Graphene is a single-atom thick, two-dimensional sheet of hexagonally arranged carbon atoms isolated from its three-dimensional parent material, graphite. Related materials include few-layer-graphene (FLG), ultrathin graphite, graphene oxide (GO), reduced graphene oxide (rGO), and graphene nanosheets (GNS). This review proposes a systematic nomenclature for this set of “Graphene-Family Nanomaterials” (GFNs) and discusses specific materials properties relevant for biomolecular and cellular interactions. The article discusses several unique modes of interaction between GFNs and nucleic acids, lipid bilayers, and conjugated small molecule drugs and dyes. Some GFNs are produced as dry powders using thermal exfoliation, and in these cases inhalation is a likely route of human exposure. Some GFNs have aerodynamic sizes that can lead to inhalation and substantial deposition in the human respiratory tract, which may impair lung defense and clearance leading to formation of granulomas and lung fibrosis. The limited literature on in vitro toxicity suggests that GFNs can be either benign or toxic to cells, and it is hypothesized that the biological response will vary across the material family depending on layer number, lateral size, stiffness, hydrophobicity, surface functionalization, and dose. Generation of reactive oxygen species (ROS) in target cells is a potential mechanism for toxicity, although the extremely high hydrophobic surface area of some GFNs may also lead to significant interactions with membrane lipids leading to direct physical toxicity or adsorption of biological molecules leading to indirect toxicity. Limited in vivo studies demonstrate systemic biodistribution and biopersistence of GFNs following intravenous delivery. Similar to other smooth, continuous, biopersistent implants or foreign bodies, GFNs have the potential to induce foreign body tumors. Long-term adverse health impacts must be considered in design of GFNs for drug delivery, tissue engineering, and fluorescence-based biomolecular sensing. Future research is needed to explore fundamental biological responses to GFNs including systematic assessment of the physical and chemical materials properties related to toxicity. Complete materials characterization and mechanistic toxicity studies are essential for safer design and manufacturing of GFNs in order to optimize biological applications with minimal risks for environmental health and safety.

Understanding and controlling the interaction of graphene-based materials with cell membranes is key to the development of graphene-enabled biomedical technologies and to the management of graphene health and safety issues. Very little is known about the fundamental behavior of cell membranes exposed to ultrathin 2D synthetic materials. Here we investigate the interactions of graphene and few-layer graphene (FLG) microsheets with three cell types and with model lipid bilayers by combining coarse-grained molecular dynamics (MD), all-atom MD, analytical modeling, confocal fluorescence imaging, and electron microscopic imaging. The imaging experiments show edge-first uptake and complete internalization for a range of FLG samples of 0.5-to 10-μm lateral dimension. In contrast, the simulations show large energy barriers relative to k B T for membrane penetration by model graphene or FLG microsheets of similar size. More detailed simulations resolve this paradox by showing that entry is initiated at corners or asperities that are abundant along the irregular edges of fabricated graphene materials. Local piercing by these sharp protrusions initiates membrane propagation along the extended graphene edge and thus avoids the high energy barrier calculated in simple idealized MD simulations. We propose that this mechanism allows cellular uptake of even large multilayer sheets of micrometer-scale lateral dimension, which is consistent with our multimodal bioimaging results for primary human keratinocytes, human lung epithelial cells, and murine macrophages. molecular dynamics simulation | graphene-cell interaction | lipid membrane | edge cutting | corner penetration

Primary cultures of adult rat hepatocytes were treated in the presence or absence of extracellular calcium with ten different membrane-active toxins. In all cases more than half the cells were killed in 1 to 6 hours in the presence but not in the absence of extracellular calcium. An effect of calcium on the primary mechanism of membrane injury by any of the agents cannot be implicated. Viability, as determined by trypan blue exclusion correlated well with other indices of viability such as plating efficiency and the hydrolysis of fluorescein diacetate. It is concluded that the cells are killed by processes that involve at least two steps. In each type of injury, disruption of the integrity of the plasma membrane by widely differing mechanisms is followed by a common functional consequence involving extracellular calcium, and most likely representing an influx of calcium across the damaged plasma membrane and down a steep concentration gradient. This later step represents, or at least initiates, a final common pathway for the toxic death of these cells.