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Abstract Background Inorganic ultraviolet filters such as titanium dioxide nanoparticles are frequently used in sunscreens. Numerous toxicological studies in vitro and in vivo have been conducted using pristine standard reference nanomaterials of these inorganic filters. While convenient, this approach is not realistic because the complex environment of sunscreen formulations could change the physicochemical properties of the nanoparticles and lead to vastly different toxicological outcomes. Therefore, this study focused on characterizing nanoparticles extracted from commercial sunscreen and evaluating the associated toxicological impacts upon exposure to human keratinocytes and human skin explants. Results Titanium dioxide nanoparticles were extracted from commercial sunscreens and thoroughly characterized. The identity of the associated molecular corona on the extracted nanoparticles was also evaluated. Cell metabolic and proliferation profiles, mitochondrial superoxide activity, reactive oxygen species levels, and genotoxicity induced through exposure to the nanoparticles were studied in vitro using a human keratinocyte cell line. The cell response was significantly different after treatment with pristine nanoparticles compared to corresponding sunscreen-extracted nanoparticles. Pristine spherical nanoparticles resulted in more pronounced toxicity in 2D cultured keratinocytes compared to extracted nanoparticles but did not impact wound-edge migration significantly in 3D ex vivo human skin explant models. Additionally, extracted rod-shaped nanoparticles had greater toxic impacts in keratinocytes in vitro and retarded wound-edge migration in the ex vivo model compared to the extracted spherical nanoparticles. Nevertheless, these heightened cell responses were not associated with any increase in phosphorylated γH2AX (which is indicative of DNA damage) both in vitro and ex vivo. Conclusions This study shows the feasibility of extracting nanoparticles from personal care products such as sunscreens to obtain relevant forms to model real-life exposure scenarios. Overall, sunscreen-extracted nanoparticles were found to be less toxic compared to pristine equivalents but retarded wound-edge migration more significantly. Skin explant cultures provide a more realistic alternative to monolayer cell cultures, although the differential outcomes between the models need more in-depth evaluation.

Abstract Atherosclerosis is one of the most common types of cardiovascular disease and is driven by lipid accumulation and chronic inflammation in the arteries, which leads to stenosis and thrombosis. Researchers have been working to design multifunctional nanomedicines with the ability to target, diagnose, and treat atherosclerosis, but recent studies have also identified that nanomaterials can cause atherosclerosis. Therefore, this review aims to outline the molecular mechanisms and physicochemical properties of nanomaterials that promote atherosclerosis. By analyzing the toxicological effects of nanomaterials on cells involved in the pathogenesis of atherosclerosis such as vascular endothelial cells, vascular smooth muscle cells and immune cells, we aim to provide new perspectives for the prevention and treatment of atherosclerosis, and raise awareness of nanotoxicology to advance the clinical translation and sustainable development of nanomaterials. Graphical Abstract

Abstract Background Glycol chitosan nanoparticles (CNPs) have emerged as an effective drug delivery system for cancer diagnosis and treatment. Although they have great biocompatibility owing to biodegradable chemical structure and low immunogenicity, sufficient information on in vivo toxicity to understand the potential risks depending on the repeated high-dose have not been adequately studied. Herein, we report the results of in vivo toxicity evaluation for CNPs focused on the number and dose of administration in healthy mice to provide a toxicological guideline for a better clinical application of CNPs. Results The CNPs were prepared by conjugating hydrophilic glycol chitosan with hydrophobic 5β-cholanic acid and the amphiphilic glycol chitosan-5β-cholanic acid formed self-assembled nanoparticles with its concentration-dependent homogeneous size distributions (265.36–288.3 nm) in aqueous condition. In cell cultured system, they showed significantly high cellular uptake in breast cancer cells (4T1) and cardiomyocytes (H9C2) than in fibroblasts (L929) and macrophages (Raw264.7) in a dose- and time-dependent manners, resulting in severe necrotic cell death in H9C2 at a clinically relevant highly concentrated condition. In particular, when the high-dose (90 mg/kg) of CNPs were intravenously injected into the healthy mice, considerable amount was non-specifically accumulated in major organs (liver, lung, spleen, kidney and heart) after 6 h of injection and sustainably retained for 72 h. Finally, repeated high-dose of CNPs (90 mg/kg, three times) induced severe cardiotoxicity accompanying inflammatory responses, tissue damages, fibrotic changes and organ dysfunction. Conclusions This study demonstrates that repeated high-dose CNPs induce severe cardiotoxicity in vivo. Through the series of toxicological assessments in the healthy mice, this study provides a toxicological guideline that may expedite the application of CNPs in the clinical settings.

