Graphene oxide activates canonical TGFß signalling in a human chondrocyte cell line via increased plasma membrane tension.

Graphene Oxide (GO) has been shown to increase the expression of key cartilage genes and matrix components within 3D scaffolds. Understanding the mechanisms behind the chondroinductive ability of GO is critical for developing articular cartilage regeneration therapies but remains poorly understood. The objectives of this work were to elucidate the effects of GO on the key chondrogenic signalling pathway - TGFß and identify the mechanism through which signal activation is achieved in human chondrocytes. Activation of canonical signalling was validated through GO-induced SMAD-2 phosphorylation and upregulation of known TGFß response genes, while the use of a TGFß signalling reporter assay allowed us to identify the onset of GO-induced signal activation which has not been previously reported. Importantly, we investigate the cell-material interactions and molecular mechanisms behind these effects, establishing a novel link between GO, the plasma membrane and intracellular signalling. By leveraging fluorescent lifetime imaging (FLIM) and a membrane tension probe, we reveal GO-mediated increases in plasma membrane tension, in real-time for the first time. Furthermore, we report the activation of mechanosensory pathways which are known to be regulated by changes in plasma membrane tension and reveal the activation of endogenous latent TGFß in the presence of GO, providing a mechanism for signal activation. The data presented here are critical to understanding the chondroinductive properties of GO and are important for the implementation of GO in regenerative medicine.

Environmental and Health Impacts of Graphene and Other Two-Dimensional Materials: A Graphene Flagship Perspective.

Two-dimensional (2D) materials have attracted tremendous interest ever since the isolation of atomically thin sheets of graphene in 2004 due to the specific and versatile properties of these materials. However, the increasing production and use of 2D materials necessitate a thorough evaluation of the potential impact on human health and the environment. Furthermore, harmonized test protocols are needed with which to assess the safety of 2D materials. The Graphene Flagship project (2013-2023), funded by the European Commission, addressed the identification of the possible hazard of graphene-based materials as well as emerging 2D materials including transition metal dichalcogenides, hexagonal boron nitride, and others. Additionally, so-called green chemistry approaches were explored to achieve the goal of a safe and sustainable production and use of this fascinating family of nanomaterials. The present review provides a compact survey of the findings and the lessons learned in the Graphene Flagship.

Defect-free graphene enhances enzyme delivery to fibroblasts derived from patients with lysosomal storage disorders.

Enzyme replacement therapy shows remarkable clinical improvement in treating lysosomal storage disorders. However, this therapeutic approach is hampered by limitations in the delivery of the enzyme to cells and tissues. Therefore, there is an urgent, unmet clinical need to develop new strategies to enhance the enzyme delivery to diseased cells. Graphene-based materials, due to their dimensionality and favourable pattern of interaction with cells, represent a promising platform for the loading and delivery of therapeutic cargo. Herein, the potential use of graphene-based materials, including defect-free graphene with positive or negative surface charge and graphene oxide with different lateral dimensions, was investigated for the delivery of lysosomal enzymes in fibroblasts derived from patients with Mucopolysaccharidosis VI and Pompe disease. We report excellent biocompatibility of all graphene-based materials up to a concentration of 100 µg mL-1 in the cell lines studied. In addition, a noticeable difference in the uptake profile of the materials was observed. Neither type of graphene oxide was taken up by the cells to a significant extent. In contrast, the two types of graphene were efficiently taken up, localizing in the lysosomes. Furthermore, we demonstrate that cationic graphene flakes can be used as carriers for arylsulfatase B enzyme, for the delivery of the lacking enzyme to the lysosomes of Mucopolysaccharidosis VI fibroblasts. Arylsulfatase B complexed with cationic graphene flakes not only retained the enzymatic activity, but also exerted biological effects almost twice as high as arylsulfatase B alone in the clearance of the substrate in Mucopolysaccharidosis VI fibroblasts. This study lays the groundwork for the potential use of graphene-based materials as carriers for enzyme replacement therapy in lysosomal storage disorders.

