Towards the Identification of an In Vitro Tool for Assessing the Biological Behavior of Aerosol Supplied Nanomaterials

Nanoparticles (NP)-based inhalation systems for drug delivery can be administered in liquid form, by nebulization or using pressurized metered dose inhalers, and in solid form by means of dry powder inhalers. However, NP delivery to the lungs has many challenges including the formulation instability due to particle-particle interactions and subsequent aggregation, causing poor deposition in the small distal airways and subsequent alveolar macrophages activity, which could lead to inflammation. This work aims at providing an in vitro experimental design for investigating the correlation between the physico-chemical properties of NP, and their biological behavior, when they are used as NP-based inhalation treatments, comparing two different exposure systems. By means of an aerosol drug delivery nebulizer, human lung cells cultured at air-liquid interface (ALI) were exposed to two titanium dioxide NP (NM-100 and NM-101), obtained from the JRC repository. In parallel, ALI cultures were exposed to NP suspension by direct inoculation, i.e., by adding the NP suspensions on the apical side of the cell cultures with a pipette. The formulation stability of NP, measured as hydrodynamic size distributions, the cell viability, cell monolayer integrity, cell morphology and pro-inflammatory cytokines secretion were investigated. Our results demonstrated that the formulation stability of NM-100 and NM-101 was strongly dependent on the aggregation phenomena that occur in the conditions adopted for the biological experiments. Interestingly, comparable biological data between the two exposure methods used were observed, suggesting that the conventional exposure coupled to ALI culturing conditions offers a relevant in vitro tool for assessing the correlation between the physico-chemical properties of NP and their biological behavior, when NP are used as drug delivery systems

Comprehensive In Vitro Toxicity Testing of a Panel of Representative Oxide Nanomaterials: First Steps towards an Intelligent Testing Strategy

Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO2 NMs with different surface chemistry – hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO – uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques – precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO2>TiO2). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially ‘weak-embryotoxic’ and ZnO and SiO2 NMs as ‘non-embryotoxic’. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103). This ranking was different in the case of embryonic tissues, for which TiO2 displayed higher toxicity compared with ZnO and SiO2. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects

Grouping Hypotheses and an Integrated Approach to Testing and Assessment of Nanomaterials Following Oral Ingestion

The risk assessment of ingested nanomaterials (NMs) is an important issue. Here we present nine integrated approaches to testing and assessment (IATAs) to group ingested NMs following predefined hypotheses. The IATAs are structured as decision trees and tiered testing strategies for each decision node to support a grouping decision. Implications (e.g., regulatory or precautionary) per group are indicated. IATAs integrate information on durability and biopersistence (dissolution kinetics) to specific hazard endpoints, e.g., inflammation and genotoxicity, which are possibly indicative of toxicity. Based on IATAs, groups of similar nanoforms (NFs) of a NM can be formed, such as very slow dissolving, highly biopersistent and systemically toxic NFs. Reference NMs (ZnO, SiO2 and TiO2) along with related NFs are applied as case studies to testing the oral IATAs. Results based on the Tier 1 level suggest a hierarchy of biodurability and biopersistence of TiO2 > SiO2 > ZnO, and are confirmed by in vivo data (Tier 3 level). Interestingly, our analysis suggests that TiO2 and SiO2 NFs are able to induce both local and systemic toxicity along with microbiota dysbiosis and can be grouped according to the tested fate and hazard descriptors. This supports that the decision nodes of the oral IATAs are suitable for classification and assessment of the toxicity of NFs

Verso l'identificazione dei determinanti strutturali di tossicità di nanoparticelle di silice amorfa e nanotubi di carbonio: uno studio in vitro

