Exploration of potential mechanism of interleukin-33 up-regulation caused by 1,4-naphthoquinone black carbon in RAW264.7 cells.

BACKGROUND: As air pollution has been paid more attention to by public in recent years, effects and mechanism in particulate matter-triggered health problems become a focus of research. Lysosomes and mitochondria play an important role in regulation of inflammation. Interleukin-33 (IL-33) has been proved to promote inflammation in our previous studies. In this research, macrophage cell line RAW264.7 was used to explore the potential mechanism of upregulation of IL-33 induced by 1,4-naphthoquinone black carbon (1,4-NQ-BC), and to explore changes of lysosomes and mitochondria during the process. RESULTS: 50 µg/mL 1,4-NQ-BC exposure for 24 h dramatically increased expression of IL-33 in RAW264.7 cells. Lysosomal membrane permeability was damaged by 1,4-NQ-BC treatment, and higher mitochondrial membrane potential and ROS level were induced by 1,4-NQ-BC. The results of proteomics suggested that expression of ferritin light chain was increased after cells were challenged with 1,4-NQ-BC, and it was verified by Western blot. Meanwhile, expressions of p62 and LC3B-II were increased by 50 µg/mL 1,4-NQ-BC in RAW264.7 cells. Ultimately, expression of IL-33 could return to same level as control in cells treated with 50 µg/mL 1,4-NQ-BC and 50 µM deferoxamine combined. CONCLUSIONS: 1,4-NQ-BC induces IL-33 upregulation in RAW264.7 cells, and it is responsible for higher lysosomal membrane permeability and ROS level, lower mitochondrial membrane potential, and inhibition of autophagy. Ferritin light chain possibly plays an important role in the upregulation of IL-33 evoked by 1,4-NQ-BC.

Phototoxic versus photoprotective effects of tattoo pigments in reconstructed human skin models: In vitro phototoxicity testing of tattoo pigments: 3D versus 2D.

The increasing number of tattooed persons urges the development of reliable test systems to assess tattoo associated risks. The alarming prevalence of 60 % phototoxic reactions in tattoos ask for a more comprehensive investigation of phototoxic reactions in tattooed skin. Here, we aimed to compare the cellular responses of human skin cells to ultraviolet (UV)A and UVB irradiation in doses of short to intermitted sun exposure (3-48 J/cm² and 0.05-5 J/cm², respectively) in the presence of tattoo pigments. Therefore, we used fibroblast monolayer culture (2D), our recently developed three dimensional full-thickness skin model with dermal-located tattoo pigments (TatSFT) and its dermal equivalents (TatSDE) that lack keratinocytes. We tested the most frequently used tattoo pigments carbon black, titanium dioxide (TiO2) anatase and rutile as well as Pigment Orange (P.O.)13 in ranges from 0.067 to 2.7 ng/cell in 2D. For TatSDE and TatSFT, concentrations were 1.3 ng/cell for TiO2, 0.67 ng/cell for P.O.13 and 0.067 ng/cell for carbon black. We assessed cell viability and cytokine release in all systems, and cyclobutane pyrimidine dimer (CPD) formation in TatSFT. Phototoxicity of tattoo pigments was exclusively observed in 2D, where especially TiO2 anatase induced phototoxic effects in all concentrations (0.067-2.7 ng/cell). In contrast, fibroblasts were protected from UV irradiation in TatSDE by TiO2 and carbon black. Neither toxic nor protective effects were recorded in TatSFT. P.O.13 showed altered cytokine secretion in 2D (0.067-1.3 ng/cell) and TatSDE, despite the absence of significant effects on viability in all systems. All pigments reduced the number of CPDs in TatSFT compared to the pigment-free controls. In conclusion, our study shows that within a 3D arrangement, intradermal tattoo pigments may act photoprotective despite intrinsic phototoxic properties in 2D. Thus, dermal 3D equivalents should be considered to evaluate acute tattoo pigment toxicology.

Optimization of intracellular macromolecule delivery by nanoparticle-mediated photoporation.