Abstract Background Short-term inhalation of occupationally relevant ultrafine zinc/copper (Zn/Cu) containing welding fumes has been shown to induce subclinical systemic inflammation, associated with an elevated risk for cardiovascular diseases. The involvement of noncoding RNAs (lncRNAs) in this setting is currently unknown. However, lncRNAs have been reported to fulfill essential roles in, e.g., cardiovascular diseases, inflammation, infectious diseases, and pollution-related lung disorders. Methods In this study, the specific lncRNAs levels of the 4 lncRNAs CoroMarker, MALAT1, CDR1as and LINC00460 were determined by RT-qPCR in THP-1 macrophages exposed to Zn/Cu metal fume suspensions for 1, 2, and 4 hours in vitro. Furthermore, 14 subjects were exposed to Zn/Cu containing welding fumes (at 2.5 mg/m3) for 6 hours. Before, 6, 10, and 29 hours after exposure start, whole blood cell lncRNAs levels were determined by RT-qPCR. Results In THP-1 macrophages, we observed a 2.3-fold increase of CDR1as at 1 h (Wilcoxon p = 0.03), a non-significant increase of CoroMarker at 1 h, and an increase of LINC00460 at 2 h (p = 0.03) and at 4 h (p = 0.06). In whole blood cells, we determined a non-significant upregulation of CDR1as at 6 h (p = 0.2), a significant downregulation of CoroMarker at 6 h (p = 0.04), and a significant upregulation of LINC00460 levels at 10 h (p = 0.04) and 29 h (p = 0.04). MALAT-1 remained unchanged in both settings. Conclusion The orientation of regulation of the lncRNAs is (except for CoroMarker) similar in the in vitro and in vivo experiments and in line with their described functions. Therefore, these results, e.g. the upregulation of the potential risk marker for cardiovascular diseases, CDR1as, contribute to understanding the underlying mechanisms of Zn/Cu-induced subclinical inflammation in metal workers.

Abstract Background Physiologically based pharmacokinetic (PBPK) modeling is an important tool in predicting target organ dosimetry and risk assessment of nanoparticles (NPs). The methodology of building a multi-route PBPK model for NPs has not been established, nor systematically evaluated. In this study, we hypothesized that the traditional route-to-route extrapolation approach of PBPK modeling that is typically used for small molecules may not be appropriate for NPs. To test this hypothesis, the objective of this study was to develop a multi-route PBPK model for different sizes (1.4–200 nm) of gold nanoparticles (AuNPs) in adult rats following different routes of administration (i.e., intravenous (IV), oral gavage, intratracheal instillation, and endotracheal inhalation) using two approaches: a traditional route-to-route extrapolation approach for small molecules and a new approach that is based on route-specific data that we propose to be applied generally to NPs. Results We found that the PBPK model using this new approach had superior performance than the traditional approach. The final PBPK model was optimized rigorously using a Bayesian hierarchical approach with Markov chain Monte Carlo simulations, and then converted to a web-based interface using R Shiny. In addition, quantitative structure–activity relationships (QSAR) based multivariate linear regressions were established to predict the route-specific key biodistribution parameters (e.g., maximum uptake rate) based on the physicochemical properties of AuNPs (e.g., size, surface area, dose, Zeta potential, and NP numbers). These results showed the size and surface area of AuNPs were the main determinants for endocytic/phagocytic uptake rates regardless of the route of administration, while Zeta potential was an important parameter for the estimation of the exocytic release rates following IV administration. Conclusions This study suggests that traditional route-to-route extrapolation approaches for PBPK modeling of small molecules are not applicable to NPs. Therefore, multi-route PBPK models for NPs should be developed using route-specific data. This novel PBPK-based web interface serves as a foundation for extrapolating to other NPs and to humans to facilitate biodistribution estimation, safety, and risk assessment of NPs.