Graphene oxide modulates dendritic cell ability to promote T cell activation and cytokine production.

An important aspect of immunotherapy is the ability of dendritic cells (DCs) to prime T cell immunity, an approach that has yielded promising results in some early phase clinical trials. However, novel approaches are required to improve DC therapeutic efficacy by enhancing their uptake of, and activation by, disease relevant antigens. The carbon nano-material graphene oxide (GO) may provide a unique way to deliver antigen to innate immune cells and modify their ability to initiate effective adaptive immune responses. We have assessed whether GO of various lateral sizes affects DC activation and function in vitro and in vivo, including their ability to take up, process and present the well-defined model antigen ovalbumin (OVA). We have found that GO flakes are internalised by DCs, while having minimal effect on their viability, activation phenotype or cytokine production. Although adsorption of OVA protein to either small or large GO flakes promoted its uptake into DCs, large GO interfered with OVA processing. In terms of modulation of DC function, delivery of OVA via small GO flakes significantly enhanced DC ability to induce proliferation of OVA-specific CD4+ T cells, promoting granzyme B secretion in vitro. On the other hand, delivery of OVA via large GO flakes augmented DC ability to induce proliferation of OVA-specific CD8+ T cells, and their production of IFN-γ and granzyme B. Together, these data demonstrate the capacity of GO of different lateral dimensions to act as a promising delivery platform for DC modulation of distinct facets of the adaptive immune response, information that could be exploited for future development of targeted immunotherapies.

Effects of Lateral Size, Thickness, and Stabilizer Concentration on the Cytotoxicity of Defect-Free Graphene Nanosheets: Implications for Biological Applications.

In this work, we apply liquid cascade centrifugation to highly concentrated graphene dispersions produced by liquid-phase exfoliation in water with an insoluble bis-pyrene stabilizer to obtain fractions containing nanosheets with different lateral size distributions. The concentration, stability, size, thickness, and the cytotoxicity profile are studied as a function of the initial stabilizer concentration for each fraction. Our results show that there is a critical initial amount of stabilizer (0.4 mg/mL) above which the dispersions show reduced concentration, stability, and biocompatibility, no matter the lateral size of the flakes.

Dynamic interactions and intracellular fate of label-free, thin graphene oxide sheets within mammalian cells: role of lateral sheet size.

Graphene oxide (GO) holds great potential for biomedical applications, however fundamental understanding of the way it interacts with biological systems is still lacking even though it is essential for successful clinical translation. In this study, we exploit intrinsic fluorescent properties of thin GO sheets to establish the relationship between lateral dimensions of the material, its cellular uptake mechanisms and intracellular fate over time. Label-free GO with distinct lateral dimensions, small (s-GO) and ultra-small (us-GO) were thoroughly characterised both in water and in biologically relevant cell culture medium. Interactions of the material with a range of non-phagocytic mammalian cell lines (BEAS-2B, NIH/3T3, HaCaT, 293T) were studied using a combination of complementary analytical techniques (confocal microscopy, flow cytometry and TEM). The uptake mechanism was initially interrogated using a range of pharmaceutical inhibitors and validated using polystyrene beads of different diameters (0.1 and 1 µm). Subsequently, RNA-Seq was used to follow the changes in the uptake mechanism used to internalize s-GO flakes over time. Regardless of lateral dimensions, both types of GO were found to interact with the plasma membrane and to be internalized by a panel of cell lines studied. However, s-GO was internalized mainly via macropinocytosis while us-GO was mainly internalized via clathrin- and caveolae-mediated endocytosis. Importantly, we report the shift from macropinocytosis to clathrin-dependent endocytosis in the uptake of s-GO at 24 h, mediated by upregulation of mTORC1/2 pathway. Finally, we show that both s-GO and us-GO terminate in lysosomal compartments for up to 48 h. Our results offer an insight into the mechanism of interaction of GO with non-phagocytic cell lines over time that can be exploited for the design of biomedically-applicable 2D transport systems.