Nanoparticle-mediated photoporation is a novel delivery platform for intracellular molecule delivery. We studied the dependence of macromolecular delivery on molecular weight and sought to enhance delivery efficiency. DU145 prostate cancer cells were exposed to pulsed laser beam in the presence of carbon-black nanoparticles. Intracellular uptake of molecules decreased with increasing molecular weight. Attributing this dependence to molecular diffusivity, we hypothesized that macromolecular delivery efficiency could be enhanced by increasing either laser fluence or laser exposure duration at low fluence. We observed increased percentages of macromolecule uptake by cells in both cases. However, trade-off between cell uptake and viability loss was most favorable at low laser fluence (25-29 mJ/cm2) and longer exposure durations (4-5 min). We conclude that long exposure at low laser fluence optimizes intracellular macromolecule delivery by nanoparticle-mediated photoporation, which may be explained by longer time for macromolecules to diffuse into cells, during and between laser pulses.

Apoptosis and Oxidative Stress Triggered by Carbon Black Nanoparticle in the LA-9 Fibroblast.

BACKGROUND/AIMS: A new type of nanoparticle, called NP CB-EDA (Black Carbon modified with ethylenediamine), is commonly used in the oil industry. In the literature, few studies are found in biological models, making NP-EDA potential cytotoxicity in organisms unclear. As its large surface area is capable of interacting with the biological system, that interaction could lead to factors harmful to health. The objective of this study was to investigate the cytotoxic effect of NP CB-EDA on fibroblasts LA-9 at 24 and 48 hours, at different concentrations of the nanoparticle (1, 50, 250, 500 and 1000 µg/ml). METHODS: NP CB-EDA was characterized by TEM microscopy and its effect on cell viability (MTT method), cell morphology (optical microscopy), cell membrane (lactate dehydrogenase release - LDH), oxidative stress pathways (species levels reactive oxygen, ROS and nitrogen, NOS) and apoptosis/necrosis (flow cytometry) were evaluated. RESULTS: The results show that NP CB-EDA at concentrations of 500 and 1000 µg/ml form clusters. The nanoparticle can be absorbed by cells decreasing cell viability. There was damage to the cell membrane of fibroblasts LA 9, an increase in the production of ROS, NOS and pro-inflammatory interleukins TNF-α and IL-6; it was also observed an increase in % of cells in the state of apoptosis in the two periods analyzed, being this response more significant in 24 hours, and concentrations of 250, 500 and 1000 µg/ml presenting higher cytotoxicity. CONCLUSION: The data suggest that NP CB-EDA in fibroblasts LA9 presents cytotoxic potential, which is associated with oxidative stress and apoptosis.

When condensed matter physics meets biology: Does superhydrophobicity benefiting the cryopreservation of human spermatozoa?

With the increasing demand in regenerative and reproductive medicine for successful conservation of living matter, the need of reliable platform in cell banking seems inevitable. Whilst the cells storage at cryogenic temperatures is a well-developed method, far less is known about the efficiency of nanotechnology in cryogenics. The primary objective of this study is to represent the first of its kind experimental results related to cryopreservation of human spermatozoa by means of superhydrophobic carbon soot coatings. The inclusion of soot-based water repellent interface during the freezing and thawing of human semen minimizes the solid-liquid interfacial area, retards the heat transfer rate and promotes the recovery of up to 80% of initial motility of post-thaw sperm cells. Our discoveries reveal a fundamentally new and exciting direction of development of cryopreservation technologies in the battle against painful biopsies and repetitive surgeries.

Real time and in vivo pharmaceutical and environmental studies with SpiderMass instrument.

Remote Infrared Matrix Assisted Laser Desorption Ionization (Remote IR MALDI) system (SpiderMass) with endogenous water as matrix allows to perform real-time DMPK in vivo. In this work, SpiderMass was used to analyze the impact on metabolite production or release of invalidated pro-protein PC1/3 macrophages by Short RNA (shRNA) versus scramble shRNA with Paclitaxel. Time course in vivo experiments were then performed on the inner and outer faces of patients' forearms or comedo treated with Melascreen (Ducray) containing ascorbyl glucoside. Finally, the impact of car pollution (emitted soot) on skin was also investigated. Taken together, we demonstrate that the SpiderMass instrument opens the door to clinical, pharmaceutical and environmental domains for real-time, in vivo pharmacokinetic (Drug Metabolism and PharmacoKinetics, DMPK) analysis.

Effect of single-walled carbon nanotubes on cytochrome P450 activity in human liver microsomes in vitro.