Abstract Background TiO2 nanomaterials (NMs) are present in a variety of food and personal hygiene products, and consumers are exposed daily to these NMs through oral exposition. While the bulk of ingested TiO2 NMs are eliminated rapidly in stool, a fraction is able to cross the intestinal epithelial barrier and enter systemic circulation from where NMs can be distributed to tissues, primarily liver and spleen. Daily exposure to TiO2 NMs, in combination with a slow rate of elimination from tissues, results in their accumulation within different tissues. Considerable evidence suggests that following oral exposure to TiO2 NMs, the presence of NMs in tissues is associated with a number of adverse effects, both in intestine and liver. Although numerous studies have been performed in vitro investigating the acute effects of TiO2 NMs in intestinal and hepatic cell models, considerably less is known about the effect of repeated exposure on these models. In this study, we investigated the cytotoxic effects of repeated exposure of relevant models of intestine and liver to two TiO2 NMs differing in hydrophobicity for 24 h, 1 week and 2 weeks at concentrations ranging from 0.3 to 80 µg/cm2. To study the persistence of these two NMs in cells, we included a 1-week recovery period following 24 h and 1-week treatments. Cellular uptake by TEM and ToF–SIMS analyses, as well as the viability and pro-inflammatory response were evaluated. Changes in the membrane composition in Caco-2 and HepaRG cells treated with TiO2 NMs for up to 2 weeks were also studied. Results Despite the uptake of NM-103 and NM-104 in cells, no significant cytotoxic effects were observed in either Caco-2 or HepaRG cells treated for up to 2 weeks at NM concentrations up to 80 µg/cm2 . In addition, no significant effects on IL-8 secretion were observed. However, significant changes in membrane composition were observed in both cell lines. Interestingly, while most of these phospholipid modifications were reversed following a 1-week recovery, others were not affected by the recovery period. Conclusion These findings indicate that although no clear effects on cytotoxicity were observed following repeated exposure of differentiated Caco-2 and HepaRG cells to TiO2 NMs, subtle effects on membrane composition could induce potential adverse effects in the long-term.

Abstract Background Precisely how silver nanoparticles (AgNPs) kill mammalian cells still is not fully understood. It is not clear if AgNP-induced damage differs from silver cation (Ag+), nor is it known how AgNP damage is transmitted from cell membranes, including endosomes, to other organelles. Cells can differ in relative sensitivity to AgNPs or Ag+, which adds another layer of complexity to identifying specific mechanisms of action. Therefore, we determined if there were specific effects of AgNPs that differed from Ag+ in cells with high or low sensitivity to either toxicant. Methods Cells were exposed to intact AgNPs, Ag+, or defined mixtures of AgNPs with Ag+, and viability was assessed. The level of dissolved Ag+ in AgNP suspensions was determined using inductively coupled plasma mass spectrometry. Changes in reactive oxygen species following AgNP or Ag+ exposure were quantified, and treatment with catalase, an enzyme that catalyzes the decomposition of H2O2 to water and oxygen, was used to determine selectively the contribution of H2O2 to AgNP and Ag+ induced cell death. Lipid peroxides, formation of 4-hydroxynonenol protein adducts, protein thiol oxidation, protein aggregation, and activation of the integrated stress response after AgNP or Ag+ exposure were quantified. Lastly, cell membrane integrity and indications of apoptosis or necrosis in AgNP and Ag+ treated cells were examined by flow cytometry. Results We identified AgNPs with negligible Ag+ contamination. We found that SUM159 cells, which are a triple-negative breast cancer cell line, were more sensitive to AgNP exposure less sensitive to Ag+ compared to iMECs, an immortalized, breast epithelial cell line. This indicates that high sensitivity to AgNPs was not predictive of similar sensitivity to Ag+. Exposure to AgNPs increased protein thiol oxidation, misfolded proteins, and activation of the integrated stress response in AgNP sensitive SUM159 cells but not in iMEC cells. In contrast, Ag+ cause similar damage in Ag+ sensitive iMEC cells but not in SUM159 cells. Both Ag+ and AgNP exposure increased H2O2 levels; however, treatment with catalase rescued cells from Ag+ cytotoxicity but not from AgNPs. Instead, our data support a mechanism by which damage from AgNP exposure propagates through cells by generation of lipid peroxides, subsequent lipid peroxide mediated oxidation of proteins, and via generation of 4-hydroxynonenal (4-HNE) protein adducts. Conclusions There are distinct differences in the responses of cells to AgNPs and Ag+. Specifically, AgNPs drive cell death through lipid peroxidation leading to proteotoxicity and necrotic cell death, whereas Ag+ increases H2O2, which drives oxidative stress and apoptotic cell death. This work identifies a previously unknown mechanism by which AgNPs kill mammalian cells that is not dependent upon the contribution of Ag+ released in extracellular media. Understanding precisely which factors drive the toxicity of AgNPs is essential for biomedical applications such as cancer therapy, and of importance to identifying consequences of unintended exposures.