Nanomaterials (NM) contain particles, in an unbound state or as an aggregate or as an agglomerate, which, for a percentage of 50% or more, have one or more external dimensions in the range 1-100 nm. The great development of nanotechnology has produced an increasing quantity of nanomaterials of different types in several productive sectors (food, chemicals, pharmaceuticals). For this reason, several studies are aimed at characterizing the physical and chemical properties of nanomaterials and the determination of their effects on human health and the environment. Multi walled carbon nanotubes (MWCNT) and amorphous silica nanoparticles (ASNP) are examples of nanomaterials widely used in many industrial fields. The overall aim of this thesis is the elucidation of the potential hazards of MWCNT and ASNP, evaluating their interaction with relevant cell models. Attention is given to the assessment of potential toxic effects on cells of innate immunity and to the identification of structural determinants of toxicity. Since inhalation is the major way of interaction with nanomaterials, we decided to study the biological effects of MWCNT and ASNP on two cell lines (MH-S and RAW264.7), as representative models of macrophages, which are the first to contact the inhaled particles, and on airway epithelial cells (Calu-3), which represents one of the first body barriers encountered by nanomaterials dispersed in the environment. The first part of the thesis is focused on the identification of structural determinants of toxicity, in vitro, of four preparations of multi-walled carbon nanotubes with different length, morphology (rigid, needle-like or flexible, tangle-like shape), and level of metal contaminants. We have assessed the biological effects of the four MWCNT preparations (NM400, NM401, NM402 and MWCNT-SA) on macrophages and airway epithelial cells, in order to identify the determinants of toxicity, thus far incompletely elucidated. To study the biological effects of MWCNT on macrophage cell lines we analyzed different endpoints, such as cell viability, phagocytic activity and pro-inflammatory M1 macrophage activation. We found that the main determinants of toxicity for macrophages are the length and the needle-like shape, which hinder, or even prevent, phagocytosis. Indeed, the greater toxicity of NM401 and MWCNT-SA, as demonstrated by the decrease in cell viability and the alteration of functional activity, are ascribable to their greater length and to their morphological features. On the contrary, reduced length and tangle-like shape (NM400 and NM402) promote M1 macrophage activation. Since these materials can be engulfed by macrophages, these results suggest that phagocytosis is a main step for the M1 macrophage activation by nanomaterials, endowed with low acute toxicity. Given the high tendency of MWCNT to aggregate and the presence of aggregates in the airway walls of exposed animals, as reported in several in vivo studies, we have investigated if MWCNT produced a barrier impairment. The behavior of epithelial cells was studied both at the monolayer (cell population) and at the single-cell level. At a cell-population level, Trans-Epithelial Electrical Resistance (TEER) was used as a synthetic indicator of barrier competence, caspase activity was assessed with standard biochemical assays, and cell viability was investigated with both standard biochemical techniques and an high throughput (HTP) technique, based on automated epifluorescence microscopy; at single-cell level, cell responses to MWCNT were investigated with confocal microscopy, by evaluating cell death (calcein/propidium iodide), proliferation (Ki-67), inflammation triggering (NF-B) and apoptosis (caspase activity). We found that the main determinant of toxicity for epithelial cells depends on the actual shape in which MWCNT get in contact with the cells and, in particular, if they form aggregates. The second part of the thesis is focused on the identification of structural determinants of toxicity of two preparations of amorphous silica nanoparticles (ASNP, a material usually considered endowed with modest toxicity). This study has evaluated the capability of ASNP, of comparable size and specific surface area, but produced through different synthetic procedures (colloidal NM200 vs pyrogenic NM203), to induce macrophage activation in MH-S and RAW264.7 cell lines. To study the biological effects of ASNP we analyzed different endpoints, such as cell viability, oxidative stress (ROS formation and the induction of Hmox-1), the induction of the inducible nitric oxide synthase Nos2, the production of NO and the secretion of cytokines like TNF-α, IL-6 and IL-1β. Helium Ion microscopy (HIM) and confocal microscopy were adopted for imaging the interaction between ASNP and the cell surface. The results demonstrate that pyrogenic ASNP are more potentially inflammogenic than colloidal ASNP. Moreover, an additional mechanism of toxicity is proposed, consisting in the greater capability of pyrogenic ASNP to bind biologically active compounds, such as LPS, enhancing their effects. Thus we found that the preparation route procedure may constitute a main determinant of toxicity of ASNP, likely because of the different surface chemistry established by high-temperature synthesis. In conclusion, this thesis highlights that determinants of toxicity of nanomaterials are strongly dependent on several parameters. The identification of these determinants, which appear essential for a “safety-by-design” approach, will therefore require an in-depth characterization of the toxicological properties of each type of nanomaterial.Con il termine “nanomateriali” si intendono materiali contenenti particelle in stato libero o aggregato delle quali almeno il 