First-in-human controlled inhalation of thin graphene oxide nanosheets to study acute cardiorespiratory responses.

Graphene oxide nanomaterials are being developed for wide-ranging applications but are associated with potential safety concerns for human health. We conducted a double-blind randomized controlled study to determine how the inhalation of graphene oxide nanosheets affects acute pulmonary and cardiovascular function. Small and ultrasmall graphene oxide nanosheets at a concentration of 200 µg m-3 or filtered air were inhaled for 2 h by 14 young healthy volunteers in repeated visits. Overall, graphene oxide nanosheet exposure was well tolerated with no adverse effects. Heart rate, blood pressure, lung function and inflammatory markers were unaffected irrespective of graphene oxide particle size. Highly enriched blood proteomics analysis revealed very few differential plasma proteins and thrombus formation was mildly increased in an ex vivo model of arterial injury. Overall, acute inhalation of highly purified and thin nanometre-sized graphene oxide nanosheets was not associated with overt detrimental effects in healthy humans. These findings demonstrate the feasibility of carefully controlled human exposures at a clinical setting for risk assessment of graphene oxide, and lay the foundations for investigating the effects of other two-dimensional nanomaterials in humans. Clinicaltrials.gov ref: NCT03659864.

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.

Pulmonary Toxicity of Boron Nitride Nanomaterials Is Aspect Ratio Dependent.

Boron nitride (BN) nanomaterials have drawn a lot of interest in the material science community. However, extensive research is still needed to thoroughly analyze their safety profiles. Herein, we investigated the pulmonary impact and clearance of two-dimensional hexagonal boron nitride (h-BN) nanosheets and boron nitride nanotubes (BNNTs) in mice. Animals were exposed by single oropharyngeal aspiration to h-BN or BNNTs. On days 1, 7, and 28, bronchoalveolar lavage (BAL) fluids and lungs were collected. On one hand, adverse effects on lungs were evaluated using various approaches (e.g., immune response, histopathology, tissue remodeling, and genotoxicity). On the other hand, material deposition and clearance from the lungs were assessed. Two-dimensional h-BN did not cause any significant immune response or lung damage, although the presence of materials was confirmed by Raman spectroscopy. In addition, the low aspect ratio h-BN nanosheets were internalized rapidly by phagocytic cells present in alveoli, resulting in efficient clearance from the lungs. In contrast, high aspect ratio BNNTs caused a strong and long-lasting inflammatory response, characterized by sustained inflammation up to 28 days after exposure and the activation of both innate and adaptive immunity. Moreover, the presence of granulomatous structures and an indication of ongoing fibrosis as well as DNA damage in the lung parenchyma were evidenced with these materials. Concurrently, BNNTs were identified in lung sections for up to 28 days, suggesting long-term biopersistence, as previously demonstrated for other high aspect ratio nanomaterials with poor lung clearance such as multiwalled carbon nanotubes (MWCNTs). Overall, we reveal the safer toxicological profile of BN-based two-dimensional nanosheets in comparison to their nanotube counterparts. We also report strong similarities between BNNTs and MWCNTs in lung response, emphasizing their high aspect ratio as a major driver of their toxicity.

Lung Persistence, Biodegradation, and Elimination of Graphene-Based Materials are Predominantly Size-Dependent and Mediated by Alveolar Phagocytes.

Graphene-based materials (GBMs) have promising applications in various sectors, including pulmonary nanomedicine. Nevertheless, the influence of GBM physicochemical characteristics on their fate and impact in lung has not been thoroughly addressed. To fill this gap, the biological response, distribution, and bio-persistence of four different GBMs in mouse lungs up to 28 days after single oropharyngeal aspiration are investigated. None of the GBMs, varying in size (large versus small) and carbon to oxygen ratio as well as thickness (few-layers graphene (FLG) versus thin graphene oxide (GO)), induce a strong pulmonary immune response. However, recruited neutrophils internalize nanosheets better and degrade GBMs faster than macrophages, revealing their crucial role in the elimination of small GBMs. In contrast, large GO sheets induce more damages due to a hindered degradation and long-term persistence in macrophages. Overall, small dimensions appear to be a leading feature in the design of safe GBM pulmonary nanovectors due to an enhanced degradation in phagocytes and a faster clearance from the lungs for small GBMs. Thickness also plays an important role, since decreased material loading in alveolar phagocytes and faster elimination are found for FLGs compared to thinner GOs. These results are important for designing safer-by-design GBMs for biomedical application.