Abstract Background Safe and rational development of nanomaterials for clinical translation requires the assessment of potential biocompatibility. Autophagy, a critical homeostatic pathway intrinsically linked to cellular health and inflammation, has been shown to be affected by nanomaterials. It is, therefore, important to be able to assess possible interactions of nanomaterials with autophagic processes. Results CEM (T cell), Raji (B lymphocyte), and THP-1 (human monocyte) cell lines were subject to treatment with rapamycin and chloroquine, known to affect the autophagic process, in order to evaluate cell line-specific responses. Flow cytometric quantification of a fluorescent autophagic vacuole stain showed that maximum observable effects (105%, 446%, and 149% of negative controls) were achieved at different exposure durations (8, 6, and 24 h for CEM, Raji, and THP-1, respectively). THP-1 was subsequently utilised as a model to assess the autophagic impact of a small library of nanomaterials. Association was observed between hydrodynamic size and autophagic impact (r2 = 0.11, p = 0.004). An ELISA for p62 confirmed the greatest impact by 10 nm silver nanoparticles, abolishing p62, with 50 nm silica and 180 nm polystyrene also lowering p62 to a significant degree (50%, 74%, and 55%, respectively, p

Abstract Background With the continued integration of engineered nanomaterials (ENMs) into everyday applications, it is important to understand their potential for inducing adverse human health effects. However, standard in vitro hazard characterisation approaches suffer limitations for evaluating ENM and so it is imperative to determine these potential hazards under more physiologically relevant and realistic exposure scenarios in target organ systems, to minimise the necessity for in vivo testing. The aim of this study was to determine if acute (24 h) and prolonged (120 h) exposures to five ENMs (TiO2, ZnO, Ag, BaSO4 and CeO2) would have a significantly different toxicological outcome (cytotoxicity, (pro-)inflammatory and genotoxic response) upon 3D human HepG2 liver spheroids. In addition, this study evaluated whether a more realistic, prolonged fractionated and repeated ENM dosing regime induces a significantly different toxicity outcome in liver spheroids as compared to a single, bolus prolonged exposure. Results Whilst it was found that the five ENMs did not impede liver functionality (e.g. albumin and urea production), induce cytotoxicity or an IL-8 (pro-)inflammatory response, all were found to cause significant genotoxicity following acute exposure. Most statistically significant genotoxic responses were not dose-dependent, with the exception of TiO2. Interestingly, the DNA damage effects observed following acute exposures, were not mirrored in the prolonged exposures, where only 0.2–5.0 µg/mL of ZnO ENMs were found to elicit significant (p ≤ 0.05) genotoxicity. When fractionated, repeated exposure regimes were performed with the test ENMs, no significant (p ≥ 0.05) difference was observed when compared to the single, bolus exposure regime. There was

Abstract Background Nanotoxicology is an increasingly relevant field and sound paradigms on how inhaled nanoparticles (NPs) interact with organs at the cellular level, causing harmful conditions, have yet to be established. This is particularly true in the case of the cardiovascular system, where experimental and clinical evidence shows morphological and functional damage associated with NP exposure. Giving the increasing interest on cobalt oxide (Co3O4) NPs applications in industrial and bio-medical fields, a detailed knowledge of the involved toxicological effects is required, in view of assessing health risk for subjects/workers daily exposed to nanomaterials. Specifically, it is of interest to evaluate whether NPs enter cardiac cells and interact with cell function. We addressed this issue by investigating the effect of acute exposure to Co3O4-NPs on excitation-contraction coupling in freshly isolated rat ventricular myocytes. Results Patch clamp analysis showed instability of resting membrane potential, decrease in membrane electrical capacitance, and dose-dependent decrease in action potential duration in cardiomyocytes acutely exposed to Co3O4-NPs. Motion detection and intracellular calcium fluorescence highlighted a parallel impairment of cell contractility in comparison with controls. Specifically, NP-treated cardiomyocytes exhibited a dose-dependent decrease in the fraction of shortening and in the maximal rate of shortening and re-lengthening, as well as a less efficient cytosolic calcium clearing and an increased tendency to develop spontaneous twitches. In addition, treatment with Co3O4-NPs strongly increased ROS accumulation and induced nuclear DNA damage in a dose dependent manner. Finally, transmission electron microscopy analysis demonstrated that acute exposure did lead to cellular internalization of NPs. Conclusions Taken together, our observations indicate that Co3O4-NPs alter cardiomyocyte electromechanical efficiency and intracellular calcium handling, and induce ROS production resulting in oxidative stress that can be related to DNA damage and adverse effects on cardiomyocyte functionality.