Single-walled carbon nanotubes (SWCNTs) are made from a rolled single sheet of graphene with a diameter in the nanometer range. SWCNTs are potential carriers for drug delivery systems because antibodies or drugs can be loaded on their surface; however, their effect on the activities of cytochrome P450 (CYP) remains unclear. The aim of this study was to investigate the effect of two kinds of SWCNTs with different lengths (FH-P- and SO-SWCNTs) on human CYP activity. In addition, other nano-sized carbon materials, such as carbon black, fullerene-C60 , and fullerene-C70 were also evaluated to compare their effects on CYP activities. Ten CYP substrates (phenacetin, coumarin, bupropion, paclitaxel, tolbutamide, S-mephenytoin, dextromethorphan, chlorzoxazone, midazolam, and testosterone) were used. Testosterone 6ß-hydroxylation and midazolam 1'-hydroxylation, which are catalysed by both CYP3A4 and CYP3A5 in liver microsomes, were decreased by 25% and 45%, respectively, in the presence of 0.1 mg/ml SO-SWCNT. Dextromethorphan O-demethylation, which is catalysed mainly by CYP2D6, was decreased by 40% in the presence of SO-SWCNT. Other CYP activities, however, were not attenuated by SO-SWCNT. FH-P-SWCNT, carbon black, fullerene-C60 , and fullerene-C70 at 0.1 mg/ml had no effect on CYP activities. The Ki values for testosterone 6ß-hydroxylation, midazolam 1'-hydroxylation, and dextromethorphan O-demethylation in liver microsomes were 136, 34, and 56 µg/ml, respectively. SO-SWCNT was determined to be a competitive inhibitor of CYP3A4, CYP3A5, and CYP2D6. These results suggest that the effect of SO-SWCNT differs among CYP isoforms, and that the inhibition potency depends on the physicochemical properties of the nanocarbons.

Photoporation Using Carbon Nanotubes for Intracellular Delivery of Molecules and Its Relationship to Photoacoustic Pressure.

Exposure of carbon-black (CB) nanoparticles to near-infrared nanosecond-pulsed laser energy can cause efficient intracellular delivery of molecules by photoporation. Here, cellular bioeffects of multi-walled carbon nanotubes (MWCNTs) and single-walled carbon nanotubes (SWCNTs) are compared to those of CB nanoparticles. In DU145 prostate-cancer cells, photoporation using CB nanoparticles transitions from (i) cells with molecular uptake to (ii) nonviable cells to (iii) fragmented cells with increasing laser fluence, as seen previously. In contrast, photoporation with MWCNTs causes uptake and, at higher fluence, fragmentation, but does not generate nonviable cells, and SWCNTs show little evidence of bioeffects, except at extreme laser conditions, which generate nonviable cells and fragmentation, but no significant uptake. These different behaviors cannot be explained by photoacoustic pressure output from the particles. All particle types emit a single, ≈100 ns, mostly positive-pressure pulse that increases in amplitude with laser fluence. Different particle types emit different peak pressures, which are highest for SWCNTs, followed by CB nanoparticles and then MWCNTs, which does not correlate with cellular bioeffects between different particle types. This study concludes that cellular bioeffects depend strongly on the type of carbon nanoparticle used during photoporation and that photoacoustic pressure is unlikely to play a direct mechanistic role in the observed bioeffects.

Carbon Black Nanoparticles Inhibit Aromatase Expression and Estradiol Secretion in Human Granulosa Cells Through the ERK1/2 Pathway.

Secretion of 17-ß-estradiol (E2) by human granulosa cells can be disrupted by various environmental toxicants. In the current study, we investigated whether carbon black nanoparticles (CB NPs) affect the steroidogenic activity of cultured human granulosa cells. The human granulosa cell line KGN and granulosa cells from patients undergoing in vitro fertilization were treated with increasing concentrations of CB NPs (1 to 100 µg/mL) together or not with follicle-stimulating hormone (FSH). We observed that CB NPs are internalized in KGN cells without affecting cell viability. CB NPs could be localized in the cytoplasm, within mitochondria and in association with the outer face of the endoplasmic reticulum membrane. In both cell types, CB NPs reduced in a dose-dependent manner the activity of aromatase enzyme, as reflected by a decrease in E2 secretion. A significant decrease was observed in response to CB NPs concentrations from 25 and 50 µg/mL in KGN cell line and primary cultures, respectively. Furthermore, CB NPs decreased aromatase protein levels in both cells and reduced aromatase transcript levels in KGN cells. CB NPs rapidly activated extracellular signal-regulated kinase 1 and 2 in KGN cells and pharmacological inhibition of this signaling pathway using PD 98059 significantly attenuated the inhibitory effects of CB NPs on CYP19A1 gene expression and aromatase activity. CB NPs also inhibited the stimulatory effect of FSH on aromatase expression and activity. Altogether, our study on cultured ovarian granulosa cells reveals that CB NPs decrease estrogens production and highlights possible detrimental effect of these common NPs on female reproductive health.

