Multi-walled carbon nanotubes elicit concordant changes in DNA methylation and gene expression following long-term pulmonary exposure in mice.

Pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) causes inflammation and fibrosis. Our previous work has shown that industrially produced MWCNTs trigger specific changes in gene expression in the lungs of exposed animals. To elucidate whether epigenetic effects play a role for these gene expression changes, we performed whole genome bisulphite sequencing to assess DNA methylation patterns in the lungs 56 days after exposure to MWCNTs. Lung tissues were also evaluated with respect to histopathological changes and cytokine profiling of bronchoalveolar lavage (BAL) fluid was conducted using a multi-plex array. Integrated analysis of transcriptomics data and DNA methylation data revealed concordant changes in gene expression. Functional analysis showed that the muscle contraction, immune system/inflammation, and extracellular matrix pathways were the most affected pathways. Taken together, the present study revealed that MWCNTs exert epigenetic effects in the lungs of exposed animals, potentially driving the subsequent gene expression changes.

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.

Next-Generation Sequencing Reveals Differential Responses to Acute versus Long-Term Exposures to Graphene Oxide in Human Lung Cells.

Numerous studies have addressed the biological impact of graphene-based materials including graphene oxide (GO), yet few have focused on long-term effects. Here, RNA sequencing is utilized to unearth responses of human lung cells to GO. To this end, the BEAS-2B cell line derived from normal human bronchial epithelium is subjected to repeated, low-dose exposures of GO (1 or 5 µg mL-1 ) for 28 days or to the equivalent, cumulative amount of GO for 48 h. Then, samples are analyzed by using the NovaSeq 6000 sequencing system followed by pathway analysis and gene ontology enrichment analysis of the differentially expressed genes. Significant differences are seen between the low-dose, long-term exposures and the high-dose, short-term exposures. Hence, exposure to GO for 48 h results in mitochondrial dysfunction. In contrast, exposure to GO for 28 days is characterized by engagement of apoptosis pathways with downregulation of genes belonging to the inhibitor of apoptosis protein (IAP) family. Validation experiments confirm that long-term exposure to GO affects the apoptosis threshold in lung cells, accompanied by a loss of IAPs. These studies reveal the sensitivity of RNA-sequencing approaches and show that acute exposure to GO is not a good predictor of the long-term effects of GO.

Degradation of Single-Layer and Few-Layer Graphene by Neutrophil Myeloperoxidase.

Biodegradability of graphene is one of the fundamental parameters determining the fate of this material in vivo. Two types of aqueous dispersible graphene, corresponding to single-layer (SLG) and few-layer graphene (FLG), devoid of either chemical functionalization or stabilizing surfactants, were subjected to biodegradation by human myeloperoxidase (hMPO) mediated catalysis. Graphene biodegradation was also studied in the presence of activated, degranulating human neutrophils. The degradation of both FLG and SLG sheets was confirmed by Raman spectroscopy and electron microscopy analyses, leading to the conclusion that highly dispersed pristine graphene is not biopersistent.

Nano⁻Bio Interactions: Nanomedicine and Nanotoxicology.

The 21st century has truly become the age of nanotechnology. Nanomaterials, design strategies, and processing have already made a significant impact in areas of materials science and electronics, with many commercial applications already being available on the consumer market[...].

Toxicology of Engineered Nanoparticles: Focus on Poly(amidoamine) Dendrimers.

Engineered nanomaterials are increasingly being developed for paints, sunscreens, cosmetics, industrial lubricants, tyres, semiconductor devices, and also for biomedical applications such as in diagnostics, therapeutics, and contrast agents. As a result, nanomaterials are being manufactured, transported, and used in larger and larger quantities, and potential impacts on environmental and human health have been raised. Poly(amidoamine) (PAMAM) dendrimers are specifically suitable for biomedical applications. They are well-defined nanoscale molecules which contain a 2-carbon ethylenediamine core and primary amine groups at the surface. The systematically variable structural architecture and the large internal free volume make these dendrimers an attractive option for drug delivery and other biomedical applications. Due to the wide range of applications, the Organisation for Economic Co-Operation and Development (OECD) have included them in their list of nanoparticles which require toxicological assessment. Thus, the toxicological impact of these PAMAM dendrimers on human health and the environment is a matter of concern. In this review, the potential toxicological impact of PAMAM dendrimers on human health and environment is assessed, highlighting work to date exploring the toxicological effects of PAMAM dendrimers.

Macrophage sensing of single-walled carbon nanotubes via Toll-like receptors.

Carbon-based nanomaterials including carbon nanotubes (CNTs) have been shown to trigger inflammation. However, how these materials are 'sensed' by immune cells is not known. Here we compared the effects of two carbon-based nanomaterials, single-walled CNTs (SWCNTs) and graphene oxide (GO), on primary human monocyte-derived macrophages. Genome-wide transcriptomics assessment was performed at sub-cytotoxic doses. Pathway analysis of the microarray data revealed pronounced effects on chemokine-encoding genes in macrophages exposed to SWCNTs, but not in response to GO, and these results were validated by multiplex array-based cytokine and chemokine profiling. Conditioned medium from SWCNT-exposed cells acted as a chemoattractant for dendritic cells. Chemokine secretion was reduced upon inhibition of NF-κB, as predicted by upstream regulator analysis of the transcriptomics data, and Toll-like receptors (TLRs) and their adaptor molecule, MyD88 were shown to be important for CCL5 secretion. Moreover, a specific role for TLR2/4 was confirmed by using reporter cell lines. Computational studies to elucidate how SWCNTs may interact with TLR4 in the absence of a protein corona suggested that binding is guided mainly by hydrophobic interactions. Taken together, these results imply that CNTs may be 'sensed' as pathogens by immune cells.

