Key Role of Choline Head Groups in Large Unilamellar Phospholipid Vesicles for the Interaction with and Rupture by Silica Nanoparticles.

For highly abundant silica nanomaterials, detrimental effects on proteins and phospholipids are postulated as critical molecular initiating events that involve hydrogen-bonding, hydrophobic, and/or hydrophilic interactions. Here, large unilamellar vesicles with various well-defined phospholipid compositions are used as biomimetic models to recapitulate membranolysis, a process known to be induced by silica nanoparticles in human cells. Differential analysis of the dominant phospholipids determined in membranes of alveolar lung epithelial cells demonstrates that the quaternary ammonium head groups of phosphatidylcholine and sphingomyelin play a critical and dose-dependent role in vesicle binding and rupture by amorphous colloidal silica nanoparticles. Surface modification by either protein adsorption or by covalent coupling of carboxyl groups suppresses the disintegration of these lipid vesicles, as well as membranolysis in human A549 lung epithelial cells by the silica nanoparticles. Furthermore, molecular modeling suggests a preferential affinity of silanol groups for choline head groups, which is also modulated by the pH value. Biomimetic lipid vesicles can thus be used to better understand specific phospholipid-nanoparticle interactions at the molecular level to support the rational design of safe advanced materials.

Comparing the Toxicological Responses of Pulmonary Air-Liquid Interface Models upon Exposure to Differentially Treated Carbon Fibers.

In recent years, the use of carbon fibers (CFs) in various sectors of industry has been increasing. Despite the similarity of CF degradation products to other toxicologically relevant materials such as asbestos fibers and carbon nanotubes, a detailed toxicological evaluation of this class of material has yet to be performed. In this work, we exposed advanced air-liquid interface cell culture models of the human lung to CF. To simulate different stresses applied to CF throughout their life cycle, they were either mechanically (mCF) or thermo-mechanically pre-treated (tmCF). Different aspects of inhalation toxicity as well as their possible time-dependency were monitored. mCFs were found to induce a moderate inflammatory response, whereas tmCF elicited stronger inflammatory as well as apoptotic effects. Furthermore, thermal treatment changed the surface properties of the CF resulting in a presumed adhesion of the cells to the fiber fragments and subsequent cell loss. Triple-cultures encompassing epithelial, macrophage, and fibroblast cells stood out with an exceptionally high inflammatory response. Only a weak genotoxic effect was detected in the form of DNA strand breaks in mono- and co-cultures, with triple-cultures presenting a possible secondary genotoxicity. This work establishes CF fragments as a potentially harmful material and emphasizes the necessity of further toxicological assessment of existing and upcoming advanced CF-containing materials.

Gene Expression Profiling of Mono- and Co-Culture Models of the Respiratory Tract Exposed to Crystalline Quartz under Submerged and Air-Liquid Interface Conditions.

In vitro lung cell models like air-liquid interface (ALI) and 3D cell cultures have advanced greatly in recent years, being especially valuable for testing advanced materials (e.g., nanomaterials, fibrous substances) when considering inhalative exposure. Within this study, we established submerged and ALI cell culture models utilizing A549 cells as mono-cultures and co-cultures with differentiated THP-1 (dTHP-1), as well as mono-cultures of dTHP-1. After ALI and submerged exposures towards α-quartz particles (Min-U-Sil5), with depositions ranging from 15 to 60 µg/cm2, comparison was made with respect to their transcriptional cellular responses employing high-throughput RT-qPCR. A significant dose- and time-dependent induction of genes coding for inflammatory proteins, e.g., IL-1A, IL-1B, IL-6, IL-8, and CCL22, as well as genes associated with oxidative stress response such as SOD2, was observed, even more pronounced in co-cultures. Changes in the expression of similar genes were more pronounced under submerged conditions when compared to ALI exposure in the case of A549 mono-cultures. Hereby, the activation of the NF-κB signaling pathway and the NLRP3 inflammasome seem to play an important role. Regarding genotoxicity, neither DNA strand breaks in ALI cultivated cells nor a transcriptional response to DNA damage were observed. Altogether, the toxicological responses depended considerably on the cell culture model and exposure scenario, relevant to be considered to improve toxicological risk assessment.

Comparing α-Quartz-Induced Cytotoxicity and Interleukin-8 Release in Pulmonary Mono- and Co-Cultures Exposed under Submerged and Air-Liquid Interface Conditions.