Correlative radioimaging and mass spectrometry imaging: a powerful combination to study 14C-graphene oxide in vivo biodistribution.

Research on graphene based nanomaterials has flourished in the last decade due their unique properties and emerging socio-economic impact. In the context of their potential exploitation for biomedical applications, there is a growing need for the development of more efficient imaging techniques to track the fate of these materials. Herein we propose the first correlative imaging approach based on the combination of radioimaging and mass spectrometry imaging for the detection of Graphene Oxide (GO) labelled with carbon-14 in mice. In this study, 14C-graphene oxide nanoribbons were produced from the oxidative opening of 14C-carbon nanotubes, and were then intensively sonicated to provide nano-size 14C-GO flakes. After Intravenous administration in mice, 14C-GO distribution was quantified by radioimaging performed on tissue slices. On the same slices, MS-imaging provided a highly resolved distribution map of the nanomaterial based on the detection of specific radical anionic carbon clusters ranging from C2˙- to C9˙- with a base peak at m/z 72 (12C) and 74 (14C) under negative laser desorption ionization mass spectrometry (LDI-MS) conditions. This proof of concept approach synergizes the strength of each technique and could be advantageous in the pre-clinical development of future Graphene-based biomedical applications.

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.

Lung recovery from DNA damage induced by graphene oxide is dependent on size, dose and inflammation profile.

BACKGROUND: A key aspect of any new material safety assessment is the evaluation of their in vivo genotoxicity. Graphene oxide (GO) has been studied for many promising applications, but there are remaining concerns about its safety profile, especially after inhalation. Herein we tested whether GO lateral dimension, comparing micrometric (LGO) and nanometric (USGO) GO sheets, has a role in the formation of DNA double strand breaks in mouse lungs. We used spatial resolution and differential cell type analysis to measure DNA damages in both epithelial and immune cells, after either single or repeated exposure. RESULTS: GO induced DNA damages were size and dose dependent, in both exposure scenario. After single exposure to a high dose, both USGO and LGO induced significant DNA damage in the lung parenchyma, but only during the acute phase response (p < 0.05 for USGO; p < 0.01 for LGO). This was followed by a fast lung recovery at day 7 and 28 for both GOs. When evaluating the chronic impact of GO after repeated exposure, only a high dose of LGO induced long-term DNA damages in lung alveolar epithelia (at 84 days, p < 0.05). Regardless of size, low dose GO did not induce any significant DNA damage after repeated exposure. A multiparametric correlation analysis of our repeated exposure data revealed that transient or persistent inflammation and oxidative stress were associated to either recovery or persistent DNA damages. For USGO, recovery from DNA damage was correlated to efficient recovery from acute inflammation (i.e., significant secretion of SAA3, p < 0.001; infiltration of neutrophils, p < 0.01). In contrast, the persistence of LGO in lungs was associated to a long-lasting presence of multinucleated macrophages (up to 84 days, p < 0.05), an underlying inflammation (IL-1α secretion up to 28 days, p < 0.05) and the presence of persistent DNA damages at 84 days. CONCLUSIONS: Overall these results highlight the importance of the exposure scenario used. We showed that LGO was more genotoxic after repeated exposure than single exposure due to persistent lung inflammation. These findings are important in the context of human health risk assessment and toward establishing recommendations for a safe use of graphene based materials in the workplace.

Hazard assessment of abraded thermoplastic composites reinforced with reduced graphene oxide.

Graphene-related materials (GRMs) are subject to intensive investigations and considerable progress has been made in recent years in terms of safety assessment. However, limited information is available concerning the hazard potential of GRM-containing products such as graphene-reinforced composites. In the present study, we conducted a comprehensive investigation of the potential biological effects of particles released through an abrasion process from reduced graphene oxide (rGO)-reinforced composites of polyamide 6 (PA6), a widely used engineered thermoplastic polymer, in comparison to as-produced rGO. First, a panel of well-established in vitro models, representative of the immune system and possible target organs such as the lungs, the gut, and the skin, was applied. Limited responses to PA6-rGO exposure were found in the different in vitro models. Only as-produced rGO induced substantial adverse effects, in particular in macrophages. Since inhalation of airborne materials is a key occupational concern, we then sought to test whether the in vitro responses noted for these materials would translate into adverse effects in vivo. To this end, the response at 1, 7 and 28 days after a single pulmonary exposure was evaluated in mice. In agreement with the in vitro data, PA6-rGO induced a modest and transient pulmonary inflammation, resolved by day 28. In contrast, rGO induced a longer-lasting, albeit moderate inflammation that did not lead to tissue remodeling within 28 days. Taken together, the present study suggests a negligible impact on human health under acute exposure conditions of GRM fillers such as rGO when released from composites at doses expected at the workplace.