Enhanced liquid phase exfoliation of graphene in water using an insoluble bis-pyrene stabiliser.

Stabilisers, such as surfactants, polymers and polyaromatic molecules, offer an effective way to produce graphene dispersions in water by Liquid Phase Exfoliation (LPE) without degrading the properties of graphene. In particular, pyrene derivatives provide better exfoliation efficiency than traditional surfactants and polymers. A stabiliser is expected to be relatively soluble in order to disperse hydrophobic graphene in water. Here, we show that exfoliation can also be achieved with insoluble pyrene stabilisers if appropriately designed. In particular, bis-pyrene stabilisers (BPSs) functionalised with pyrrolidine provide a higher exfoliation efficiency and percentage of single layers compared to traditional pyrene derivatives under the same experimental conditions. This is attributed to the enhanced interactions between BPS and graphene, provided by the presence of two pyrene binding groups. This approach is therefore attractive not only to produce highly concentrated graphene, but also to use graphene to disperse insoluble molecules in water. The enhanced adsorption of BPS on graphene, however, is reflected in higher toxicity towards human epithelial bronchial immortalized cells, limiting the use of this material for biomedical applications.

Nitric oxide-dependent biodegradation of graphene oxide reduces inflammation in the gastrointestinal tract.

Understanding the biological fate of graphene-based materials such as graphene oxide (GO) is crucial to assess adverse effects following intentional or inadvertent exposure. Here we provide first evidence of biodegradation of GO in the gastrointestinal tract using zebrafish as a model. Raman mapping was deployed to assess biodegradation. The degradation was blocked upon knockdown of nos2a encoding the inducible nitric oxide synthase (iNOS) or by pharmacological inhibition of NOS using l-NAME, demonstrating that the process was nitric oxide (NO)-dependent. NO-dependent degradation of GO was further confirmed in vitro by combining a superoxide-generating system, xanthine/xanthine oxidase (X/XO), with an NO donor (PAPA NONOate), or by simultaneously producing superoxide and NO by decomposition of SIN-1. Finally, by using the transgenic strain Tg(mpx:eGFP) to visualize the movement of neutrophils, we could show that inhibition of the degradation of GO resulted in increased neutrophil infiltration into the gastrointestinal tract, indicative of inflammation.

Stable, concentrated, biocompatible, and defect-free graphene dispersions with positive charge.

The outstanding properties of graphene offer high potential for biomedical applications. In this framework, positively charged nanomaterials show better interactions with the biological environment, hence there is strong interest in the production of positively charged graphene nanosheets. Currently, production of cationic graphene is either time consuming or producing dispersions with poor stability, which strongly limit their use in the biomedical field. In this study, we made a family of new cationic pyrenes, and have used them to successfully produce water-based, highly concentrated, stable, and defect-free graphene dispersions with positive charge. The use of different pyrene derivatives as well as molecular dynamics simulations allowed us to get insights on the nanoscale interactions required to achieve efficient exfoliation and stabilisation. The cationic graphene dispersions show outstanding biocompatibility and cellular uptake as well as exceptional colloidal stability in the biological medium, making this material extremely attractive for biomedical applications.

Graphene oxide nanosheets modulate spinal glutamatergic transmission and modify locomotor behaviour in an in vivo zebrafish model.

