Graphene-Based Nanoplatelets: A New Risk to the Respiratory System as a Consequence of Their Unusual Aerodynamic Properties

Graphene is a new nanomaterial with unusual and useful physical and chemical properties. However, in the form of nanoplatelets this new, emerging material could pose unusual risks to the respiratory system after inhalation exposure. The graphene-based nanoplatelets used in this study are commercially available and consist of several sheets of graphene (few-layer graphene). We first derived the respirability of graphene nanoplatelets (GP) from the basic principles of the aerodynamic behavior of plate-shaped particles which allowed us to calculate their aerodynamic diameter. This showed that the nanoplatelets, which were up to 25 μm in diameter, were respirable and so would deposit beyond the ciliated airways following inhalation. We therefore utilized models of pharyngeal aspiration and direct intrapleural installation of GP, as well as an <i>in vitro</i> model, to assess their inflammatory potential. These large but respirable GP were inflammogenic in both the lung and the pleural space. MIP-1α, MCP-1, MIP-2, IL-8, and IL-1β expression in the BAL, the pleural lavage, and cell culture supernatant from THP-1 macrophages were increased with GP exposure compared to controls but not with nanoparticulate carbon black (CB). <i>In vitro</i>, macrophages exposed to GP showed expression of IL-1β. This study highlights the importance of nanoplatelet form as a driver for <i>in vivo</i> and <i>in vitro</i> inflammogenicity by virtue of their respirable aerodynamic diameter, despite a considerable 2-dimensional size which leads to frustrated phagocytosis when they deposit in the distal lungs and macrophages attempt to phagocytose them. Our data suggest that nanoplatelets pose a novel nanohazard and structure-toxicity relationship in nanoparticle toxicology

Surface charge determines the lung inflammogenicity: A study with polystyrene nanoparticles

<p>Surface functionalization is a routine process to improve the behavior of nanoparticles (NPs), but the induced surface properties, such as surface charge, can produce differential toxicity profiles. Here, we synthesized a library of covalently functionalized fluorescent polymeric NPs (<i>F-</i>PLNPs) to evaluate the role of surface charge on the acute inflammation and the localization in the lung. Guanidinium-, acetylated-, zwitterionic-, hydroxylated-, PEGylated-, carboxylated- and sulfated-<i>F-</i>PLNPs were synthesized from aminated-<i>F</i>-PLNP. The primary particle sizes were identical, but the hydrodynamic sizes ranged from 210 to 345 nm. Following surface functionalization, the <i>F</i>-PLNPs showed diverse zeta potentials from −41.2 to 31.0 mV, and each <i>F</i>-PLNP showed a single, narrow peak. Pharyngeal aspiration with these eight types of <i>F</i>-PLNPs into rats produced diverse acute lung inflammation, with zeta potentials of the <i>F</i>-PLNPs showing excellent correlation with acute pulmonary inflammation parameters including the percentage of polymorphonuclear leukocytes (<i>R</i>² = 0.90, <i>p</i> < 0.0001) and the levels of interleukin-1β (<i>R</i>² = 0.83, <i>p</i> < 0.0001) and of cytokine-induced neutrophil chemoattractant-3 (<i>R</i>² = 0.86, <i>p</i> < 0.0001). These results imply that surface charge is a key factor influencing lung inflammation by functionalized polymeric NPs, which further confirms and extends the surface charge paradigm that we reported for pristine metal oxide NPs. This demonstrates that the surface charge paradigm is a valuable tool to predict the toxicity of NPs.</p

Monitoring Intracellular Redox Potential Changes Using SERS Nanosensors

Redox homeostasis and signaling are critically important in the regulation of cell function. There are significant challenges in quantitatively measuring intracellular redox potentials, and in this paper, we introduce a new approach. Our approach is based on the use of nanosensors which comprise molecules that sense the local redox potential, assembled on a gold nanoshell. Since the Raman spectrum of the sensor molecule changes depending on its oxidation state and since the nanoshell allows a huge enhancement of the Raman spectrum, intracellular potential can be calculated by a simple optical measurement. The nanosensors can be controllably delivered to the cytoplasm, without any toxic effects, allowing redox potential to be monitored in a reversible, non-invasive manner over a previously unattainable potential range encompassing both superphysiological and physiological oxidative stress

Additional file 1: Figure S1. of Platelet activation independent of pulmonary inflammation contributes to diesel exhaust particulate-induced promotion of arterial thrombosis

Intravenous administration of DEP does not increase systemic CRP or pulmonary pro-inflammatory cytokines. Intravenous injection of DEP (black columns) or CB (light grey columns) did not increase bronchoalveolar lavage fluid concentrations of (i) TNF-α or (ii) C-reactive protein (CRP) when compared to saline (white column) collected 2 h after injection. Levels of IL-6 were below the limit of detection. (iii) Systemic inflammation was assessed by measuring CRP in plasma taken from rats 2 h (the time-point at which thrombus formation was increased by DEP) after intravenous injection of diesel exhaust particulate (DEP) or carbon black (CB). CB (light grey column), increased plasma concentrations of CRP whereas DEP did not (black column). Data are mean ± s.e.mean (n = 6) and were compared using Student’s unpaired t-test ((i) & (ii)) or one-way ANOVA (iii); ns = not significant ***P < 0.001 compared with saline-treated control. (TIF 55 kb

Additional file 3: Table S1. of Platelet activation independent of pulmonary inflammation contributes to diesel exhaust particulate-induced promotion of arterial thrombosis

Real time polymerase chain reaction primers. (TIF 53 kb

MDMs from healthy volunteers differentiated in the presence of DEP showed reduced proinflammatory responses to <i>E. coli</i>.