Innate but Not Adaptive Immunity Regulates Lung Recovery from Chronic Exposure to Graphene Oxide Nanosheets.

Graphene has drawn a lot of interest in the material community due to unique physicochemical properties. Owing to a high surface area to volume ratio and free oxygen groups, the oxidized derivative, graphene oxide (GO) has promising potential as a drug delivery system. Here, the lung tolerability of two distinct GO varying in lateral dimensions is investigated, to reveal the most suitable candidate platform for pulmonary drug delivery. Following repeated chronic pulmonary exposure of mice to GO sheet suspensions, the innate and adaptive immune responses are studied. An acute and transient influx of neutrophils and eosinophils in the alveolar space, together with the replacement of alveolar macrophages by interstitial ones and a significant activation toward anti-inflammatory subsets, are found for both GO materials. Micrometric GO give rise to persistent multinucleated macrophages and granulomas. However, neither adaptive immune response nor lung tissue remodeling are induced after exposure to micrometric GO. Concurrently, milder effects and faster tissue recovery, both associated to a faster clearance from the respiratory tract, are found for nanometric GO, suggesting a greater lung tolerability. Taken together, these results highlight the importance of dimensions in the design of biocompatible 2D materials for pulmonary drug delivery system.

Adsorption of P103 Nanoaggregates on Graphene Oxide Nanosheets: Role of Electrostatic Forces in Improving Nanosheet Dispersion.

Graphene oxide (GO) nanosheet suspension is not stable in physiological ionic fluids. To improve stability, surfactants such as Pluronic 103 (P103) have been tested. Going further, this work investigated whether conferring positive surface charge to the surfactant may improve the adsorption ability of P103 micelles on GO sheets. Positive charge on the surfactant was induced by adding dodecyltrimethylammonium bromide (DTAB, a cationic surfactant) in P103 micelles. Subsequent changes in aggregation parameters were investigated through dynamic light scattering and small-angle neutron scattering studies. DTAB incorporation was accompanied by a steady increase in the ζ potential and mixed micelle formation. At high surface charge density, the interaction between adjacent head groups was distorted, which led to dissociation of mixed micelles. Structural developments during the adsorption of mixed micelles on the sheet surface (mass fractal formation) were monitored in terms of changes in the scattering features of aggregates. These fractals emerged as a result of electrostatic interactions. Our observations point toward the existence of small-sized building blocks at low DTAB concentration (≤4 mM). With a superior adsorption, mixed micelles are expected to occupy the intersheet space and maintain a hydration layer. However, at a higher DTAB concentration (≥10 mM), micelles dissociate to produce DTAB-rich unimers and P103-rich loose aggregates. At this point, sheets tend to aggregate in the solvent, regardless of fractal formation.

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.

Intracerebral Injection of Graphene Oxide Nanosheets Mitigates Microglial Activation Without Inducing Acute Neurotoxicity: A Pilot Comparison to Other Nanomaterials.

Carbon-based nanomaterials (CNMs) are being explored for neurological applications. However, systematic in vivo studies investigating the effects of CNM nanocarriers in the brain and how brain cells respond to such nanomaterials are scarce. To address this, functionalized multiwalled carbon nanotubes and graphene oxide (GO) sheets are injected in mice brain and compared with charged liposomes. The induction of acute neuroinflammatory and neurotoxic effects locally and in brain structures distant from the injection site are assessed up to 1 week postadministration. While significant neuronal cell loss and sustained microglial cell activation are observed after injection of cationic liposomes, none of the tested CNMs induces either neurodegeneration or microglial activation. Among the candidate nanocarriers tested, GO sheets appear to elicit the least deleterious neuroinflammatory profile. At molecular level, GO induces moderate activation of proinflammatory markers compared to vehicle control. At histological level, brain response to GO is lower than after vehicle control injection, suggesting some capacity for GO to reduce the impact of stereotactic injection on brain. While these findings are encouraging and valuable in the selection and design of nanomaterial-based brain delivery systems, they warrant further investigations to better understand the mechanisms underlying GO immunomodulatory properties in brain.