The occupational exposure to particles such as crystalline quartz and its impact on the respiratory tract have been studied extensively in recent years. For hazard assessment, the development of physiologically more relevant in-vitro models, i.e., air-liquid interface (ALI) cell cultures, has greatly progressed. Within this study, pulmonary culture models employing A549 and differentiated THP-1 cells as mono-and co-cultures were investigated. The different cultures were exposed to α-quartz particles (Min-U-Sil5) with doses ranging from 15 to 66 µg/cm2 under submerged and ALI conditions and cytotoxicity as well as cytokine release were analyzed. No cytotoxicity was observed after ALI exposure. Contrarily, Min-U-Sil5 was cytotoxic at the highest dose in both submerged mono- and co-cultures. A concentration-dependent release of interleukin-8 was shown for both exposure types, which was overall stronger in co-cultures. Our findings showed considerable differences in the toxicological responses between ALI and submerged exposure and between mono- and co-cultures. A substantial influence of the presence or absence of serum in cell culture media was noted as well. Within this study, the submerged culture was revealed to be more sensitive. This shows the importance of considering different culture and exposure models and highlights the relevance of communication between different cell types for toxicological investigations.

Air-Liquid Interface Exposure of Lung Epithelial Cells to Low Doses of Nanoparticles to Assess Pulmonary Adverse Effects.

Reliable and predictive in vitro assays for hazard assessments of manufactured nanomaterials (MNMs) are still limited. Specifically, exposure systems which more realistically recapitulate the physiological conditions in the lung are needed to predict pulmonary toxicity. To this end, air-liquid interface (ALI) systems have been developed in recent years which might be better suited than conventional submerged exposure assays. However, there is still a need for rigorous side-by-side comparisons of the results obtained with the two different exposure methods considering numerous parameters, such as different MNMs, cell culture models and read outs. In this study, human A549 lung epithelial cells and differentiated THP-1 macrophages were exposed under submerged conditions to two abundant types of MNMs i.e., ceria and titania nanoparticles (NPs). Membrane integrity, metabolic activity as well as pro-inflammatory responses were recorded. For comparison, A549 monocultures were also exposed at the ALI to the same MNMs. In the case of titania NPs, genotoxicity was also investigated. In general, cells were more sensitive at the ALI compared to under classical submerged conditions. Whereas ceria NPs triggered only moderate effects, titania NPs clearly initiated cytotoxicity, pro-inflammatory gene expression and genotoxicity. Interestingly, low doses of NPs deposited at the ALI were sufficient to drive adverse outcomes, as also documented in rodent experiments. Therefore, further development of ALI systems seems promising to refine, reduce or even replace acute pulmonary toxicity studies in animals.

Silica Nanoparticles Provoke Cell Death Independent of p53 and BAX in Human Colon Cancer Cells.

Several in vitro studies have suggested that silica nanoparticles (NPs) might induce adverse effects in gut cells. Here, we used the human colon cancer epithelial cell line HCT116 to study the potential cytotoxic effects of ingested silica NPs in the presence or absence of serum. Furthermore, we evaluated different physico-chemical parameters important for the assessment of nanoparticle safety, including primary particle size (12, 70, 200, and 500 nm) and surface modification (-NH2 and -COOH). Silica NPs triggered cytotoxicity, as evidenced by reduced metabolism and enhanced membrane leakage. Automated microscopy revealed that the silica NPs promoted apoptosis and necrosis proportional to the administered specific surface area dose. Cytotoxicity of silica NPs was suppressed by increasing amount of serum and surface modification. Furthermore, inhibition of caspases partially prevented silica NP-induced cytotoxicity. In order to investigate the role of specific cell death pathways in more detail, we used isogenic derivatives of HCT116 cells which lack the pro-apoptotic proteins p53 or BAX. In contrast to the anticancer drug cisplatin, silica NPs induced cell death independent of the p53-BAX axis. In conclusion, silica NPs initiated cell death in colon cancer cells dependent on the specific surface area and presence of serum. Further studies in vivo are warranted to address potential cytotoxic actions in the gut epithelium. The unintended toxicity of silica NPs as observed here could also be beneficial. As loss of p53 in colon cancer cells contributes to resistance against anticancer drugs, and thus to reoccurrence of colon cancer, targeted delivery of silica NPs could be envisioned to also deplete p53 deficient tumor cells.

The protein corona suppresses the cytotoxic and pro-inflammatory response in lung epithelial cells and macrophages upon exposure to nanosilica.

