Critical aspects in occupational exposure assessment with different aerosol metrics in an industrial spray coating process.

Engineered Nanomaterials (ENMs) have several uses in various industrial fields and are embedded in a myriad of consumer products. However, there is continued concern over the potential adverse health effects and environmental impacts of ENMs due to their unique physico-chemical characteristics. Currently, there are no specific international regulations for various ENMs. There are also no Occupational Exposure Limits (OEL) regulated by the European Union (EU) for nanomaterials in the form of nano-objects, their aggregates or agglomerates (NOAA). For ENMs the question of which metric to be used (i.e., mass, surface area, number concentrations) to determine the exposure is still not resolved. The aim of this work is to assess the worker exposure by inhalation in an industrial spray coating process by using all three metrics mentioned above. Two target ENMs (N-doped TiO2, TiO2N and AgNPs capped with a quaternized hydroxyethyl-cellulose, AgHEC) generated for industrial-scale spraying processes were considered. Results showed that the averaged particle number concentration (10-100 nm) was below 2.7 104 cm-3 for both materials. The Lung Deposited Surface Area (LDSA) was in the range between 73 and 98 µm2cm-3 and the particle mass concentration (obtained by means of ICP-EOS off-line analysis) resulted below 70 µg m-3 and 0.4 µg m-3 for TiO2 and Ag, respectively. Although, the airborne particles concentration compared well with the NIOSH Recommended Exposure Level (REL) limits the contribution to the background, according to EN 17058 (Annex E) was significant (particularly in the particle number and PM1 mass concentrations). We successfully evaluated the worker exposure by means of the different airborne particles' metrics (number, surface and mass concentrations). We concluded that worker exposure assessment involving ENMs is a complex procedure with requires both real time and off-line measurements and a deep investigation of the background.

Design of TiO2-Surfactin Hybrid Systems with Multifunctional Properties.

In recent years, multifunctional inorganic-organic hybrid materials have been widely investigated in order to determine their potential synergetic, antagonist, or independent effects in terms of reactivity. The aim of this study was to design and characterize a new hybrid material by coupling well-known photocatalytic TiO2 nanoparticles with sodium surfactin (SS), a biosurfactant showing high binding affinity for metal cations as well as the ability to interact with and disrupt microorganisms' cell membranes. We used both chemical and colloidal synthesis methodologies and investigated how different TiO2:SS weight ratios affected colloidal, physicochemical, and functional properties. We discovered a clear breaking point between TiO2 and SS single-component trends and identified different ranges of applicability by considering different functional properties such as photocatalytic, heavy metal sorption capacity, and antibacterial properties. At low SS contents, the photocatalytic properties of TiO2 are preserved (conversion of organic dye = 99% after 40 min), and the hybrid system can be used in advanced oxidation processes, taking advantage of the additional antimicrobial SS properties. At high SS contents, the TiO2 photoactivity is inhibited, and the hybrid can be usefully exploited as a UV blocker in cosmetics, avoiding undesired oxidative effects (UV adsorption in the range between 300-400 nm). Around the breaking point (TiO2:SS 1:1), the hybrid material preserves the high surface area of TiO2 (specific surface area around 180 m2/g) and demonstrates NOx depletion of up to 100% in 80 min, together with improved adhesion of hybrid antibacterial coating. The last design demonstrated the best results for the concurrent removal of inorganic, organic, and biological pollutants in water/soil remediation applications.

Native Study of the Behaviour of Magnetite Nanoparticles for Hyperthermia Treatment during the Initial Moments of Intravenous Administration.