<p>MDMs from healthy volunteers, COPD and age matched controls were differentiated from purified monocytes in the presence or absence of varied concentrations of DEP as described in the methods. At day 14 MDMs were washed and stimulated with heat killed <i>E. coli</i> as shown. After 24 hours, levels of CXCL8 in the supernatant were determined by ELISA. Data shown are mean±SEM of n = 5, performed on different donors with significant differences denoted by *p<0.05, **p<0.01 and ***p<0.001, compared to MDMs differentiated in the absence of DEP, as measured by one way ANOVA and Dunnett’s post test.</p

MDMs differentiated in the presence of DEP showed loss of lysosomal acidification.

<p>MDMs were differentiated from purified monocytes in the presence or absence of varied concentrations of DEP as described in the methods. At day 14 cells from healthy volunteers were stained with 5 µM acridine orange (AO) for 30 minutes at 37°C. The cells were harvested and resuspended in ice cold PBS for analysis using a FACSCalibur flow cytometer. Loss of lysosomal acidification was measured in the FL-3 channel. Viable MDMs were identified based on size and granularity on the forward and side scatter plots and ten thousand events were recorded. Data were presented as percentage of cells showing loss of green staining compared to control cells (A) and geomean fluorescence (B) and data analysis was performed using FlowJo software. Figure C shows a representative histogram flow analysis plot from one donor. Data shown are mean±SEM of n = 4–5, performed on different donors with significant differences denoted by *p<0.05, **p<0.01 and ***p<0.001, compared to MDMs differentiated in the absence of DEP, as measured by one way ANOVA and Dunnett’s post test.</p

MDMs differentiated in the presence of DEP showed reduced CD14, CD11b and CD86 surface marker expression.

<p>MDMs from healthy volunteers were differentiated from purified monocytes in the presence or absence of varied concentrations of DEP as described in the methods. At day 14 MDMs were harvested and non specific binding was blocked by incubating at room temperature for 10 minutes with 1∶50 IgG from murine serum reagent grade in FACS buffer. Cells were then incubated with 1∶10 PE conjugated mouse anti-human CD14, CD11b, HLA-DR, TLR2, TLR4, CD80 or CD86 or the respective isotype controls. PE fluorescence was measured on the FL-2 channel of a FACSCalibur flow cytometer. Data are presented as a mean±SEM of n = 4 performed on different donors and representative histogram flow analysis plots from one donor. Significant differences between MDMs and DEP-MDMs cell surface expression are denoted by *p<0.05 and **p<0.01 as measured by one way ANOVA and Dunnett’s post test.</p

DEP induced cell loss of MDMs from COPD and age matched controls.

<p>MDMs were differentiated from purified monocytes obtained from stage II GOLD criteria COPD and age matched controls in the presence or absence of varied concentrations of DEP as described in the methods. A control well of MDMs was incubated overnight in 1 µM staurosporine (ST). MDMs were visualised by microscopy at the stated time points. A total of 4 randomly selected 40 magnification fields were visualised and cell counts were performed and denoted as total cell count. Data shown are mean±SEM of n = 4, performed on different donors with significant differences denoted by *p<0.05, **p<0.01 and ***p<0.001 compared to MDMs differentiated in the absence of DEP, as measured by one way ANOVA and Dunnett’s post test. To facilitate comparison the percentage cell loss was calculated compared to MDMs differentiated in the absence of DEP.</p

MDMs differentiated in the presence of DEP showed loss of mitochondrial membrane potential (ΔΨm).

<p>MDMs were differentiated from purified monocytes in the presence or absence of varied concentrations of DEP as described in the methods. At day 14 MDMs and DEP-MDMs from healthy volunteers were washed three times in PBS. Cells were stained with 10 µM JC-1 in serum free RPMI 1640 for 30 minutes at 37°C. The cells were harvested and resuspended in ice cold PBS for analysis by flow cytometry. Loss of Δψ_m was detected using a FACSCalibur flow cytometer and was indicated by a decrease in red fluorescence (FL-2). Viable MDMs were identified based on size and granularity on the forward and side scatter plots and ten thousand events were recorded. Data were presented as percentage of cells showing loss of red staining compared to control cells (A) and geomean fluorescence (B) and data analysis was performed using FlowJo software. Figure C shows a representative histogram flow analysis plot from one donor. Data shown are mean±SEM of n = 4–5, performed on different donors with significant differences denoted by *p<0.05, **p<0.01 and ***p<0.001, compared to MDMs differentiated in the absence of DEP, as measured by one way ANOVA and Dunnett’s post test.</p