Engineered amorphous silica nanoparticles (nanosilica) are one of the most abundant nanomaterials and are widely used in industry. Furthermore, novel nanosilica materials are promising theranostic tools for biomedicine. However, hazardous effects of nanosilica especially after inhalation into the lung have been documented. Therefore, the safe development of nanosilica materials urgently requires predictive assays to monitor toxicity. Here, we further investigate the impact of the protein corona on the biological activity of two different types of nanosilica (colloidal and pyrogenic) in lung cells. As previously described, adsorption of serum proteins to the nanosilica surface suppresses cytotoxicity in macrophages and lung epithelial cells. As the increase of pro-inflammatory mediators is a hallmark of inflammation in the lung upon nanosilica exposure, we studied the potential coupling of the cytotoxic and pro-inflammatory response in A549 human lung epithelial cells and RAW264.7 mouse macrophages. Indeed, cytotoxicity precedes the onset of pro-inflammatory gene expression and cytokine release as exemplified for IL-8 in A549 cells and TNF-alpha in RAW264.7 macrophages after exposure to 0-100 µg/mL nanosilica in medium without serum. Formation of a protein corona not only inhibited cellular toxicity, but also the pro-inflammatory response. Of note, uptake of nanosilica into cells was negligible in the absence, but enhanced in the presence of a protein corona. Hence, the prevailing explanation that the protein corona simply interferes with cellular uptake thus preventing adverse effects needs to be revisited. In conclusion, for the reliable prediction of adverse effects of nanosilica in the lung, in vitro assays should be performed in media not complemented with complete serum. However, in case of different exposure routes, e.g., injection into the blood stream as intended for biomedicine, the protein corona prevents acute toxic actions of nanosilica.

Biocompatibility of Amine-Functionalized Silica Nanoparticles: The Role of Surface Coverage.

Here, amorphous silica nanoparticles (NPs), one of the most abundant nanomaterials, are used as an example to illustrate the utmost importance of surface coverage by functional groups which critically determines biocompatibility. Silica NPs are functionalized with increasing amounts of amino groups, and the number of surface exposed groups is quantified and characterized by detailed NMR and fluorescamine binding studies. Subsequent biocompatibility studies in the absence of serum demonstrate that, irrespective of surface modification, both plain and amine-modified silica NPs trigger cell death in RAW 264.7 macrophages. The in vitro results can be confirmed in vivo and are predictive for the inflammatory potential in murine lungs. In the presence of serum proteins, on the other hand, a replacement of only 10% of surface-active silanol groups by amines is sufficient to suppress cytotoxicity, emphasizing the relevance of exposure conditions. Mechanistic investigations identify a key role of lysosomal injury for cytotoxicity only in the presence, but not in the absence, of serum proteins. In conclusion, this work shows the critical need to rigorously characterize the surface coverage of NPs by their constituent functional groups, as well as the impact of serum, to reliably establish quantitative nanostructure activity relationships and develop safe nanomaterials.

Assessment of in vitro particle dosimetry models at the single cell and particle level by scanning electron microscopy.

BACKGROUND: Particokinetic models are important to predict the effective cellular dose, which is key to understanding the interactions of particles with biological systems. For the reliable establishment of dose-response curves in, e.g., the field of pharmacology and toxicology, mostly the In vitro Sedimentation, Diffusion and Dosimetry (ISDD) and Distorted Grid (DG) models have been employed. Here, we used high resolution scanning electron microscopy to quantify deposited numbers of particles on cellular and intercellular surfaces and compare experimental findings with results predicted by the ISDD and DG models. RESULTS: Exposure of human lung epithelial A549 cells to various concentrations of differently sized silica particles (100, 200 and 500 nm) revealed a remarkably higher dose deposited on intercellular regions compared to cellular surfaces. The ISDD and DG models correctly predicted the areal densities of particles in the intercellular space when a high adsorption ("stickiness") to the surface was emulated. In contrast, the lower dose on cells was accurately inferred by the DG model in the case of "non-sticky" boundary conditions. Finally, the presence of cells seemed to enhance particle deposition, as aerial densities on cell-free substrates were clearly reduced. CONCLUSIONS: Our results further validate the use of particokinetic models but also demonstrate their limitations, specifically, with respect to the spatial distribution of particles on heterogeneous surfaces. Consideration of surface properties with respect to adhesion and desorption should advance modelling approaches to ultimately predict the cellular dose with higher precision.

Revisiting the stress paradigm for silica nanoparticles: decoupling of the anti-oxidative defense, pro-inflammatory response and cytotoxicity.