Graphene oxide (GO), an oxidised form of graphene, is widely used for biomedical applications, due to its dispersibility in water and simple surface chemistry tunability. In particular, small (less than 500 nm in lateral dimension) and thin (1-3 carbon monolayers) graphene oxide nanosheets (s-GO) have been shown to selectively inhibit glutamatergic transmission in neuronal cultures in vitro and in brain explants obtained from animals injected with the nanomaterial. This raises the exciting prospect that s-GO can be developed as a platform for novel nervous system therapeutics. It has not yet been investigated whether the interference of the nanomaterial with neurotransmission may have a downstream outcome in modulation of behaviour depending specifically on the activation of those synapses. To address this problem we use early stage zebrafish as an in vivo model to study the impact of s-GO on nervous system function. Microinjection of s-GO into the embryonic zebrafish spinal cord selectively reduces the excitatory synaptic transmission of the spinal network, monitored in vivo through patch clamp recordings, without affecting spinal cell survival. This effect is accompanied by a perturbation in the swimming activity of larvae, which is the locomotor behaviour generated by the neuronal network of the spinal cord. Such results indicate that the impact of s-GO on glutamate based neuronal transmission is preserved in vivo and can induce changes in animal behaviour. These findings pave the way for use of s-GO as a modulator of nervous system function.

Graphene and other 2D materials: a multidisciplinary analysis to uncover the hidden potential as cancer theranostics.

Cancer represents one of the main causes of death in the world; hence the development of more specific approaches for its diagnosis and treatment is urgently needed in clinical practice. Here we aim at providing a comprehensive review on the use of 2-dimensional materials (2DMs) in cancer theranostics. In particular, we focus on graphene-related materials (GRMs), graphene hybrids, and graphdiyne (GDY), as well as other emerging 2DMs, such as MXene, tungsten disulfide (WS2), molybdenum disulfide (MoS2), hexagonal boron nitride (h-BN), black phosphorus (BP), silicene, antimonene (AM), germanene, biotite (black mica), metal organic frameworks (MOFs), and others. The results reported in the scientific literature in the last ten years (>200 papers) are dissected here with respect to the wide variety of combinations of imaging methodologies and therapeutic approaches, including drug/gene delivery, photothermal/photodynamic therapy, sonodynamic therapy, and immunotherapy. We provide a unique multidisciplinary approach in discussing the literature, which also includes a detailed section on the characterization methods used to analyze the material properties, highlighting the merits and limitations of the different approaches. The aim of this review is to show the strong potential of 2DMs for use as cancer theranostics, as well as to highlight issues that prevent the clinical translation of these materials. Overall, we hope to shed light on the hidden potential of the vast panorama of new and emerging 2DMs as clinical cancer theranostics.

Functionalized Surface-Charged SiO2 Nanoparticles Induce Pro-Inflammatory Responses, but Are Not Lethal to Caco-2 Cells.

Nanoparticles (NPs) are widely used in food, and analysis of their potential gastrointestinal toxicity is necessary. The present study was designed to determine the effects of silica dioxide (SiO2), titanium dioxide (TiO2), and zinc oxide (ZnO) NPs on cultured THP-1 monocyte-derived macrophages and human epithelial colorectal adenocarcinoma (Caco-2) cells. Exposure to ZnO NPs for 24 h increased the production of redox response species (ROS) and reduced cell viability in a dose-dependent manner in THP-1 macrophages and Caco-2 cells. Although TiO2 and SiO2 NPs induced oxidative stress, they showed no apparent cytotoxicity against both cell types. The effects of functionalized SiO2 NPs on undifferentiated and differentiated Caco-2 cells were investigated using fluorescently labeled SiO2 NPs with neutral, positive, or negative surface charge. Exposure of both types of cells to the three kinds of SiO2 NPs significantly increased their interaction in a dose-dependent manner. The largest interaction with both types of cells was noted with exposure to more negatively surface-charged SiO2 NPs. Exposure to either positively or negatively, but not neutrally, surface-charged SiO2 NPs increased NO levels in differentiated Caco-2 cells. Exposure of differentiated Caco-2 cells to positively or negatively surface-charged SiO2 NPs also upregulated interleukin-8 expression. We conclude that functionalized surface-charged SiO2 NPs can induce pro-inflammatory responses but are noncytotoxic.

Hampering brain tumor proliferation and migration using peptide nanofiber:siPLK1/MMP2 complexes.