Air pollution alters Staphylococcus aureus and Streptococcus pneumoniae biofilms, antibiotic tolerance and colonisation.

Air pollution is the world's largest single environmental health risk (WHO). Particulate matter such as black carbon is one of the main components of air pollution. The effects of particulate matter on human health are well established however the effects on bacteria, organisms central to ecosystems in humans and in the natural environment, are poorly understood. We report here for the first time that black carbon drastically changes the development of bacterial biofilms, key aspects of bacterial colonisation and survival. Our data show that exposure to black carbon induces structural, compositional and functional changes in the biofilms of both S. pneumoniae and S. aureus. Importantly, the tolerance of the biofilms to multiple antibiotics and proteolytic degradation is significantly affected. Additionally, our results show that black carbon impacts bacterial colonisation in vivo. In a mouse nasopharyngeal colonisation model, black carbon caused S. pneumoniae to spread from the nasopharynx to the lungs, which is essential for subsequent infection. Therefore our study highlights that air pollution has a significant effect on bacteria that has been largely overlooked. Consequently these findings have important implications concerning the impact of air pollution on human health and bacterial ecosystems worldwide.

Revealing the role of oxidation state in interaction between nitro/amino-derived particulate matter and blood proteins.

Surface oxidation states of ultrafine particulate matter can influence the proinflammatory responses and reactive oxygen species levels in tissue. Surface active species of vehicle-emission soot can serve as electron transfer-mediators in mitochondrion. Revealing the role of surface oxidation state in particles-proteins interaction will promote the understanding on metabolism and toxicity. Here, the surface oxidation state was modeled by nitro/amino ligands on nanoparticles, the interaction with blood proteins were evaluated by capillary electrophoresis quantitatively. The nitro shown larger affinity than amino. On the other hand, the affinity to hemoglobin is 10(3) times larger than that to BSA. Further, molecular docking indicated the difference of binding intensity were mainly determined by hydrophobic forces and hydrogen bonds. These will deepen the quantitative understanding of protein-nanoparticles interaction from the perspective of surface chemical state.

Cancer Therapeutic Effects of Titanium Dioxide Nanoparticles Are Associated with Oxidative Stress and Cytokine Induction.

Nanoparticles (NPs) are considered to influence the inflammatory process; however, the precise mechanism and the significance in tumors are still not clear. In this study, when CT26 and LL2 mouse cancer cells were treated with 6-nm anatase titanium dioxide NPs (TDNPs) without ultraviolet irradiation, oxidative stress and induction of inflammatory cytokines were observed. Oxidative stress was further increased by disease-associated conditions such as high glucose concentrations and hypoxia. Inhaled or orally administered TDNPs generated granulomatous lesions in the lungs and colon of the rodent models tested, with increased oxidative stress and inflammatory cytokines. Oxidative stress and inflammatory cytokines were also found in cancer cells treated with gold or carbon black NPs. Treatment of CT26 cells with 10- to 70-nm rutile TDNPs showed that smaller NPs produced more oxidative stress and inflammatory cytokines than larger ones did. To avoid diffusion of TDNPs and to minimize toxicity, 10-nm TDNPs were suspended in a collagen gel inserted into a subcutaneous tumor in a CT26 mouse. A single TDNP treatment via this method inhibited tumor growth in a size- and dose-dependent manner, and resulted in lower levels of urinary 8-OHdG when compared to systemically administered TDNPs. These findings suggest that TDNPs might be useful for the local treatment of tumors.

Effect of carbon black nanomaterial on biological membranes revealed by shape of human erythrocytes, platelets and phospholipid vesicles.