Cytokine Profiling of Primary Human Macrophages Exposed to Endotoxin-Free Graphene Oxide: Size-Independent NLRP3 Inflammasome Activation.

Graphene-based materials including graphene oxide (GO) are envisioned for a variety of biomedical applications. However, there are conflicting results concerning the biocompatibility of these materials. Here, a question is raised whether GO with small or large lateral dimensions triggers cytotoxicity and/or cytokine responses in primary human monocyte-derived macrophages. GO sheets produced under sterile conditions by a modified Hummers' method are found to be taken up by macrophages without signs of cytotoxicity. Then, multiplex arrays are used for profiling of proinflammatory and anti-inflammatory responses. Notably, GO suppresses the lipopolysaccharide (LPS)-triggered induction of several chemokines and cytokines, including the anti-inflammatory cytokine, interleukin-10 (IL-10). No production of proinflammatory TNF-α is observed. However, GO elicits caspase-dependent IL-1 ß expression, a hallmark of inflammasome activation, in LPS-primed macrophages. Furthermore, GO-triggered IL-1 ß production requires NADPH oxidase-generated reactive oxygen species and cellular uptake of GO and is accompanied by cathepsin B release and K+ efflux. Using THP-1 knockdown cells, a role for the inflammasome sensor, NLRP3, the adaptor protein, ASC, and caspase-1 for GO-induced IL-1ß secretion is demonstrated. Finally, these studies show that inflammasome activation is independent of the lateral dimensions of the GO sheets. These studies provide novel insights regarding the immunomodulatory properties of endotoxin-free GO.

Graphene oxide is degraded by neutrophils and the degradation products are non-genotoxic.

Neutrophils were previously shown to digest oxidized carbon nanotubes through a myeloperoxidase (MPO)-dependent mechanism, and graphene oxide (GO) was found to undergo degradation when incubated with purified MPO, but there are no studies to date showing degradation of GO by neutrophils. Here we produced endotoxin-free GO by a modified Hummers' method and asked whether primary human neutrophils stimulated to produce neutrophil extracellular traps or activated to undergo degranulation are capable of digesting GO. Biodegradation was assessed using a range of techniques including Raman spectroscopy, transmission electron microscopy, atomic force microscopy, and mass spectrometry. GO sheets of differing lateral dimensions were effectively degraded by neutrophils. As the degradation products could have toxicological implications, we also evaluated the impact of degraded GO on the bronchial epithelial cell line BEAS-2B. MPO-degraded GO was found to be non-cytotoxic and did not elicit any DNA damage. Taken together, these studies have shown that neutrophils can digest GO and that the biodegraded GO is non-toxic for human lung cells.

Graphene and the Immune System: A Romance of Many Dimensions.

Graphene-based materials (GBMs) are emerging as attractive materials for biomedical applications. Understanding how these materials are perceived by and interact with the immune system is of fundamental importance. Phagocytosis is a major mechanism deployed by the immune system to remove pathogens, particles, and cellular debris. Here, we discuss recent studies on the interactions of GBMs with different phagocytic cells, including macrophages, neutrophils, and dendritic cells. The importance of assessing GBMs for endotoxin contamination is discussed as this may skew results. We also explore the role of the bio-corona for interactions of GBMs with immune cells. Finally, we highlight recent evidence for direct plasma membrane interactions of GBMs.

Detection of Endotoxin Contamination of Graphene Based Materials Using the TNF-α Expression Test and Guidelines for Endotoxin-Free Graphene Oxide Production.

Nanomaterials may be contaminated with bacterial endotoxin during production and handling, which may confound toxicological testing of these materials, not least when assessing for immunotoxicity. In the present study, we evaluated the conventional Limulus amebocyte lysate (LAL) assay for endotoxin detection in graphene based material (GBM) samples, including graphene oxide (GO) and few-layered graphene (FLG). Our results showed that some GO samples interfered with various formats of the LAL assay. To overcome this problem, we developed a TNF-α expression test (TET) using primary human monocyte-derived macrophages incubated in the presence or absence of the endotoxin inhibitor, polymyxin B sulfate, and found that this assay, performed with non-cytotoxic doses of the GBM samples, enabled unequivocal detection of endotoxin with a sensitivity that is comparable to the LAL assay. FLG also triggered TNF-α production in the presence of the LPS inhibitor, pointing to an intrinsic pro-inflammatory effect. Finally, we present guidelines for the preparation of endotoxin-free GO, validated by using the TET.

Biological interactions of carbon-based nanomaterials: From coronation to degradation.

Carbon-based nanomaterials including carbon nanotubes, graphene oxide, fullerenes and nanodiamonds are potential candidates for various applications in medicine such as drug delivery and imaging. However, the successful translation of nanomaterials for biomedical applications is predicated on a detailed understanding of the biological interactions of these materials. Indeed, the potential impact of the so-called bio-corona of proteins, lipids, and other biomolecules on the fate of nanomaterials in the body should not be ignored. Enzymatic degradation of carbon-based nanomaterials by immune-competent cells serves as a special case of bio-corona interactions with important implications for the medical use of such nanomaterials. In the present review, we highlight emerging biomedical applications of carbon-based nanomaterials. We also discuss recent studies on nanomaterial 'coronation' and how this impacts on biodistribution and targeting along with studies on the enzymatic degradation of carbon-based nanomaterials, and the role of surface modification of nanomaterials for these biological interactions. FROM THE CLINICAL EDITOR: Advances in technology have produced many carbon-based nanomaterials. These are increasingly being investigated for the use in diagnostics and therapeutics. Nonetheless, there remains a knowledge gap in terms of the understanding of the biological interactions of these materials. In this paper, the authors provided a comprehensive review on the recent biomedical applications and the interactions of various carbon-based nanomaterials.