Abstract Nanoparticles exhibit a specific diffusion and sedimentation behavior under cell culture conditions as used in nantoxicological in vitro testing. How a particular particle suspension behaves depends on the particular physicochemical characteristics of the particles and the cell culture system. Only a fraction of the nanoparticles applied to a cell culture will thus reach the cells within a given time frame. Therefore, dosimetric calculations are essential not only to determine the exact fraction of nanoparticles that has come into contact with the cells, but also to ensure experimental comparability and correct interpretation of results, respectively. Yet, the use of published dosimetry models is limited. Not the least because the correct application of these in silico tools usually requires bioinformatics knowledge, which often is perceived a hurdle. Moreover, not all models are freely available and accessible. In order to overcome this obstacle, we have now developed an easy-to-use interface for our recently published 3DSDD dosimetry model, called NanoPASS (NanoParticle Administration Sedimentation Simulator). The interface is freely available to all researchers. It will facilitate the use of in silico dosimetry in nanotoxicology and thus improve interpretation and comparability of in vitro results in the field.

Abstract Background Accurate knowledge of cell−/tissue-delivered dose plays a pivotal role in inhalation toxicology studies, since it is the key parameter for hazard assessment and translation of in vitro to in vivo dose-response. Traditionally, (nano-)particle toxicological studies with in vivo and in vitro models of the lung rely on in silio computational or off-line analytical methods for dosimetry. In contrast to traditional in vitro testing under submerged cell culture conditions, the more physiologic air-liquid interface (ALI) conditions offer the possibility for real-time dosimetry using quartz crystal microbalances (QCMs). However, it is unclear, if QCMs are sensitive enough for nanotoxicological studies. We investigated this issue for two commercially available VITROCELL®Cloud ALI exposure systems. Results Quantitative fluorescence spectroscopy of fluorescein-spiked saline aerosol was used to determine detection limit, precision and accuracy of the QCMs implemented in a VITROCELL®Cloud 6 and Cloud 12 system for dose-controlled ALI aerosol-cell exposure experiments. Both QCMs performed linearly over the entire investigated dose range (200 to 12,000 ng/cm2) with an accuracy of 3.4% (Cloud 6) and 3.8% (Cloud 12). Their precision (repeatability) decreased from 2.5% for large doses (> 9500 ng/cm2) to values of 10% and even 25% for doses of 1000 ng/cm2 and 200 ng/cm2, respectively. Their lower detection limit was 170 ng/cm2 and 169 ng/cm2 for the Cloud 6 and Cloud 12, respectively. Dose-response measurements with (NM110) ZnO nanoparticles revealed an onset dose of 3.3 μg/cm2 (or 0.39 cm2/cm2) for both cell viability (WST-1) and cytotoxicity (LDH) of A549 lung epithelial cells. Conclusions The QCMs of the Cloud 6 and Cloud 12 systems show similar performance and are highly sensitive, accurate devices for (quasi-) real-time dosimetry of the cell-delivered particle dose in ALI cell exposure experiments, if operated according to manufacturer specifications. Comparison with in vitro onset doses from this and previously published ALI studies revealed that the detection limit of 170 ng/cm2 is sufficient for determination of toxicological onset doses for all particle types with low (e.g. polystyrene) or high mass-specific toxicity (e.g. ZnO and Ag) investigated here. Hence, in principle QCMs are suitable for in vitro nanotoxciological studies, but this should be investigated for each QCM and ALI exposure system under the specific exposure conditions as described in the present study.

