Threshold Rigidity Values for the Asbestos-like Pathogenicity of High-Aspect-Ratio Carbon Nanotubes in a Mouse Pleural Inflammation Model.

The qualitative and quantitative evaluation of the physicochemical parameters associated with the pathogenicity of high-aspect-ratio nanomaterials is important for comprehensive regulation efforts and safety-by-design approaches. Here, we report quantitative data on the correlations between the rigidity of these nanomaterials and toxicity endpoints in vitro and in vivo. As measured by new ISO standards published in 2017, rigidity shows a strong positive correlation with inflammogenic potential, as indicated by inflammatory cell counts and IL-1ß (a biomarker for frustrated phagocytosis) levels in both the acute and chronic phases. In vitro experiments using differentiated THP-1 cells find that only highly rigid multiwalled carbon nanotubes (MWCNTs) and asbestos fibers lead to piercing and frustrated phagocytosis. Thus, this study suggests a bending ratio of 0.97 and a static bending persistence length of 1.08 as threshold rigidity values for asbestos-like pathogenicity. However, additional research using MWCNTs with rigidity values that lie between those of non-inflammogenic ( Db = 0.66 and SBPL = 0.87) and inflammogenic fibers ( Db = 0.97 and SBPL = 1.09) is required to identify more accurate threshold values, which would be useful for comprehensive regulation and safety-by-design approaches based on MWCNTs.

High inflammogenic potential of rare earth oxide nanoparticles: the New Hazardous Entity.

Due to the exponential increase in the development and utilization of rare earth oxide nanoparticles (REO NPs) in various fields, the possibility of exposure in humans by inhalation has increased. However, there are little information about hazards of REO NPs and its mechanisms of toxicity. In this study, we evaluated the acute pulmonary inflammation using 10 REO NPs (Dy2O3, Er2O3, Eu2O3, Gd2O3 La2O3, Nd2O3, Pr6O11, Sm2O3, Tb4O7, and Y2O3) and four well-known toxic particles (CuO, NiO, ZnO, and DQ12). Minimum three doses per NP were instilled into the lungs of female Wistar rats at surface area dose metric and lung inflammation was evaluated at 24 h post-instillation by bronchoalveolar lavage fluid (BALF) analysis and histopathological observation. All types of REO NPs showed common pathological changes including mild to moderate infiltration of neutrophils and activated macrophages in the alveoli, peribronchial, and perivascular region. The inflammogenic potential evaluated by the number of granulocytes divided by the treated surface area dose showed all types of REO NPs has much higher inflammogenic potential than DQ12, ZnO, and NiO NPs. The correlation plot between the number of granulocytes and the potential for reactive oxygen species (ROS) generation showed a good correlation with exception of Pr6O11. The higher inflammogenic potential of REO NPs than that of well-known highly toxic particles imply that REO NPs need special attention for inhalation exposure and more studies are needed. In addition, the potential of ROS generation is one of the key factors producing lung inflammation by REO NPs.

Differential Contribution of Constituent Metal Ions to the Cytotoxic Effects of Fast-Dissolving Metal-Oxide Nanoparticles.