BACKGROUND: We studied the effect of carbon black (CB) agglomerated nanomaterial on biological membranes as revealed by shapes of human erythrocytes, platelets and giant phospholipid vesicles. Diluted human blood was incubated with CB nanomaterial and observed by different microscopic techniques. Giant unilamellar phospholipid vesicles (GUVs) created by electroformation were incubated with CB nanomaterial and observed by optical microscopy. Populations of erythrocytes and GUVs were analyzed: the effect of CB nanomaterial was assessed by the average number and distribution of erythrocyte shape types (discocytes, echinocytes, stomatocytes) and of vesicles in test suspensions, with respect to control suspensions. Ensembles of representative images were created and analyzed using computer aided image processing and statistical methods. In a population study, blood of 14 healthy human donors was incubated with CB nanomaterial. Blood cell parameters (concentration of different cell types, their volumes and distributions) were assessed. RESULTS: We found that CB nanomaterial formed micrometer-sized agglomerates in citrated and phosphate buffered saline, in diluted blood and in blood plasma. These agglomerates interacted with erythrocyte membranes but did not affect erythrocyte shape locally or globally. CB nanomaterial agglomerates were found to mediate attractive interaction between blood cells and to present seeds for formation of agglomerate - blood cells complexes. Distortion of disc shape of resting platelets due to incubation with CB nanomaterial was not observed. CB nanomaterial induced bursting of GUVs while the shape of the remaining vesicles was on the average more elongated than in control suspension, indicating indirect osmotic effects of CB nanomaterial. CONCLUSIONS: CB nanomaterial interacts with membranes of blood cells but does not have a direct effect on local or global membrane shape in physiological in vitro conditions. Blood cells and GUVs are convenient and ethically acceptable methods for the study of effects of various substances on biological membranes and therefrom derived effects on organisms.

Poloxamer surfactant preserves cell viability during photoacoustic delivery of molecules into cells.

Efficient intracellular delivery of molecules is needed to modulate cellular behavior for laboratory and medical applications, but is often limited by trade-offs between achieving high intracellular delivery and maintaining high cell viability. Here, we studied photoacoustic delivery of molecules into cells by exposing DU145 human prostate carcinoma cells to nanosecond laser pulses in the presence of carbon black nanoparticles. Under strong laser exposure conditions, less than 30% of cells were viable and exhibited uptake. Addition of poloxamer surfactant at those laser exposure conditions increased cell viability to almost 90%, with intracellular uptake in >80% of cells. This remarkable increase in efficiency of intracellular delivery and cell viability may be attributed to enhanced cell membrane resealing by poloxamer surfactant after photoacoustic delivery. While F-68 poloxamer was effective, the larger, more-hydrophobic F-127 poloxamer provided the best results. There was no significant protective effect from addition of Ca(2+) , BAPTA-AM, ATP, fetal bovine serum or glycine betaine, which were expected to promote active cell membrane repair mechanisms and other active intracellular protective processes. We conclude that poloxamer surfactant preserves cell viability during photoacoustic delivery of molecules into cells, thereby enabling highly efficient intracellular delivery.

DNA damage following pulmonary exposure by instillation to low doses of carbon black (Printex 90) nanoparticles in mice.

We previously observed genotoxic effects of carbon black nanoparticles at low doses relative to the Danish Occupational Exposure Limit (3.5 mg/m(3)). Furthermore, DNA damage occurred in broncho-alveolar lavage (BAL) cells in the absence of inflammation, indicating that inflammation is not required for the genotoxic effects of carbon black. In this study, we investigated inflammatory and acute phase response in addition to genotoxic effects occurring following exposure to nanoparticulate carbon black (NPCB) at even lower doses. C57BL/6JBomTac mice were examined 1, 3, and 28 days after a single instillation of 0.67, 2, 6, and 162 µg Printex 90 NPCB and vehicle. Cellular composition and protein concentration was evaluated in BAL fluid as markers of inflammatory response and cell damage. DNA strand breaks in BAL cells, lung, and liver tissue were assessed using the alkaline comet assay. The pulmonary acute phase response was analyzed by Saa3 mRNA real-time quantitative PCR. Instillation of the low doses of NPCB induced a slight neutrophil influx one day after exposure. Pulmonary exposure to small doses of NPCB caused an increase in DNA strand breaks in BAL cells and lung tissue measured using the comet assay. We interpret the increased DNA strand breaks occurring following these low exposure doses of NPCB as DNA damage caused by primary genotoxicity in the absence of substantial inflammation, cell damage, and acute phase response.

