Is Graphene Toxic? A Look at Its Potential Health Effects

Graphene’s remarkable properties have made it a focus of scientific and industrial interest, with applications ranging from electronics to medicine. However, as its use expands, concerns about potential health risks have emerged, particularly regarding its interactions with biological systems.

Determining graphene’s toxicity requires examining its physicochemical characteristics, cellular interactions, and the methods used to assess its effects.

Physicochemical Traits That Affect Toxicity

Graphene’s biological impact is shaped by its physicochemical properties, which influence how it interacts with cells and tissues. Size plays a crucial role in toxicity. Larger graphene sheets in the micrometer range exhibit lower cellular uptake, whereas nanoscale fragments penetrate biological membranes more easily. A study in ACS Nano (2018) found that smaller graphene oxide sheets (<100 nm) were more readily internalized by human lung epithelial cells, leading to increased oxidative stress and cytotoxicity. This suggests that controlling graphene’s size distribution is key to mitigating potential health risks. Surface chemistry also affects graphene’s biological behavior, particularly its functional groups and charge distribution. Oxygen-containing functional groups like hydroxyl, carboxyl, and epoxy influence hydrophilicity and reactivity. Graphene oxide, with abundant oxygen functionalities, disperses well in aqueous environments, increasing bioavailability. However, these same groups can generate reactive oxygen species (ROS), contributing to oxidative damage. A 2021 review in Environmental Science: Nano highlighted that graphene derivatives with high oxygen content exhibited greater ROS generation, leading to DNA fragmentation and mitochondrial dysfunction. In contrast, reduced graphene oxide, with fewer oxygen groups, tends to aggregate in biological fluids, altering biodistribution and clearance. Graphene’s structural integrity and layer number also influence toxicity. Single-layer graphene is more flexible and can wrap around cellular structures, potentially disrupting membranes. Multilayer graphene, being more rigid, reduces direct penetration but may cause mechanical irritation. A Nano Letters (2019) study found that single-layer graphene induced greater cytoskeletal disruptions in fibroblast cultures than multilayer graphene. Additionally, defects in graphene’s lattice structure serve as active sites for biochemical reactions, affecting its reactivity with biomolecules. Dispersion and aggregation in biological media further shape graphene’s toxicity. Pristine graphene tends to aggregate in physiological fluids due to its hydrophobic nature, reducing its effective surface area for cellular interactions. However, when stabilized with surfactants or protein coronas, graphene remains more bioavailable, increasing cellular uptake. A Toxicology Research (2022) study showed that graphene nanosheets coated with bovine serum albumin exhibited enhanced internalization and higher intracellular ROS levels. This underscores the importance of considering graphene’s behavior in biological environments rather than just its initial properties.

Mechanisms Of Cellular Interaction

Graphene’s interactions with cells depend on physical and biochemical mechanisms that influence uptake, intracellular effects, and toxicity. Size, surface charge, and functionalization dictate how graphene engages with lipid bilayers. Its two-dimensional structure and high surface area allow adhesion to membranes through hydrophobic interactions and π-π stacking with lipids. This adhesion can lead to membrane wrapping, facilitating endocytosis. Studies in Biomaterials (2020) found that graphene oxide enters cells primarily via clathrin-mediated endocytosis, while reduced graphene oxide favors caveolae-dependent pathways, demonstrating how surface chemistry affects internalization.

Once inside, graphene’s behavior depends on its properties and the intracellular environment. Graphene oxide, being hydrophilic, disperses in the cytoplasm and interacts with organelles like lysosomes and mitochondria. The acidic lysosomal environment can promote oxidative stress by degrading graphene oxide and releasing ROS. A Nature Nanotechnology (2021) study observed that lysosomal degradation of graphene oxide led to mitochondrial damage, triggering apoptosis in macrophages. Reduced graphene oxide, being less soluble, tends to aggregate in the cytosol, potentially interfering with intracellular trafficking and protein interactions.

Graphene’s high affinity for aromatic amino acids allows it to disrupt protein folding and enzymatic activity. Proteomic analyses in ACS Nano (2019) showed that graphene oxide binds to cytoskeletal proteins like actin and tubulin, impairing cell motility and altering morphology in fibroblast cultures. Additionally, graphene’s interaction with nuclear proteins raises concerns about genotoxicity. A Toxicology Letters (2022) study found that graphene oxide can enter the nucleus in dividing cells, binding to DNA and inducing strand breaks, which may contribute to mutagenesis and genomic instability.