The main mechanism of toxicity for fast-dissolving nanoparticles (NPs) is relatively simple as it originates from the intrinsic toxicity of their constituent elements rather than complicated surface reactivity. However, there is little information about the compared toxicity of fast-dissolving NP and its constituent ion, which is essential for understanding the mechanism of NP toxicity and the development of a structure-toxicity relationship (STR) model. Herein, we selected three types of fast-dissolving metal-oxide NPs (CoO, CuO, and ZnO) and constituent metal chlorides (CoCl2, CuCl2, and ZnCl2) to compare dose-response curves between NP and its constituent metal. These materials were treated relevant cell lines for inhalation setting (i.e., differentiated THP-1 cells for macrophages and A549 cells for alveolar epithelial cells) and cytotoxicity as an endpoint was evaluated at 24 h post-incubation. The results showed that CoO and CuO NPs in both cell types showed similar patterns of dose-response curves and cytotoxic potential compared to that of their respective metal chloride. On the other hand, ZnO NPs in both cell types showed a completely different dose-response curve compared to that of ZnCl2: ZnO NPs showed modest slope and much less potential for cytotoxicity compared to that of ZnCl2. These results imply that fast-dissolving metal-oxide NPs are not always have similar dose-response curves and toxic potentials compared to their constituent metal chlorides and this may be due to the differential mechanism of intracellular uptake of these substances and their interaction with intracellular detoxification molecules. Further investigations are needed for the use of toxic potential of metal ions as a predicting factors of fast-dissolving NPs toxicity. In addition, chelating agent specific for dissolved metal ions can be applied for the treatment of these fast-dissolving NPs.

Surface functionalization-specific binding of coagulation factors by zinc oxide nanoparticles delays coagulation time and reduces thrombin generation potential in vitro.

Zinc oxide nanoparticles (ZnO NPs) have many biomedical applications such as chemotherapy agents, vaccine adjuvants, and biosensors but its hemocompatibility is still poorly understood, especially in the event of direct contact of NPs with blood components. Here, we investigated the impact of size and surface functional groups on the platelet homeostasis. ZnO NPs were synthesized in two different sizes (20 and 100 nm) and with three different functional surface groups (pristine, citrate, and L-serine). ZnO NPs were incubated with plasma collected from healthy rats to evaluate the coagulation time, kinetics of thrombin generation, and profile of levels of coagulation factors in the supernatant and coronated onto the ZnO NPs. Measurements of plasma coagulation time showed that all types of ZnO NPs prolonged both active partial thromboplastin time and prothrombin time in a dose-dependent manner but there was no size- or surface functionalization-specific pattern. The kinetics data of thrombin generation showed that ZnO NPs reduced the thrombin generation potential with functionalization-specificity in the order of pristine > citrate > L-serine but there was no size-specificity. The profile of levels of coagulation factors in the supernatant and coronated onto the ZnO NPs after incubation of platelet-poor plasma with ZnO NPs showed that ZnO NPs reduced the levels of coagulation factors in the supernatant with functionalization-specificity. Interestingly, the pattern of coagulation factors in the supernatant was consistent with the levels of coagulation factors adsorbed onto the NPs, which might imply that ZnO NPs simply adsorb coagulation factors rather than stimulating these factors. The reduced levels of coagulation factors in the supernatant were consistent with the delayed coagulation time and reduced potential for thrombin generation, which imply that the adsorbed coagulation factors are not functional.

The role of surface functionalization on the pulmonary inflammogenicity and translocation into mediastinal lymph nodes of graphene nanoplatelets in rats.

Graphene, a two-dimensional monocrystalline layer of carbon atoms, has potential in many applications not only in material sciences, but also in the biomedical fields, but there is little information about the role of surface modification on the toxicity of graphene-based nanomaterials. Here, we evaluated the role of surface functionalization of the graphene nanoplatelets (GNPs) on the pulmonary inflammogenicity and translocation into mediastinal lymph nodes using a rat intratracheal instillation model. Six types of GNPs were used: All types of GNPs were based on the pristine GNPs (GNPdot), and different functional groups were conjugated onto them including a COOH (GNPCOOH), COH [Formula: see text], N-H [Formula: see text], F x (GNPF), and N=H [Formula: see text]. All types of GNPs showed very high potential for the generation of reactive oxygen species (ROS) in a dose-dependent manner when measured by a 2'7'-dichlorofluorescin diacetate assay. GNPs were instilled into the lungs of rats at 0.3 and 1 mg/rat for the evaluation of acute (24 h) inflammation and at 3 mg/rat for chronic (1 and 4 weeks) inflammation. At 24 h after instillation, all types of GNPs showed good dose-dependent increases in polymorphonuclear leukocytes with a clear dose-dependency although significant increases compared to vehicle control were found only in positively charged GNPs [Formula: see text]. While the acute inflammation in all treatment groups was returned to control levels at 1 and 4 weeks after instillation, GNPs showed similar patterns of translocation into the mediastinal lymph nodes with a higher degree over time. This study implies that the main factors of GNPs for producing lung inflammation are the potential for ROS generation and surface charge. In addition, functional groups on the GNPs might not play an important role in the extrapulmonary translocation into the mediastinal lymph nodes.