Abstract Toxicity testing and regulation of advanced materials at the nanoscale, i.e. nanosafety, is challenged by the growing number of nanomaterials and their property variants requiring assessment for potential human health impacts. The existing animal-reliant toxicity testing tools are onerous in terms of time and resources and are less and less in line with the international effort to reduce animal experiments. Thus, there is a need for faster, cheaper, sensitive and effective animal alternatives that are supported by mechanistic evidence. More importantly, there is an urgency for developing alternative testing strategies that help justify the strategic prioritization of testing or targeting the most apparent adverse outcomes, selection of specific endpoints and assays and identifying nanomaterials of high concern. The Adverse Outcome Pathway (AOP) framework is a systematic process that uses the available mechanistic information concerning a toxicological response and describes causal or mechanistic linkages between a molecular initiating event, a series of intermediate key events and the adverse outcome. The AOP framework provides pragmatic insights to promote the development of alternative testing strategies. This review will detail a brief overview of the AOP framework and its application to nanotoxicology, tools for developing AOPs and the role of toxicogenomics, and summarize various AOPs of relevance to inhalation toxicity of nanomaterials that are currently under various stages of development. The review also presents a network of AOPs derived from connecting all AOPs, which shows that several adverse outcomes induced by nanomaterials originate from a molecular initiating event that describes the interaction of nanomaterials with lung cells and involve similar intermediate key events. Finally, using the example of an established AOP for lung fibrosis, the review will discuss various in vitro tests available for assessing lung fibrosis and how the information can be used to support a tiered testing strategy for lung fibrosis. The AOPs and AOP network enable deeper understanding of mechanisms involved in inhalation toxicity of nanomaterials and provide a strategy for the development of alternative test methods for hazard and risk assessment of nanomaterials.

Abstract Background Non-communicable diseases, intended as the results of a combination of inherited, environmental and biological factors, kill 40 million people each year, equivalent to roughly 70% of all premature deaths globally. The possibility that manufactured nanoparticles (NPs) may affect cardiac performance, has led to recognize NPs-exposure not only as a major Public Health concern, but also as an occupational hazard. In volunteers, NPs-exposure is problematic to quantify. We recently found that inhaled titanium dioxide NPs, one of the most produced engineered nanomaterials, acutely increased cardiac excitability and promoted arrhythmogenesis in normotensive rats by a direct interaction with cardiac cells. We hypothesized that such scenario can be exacerbated by latent cardiovascular disorders such as hypertension. Results We monitored cardiac electromechanical performance in spontaneously hypertensive rats (SHRs) exposed to titanium dioxide NPs for 6 weeks using a combination of cardiac functional measurements associated with toxicological, immunological, physical and genetic assays. Longitudinal radio-telemetry ECG recordings and multiple-lead epicardial potential mapping revealed that atrial activation times significantly increased as well as proneness to arrhythmia. At the third week of nanoparticles administration, the lung and cardiac tissue encountered a maladaptive irreversible structural remodelling starting with increased pro-inflammatory cytokines levels and lipid peroxidation, resulting in upregulation of the main pro-fibrotic cardiac genes. At the end of the exposure, the majority of spontaneous arrhythmic events terminated, while cardiac hemodynamic deteriorated and a significant accumulation of fibrotic tissue occurred as compared to control untreated SHRs. Titanium dioxide nanoparticles were quantified in the heart tissue although without definite accumulation as revealed by particle-induced X-ray emission and ultrastructural analysis. Conclusions The co-morbidity of hypertension and inhaled nanoparticles induces irreversible hemodynamic impairment associated with cardiac structural damage potentially leading to heart failure. The time-dependence of exposure indicates a non-return point that needs to be taken into account in hypertensive subjects daily exposed to nanoparticles.

Abstract Our development and usage of engineered nanomaterials has grown exponentially despite concerns about their unfavourable cardiorespiratory consequence, one that parallels ambient ultrafine particle exposure from vehicle emissions. Most research in the field has so far focused on airway inflammation in response to nanoparticle inhalation, however, little is known about nanoparticle-microbiome interaction in the human airway and the environment. Emerging evidence illustrates that the airway, even in its healthy state, is not sterile. The resident human airway microbiome is further altered in chronic inflammatory respiratory disease however little is known about the impact of nanoparticle inhalation on this airway microbiome. The composition of the airway microbiome, which is involved in the development and progression of respiratory disease is dynamic, adding further complexity to understanding microbiota-host interaction in the lung, particularly in the context of nanoparticle exposure. This article reviews the size-dependent properties of nanomaterials, their body deposition after inhalation and factors that influence their fate. We evaluate what is currently known about nanoparticle-microbiome interactions in the human airway and summarise the known clinical, immunological and toxicological consequences of this relationship. While associations between inhaled ambient ultrafine particles and host immune-inflammatory response are known, the airway and environmental microbiomes likely act as intermediaries and facilitate individual susceptibility to inhaled nanoparticles and toxicants. Characterising the precise interaction between the environment and airway microbiomes, inhaled nanoparticles and the host immune system is therefore critical and will provide insight into mechanisms promoting nanoparticle induced airway damage.

