Cellular internalization assessment

Flow cytometry based cellular uptake analysis: Flow cytometry based GD internalization assessment in cultured cells was carried out according to the defined protocol⁵³. According to this method, increase in the intensity of side scattered (SSC) light with constant intensity of forward scattered (FSC) light in exposed cells is the remark of cellular uptake of NMs in cell. In brief, 1 × 10⁵ cells/ml/well were seeded in 12 well culture plate and exposed to varying concentrations (1–100 μg/ml) of GD for 24 h and 48 h. After completion of exposure time, cells were harvested using 0.25% trypsin and centrifuged at 1000 rpm for 10 min. Supernatant was discarded and the pellet was re-suspended in 1X PBS (500 μl). Acquisition and analysis was made using flow cytometer equipped with 488 nm laser (FACS Canto^TM II, BD Biosciences, San Jose, CA, USA) instrument.

TEM analysis: Ultrathin section of cells exposed to different GD (GO, TRGO and CRGO) were analysed using TEM for cellular internalization. Briefly, cells were fixed, embedded and thin sections were cut, stained, analysed using Tecnai^TM G2 spirit (FEI, Netherland) instrument at an accelerating voltage of 80 kV.