Evaluation of the dose metric for acute lung inflammogenicity of fast-dissolving metal oxide nanoparticles.

Although surface area metric was suggested as an appropriate dose metric for acute lung inflammation of NPs, it might not be effective for fast-dissolving NPs because they lose their reactive surface when dissolved in the phagolysosomes. Herein, we evaluated the dose metric for fast-dissolving NPs using a rat intratracheal instillation model. A panel of fast-dissolving NPs (CoO, CuO and ZnO) and their constituent metal ions (CoCl2, CuCl2 and ZnCl2) were compiled and each compound was intratracheally instilled into the lungs of female Wistar rats at the same molar concentrations in the NP doses (40, 100 and 400 µg/rat). The toxicity endpoints including cytological and biochemical data in bronchoalveolar lavage fluid were evaluated at 24 h after instillation. To evaluate the dose metric, each toxicity endpoint was plotted against the instilled dose (mass or surface area) or the equivalent dose (mass or surface area) that was weighted by the ratio of specific dose-generated responses between metal chlorides. Dose-response curves of fast-dissolving NPs about percentage of granulocytes, lactate dehydrogenase levels and total protein levels showed similar pattern but slightly less potential than those of their respective metal chlorides. When each toxicity endpoint was plotted against the equivalent mass dose, three types of NPs showed more overlapping dose-response curves than other dose metrics. In conclusion, this study implies that the equivalent mass dose is an appropriate dose metric for fast-dissolving NPs and the main factor determining the slope of the dose-response curve is the intrinsic toxicity of the their constituent ions.

Dual contribution of surface charge and protein-binding affinity to the cytotoxicity of polystyrene nanoparticles in nonphagocytic A549 cells and phagocytic THP-1 cells.

Knowledge that links the physicochemical properties of nanoparticles (NP) to their toxicity is key to evaluating and understanding mechanisms underlying toxicity and developing appropriate testing methods for NP; however, this is currently limited since only a small set of NP have been used, with typically poor control of their physical properties. In this study, eight types of polystyrene NP (PLNP) were synthesized with different functional groups, but all based on an identical core. In vitro cell-based assays were performed to determine the influence of changes in physicochemical properties, such as charge, hydrodynamic size, and protein binding potential, in relation to NP-mediated toxicity. The PLNP were incubated with nonphagocytic A549 cells or phagocytic differentiated THP-1 cells for 4 h with/without fetal bovine serum (FBS), followed by incubation for 20 h in FBS-supplemented medium with/without a washing step, to assess cell-type specificity and impact of protein corona formation. The effect of surface charge on cytotoxicity differed between A549 cells and THP-1 cells. In nonphagocytic A549 cells, the zeta potential of PLNP exhibited a negative correlation with cytotoxicity, partly due to the level of coronated protein that might affect cellular uptake. In phagocytic THP-1 cells, the zeta potential of PLNP showed a positive correlation with cytotoxicity but coronated protein levels displayed no marked association with cytotoxicity, owing to the professional uptake efficacy of phagocytic cells. The consistency of our data with THP-1 cells with the surface charge paradigm in nanotoxicology suggests that phagocytic cells are the predominant targets for lung inflammatory reactions induced by PLNP.

Nickel oxide nanoparticles can recruit eosinophils in the lungs of rats by the direct release of intracellular eotaxin.