Carbon black nanoparticles promote endothelial activation and lipid accumulation in macrophages independently of intracellular ROS production.

Exposure to nanoparticles (NPs) may cause vascular effects including endothelial dysfunction and foam cell formation, with oxidative stress and inflammation as supposed central mechanisms. We investigated oxidative stress, endothelial dysfunction and lipid accumulation caused by nano-sized carbon black (CB) exposure in cultured human umbilical vein endothelial cells (HUVECs), THP-1 (monocytes) and THP-1 derived macrophages (THP-1a). The proliferation of HUVECs or co-cultures of HUVECs and THP-1 cells were unaffected by CB exposure, whereas there was increased cytotoxicity, assessed by the LDH and WST-1 assays, especially in THP-1 and THP-1a cells. The CB exposure decreased the glutathione (GSH) content in THP-1 and THP-1a cells, whereas GSH was increased in HUVECs. The reactive oxygen species (ROS) production was increased in all cell types after CB exposure. A reduction of the intracellular GSH concentration by buthionine sulfoximine (BSO) pre-treatment further increased the CB-induced ROS production in THP-1 cells and HUVECs. The expression of adhesion molecules ICAM-1 and VCAM-1, but not adhesion of THP-1 to HUVECs or culture dishes, was elevated by CB exposure, whereas these effects were unaffected by BSO pre-treatment. qRT-PCR showed increased VCAM1 expression, but no change in GCLM and HMOX1 expression in CB-exposed HUVECs. Pre-exposure to CB induced lipid accumulation in THP-1a cells, which was not affected by the presence of the antioxidant N-acetylcysteine. In addition, the concentrations of CB to induce lipid accumulation were lower than the concentrations to promote intracellular ROS production in THP-1a cells. In conclusion, exposure to nano-sized CB induced endothelial dysfunction and foam cell formation, which was not dependent on intracellular ROS production.

Atomic layer deposition coating of carbon nanotubes with aluminum oxide alters pro-fibrogenic cytokine expression by human mononuclear phagocytes in vitro and reduces lung fibrosis in mice in vivo.

BACKGROUND: Multi-walled carbon nanotubes (MWCNTs) pose a possible human health risk for lung disease as a result of inhalation exposure. Mice exposed to MWCNTs develop pulmonary fibrosis. Lung macrophages engulf MWCNTs and produce pro-fibrogenic cytokines including interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and osteopontin (OPN). Atomic layer deposition (ALD) is a novel process used to enhance functional properties of MWCNTs, yet the consequence of ALD-modified MWCNTs on macrophage biology and fibrosis is unknown. METHODS: The purpose of this study was to determine whether ALD coating with aluminum oxide (Al2O3) would alter the fibrogenic response to MWCNTs and whether cytokine expression in human macrophage/monocytes exposed to MWCNTs in vitro would predict the severity of lung fibrosis in mice. Uncoated (U)-MWCNTs or ALD-coated (A)-MWCNTs were incubated with THP-1 macrophages or human peripheral blood mononuclear cells (PBMC) and cell supernatants assayed for cytokines by ELISA. C57BL6 mice were exposed to a single dose of A- or U-MWCNTs by oropharyngeal aspiration (4 mg/kg) followed by evaluation of histopathology, lung inflammatory cell counts, and cytokine levels at day 1 and 28 post-exposure. RESULTS: ALD coating of MWCNTs with Al2O3 enhanced IL-1ß secretion by THP-1 and PBMC in vitro, yet reduced protein levels of IL-6, TNF-α, and OPN production by THP-1 cells. Moreover, Al2O3 nanoparticles, but not carbon black NPs, increased IL-1ß but decreased OPN and IL-6 in THP-1 and PBMC. Mice exposed to U-MWCNT had increased levels of all four cytokines assayed and developed pulmonary fibrosis by 28 days, whereas ALD-coating significantly reduced fibrosis and cytokine levels at the mRNA or protein level. CONCLUSION: These findings indicate that ALD thin film coating of MWCNTs with Al2O3 reduces fibrosis in mice and that in vitro phagocyte expression of IL-6, TNF-α, and OPN, but not IL-1ß, predict MWCNT-induced fibrosis in the lungs of mice in vivo.