50% abbia dimensioni comprese tra 1 e 100 nm. Il grande sviluppo delle nanotecnologie ha immesso sul mercato nanomateriali di vario genere in diversi settori produttivi (alimentare, chimico, farmaceutico) ed in numero sempre crescente. Per questo motivo, diversi studi si pongono come obiettivo la caratterizzazione delle proprietà chimico-fisiche dei nanomateriali e la determinazione dei loro effetti sull’ambiente e sulla salute umana. Esempi di nanomateriali ampiamente usati in campo industriale sono i nanotubi di carbonio a parete multipla (MWCNT) e nanoparticelle di silice amorfa (ASNP). Lo scopo di questa tesi è quello di chiarire i potenziali rischi degli MWCNT ed ASNP, valutando la loro interazione con determinati modelli cellulari. In particolare, l’attenzione è stata focalizzata sulla valutazione dei potenziali effetti tossici su cellule dell’immunità innata e sull’identificazione dei determinanti strutturali di tossicità. Dal momento che l’inalazione è la maggiore via d’interazione con i nanomateriali, le prime cellule che interagiscono con queste particelle sono i macrofagi e le cellule dell’epitelio respiratorio. Per questo motivo, gli effetti biologici degli MWCNT ed ASNP sono stati studiati su tre linee cellulari, rappresentativi di questi tipi cellulari: i macrofagi MH-S e RAW264.7 e le cellule epiteliali bronchiali Calu-3. La prima parte della tesi è incentrata sull’identificazione dei determinanti strutturali di tossicità, in vitro, di quattro preparazioni di MWCNT (NM400, NM401, NM402 e MWCNT-SA) che differiscono per lunghezza, morfologia (rigida, needle-like o flessibile, tangle-like) e livelli di contaminanti metallici. Abbiamo valutato gli effetti biologici degli MWCNT sui macrofagi e cellule epiteliali, allo scopo di identificare i determinanti di tossicità ancora non completamente chiariti. Per studiare gli effetti biologici sui macrofagi, sono stati valutati i seguenti endpoints: vitalità cellulare, funzionalità macrofagica ed attivazione macrofagica di tipo classico (M1). Nel complesso i risultati ottenuti mostrano che, i principali determinanti di tossicità per i macrofagi, sono la lunghezza e la conformazione needle-like, la quale ostacola o addirittura impedisce la fagocitosi. Infatti, la maggiore tossicità di NM401 e degli MWCNT-SA, come dimostrato dalla diminuzione di vitalità cellulare e dall’alterazione dell’attività funzionale dei macrofagi, è riconducibile alla loro maggiore lunghezza e alla loro morfologia. Al contrario, la lunghezza ridotta e la conformazione tangle-like (NM400 e NM402) promuovono l’attivazione macrofagica M1. Dal momento che questi materiali sono fagocitabili dai macrofagi, questi risultati suggeriscono che la fagocitosi rappresenta il principale step per l’attivazione macrofagica di tipo M1, per i materiali con bassa tossicità. In seguito abbiamo verificato se gli MWCNT producessero un’alterazione della barriera epiteliale, in quanto è ben noto in letteratura che questi tendono ad aggregare e in tale forma persistere sulla parete dell’epitelio respiratorio. Il comportamento delle cellule epiteliali è stato studiato sia a livello di monostrato (popolazione cellulare) che a livello della singola cellula. A livello di popolazione cellulare, la resistenza elettrica trans-epiteliale (TEER) è stata usata come indicatore delle competenze di barriera; l’attività caspasica è stata misurata con un saggio biochimico e la vitalità cellulare è stata misurata sia con un saggio biochimico sia con tecniche di alta risoluzione (HTP), basate sulla microscopia automatizzata a epifluorescenza. A livello della singola cellula, gli effetti biologici degli MWCNT sono stati studiati utilizzando la microscopia confocale valutando la morte cellulare (calceina/propidio), la proliferazione (Ki-67), l’innesco di fenomeni infiammatori (NF-κB) e l’apoptosi ( attività caspasica). I nostri dati mostrano che il principale determinante di tossicità per le cellule epiteliali dipende dalla conformazione con la quale gli MWCNT entrano in contatto con le cellule, in particolare, se gli MWCNT formano aggregati. La seconda parte della tesi è incentrata sull’identificazione dei determinanti strutturali di tossicità di due preparazioni di ASNP ( materiali con modesta tossicità). Questo studio, ha valutato la capacità delle ASNP, con dimensioni ed area di superficie simili ma prodotti tramite processi diversi, (colloidale NM200 vs. pirogenica NM203), di indurre attivazione macrofagica nelle linee cellulari MH-S e RAW264.7. Per studiare gli effetti biologici di queste due preparazioni sono stati valutati i seguenti endpoints: vitalità cellulare, lo stress ossidativo (produzione di ROS e induzione di Hmox-1), l’induzione dell’ossido nitrico sintetasi (Nos2), la produzione di ossido nitrico (NO), la secrezione delle citochine TNF-α, IL-6 e IL-1β. Il microscopio confocale e quello ad elio (HIM) sono stati utilizzati per studiare l’interazione tra ASNP e superficie cellulare. I risultati dimostrano che le ASNP pirogeniche sono molto più inflammogeniche delle colloidali. Inoltre, è stato proposto un altro meccanismo di tossicità, che consiste nella maggiore capacità delle ASNP pirogeniche di legare composti biologicamente attivi, come l’LPS, aumentandone i loro effetti. Per questo motivo, il principale determinante di tossicità delle ASNP, è il procedimento al alta temperatura con la quale vengono prodotte le ASNP pirogeniche. In conclusione, questa tesi evidenzia che i determinati di tossicità dei nanomateriali sono fortemente dipendenti da diversi parametri. L’identificazione di questi determinanti, che appare essenziale per il cosidetto “safety-by-design approch”, richiede, quindi, una profonda caratterizzazione delle proprietà tossicologiche di ogni tipo di nanomateriale

