great potential in bio-imaging applications due to their excellent biocompatibility, low cytotoxicity, feasibility for surface functionalization, physiological stability, and tunable fluorescence properties. htsFLT۰۱ plasmids coloned in XL۱۰ bacteria and extracted using an anion exchange affinity column according to the Favorgen Maxi preparation kit protocol. BL۲۱ bacteria containing the MiRGD
peptide gene were cultured in a ۲xyt medium and IPTG with a final concentration of ۰.۵ mM. Then, the bacterial lysate was transferred to the Ni-NTA chromatography column with increasing imidazole and decreasing urea gradient. After observing the
peptide bands on ۱۵% SDS-PAGE, the purified
peptide was desalted by dialysis. The graphene quantum dot was produced using the hydrothermal method using citric acid and urea. Bio Tek cytation examined GQDs absorption and emission wavelength, and FTIR showed their functional surface group. ۱.۰ μg plasmid was mixed with different amounts of purified
peptide in different nitrogen to phosphate rates and incubated at room temperature for ۴۰ min. Then, different concentrations of GQDs were mixed with prepared pDNA/MiRGD complexes. This step was performed on ice. Next, these mixtures were incubated at ۴ °C under controlled agitation overnight. Preliminary investigation of the complex formation was performed by gel retardation assay. Finally, DLS was performed to determine the size and charge of GQDs, MiRGD, and complexes. The human RB cell line Y۷۹ was preserved in RPMI ۱۶۴۰ medium containing ۱۰?tal bovine serum and ۱% penicillin/streptomycin and was placed in a ۳۷℃, ۵% CO۲ incubator. Agarose electrophoresis results showed the plasmid's good quality. The
peptide band was seen and confirmed using ۱۵% Tris-Glycine SDS PAGE. Cytation revealed that GQDs have ۳۳۰ nm absorption and ۴۴۰ nm emission wavelengths. DLS determined the charge of GQDs, MiRGDs, and complexes as -۲۳, +۶, and +۱۱, respectively. Acrylamide-based gel retardation demonstrated that the dual and ternary stable complexes. agarose-based gel retardation assay followed by ethidium bromide staining confirmed pDNA attached to the complexes. After determining the best dose and time to treat cells with the nano complex using the MTT assay, further molecular investigations, such as apoptosis flow cytometry and real-time PCR, will be conducted to determine the final impact of the targeted
gene delivery by the nano complex on inhibiting the retinoblastoma cell line by reducing angiogenesis.