Aim: To develop a nonviral tool for the delivery of siRNA to brain tumor cells using peptide nanofibers (PNFs). Materials & methods: Uptake of PNFs was evaluated by confocal microscopy and flow cytometry. Gene silencing was determined by RT-qPCR and cell invasion assay. Results: PNFs enter phagocytic (BV-2) and nonphagocytic (U-87 MG) cells via endocytosis and passive translocation. siPLK1 delivered using PNFs reduced the expression of polo-like kinase 1 mRNA and induced cell death in a panel of immortalized and glioblastoma-derived stem cells. Moreover, targeting MMP2 using PNF:siMMP2 reduced the invasion capacity of U-87 MG cells. We show that stereotactic intra-tumoral administration of PNF:siPLK1 significantly extends the survival of tumor bearing mice comparing with the untreated tumor bearing animals. Conclusion: Our results suggest that this nanomedicine-based RNA interference approach deserves further investigation as a potential brain tumor therapeutic tool.

Thermal monitoring during photothermia: hybrid probes for simultaneous plasmonic heating and near-infrared optical nanothermometry.

The control of temperature during photothermal therapy is key to preventing unwanted damage in surrounding tissue or post-treatment inflammatory responses. Lack of accurate thermal control is indeed one of the main limitations that hyperthermia techniques present to allow their translation into therapeutic applications. We developed a nanoprobe that allows controlled local heating, combined with in situ nanothermometry. The design of the probe follows a practical rationale that aims at simplifying experimental requirements and exploits exclusively optical wavelengths matching the first and second biological windows in the near-infrared. Methods: Hybrid nanostructures were chemically synthesized, and combine gold nanostars (photothermal agents) with CaF2:Nd3+,Y3+ nanoparticles (luminescent nanothermometers). Both components were simultaneously excited in the near-infrared range, at 808 nm. Following the goal of simplifying the thermal monitoring technique, the luminescent signal was recorded with a portable near-infrared detector. The performance of the probes was tested in 3D tumor spheroids from a human glioblastoma (U87MG) cell line. The location of the beads within the spheroids was determined measuring Nd3+ emission in a commercial Lightsheet microscope, modified in-house to be able to select the required near-infrared wavelengths. The temperature achieved inside the tumor spheroids was deduced from the luminescence of Nd3+, following a protocol that we developed to provide reliable thermal readings. Results: The choice of materials was shown to work as an optically excited hybrid probe. Depending on the illumination parameters, temperature can be controlled in a range between 37 ºC and 100 ºC. The near-infrared emission of nanothermometers also allows microscopic tracking of the hybrid nanostructures, confirming that the probes can penetrate deeper into the spheroid mass. We observed that, application of optical thermometry in biological environments requires often neglected considerations, since the optical signal changes along the optical path. Accordingly, we developed data analysis protocols that guarantee reliable thermal readings. Conclusions: The prepared hybrid probes are internalized in 3D tumor spheroids and can be used to induce cell death through photothermal effects, while simultaneously measuring the local temperature in situ. We show that luminescent thermometry in biomedical applications requires the development of protocols that guarantee accurate readings. Regarding photothermal treatments, we observe a sharp thermal threshold at around 55 ºC (for 10 min treatments) that separates high survival ratio from complete cell death.

Graphene oxide as a 2D platform for complexation and intracellular delivery of siRNA.