Proinflammatory effects of diesel exhaust nanoparticles on scleroderma skin cells.

Autoimmune diseases are complex disorders of unknown etiology thought to result from interactions between genetic and environmental factors. We aimed to verify whether environmental pollution from diesel engine exhaust nanoparticulate (DEP) of actually operating vehicles could play a role in the development of a rare immune-mediated disease, systemic sclerosis (SSc), in which the pathogenetic role of environment has been highlighted. The effects of carbon-based nanoparticulate collected at the exhaust of newer (Euro 5) and older (Euro 4) diesel engines on SSc skin keratinocytes and fibroblasts were evaluated in vitro by assessing the mRNA expression of inflammatory cytokines (IL-1 α , IL-6, IL-8, and TNF-α) and fibroblast chemical mediators (metalloproteases 2, 3, 7, 9, and 12; collagen types I and III; VEGF). DEP was shown to stimulate cytokine gene expression at a higher extent in SSc keratinocytes versus normal cells. Moreover, the mRNA gene expression of all MMPs, collagen types, and VEGF genes was significantly higher in untreated SSc fibroblasts versus controls. Euro 5 particle exposure increased the mRNA expression of MMP-2, -7, and -9 in SSc fibroblasts in a dose dependent manner and only at the highest concentration in normal cells. We suggest that environmental DEP could trigger the development of SSc acting on genetically hyperreactive cell systems.

Effects of surface-modified nano-scale carbon black on Cu and Zn fractionations in contaminated soil.

Cu contamination soil (547 mg kg(-1)) was mixed separately with the surface-modified nano-scale carbon black (MCB) and placed in the ratios (w/w) of 0, 1%, 3%, and 5% in pots, together with 0.33 g KH2PO4 and 0.35 g urea/pot. Each pot contained 20 ryegrass seedlings (Lolium multiflorum). Greenhouse cultivation experiments were conducted to examine the effect of the MCB on Cu and Zn fractionations in soil, accumulation in shoot and growth of ryegrass. The results showed that the biomass of ryegrass shoot and root increased with the increasing of MCB adding amount (p < 0.05). The Cu and Zn accumulation in ryegrass shoot and the concentrations of DTPA extractable Cu and Zn in soil were significantly decreased with the increasing of MCB adding amount (p < 0.05). The metal contents of exchangeable and bound to carbonates (EC-Cu or EC-Zn) in the treatments with MCB were generally lower than those without MCB, and decreased with the increasing of MCB adding amount (p < 0.05). There was a positive linear correlation between the Cu and Zn accumulation in ryegrass shoot and the EC-Cu and EC-Zn in soil. The present results indicated the MCB could be applied for the remediation the soils polluted by Cu and Zn.

Efficient intracellular delivery of molecules with high cell viability using nanosecond-pulsed laser-activated carbon nanoparticles.

Conventional physical and chemical methods that efficiently deliver molecules into cells are often associated with low cell viability. In this study, we evaluated the cellular effects of carbon nanoparticles believed to emit photoacoustic waves due to nanosecond-pulse laser activation to test the hypothesis that this method could achieve efficient intracellular delivery while maintaining high cell viability. Suspensions of DU145 human prostate carcinoma cells, carbon black (CB) nanoparticles, and calcein were exposed to 5-9 ns long laser pulses of near-infrared (1064 nm wavelength) light and then analyzed by flow cytometry for intracellular uptake of calcein and cell viability by propidium iodide staining. We found that intracellular uptake increased and in some cases saturated at high levels with only small losses in cell viability as a result of increasing laser fluence, laser exposure time, and as a unifying parameter, the total laser energy. Changing interpulse spacing between 0.1 and 10 s intervals showed no significant change in bioeffects, suggesting that the effects of each pulse were independent when spaced by at least 0.1 s intervals. Pretreatment of CB nanoparticles to intense laser exposure followed by mixing with cells also had no significant effect on uptake or viability. Similar uptake and viability were seen when CB nanoparticles were substituted with India ink, when DU145 cells were substituted with H9c2 rat cardiomyoblast cells, and when calcein was substituted with FITC-dextran. The best laser exposure conditions tested led to 88% of cells with intracellular uptake and close to 100% viability, indicating that nanosecond-pulse laser-activated carbon nanoparticles can achieve efficient intracellular delivery while maintaining high cell viability.