Beyond direct interactions, graphene influences intracellular signaling pathways. Exposure activates stress-related kinases such as p38 MAPK and JNK, leading to pro-inflammatory and apoptotic responses. Cell Reports (2023) reported that graphene exposure led to phosphorylation of p53, a key regulator of apoptosis, in lung epithelial cells. Additionally, graphene disrupts ion channels and membrane receptors, altering cellular homeostasis. Nano Research (2020) found that graphene oxide interfered with voltage-gated calcium channels in neuronal cells, affecting neurotransmission and excitability.

Laboratory Techniques For Toxicity Analysis

Graphene toxicity is assessed through various laboratory techniques that examine its cellular, molecular, and systemic effects.

In vitro assays provide initial insights into how graphene affects cell viability, proliferation, and function. MTT and resazurin assays, commonly used to measure metabolic activity, have evaluated graphene-induced cytotoxicity in multiple cell lines. However, graphene’s optical properties can interfere with these tests, requiring careful controls. Flow cytometry, paired with annexin V/propidium iodide staining, distinguishes between apoptotic and necrotic cell death, offering a clearer picture of cell responses.

Oxidative stress assays are crucial for toxicity evaluation. Since graphene can generate ROS, the DCFH-DA assay quantifies intracellular ROS levels, revealing oxidative imbalances that may lead to DNA damage or lipid peroxidation. High-performance liquid chromatography (HPLC) has been used to measure oxidative byproducts like malondialdehyde, a marker of lipid peroxidation. Transcriptomic and proteomic analyses further identify changes in gene expression and protein pathways linked to stress responses. RNA sequencing studies have shown that graphene exposure upregulates antioxidant defense genes, suggesting cellular adaptation to oxidative stress.

Advanced imaging techniques help visualize graphene’s interactions at a structural level. Transmission electron microscopy (TEM) and confocal laser scanning microscopy reveal graphene’s cellular uptake and intracellular distribution. TEM has shown that graphene oxide accumulates in lysosomes, forming dense agglomerates that may disrupt organelle function. Atomic force microscopy (AFM) maps changes in cell membrane stiffness after graphene exposure, indicating biomechanical stress. Raman spectroscopy provides additional insights into graphene’s structural properties and interactions with biomolecules.

In vivo studies extend toxicity assessments to whole organisms, capturing systemic effects. Rodent models have been widely used to evaluate graphene’s biodistribution, clearance, and organ toxicity. Intravenous administration studies indicate that graphene oxide primarily accumulates in the liver, spleen, and lungs, with clearance occurring through renal and hepatobiliary pathways. Histopathological analysis using hematoxylin and eosin (H&E) staining identifies tissue-level changes such as inflammation or fibrosis. Pharmacokinetic studies employing radiolabeled graphene derivatives offer quantitative insights into absorption, distribution, metabolism, and excretion (ADME) profiles, essential for understanding exposure risks in biomedical applications.

Variation In Graphene Forms

Graphene exists in multiple forms, each with distinct structural and chemical properties that influence biological interactions. Differences in oxidation state, layer number, and functionalization significantly alter their behavior in physiological environments.

Graphene Oxide

Graphene oxide (GO) is a highly oxidized form of graphene, characterized by oxygen-containing functional groups such as hydroxyl, carboxyl, and epoxy. These modifications increase hydrophilicity, enhancing dispersion in aqueous environments and bioavailability. GO strongly interacts with biomolecules, including proteins and nucleic acids, influencing cellular processes. Its oxidative properties contribute to ROS generation, leading to oxidative stress. Structural defects introduced by oxidation also affect mechanical properties, making GO more prone to fragmentation, which alters its interactions with cells and tissues.

Reduced Graphene Oxide

Reduced graphene oxide (rGO) is derived from GO through chemical or thermal reduction, removing some oxygen-containing groups. This process restores some electrical conductivity and hydrophobicity, affecting dispersion and aggregation in biological environments. Unlike GO, rGO forms larger aggregates in physiological fluids, influencing biodistribution and clearance. With lower oxygen content, rGO generates less ROS, altering its cytotoxic profile. However, residual defects and partially restored sp² hybridization still impact its interactions with cellular membranes and intracellular components. The degree of reduction plays a crucial role in determining biological effects, with partially reduced forms exhibiting intermediate properties between GO and pristine graphene.

Graphene Quantum Dots

Graphene quantum dots (GQDs) are nanoscale graphene fragments, typically less than 10 nm in diameter, with unique optical and electronic properties. Their small size allows them to penetrate biological barriers more easily, making them promising for bioimaging and drug delivery. GQDs often exhibit fluorescence, which can be tuned through surface modifications, enabling diagnostic imaging. Their high surface-to-volume ratio enhances biomolecule interactions, which can be advantageous for targeted therapies but also raises toxicity concerns. Due to their nanoscale dimensions, GQDs are more readily excreted from the body, reducing long-term accumulation risks. However, their interactions with cellular components and potential for oxidative stress remain active areas of research.