Carboxylated graphene oxide promoted axonal guidance growth by activating Netrin-1/deleted in colorectal cancer signaling in rat primary cultured cortical neurons

Nanomaterials of graphene and its derivatives have been widely applied in recent years, but whose impacts on the neuronal guidance growth are still not reported. In the present study, graphene oxide (GO) and carboxylated graphene oxide (GO-COOH) were used to investigate the potential effects on axonal guidance growth in the primary cultured cortical neurons. In addition, we characterized the structure and chemical composition of synthesized GO and GO-COOH using Fourier transform infrared spectrophotometer and scanning electron microscope assays and Raman analysis. GO is not neurotoxic and not conductive in a soluble form. However, GO-COOH has higher solubility and conductivity. Cell viability was assessed using CCK-8 assays and fluorescein diacetate after GO and GO-COOH treatment (0, 1, 2, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 50, and 100 µg/mL). There are significant increases of cell viability and axonal growth after GO (2 and 4 μg/mL) and GO-COOH treatment (2 and 4 μg/mL). We further investigated the molecular mechanism of axonal guidance growth after GO and GO-COOH (2 and 4 μg/mL) application. Additionally, GO and GO-COOH up-regulated expression of Netrin-1 and its receptor, deleted in colorectal cancer by immunofluorescence assays and western blots assay. Our study demonstrated that GO-COOH activated Cdc42 and Rac1 and dramatically decreased RhoA. Thus, GO-COOH (2 µg/mL) is much better to be nanocarriers than GO for axonal guidance and growth in this study. GO-COOH may be used to facilitate guidance for regenerating neurons in the future.