A Human-Relevant 3D In Vitro Platform for an Effective and Rapid Simulation of Workplace Exposure to Nanoparticles: Silica Nanoparticles as Case Study

In this contribution, we show the suitability of a 3D airway model, when coupled with a nebulizer system, for simulating workplace exposure to nanoparticles. As a proof of concept, workplace exposure to silica nanoparticles was experimentally measured in an occupational facility where nanoparticles are produced weekly, and compared with the official limit value for bulk silica materials. These values of potential exposure were simulated in a 3D airway model by nebulizing low doses (from 0.90 to 55 µg/cm2) of silica nanoparticles over a prolonged period (12 weeks of repeated exposure, 5 days per week). Overall, the results suggest the efficiency of the defense mechanisms of the respiratory system and the clearance of the breathed silica nanoparticles by the mucociliary apparatus in accordance with the recent in vivo data. This in vitro platform shows that the doses tested may correlate with the occupational exposure limit values. Such relationship could provide regulatory-oriented data useful for risk classification of nanomaterials

TiO2 Nanoparticles synergistically increase LPS-induced NO Production by Macrophages

Although TiO2 NPs particles are endowed with little acute toxicity in vitro, they exert pro-inflammatory effects when inhaled in vivo. In this study, we evaluated the effects of several TiO2NPs, alone or in combination with LPS, on Raw264.7 macrophages in terms of cell viability (determined up to 72h with resazurin assay), NO production (estimated by nitrite concentration in the culture medium after 48h and 72h of treatment), and Nos2 induction, assessed with RT-PCR after 24h of treatment. We tested three types of TiO2 NP: a) pristine TiO2 NP of industrial origin (84% anatase/16% brookite, average NP size 45 nm), b) citrate-coated TiO2 NP, derived from pristine TiO2 NPs (average NP size 64 nm), c) Aeroxide P25 TiO2 NPs (80% anatase/20% rutile, average NP size 25 nm). All the TiO2 NPs did not affect significantly cell viability even at the highest doses tested (IC50>80 µg/cm2 at 24,48 and 72h). On the contrary, all the NP preparations, at the dose of 20 µg/cm2, induced Nos2. In particular, Nos2 was 3-fold, 8-fold and 4-fold induced by pristine, citrate coated and Aeroxide P25 TiO2 NPs alone, while fold-induction rose to 13 for pristine and citrate-coated NPs and to 30 for Aeroxide P25 NPs in the presence of 1 ng/ml LPS. LPS alone caused a 6-fold increase in Nos2 expression. The effects of pristine, citrate-coated and Aeroxide P25 TiO2 NPs on NO production were clearly dose- and time-dependent. At 80 g/cm2 of NP, nitrites were 5-fold, 9-fold and 7-fold higher than in the untreated control at 48h and 7-fold,10-fold and 8-fold higher than control at 72h. For all the TiO2NPs tested, the No-Observed-Effect-Level (NOEL) was 20 g/cm2 at 48h and 10 g/cm2 at 72h. After 48h-incubation with LPS (1 ng/ml) and TiO2 (80 µg/cm2), nitrite medium concentration was 31-fold, 30-fold and 25-fold higher than control with, respectively, pristine, citrate-coated and Aeroxide P25. LPS alone caused a 22-fold increase in medium nitrites. Several TiO2 NPs significantly induce Nos2 gene expression and increase NO production in a dose- and time-dependent manner in Raw264.7 cells. The enhancement of Nos2 induction by LPS suggests that the pro-inflammatory effect of TiO2 NPs in vivo may be exacerbated by concomitant infectious conditions and surface interactions with bacterial endotoxins. Supported by EU Grant NMP4-SL-2012-280716 (Sanowork Project

