*2.1. Direct Physical Nucleus Damage by GFNs*

After GFNs exposure, monolayer or a few-layer GFNs (GO and rGO) sheets are able to cut and penetrate cell membranes and the cell wall (if present), resulting in direct physical membrane damage [67,68]. Moreover, small pieces of GFNs will enter the nucleus, interacting directly with DNA [69]. Generally, nuclear DNA is the main target of gene toxicity [42]. Prokaryotes (e.g., bacteria) only have naked DNA without a nuclear envelope. GFNs can directly contact bacteria RNA/DNA hydrogen groups, interrupting the replicative stage after internalization [70]. During mitosis, GFNs are likely to interact with DNA, leading to DNA aberration when the nuclear membrane ruptures [3]. As shown in Figure 2, the nuclear uptake and nuclear response related to contact with GQDs have been systematically reported by using atomic force microscopy (Figure 2a,b), confocal microscopy (Figure 2c,d), transmission electron microscopy (Figure 2e,f), and high content screening (Figure 2g,h) [69]. GQDs are mainly uptaken into cells via energy-dependent endocytosis, phagocytosis, and caveolae-mediated endocytosis. More than half of GQDs are exposed and accumulated in the nucleus by microscopy investigation. The accumulated GQDs may direct contact with DNA strand, thereby causing physical damage. After 1 h exposure, the rGO nanoplatelet can pierce the nucleus of the human mesenchymal stem cells (hMSCs), leading to DNA fragmentation and chromosomal aberrations at 0.1 and 1.0 mg/L. Notably, rGO sheets with the same size or larger size showed no genotoxicity in the hMSCs after 24 h exposure at 100 mg/L [71]. The single-layer rGO nanoribbons can penetrate into the hMSCs nucleus at 100 mg/L detected by confocal fluorescence imaging, and cells showed a high degree of DNA fragmentation. The above DNA damage is mainly related to oxidative stress caused by DNA released, rather than DNA damage within the nucleus. Interestingly, rGO nanoribbons showed no significant cytotoxicity at 1.0 mg/L but can induce genotoxicity through DNA fragmentation and chromosomal aberrations in the hMSCs [72]. In a word, GFNs can interact directly with chromatin and DNA, causing DNA damage and thus exhibiting genotoxicity.

**Figure 2.** Effects of GQDs on the nucleus and DNA in the nucleus. (**a**,**b**) DNA chain damage caused by GQDs; (**c**,**d**) accumulation of GQDs; (**e**,**f**) nuclear damage by GQDs; (**g**,**h**) effect of GQDs on nuclear viability and area; (**a**,**c**,**e**,**g**) are the blank control groups, and (**b**,**d**,**f**,**h**) are the exposed groups of 200 mg/L GQDs for 24 h, reproduced from [69], from BioMed Central, 2018.