Splenic Capture and In Vivo Intracellular Biodegradation of Biological-Grade Graphene Oxide Sheets.

Carbon nanomaterials, including 2D graphene-based materials, have shown promising applicability to drug delivery, tissue engineering, diagnostics, and various other biomedical areas. However, to exploit the benefits of these materials in some of the areas mentioned, it is necessary to understand their possible toxicological implications and long-term fate in vivo. We previously demonstrated that following intravenous administration, 2D graphene oxide (GO) nanosheets were largely excreted via the kidneys; however, a small but significant portion of the material was sequestered in the spleen. Herein, we interrogate the potential consequences of this accumulation and the fate of the spleen-residing GO over a period of nine months. We show that our thoroughly characterized GO materials are not associated with any detectable pathological consequences in the spleen. Using confocal Raman mapping of tissue sections, we determine the sub-organ biodistribution of GO at various time points after administration. The cells largely responsible for taking up the material are confirmed using immunohistochemistry coupled with Raman spectroscopy, and transmission electron microscopy (TEM). This combination of techniques identified cells of the splenic marginal zone as the main site of GO bioaccumulation. In addition, through analyses using both bright-field TEM coupled with electron diffraction and Raman spectroscopy, we reveal direct evidence of in vivo intracellular biodegradation of GO sheets with ultrastructural precision. This work offers critical information about biological processing and degradation of thin GO sheets by normal mammalian tissue, indicating that further development and exploitation of GO in biomedicine would be possible.

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.

Size-Dependent Pulmonary Impact of Thin Graphene Oxide Sheets in Mice: Toward Safe-by-Design.

Safety assessment of graphene-based materials (GBMs) including graphene oxide (GO) is essential for their safe use across many sectors of society. In particular, the link between specific material properties and biological effects needs to be further elucidated. Here, the effects of lateral dimensions of GO sheets in acute and chronic pulmonary responses after single intranasal instillation in mice are compared. Micrometer-sized GO induces stronger pulmonary inflammation than nanometer-sized GO, despite reduced translocation to the lungs. Genome-wide RNA sequencing also reveals distinct size-dependent effects of GO, in agreement with the histopathological results. Although large GO, but not the smallest GO, triggers the formation of granulomas that persists for up to 90 days, no pulmonary fibrosis is observed. These latter results can be partly explained by Raman imaging, which evidences the progressive biotransformation of GO into less graphitic structures. The findings demonstrate that lateral dimensions play a fundamental role in the pulmonary response to GO, and suggest that airborne exposure to micrometer-sized GO should be avoided in the production plant or applications, where aerosolized dispersions are likely to occur. These results are important toward the implementation of a safer-by-design approach for GBM products and applications, for the benefit of workers and end-users.

Safety Assessment of Graphene-Based Materials: Focus on Human Health and the Environment.

Graphene and its derivatives are heralded as "miracle" materials with manifold applications in different sectors of society from electronics to energy storage to medicine. The increasing exploitation of graphene-based materials (GBMs) necessitates a comprehensive evaluation of the potential impact of these materials on human health and the environment. Here, we discuss synthesis and characterization of GBMs as well as human and environmental hazard assessment of GBMs using in vitro and in vivo model systems with the aim to understand the properties that underlie the biological effects of these materials; not all GBMs are alike, and it is essential that we disentangle the structure-activity relationships for this class of materials.

Immunological impact of graphene oxide sheets in the abdominal cavity is governed by surface reactivity.