Engineered amorphous silica nanoparticles (nanosilica) are widely used in industry yet can induce adverse effects, which might be classified according to the oxidative stress model. However, the underlying mechanisms as well as the potential interactions of the three postulated different tiers of toxicity-i.e. oxidative-, pro-inflammatory- and cytotoxic-stress response-are poorly understood. As macrophages are primary targets of nanoparticles, we used several macrophage models, primarily murine RAW264.7 macrophages, and monitored pro-inflammatory and anti-oxidative reactions as well as cytotoxicity in response to nanosilica at max. 50 µg/mL. Special attention was given to the activation of mitogen-activated protein kinases (MAPKs) as potential regulators of the cellular stress response. Indeed, according to the oxidative stress model, also nanosilica elicits an, albeit modest, anti-oxidative response as well as pronounced pro-inflammatory reactions and cytotoxicity in macrophages. Interestingly however, these three tiers of toxicity seem to operate separately of each other for nanosilica. Specifically, impeding the anti-oxidative response by scavenging of reactive oxygen species does not prevent the pro-inflammatory and cytotoxic response. Furthermore, blocking the pro-inflammatory response by inhibition of MAPKs does not impair cell death. As hazard assessment has been guided by the prevailing assumption of a dose-dependent coupling of sequential tiers of toxicity, identification of critical physico-chemical parameters to assist the safe-by-design concept should be enabled by simply monitoring one of the toxicity read-outs. Our results indicate a more complex scenario in the case of nanosilica, which triggers independent pleiotropic effects possibly also related to different material properties and primary cellular targets.

Microscopy-based high-throughput assays enable multi-parametric analysis to assess adverse effects of nanomaterials in various cell lines.

Manufactured nanomaterials (MNMs) selected from a library of over 120 different MNMs with varied compositions, sizes, and surface coatings were tested by four different laboratories for toxicity by high-throughput/-content (HT/C) techniques. The selected particles comprise 14 MNMs composed of CeO2, Ag, TiO2, ZnO and SiO2 with different coatings and surface characteristics at varying concentrations. The MNMs were tested in different mammalian cell lines at concentrations between 0.5 and 250 µg/mL to link physical-chemical properties to multiple adverse effects. The cell lines are derived from relevant organs such as liver, lung, colon and the immune system. Endpoints such as viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential, lysosomal acidification and steatosis have been studied. Soluble MNMs, Ag and ZnO, were toxic in all cell types. TiO2 and SiO2 MNMs also triggered toxicity in some, but not all, cell types and the cell type-specific effects were influenced by the specific coating and surface modification. CeO2 MNMs were nearly ineffective in our test systems. Differentiated liver cells appear to be most sensitive to MNMs, Whereas most of the investigated MNMs showed no acute toxicity, it became clear that some show adverse effects dependent on the assay and cell line. Hence, it is advised that future nanosafety studies utilise a multi-parametric approach such as HT/C screening to avoid missing signs of toxicity. Furthermore, some of the cell type-specific effects should be followed up in more detail and might also provide an incentive to address potential adverse effects in vivo in the relevant organ.

Contrast of Backscattered Electron SEM Images of Nanoparticles on Substrates with Complex Structure.

This study is concerned with backscattered electron scanning electron microscopy (BSE SEM) contrast of complex nanoscaled samples which consist of SiO2 nanoparticles (NPs) deposited on indium-tin-oxide covered bulk SiO2 and glassy carbon substrates. BSE SEM contrast of NPs is studied as function of the primary electron energy and working distance. Contrast inversions are observed which prevent intuitive interpretation of NP contrast in terms of material contrast. Experimental data is quantitatively compared with Monte-Carlo- (MC-) simulations. Quantitative agreement between experimental data and MC-simulations is obtained if the transmission characteristics of the annular semiconductor detector are taken into account. MC-simulations facilitate the understanding of NP contrast inversions and are helpful to derive conditions for optimum material and topography contrast.

Autophagy induced by silica nanoparticles protects RAW264.7 macrophages from cell death.

Although the technological and economic benefits of engineered nanomaterials are obvious, concerns have been raised about adverse effects if such material is inhaled, ingested, applied to the skin or even released into the environment. Here we studied the cytotoxic effects of the most abundant nanomaterial, silica nanoparticles (SiO2-NPs), in murine RAW264.7 macrophages. SiO2-NPs dose-dependently induce membrane leakage and cell death without obvious involvement of reactive oxygen species. Interestingly, at low concentrations SiO2-NPs trigger autophagy, evidenced by morphological and biochemical hallmarks such as autophagolysosomes or increased levels of LC3-II, which serves to protect cells from cytotoxicity. Hence SiO2-NPs initiate an adaptive stress response which dependent on dose serve to balance survival and death and ultimately dictates the cellular fate.

Silica nanoparticles are less toxic to human lung cells when deposited at the air-liquid interface compared to conventional submerged exposure.