Abstract Background The special physicochemical properties of gold nanoprisms make them very useful for biomedical applications including biosensing and cancer therapy. However, it is not clear how gold nanoprisms may affect cellular physiology including viability and other critical functions. We report a multiparametric investigation on the impact of gold-nanoprisms on mice and human, transformed and primary cells as well as tissue distribution and toxicity in vivo after parental injection. Methods Cellular uptake of the gold-nanoprisms (NPRs) and the most crucial parameters of cell fitness such as generation of reactive oxygen species (ROS), mitochondria membrane potential, cell morphology and apoptosis were systematically assayed in cells. Organ distribution and toxicity including inflammatory response were analysed in vivo in mice at 3 days or 4 months after parental administration. Results Internalized gold-nanoprisms have a significant impact in cell morphology, mitochondrial function and ROS production, which however do not affect the potential of cells to proliferate and form colonies. In vivo NPRs were only detected in spleen and liver at 3 days and 4 months after administration, which correlated with some changes in tissue architecture. However, the main serum biochemical markers of organ damage and inflammation (TNFα and IFNγ) remained unaltered even after 4 months. In addition, animals did not show any macroscopic sign of toxicity and remained healthy during all the study period. Conclusion Our data indicate that these gold-nanoprisms are neither cytotoxic nor cytostatic in transformed and primary cells, and suggest that extensive parameters should be analysed in different cell types to draw useful conclusions on nanomaterials safety. Moreover, although there is a tendency for the NPRs to accumulate in liver and spleen, there is no observable negative impact on animal health.

Abstract Background The exceptional physical-chemical properties of carbon nanotubes have lead to their use in diverse commercial and biomedical applications. However, their utilization has raised concerns about human exposure that may predispose individuals to adverse health risks. The present study investigated the susceptibility to cardiac ischemic injury following a single exposure to various forms of multi-walled carbon nanotubes (MWCNTs). It was hypothesized that oropharyngeal aspiration of MWCNTs exacerbates myocardial ischemia and reperfusion injury (I/R injury). Methods Oropharyngeal aspiration was performed on male C57BL/6J mice with a single amount of MWCNT (0.01 - 100 μg) suspended in 100 μL of a surfactant saline (SS) solution. Three forms of MWCNTs were used in this study: unmodified, commercial grade (C-grade), and functionalized forms that were modified either by acid treatment (carboxylated, COOH) or nitrogenation (N-doped) and a SS vehicle. The pulmonary inflammation, serum cytokine profile and cardiac ischemic/reperfusion (I/R) injury were assessed at 1, 7 and 28 days post-aspiration. Results Pulmonary response to MWCNT oropharyngeal aspiration assessed by bronchoalveolar lavage fluid (BALF) revealed modest increases in protein and inflammatory cell recruitment. Lung histology showed modest tissue inflammation as compared to the SS group. Serum levels of eotaxin were significantly elevated in the carboxylated MWCNT aspirated mice 1 day post exposure. Oropharyngeal aspiration of all three forms of MWCNTs resulted in a time and/or dose-dependent exacerbation of myocardial infarction. The severity of myocardial injury varied with the form of MWCNTs used. The N-doped MWCNT produced the greatest expansion of the infarct at any time point and required a log concentration lower to establish a no effect level. The expansion of the I/R injury remained significantly elevated at 28 days following aspiration of the COOH and N-doped forms, but not the C-grade as compared to SS. Conclusion Our results suggest that oropharyngeal aspiration of MWCNT promotes increased susceptibility of cardiac tissue to ischemia/reperfusion injury without a significant pulmonary inflammatory response. The cardiac injury effects were observed at low concentrations of MWCNTs and presence of MWCNTs may pose a significant risk to the cardiovascular system.