Graphene oxide (GO) is an oxidised form of graphene that has attracted commercial interest in multiple applications, including inks, printed electronics and spray coatings, which all raise health concerns due to potential creation of inhalable aerosols. Although a number of studies have discussed the toxicity of GO sheets, the in vivo impact of their lateral dimensions is still not clear. Here, we compared the effects of large GO sheets (l-GO, 1-20 µm) with those of small GO sheets (s-GO, < 1 µm) in terms of mesothelial damage and peritoneal inflammation, after intraperitoneal (i.p.) injection in mice. To benchmark the outcomes, long and rigid multi-walled carbon nanotubes (MWCNTs) that were shown to be associated with asbestos-like pathogenicity on the mesothelium were also tested. Our aim was to assess whether lateral dimensions can be a predictor of inflammogenicity for GO sheets in a similar fashion as length is for MWCNTs. While long MWCNTs dispersed in 0.5% BSA induced a granulomatous response on the diaphragmatic mesothelium and immune cell recruitment to the peritoneal cavity, GO sheets dispersed under similar conditions did not cause any response, regardless of their lateral dimensions. We further interrogated whether tuning the surface reactivity of GO by testing different dispersions (5% dextrose instead of 0.5% BSA) may change the biological outcome. Although the change of dispersion did not alter the impact of GO on the mesothelium (i.e. no granuloma), we observed that, when dispersed in protein-free 5% dextrose solution, s-GO elicited a greater recruitment of monocytic cells to the peritoneal cavity than l-GO, or when dispersed in protein-containing solution. Such recruitment coincided with the greater ability of s-GO to interact in vivo with peritoneal macrophages and was associated with a greater surface reactivity in comparison to l-GO. In conclusion, large dimension was not a determining factor of the immunological impact of GO sheets after i.p. administration. For an equal dose, GO sheets with lateral dimensions similar to the length of long MWCNTs were less pathogenic than the MWCNTs. On the other hand, surface reactivity and the ability of some smaller GO sheets to interact more readily with immune cells seem to be key parameters that can be tuned to improve the safety profile of GO. In particular, the choice of dispersion modality, which affected these two parameters, was found to be of crucial importance in the assessment of GO impact in this model. Overall, these findings are essential for a better understanding of the parameters governing GO toxicity and inflammation, and the rational design of safe GO-based formulations for various applications, including 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.

Direct visualization of carbon nanotube degradation in primary cells by photothermal imaging.

Assessment of biodegradability of carbon nanotubes (CNTs) is a critically important aspect that needs to be solved before their translation into new biomedical tools. CNT biodegradation has been shown both in vitro and in vivo, but we are limited by the number of analytical techniques that can be used to follow the entire process. Photothermal imaging (PhI) is an innovative technique that enables the quantitative detection of nanometer-sized absorptive objects. In this study, we demonstrate that PhI allows the observation of the degradation process of functionalized multi-walled carbon nanotubes (MWCNTs) following their internalization by primary glial cells. The absence of interference from the biological matrix components, together with the possibility to combine PhI with other detection techniques (e.g. fluorescence, light or electron microscopy) validate the potential of this method to follow the fate and behavior of carbon nanostructures in a biological environment.

Primary microglia maintain their capacity to function despite internalisation and intracellular loading with carbon nanotubes.

Biomedical applications of functionalised carbon nanotubes (f-CNTs) for imaging as well as drug or gene delivery in the brain have recently gained interest. Several studies have demonstrated the potential of f-CNTs to offer treatment options for neurological conditions with success. However, there is also evidence that f-CNTs accumulate preferentially within microglial cells when introduced in the brain. Considering the key immunological role of these cells in the brain and the limited knowledge regarding the interaction of CNTs with microglial cells, it is imperative to understand whether accumulation of CNTs in microglial cells can alter their physiological functions or trigger pro-inflammatory signalling. The aim of the present study was to investigate the basic physiological functions of isolated primary microglial cells over time, following their exposure to multi-walled carbon nanotubes functionalized via different surface chemistries. We rationalized that some chemical strategies may be more deleterious for microglial cell functions than others. We used rat primary microglial cells that can be maintained in cell culture for a long period of time without undergoing cell division. Cell viability, phagocytosis, migration, and pro-inflammatory factor release were studied over one month, following a single exposure to a non-cytotoxic concentration of three different f-CNTs (carboxylated, aminated and both carboxylated and aminated). We show that f-CNTs do not induce inflammation and do not affect the basic functions of microglial cells under these conditions. Importantly, this was the case from day 1 to day 28 post-exposure, even though microglial cells had internalized f-CNTs in subcellular compartments and retained the nanotube load in their cytoplasm over time.