The development of efficient and safe nucleic acid delivery vectors remains an unmet need holding back translation of gene therapy approaches to the bedside. Graphene oxide (GO) could help bypass such bottlenecks, thanks to its large surface area, versatile chemistry and biocompatibility, which could overall enhance transfection efficiency while abolishing some of the limitations linked to the use of viral vectors. Here, we aimed to assess the capacity of bare GO, without any further surface modification, to complex a short double-stranded nucleic acid of biological relevance (siRNA) and mediate its intracellular delivery. GO formed stable complexes with siRNA at 10 : 1, 20 : 1 and 50 : 1 GO : siRNA mass ratios. Complexation was further corroborated by atomistic molecular dynamics simulations. GO : siRNA complexes were promptly internalized in a primary mouse cell culture, as early as 4 h after exposure. At this time point, intracellular siRNA levels were comparable to those provided by a lipid-based transfection reagent that achieved significant gene silencing. The time-lapse tracking of internalized GO and siRNA evidenced a sharp decrease of intracellular siRNA from 4 to 12 h, while GO was sequestered in large vesicles, which may explain the lack of biological effects (i.e. gene silencing) achieved by GO : siRNA complexes. This study underlines the potential of non-surface modified GO flakes to act as 2D siRNA delivery platforms, without the need for cationic functionalization, but warrants further vector optimization to allow the effective release of the nucleic acid and achieve efficient gene silencing.

Graphene Oxide Flakes Tune Excitatory Neurotransmission in Vivo by Targeting Hippocampal Synapses.

Synapses compute and transmit information to connect neural circuits and are at the basis of brain operations. Alterations in their function contribute to a vast range of neuropsychiatric and neurodegenerative disorders and synapse-based therapeutic intervention, such as selective inhibition of synaptic transmission, may significantly help against serious pathologies. Graphene is a two-dimensional nanomaterial largely exploited in multiple domains of science and technology, including biomedical applications. In hippocampal neurons in culture, small graphene oxide nanosheets (s-GO) selectively depress glutamatergic activity without altering cell viability. Glutamate is the main excitatory neurotransmitter in the central nervous system and growing evidence suggests its involvement in neuropsychiatric disorders. Here we demonstrate that s-GO directly targets the release of presynaptic vesicle. We propose that s-GO flakes reduce the availability of transmitter, via promoting its fast release and subsequent depletion, leading to a decline ofglutamatergic neurotransmission. We injected s-GO in the hippocampus in vivo, and 48 h after surgery ex vivo patch-clamp recordings from brain slices show a significant reduction in glutamatergic synaptic activity in respect to saline injections.

Toxicological Evaluation of SiO2 Nanoparticles by Zebrafish Embryo Toxicity Test.

As the use of nanoparticles (NPs) is increasing, the potential toxicity and behavior of NPs in living systems need to be better understood. Our goal was to evaluate the developmental toxicity and bio-distribution of two different sizes of fluorescently-labeled SiO2 NPs, 25 and 115 nm, with neutral surface charge or with different surface functionalization, rendering them positively or negatively charged, in order to predict the effect of NPs in humans. We performed a zebrafish embryo toxicity test (ZFET) by exposing the embryos to SiO2 NPs starting from six hours post fertilization (hpf). Survival rate, hatching time, and gross morphological changes were assessed at 12, 24, 36, 48, 60, and 72 hpf. We evaluated the effect of NPs on angiogenesis by counting the number of sub-intestinal vessels between the second and seventh intersegmental vessels and gene expression analysis of vascular endothelial growth factor (VEGF) and VEGF receptors at 72 hpf. SiO2 NPs did not show any adverse effects on survival rate, hatching time, gross morphology, or physiological angiogenesis. We found that SiO2 NPs were trapped by the chorion up until to the hatching stage. After chemical removal of the chorion (dechorionation), positively surface-charged SiO2 NPs (25 nm) significantly reduced the survival rate of the fish compared to the control group. These results indicate that zebrafish chorion acts as a physical barrier against SiO2 NPs, and removing the chorions in ZFET might be necessary for evaluation of toxicity of NPs.

Small, Thin Graphene Oxide Is Anti-inflammatory Activating Nuclear Factor Erythroid 2-Related Factor 2 via Metabolic Reprogramming.

