br Introduction In the past two

Introduction In the past two decades, graphene and graphene based nanomaterials with unique physicochemical properties have attracted great attentions in many fields including biomedicine and biotechnology. Graphene oxide (GO) and its derivatives have shown promising potential as biological and chemical sensors [1], [2], [3], [4], [5], [6], [7], gene and drug delivery carriers [8], [9], [10], [11], [12], cancer therapy agents [13], [14], [15], [16], antibacterial agents [17], [18], protein modulators [19], [20], [21], [22], as well as tissue engineering materials [23], [24], [25]. Over the years, the interactions of graphene-based nanomaterials with different biological systems have also received substantial interests. It has been shown that various parameters, such as size, morphology, and surface chemistry, play important roles in the behaviors of graphene-based nanomaterials in biological systems [26], [27], [28], [29], [30], [31]. For example, raw graphene or GO without further surface modification usually would exhibit toxicities towards cells or animals in a dose-dependent and/or size-dependent manner, yet their toxicities could be remarkably reduced or altered with appropriate surface modifications [32], [33], [34], [35], [36], [37]. Although the biological effects of GO and its derivatives on various biological systems, ranging from biomacromolecules to organisms, have been explored, how they might affect the mammalian TAK-715 has been rarely studied. Yuan et al. reported a decrease in the population of G2/M phase cells in the cell cycle for HepG2 cells treated with GO [38], whereas haemangioblasts cultured on GO showed slightly increased population of cells in G2/M phase [39]. In addition, GO coated with polyethylene glycol (PEG) could significantly induce G0/G1 phase arrest in MC3T3-E1 preosteoblasts and RAW-264.7 macrophages [40]. Since the cell cycle is one of the most important and fundamental events for majority of the eukaryotic cells, all these reports raise important issues such as biocompatibility and biosafety for GO and its derivatives. Therefore, before applying such nanomaterials in biological research or for biomedical purposes, further attentions are needed to investigate in more detail any potential effects of GO and its derivatives on cell proliferation and the cell cycle, and more importantly, to unveil the underlying mechanisms and signaling pathway(s) involved. In our previous studies, we functionalized GO surface with a series of combinations of various polymers, and discovered that some of these GO derivatives could successfully serve as novel, highly effective protein modulators [19], [22], gene carrier [41], [42], vaccine nano-adjuvants [43], as well as a bacterial growth stimulator which accelerates the Phase 1 of the bacterial cell cycle [44]. In this work, we functionalized GO with 10 kDa amine-terminated six-arm-branched PEG (10k-6br-PEG-NH2) and 25 kDa polyethylenimine (PEI) (named GO-PEG-PEI), and carried out detailed investigations of its interaction with mammalian cells. Our results have shown that GO-PEG-PEI could induce cell cycle defect in all five mammalian cell lines tested, leading to increased population of cells detained in S phase of the cell cycle. Even at a rather safe concentration (∼90% cells viable), GO-PEG-PEI could induce S phase defect in live cells, whereas at higher concentrations, cell necrosis would be induced in a time-dependent manner, and nearly 80% of the dead/dying cells would be arrested in S phase. Decreased DNA synthesis and abnormal cytoskeleton structure were also observed upon GO-PEG-PEI treatment. Further analysis uncovered that treatments with GO-PEG-PEI would result in genomic DNA damage, activating the intra-S-phase checkpoint control via both the ATM and the ATR signaling pathways [45], [46], [47]. The cell cycle defect induced by GO-PEG-PEI could be blocked by the checkpoint kinase inhibitor AZD7762 [48], [49], further confirming the involvement of the ATM and the ATR signaling pathways. Interestingly, the coating polymer PEI, although cytotoxic, does not possess such effect on the cell cycle, demonstrating that this phenomenon may be unique for GO-PEG-PEI. Our work highlights the critical roles of surface chemistry in biological effects of nanomaterials. Furthermore, for nanomaterials with promising biomedical potentials and seemingly little/low cytotoxicity during preliminary evaluations, our results point out the necessity and importance of detailed, comprehensive investigations of their potential genotoxicity as well as their effects on the cell cycle and other cellular pathways.