Silver nanoparticles as a medical device in healthcare settings: A five-step approach for candidate screening of coating agents

Silver nanoparticle-based antimicrobials can promote a long lasting bactericidal effect without detrimental toxic side effects. However, there is not a clear and complete protocol to define and relate the properties of the particles (size, shape, surface charge, ionic content) with their specific activity. In this paper, we propose an effective multi-step approach for the identification of a â\u80\u98purpose-specific active applicability windowâ\u80\u99 to maximize the antimicrobial activity of medical devices containing silver nanoparticles (Ag NPs) (such as surface coaters), minimizing any consequent risk for human health (safety by design strategy). The antimicrobial activity and the cellular toxicity of four types of Ag NPs, differing in their coating composition and concentration have been quantified. Through the implementation of flow-field flow fractionation, Ag NPs have been characterized in terms of metal release, size and shape. The particles are fractionated in the process while being left unmodified, allowing for the identification of biological particle-specific contribution. Toxicity and inflammatory response in vitro have been assessed on human skin models, while antimicrobial activity has been monitored with both non-pathogenic and pathogenic Escherichia coli. The main benefit associated with such approach is the comprehensive assessment of the maximal effectiveness of candidate nanomaterials, while simultaneously indexing their properties against their safety

Identifying contact-mediated, localized toxic effects of MWCNT aggregates on epithelial monolayers: A single-cell monitoring toxicity assay

Aggregates of multiwalled carbon nanotubes (MWCNT) impair the barrier properties of human airway cell monolayers. To resolve the mechanism of the barrier alteration, monolayers of Calu-3 human airway epithelial cells were exposed to aggregated MWCNT. At the cell-population level, trans-epithelial electrical resistance (TEER) was used as an indicator of barrier competence, caspase activity was assessed with standard biochemical assays, and cell viability was investigated by biochemical techniques and high-throughput (HTP) technique based on automated epifluorescence microscopy. At cell level, the response to MWCNT was investigated with confocal microscopy, by evaluating cell death (calcein/propidium iodide (PI)), proliferation (Ki-67), and apoptosis (caspase activity). At the cell-population level, exposure to aggregated MWCNT caused a decrease in TEER, which was not associated with a decrease in cell viability or onset of apoptosis even after an 8-d exposure. In contrast, confocal imaging demonstrated contact with MWCNT aggregates triggered cell death after 24 h of exposure. In the presence of a natural surfactant, both TEER decrease and contact-mediated toxicity were mitigated. With confocal imaging, increased proliferation and apoptosis were detected in Calu-3 cells next to the aggregates. Contact-mediated cytotoxicity was recorded in two additional cell lines (BEAS-2B and A549) derived from human airways. Similar results were confirmed by adopting two additional MWCNT preparations with different physico-chemical features. This indicates MWCNT caused localized damage to airway epithelial monolayers in vitro and altered the apoptotic and proliferative rate of epithelial cells in close proximity to the aggregates. These findings provide evidence on the pathway by which MWCNT aggregates impair airway barrier function, and support the use of imaging techniques as a possible regulatory-decision supporting tool to identify effects of aggregated nanomaterials not readily detected at cell population level