BACKGROUND: Instillation of highly soluble nanoparticles (NPs) into the lungs of rodents can cause acute eosinophilia without any previous sensitizations by the role of dissolved ions. However, whether gradually dissolving NPs can cause the same type of eosinophilia remains to be elucidated. We selected nickel oxide (NiO) as a gradually dissolving NP and evaluated the time course pulmonary inflammation pattern as well as its mechanisms. METHODS: NiO NPs were intratracheally instilled into female Wistar rats at various concentrations (50, 100, and 200 cm(2)/rat) and the lung inflammation was evaluated at various time-points (1, 2, 3, and 4 days). As positive controls, NiCl2 and the ovalbumin-induced allergic airway inflammation model was applied. NiCl2 was instilled at 171.1 µg Ni/rat, which is equivalent nickel concentration of 200 cm(2)/rat of NiO NPs. Cytological analysis and biochemical analysis including lactate dehydrogenase (LDH), total protein, and pro-inflammatory cytokines were measured in bronchoalveolar lavage fluid (BALF). The levels of total immunoglobulin E (IgE) and anaphylatoxins (C3a and C5a) were measured in BALF and serum. The levels of eotaxin were measured in the alveolar macrophages and normal lung tissue before and after addition of cell lysis buffer to evaluate whether the direct lysis of cells can release intracellular eotaxin. RESULTS: NiO NPs produced acute neutrophilic inflammation throughout the study. However, eosinophils were recruited at 3 and 4 days post-instillation of NiO NPs and the magnitude and pattern of inflammation was similar with NiCl2 at 24 h post-instillation. The eosinophil recruitment by NiO NPs was not related with either the levels of total IgE or anaphylatoxins. The lysis of alveolar macrophages and normal lung tissue showed high levels of intracellular eotaxin and the levels of LDH showed positive correlation with the levels of eotaxin. CONCLUSIONS: Instillation of NiO NPs produced neutrophilia at 1 and 2 days after instillation, while the mixed type of neutrophilic and eosinophilic inflammation was produced at 3 and 4 days post-instillation, which was consistent with NiCl2. The mechanism of the eosinophilia involves the direct release of intracellular eotaxin due to the rupture of cells by the accumulated solubilized nickel ions in the phagolysosome.

Response-metrics for acute lung inflammation pattern by cobalt-based nanoparticles.

BACKGROUND: Although the surface area metric has been proposed as a possible dose-metric for nanoparticles (NPs), it is limited to low-solubility NPs and the dose-metric for high-solubility NPs is poorly understood. In this study, we aimed to assess the appropriate dose-metric or response-metric for NPs using two cobalt (Co)-based NPs, cobalt monoxide (CoO) and cobalt oxide (Co3O4), which both show distinctive solubility, and determine the role of their soluble Co ions in inflammation. METHODS: We evaluated the physicochemical properties of NPs, including solubility in artificial lysosomal fluid (ALF, pH 5.5). Acute lung inflammogenicity was evaluated by bronchoalveolar lavage fluid analysis using the rat intratracheal instillation model. The appropriate response-metric was then determined by plotting several dose-metrics against parameters for lung inflammation. To investigate the effect of the soluble fraction of CoO NPs, the equivalent doses of Co ions from CoCl2 were instilled. RESULTS: The Co3O4 and CoO NPs showed about 11.46% and 92.65% solubility in ALF, respectively. Instillation of Co3O4 NPs produced neutrophilic inflammation, but CoO NPs induced eosinophilic inflammation. The number of eosinophils showed good correlation with the soluble Co ions dose from NPs (r2=0.987, p<0.001), while the number of neutrophils showed good correlation with the surface area dose of the biopersistent NPs (r2=0.876, p<0.001). Instillation of CoCl2 showed a similar type and magnitude of inflammation as CoO NPs. CONCLUSIONS: In the Co-based NPs, the eosinophilic inflammation was produced by Co ions based on the ion metric, while the neutrophilic inflammation was developed based on the surface area metric of the biopersistent NPs.