Graphene oxide (GO), an oxidized form of graphene, has potential applications in biomedical research. However, how GO interacts with biological systems, including the innate immune system, is poorly understood. Here, we elucidate the effects of GO sheets on macrophages, identifying distinctive effects of GO on the inflammatory phenotype. Small, thin (s)-GO dose-dependently inhibited release of interleukin (IL)-1ß and IL-6 but not tumor necrosis factor α. NLRP3 inflammasome and caspase-1 activation was not affected. The effect of s-GO was pretranslational, as s-GO blocked Toll-like receptor 4-dependent expression of Il1b and Il6 but not Nlrp3 or Tnf mRNA transcripts. s-GO was internalized by immortalized bone-marrow-derived macrophages, suggesting a potential intracellular action. Uptake of polystyrene beads with similar lateral dimensions and surface charge did not phenocopy the effects of s-GO, suggesting that s-GO-mediated inhibition of interleukin expression was not simply due to particle phagocytosis. RNA-Seq analysis established that s-GO had profound effects on the immunometabolism of the cells, leading to activation of the transcription factor nuclear factor erythroid 2-related factor 2, which inhibited expression of cytokines such as IL-1ß and IL-6. Thus, we have identified immunometabolic effects of GO that reveal another dimension to its effects on cells. These findings suggest that s-GO may be used as a valuable tool to generate further insights into inflammatory mechanisms and indicate its potential applications in biomedicine.

Live Imaging of Label-Free Graphene Oxide Reveals Critical Factors Causing Oxidative-Stress-Mediated Cellular Responses.

The interest in graphene and its translation into commercial products has been expanding at a high pace. Based on previously described pulmonary safety concerns for carbon nanomaterials, there is a great need to define parameters guiding interactions between graphene-based materials and the pulmonary system. The aim of the present study was to determine the importance of two critical parameters: lateral dimensions of the material and coating with proteins in relation to each other and their pulmonary impact. Endotoxin-free materials with distinct lateral dimensions, s-GO (50-200 nm) and l-GO (5-15 µm), were produced and thoroughly characterized. Exploiting intrinsic fluorescence of graphene oxide (GO) and using confocal live-cell imaging, the behavior of the cells in response to the material was visualized in real time. Although BEAS-2B cells internalized GO efficiently, l-GO was linked to higher plasma membrane interactions correlated with elevated reactive oxygen species (ROS) levels, pro-inflammatory response, and greater cytotoxicity, in agreement with the oxidative stress paradigm. For both GO types, the presence of serum alleviated lipid peroxidation of plasma membrane and decreased intracellular ROS levels. However, protein coating was not enough to entirely mitigate toxicity and inflammatory response induced by l-GO. In vitro results were validated in vivo, as l-GO was more prone to induce pulmonary granulomatous response in mice compared to s-GO. In conclusion, the lateral dimension of GO played a more important role than serum protein coating in determining biological responses to the material. It was also demonstrated that time-lapse imaging of live cells interacting with label-free GO sheets can be used as a tool to assess GO-induced cytotoxicity.

High-Accuracy Determination of Cytotoxic Responses from Graphene Oxide Exposure Using Imaging Flow Cytometry.

Graphene and other 2D materials have received increased attention in the biomedical field due to their unique properties and potential use as carriers for targeted drug delivery or in regenerative medicine. Before the exploitation of graphene-based materials in biomedicine becomes a reality, it is necessary to establish the full toxicological profile and better understand how the material interacts with cells and tissues. Because specific properties, such as flake size and surface chemistry, might determine whether graphene can achieve therapeutic efficacy without causing toxicity, it is important to develop highly accurate and reliable screening techniques to accurately assess the biocompatibility of different types of graphene-based materials. In this protocol, we describe a method to achieve accurate determination of the cytotoxic response following in vitro exposure to large graphene oxide (L-GO) sheets using annexin V/propidium iodide staining and the Imagestream® platform. The proposed protocol is especially suitable for the toxicity assessment of carbonaceous materials that form aggregates in cell culture media, which is a common occurrence. We describe how to best gate out any interfering signals coming from the material by visual inspection and by using powerful software, thus performing the analysis of cellular death on a selected population of cells with higher accuracy and statistical relevance compared to conventional flow cytometry.