Characterization, Cytotoxicity and Genotoxicity of Graphene Oxide and Folate Coupled Chitosan Nanocomposites Loading Polyprenol and Fullerene Based Nanoemulsion Against MHCC97H Cells

Hepatocellular carcinoma (HCC) is one of the most lethal diseases worldwide. Ginkgo Biloba Leaves polyprenol (GBP) is considered potential efficacy in the treatment of HCC. This study involved oil-in-water type nanoemulsion (NE) loading GBP and Fullerene C₆₀ (C₆₀F) were prepared by dissolving GBP and C₆₀F in NE using inversed phase emulsification (EIP) with ultrasonic-assisted emulsification (UAE) method. Folic acid (FA) coupled Chitosan (CS) and Graphene oxide (GO) nanocomposites (NCs): FA-CS-GO-NCs were fabricated by ionic cross-linking of positively charged FA-CS-GO solution conjugates and negative charged NE with TPP. We combined these materials for preparing NCs loading GBP and C₆₀F (GBP-C₆₀F-FA-CS-GO-NCs) based GBP-C₆₀F-NE. GBP-C₆₀F-FA-CS-GO-NCs in the NE were the mean size was 44.9 nm, PdI was 0.267 and the mean zeta potential was 47.8 mV. And they had good drug loading efficiency, encapsulation efficiency, drug release property and storage stability. In this cytotoxic study, it demonstrated the GBP-C₆₀F-FA-CS-GO-NCs were greater inhibition capacity on MHCC97H cells compared with GBP-NE, C₆₀F-NE, GBP-C₆₀F-NE, GBP-FA-CS-GO-NCs and C₆₀F-FA-CS-GO-NCs. In this genotoxic study, GBP-C₆₀F-FA-CS-GO-NCs at low C₆₀F concentration showed low genotoxicity on MHCC97H and L02 cells. By comparison, GBP-C₆₀F-FA-CS-GO-NCs had higher cytotoxicity on MHCC97H than L02 cells. By the cell apoptosis analysis, the results revealed that GBP-C₆₀F-NE and GBP-C₆₀F-FA-CS-GO-NCs could obviously induce MHCC97H cell apoptosis, especially high concentration GBP-C₆₀F-FA-CS-GO-NCs had the strongest apoptosis inducing ability. The treatment against MHCC97H cells with moderate C₆₀F concentrations of GBP-C₆₀F-FA-CS-GO-NCs effectively inhibited the overexpressions of Akt1 and Akt2, and significantly increased the expression of PTEN. Our results suggest that PTEN, Akt1 and Akt2 might play an important role in the aspect of GBP-C₆₀F-FA-CS-GO-NCs inhibiting against HCC cells. It provides further evidence to speculation that up-regulated PTEN expression and down-regulated Akt1 and Akt2 expression might be one of the important mechanisms for GBP-C₆₀F-FA-CS-GO-NCs inhibiting HCC cells. All these results suggest that GBP-C₆₀F-FA-CS-GO-NCs effectively inhibited the HCC malignant development and has potentially important value in the HCC treatment.