BACKGROUND: Investigations on adverse biological effects of nanoparticles (NPs) in the lung by in vitro studies are usually performed under submerged conditions where NPs are suspended in cell culture media. However, the behaviour of nanoparticles such as agglomeration and sedimentation in such complex suspensions is difficult to control and hence the deposited cellular dose often remains unknown. Moreover, the cellular responses to NPs under submerged culture conditions might differ from those observed at physiological settings at the air-liquid interface. RESULTS: In order to avoid problems because of an altered behaviour of the nanoparticles in cell culture medium and to mimic a more realistic situation relevant for inhalation, human A549 lung epithelial cells were exposed to aerosols at the air-liquid interphase (ALI) by using the ALI deposition apparatus (ALIDA). The application of an electrostatic field allowed for particle deposition efficiencies that were higher by a factor of more than 20 compared to the unmodified VITROCELL deposition system. We studied two different amorphous silica nanoparticles (particles produced by flame synthesis and particles produced in suspension by the Stöber method). Aerosols with well-defined particle sizes and concentrations were generated by using a commercial electrospray generator or an atomizer. Only the electrospray method allowed for the generation of an aerosol containing monodisperse NPs. However, the deposited mass and surface dose of the particles was too low to induce cellular responses. Therefore, we generated the aerosol with an atomizer which supplied agglomerates and thus allowed a particle deposition with a three orders of magnitude higher mass and of surface doses on lung cells that induced significant biological effects. The deposited dose was estimated and independently validated by measurements using either transmission electron microscopy or, in case of labelled NPs, by fluorescence analyses. Surprisingly, cells exposed at the ALI were less sensitive to silica NPs as evidenced by reduced cytotoxicity and inflammatory responses. CONCLUSION: Amorphous silica NPs induced qualitatively similar cellular responses under submerged conditions and at the ALI. However, submerged exposure to NPs triggers stronger effects at much lower cellular doses. Hence, more studies are warranted to decipher whether cells at the ALI are in general less vulnerable to NPs or specific NPs show different activities dependent on the exposure method.

Regulation of the arachidonic acid mobilization in macrophages by combustion-derived particles.

BACKGROUND: Acute exposure to elevated levels of environmental particulate matter (PM) is associated with increasing morbidity and mortality rates. These adverse health effects, e.g. culminating in respiratory and cardiovascular diseases, have been demonstrated by a multitude of epidemiological studies. However, the underlying mechanisms relevant for toxicity are not completely understood. Especially the role of particle-induced reactive oxygen species (ROS), oxidative stress and inflammatory responses is of particular interest.In this in vitro study we examined the influence of particle-generated ROS on signalling pathways leading to activation of the arachidonic acid (AA) cascade. Incinerator fly ash particles (MAF02) were used as a model for real-life combustion-derived particulate matter. As macrophages, besides epithelial cells, are the major targets of particle actions in the lung murine RAW264.7 macrophages and primary human macrophages were investigated. RESULTS: The interaction of fly ash particles with macrophages induced both the generation of ROS and as part of the cellular inflammatory responses a dose- and time-dependent increase of free AA, prostaglandin E2/thromboxane B2 (PGE2/TXB2), and 8-isoprostane, a non-enzymatically formed oxidation product of AA. Additionally, increased phosphorylation of the mitogen-activated protein kinases (MAPK) JNK1/2, p38 and ERK1/2 was observed, the latter of which was shown to be involved in MAF02-generated AA mobilization and phosphorylation of the cytosolic phospolipase A2. Using specific inhibitors for the different phospolipase A2 isoforms the MAF02-induced AA liberation was shown to be dependent on the cytosolic phospholipase A2, but not on the secretory and calcium-independent phospholipase A2. The initiation of the AA pathway due to MAF02 particle exposure was demonstrated to depend on the formation of ROS since the presence of the antioxidant N-acetyl-cysteine (NAC) prevented the MAF02-mediated enhancement of free AA, the subsequent conversion to PGE2/TXB2 via the induction of COX-2 and the ERK1/2 and JNK1/2 phosphorylation. Finally we showed that the particle-induced formation of ROS, liberation of AA and PGE2/TXB2 together with the phosphorylation of ERK1/2 and JNK1/2 proteins was decreased after pre-treatment of macrophages with the metal chelator deferoxamine mesylate (DFO). CONCLUSIONS: These results indicate that one of the primary mechanism initiating inflammatory processes by incinerator fly ash particles seems to be the metal-mediated generation of ROS, which triggers via the MAPK cascade the activation of AA signalling pathway.