Magnetic nanoparticles (MNPs) present outstanding properties making them suitable as therapeutic agents for hyperthermia treatments. Since the main safety concerns of MNPs are represented by their inherent instability in a biological medium, strategies to both achieve long-term stability and monitor hazardous MNP degradation are needed. We combined a dynamic approach relying on flow field flow fractionation (FFF)-multidetection with conventional techniques to explore frame-by-frame changes of MNPs injected in simulated biological medium, hypothesize the interaction mechanism they are subject to when surrounded by a saline, protein-rich environment, and understand their behaviour at the most critical point of intravenous administration. In the first moments of MNPs administration in the patient, MNPs change their surrounding from a favorable to an unfavorable medium, i.e., a complex biological fluid such as blood; the particles evolve from a synthetic identity to a biological identity, a transition that needs to be carefully monitored. The dynamic approach presented herein represents an optimal alternative to conventional batch techniques that can monitor only size, shape, surface charge, and aggregation phenomena as an averaged information, given that they cannot resolve different populations present in the sample and cannot give accurate information about the evolution or temporary instability of MNPs. The designed FFF method equipped with a multidetection system enabled the separation of the particle populations providing selective information on their morphological evolution and on nanoparticle-proteins interaction in the very first steps of infusion. Results showed that in a dynamic biological setting and following interaction with serum albumin, PP-MNPs retain their colloidal properties, supporting their safety profile for intravenous administration.

Measuring TiO2N and AgHEC Airborne Particle Density during a Spray Coating Process.

Effective particle density is a key parameter for assessing inhalation exposure of engineered NPs in occupational environments. In this paper, particle density measurements were carried out using two different techniques: one based on the ratio between mass and volumetric particle concentrations; the other one based on the ratio between aerodynamic and geometric particle diameter. These different approaches were applied to both field- and laboratory-scale atomization processes where the two target NPs (N-doped TiO2, TiO2N and AgNPs capped with a quaternized hydroxyethylcellulose, AgHEC) were generated. Spray tests using TiO2N were observed to release more and bigger particles than tests with AgHEC, as indicated by the measured particle mass concentrations and volumes. Our findings give an effective density of TiO2N particle to be in a similar range between field and laboratory measurements (1.8 ± 0.5 g/cm3); while AgHEC particle density showed wide variations (3.0 ± 0.5 g/cm3 and 1.2 + 0.1 g/cm3 for field and laboratory campaigns, respectively). This finding leads to speculation regarding the composition of particles emitted because atomized particle fragments may contain different Ag-to-HEC ratios, leading to different density values. A further uncertainty factor is probably related to low process emissions, making the subtraction of background concentrations from AgHEC process emissions unreliable.

Particles Emission from an Industrial Spray Coating Process Using Nano-Materials.

Industrial spray coating processes are known to produce excellent coatings on large surfaces and are thus often used for in-line production. However, they could be one of the most critical sources of worker exposure to ultrafine particles (UFPs). A monitoring campaign at the Witek s.r.l. (Florence, Italy) was deployed to characterize the release of TiO2 NPs doped with nitrogen (TiO2-N) and Ag capped with hydroxyethyl cellulose (AgHEC) during automatic industrial spray-coating of polymethyl methacrylate (PMMA) and polyester. Aerosol particles were characterized inside the spray chamber at near field (NF) and far field (FF) locations using on-line and off-line instruments. Results showed that TiO2-N suspension produced higher particle number concentrations than AgHEC in the size range 0.3-1 µm (on average 1.9 102 p/cm3 and 2.5 101 p/cm3, respectively) after background removing. At FF, especially at worst case scenario (4 nozzles, 800 mL/min flow rate) for TiO2-N, the spray spikes were correlated with NF, with an observed time lag of 1 minute corresponding to a diffusion speed of 0.1 m/s. The averaged ratio between particles mass concentrations in the NF position and inside the spray chamber was 1.7% and 1.5% for TiO2-N and for AgHEC suspensions, respectively. The released particles' number concentration of TiO2-N in the size particles range 0.3-1 µm was comparable for both PMMA and polyester substrates, about 1.5 and 1.6 102 p/cm3. In the size range 0.01-30 µm, the aerosol number concentration at NF for both suspensions was lower than the nano reference values (NRVs) of 16·103 p/cm-3.

Monitoring and Optimisation of Ag Nanoparticle Spray-Coating on Textiles.

An automatic lab-scaled spray-coating machine was used to deposit Ag nanoparticles (AgNPs) on textile to create antibacterial fabric. The spray process was monitored for the dual purpose of (1) optimizing the process by maximizing silver deposition and minimizing fluid waste, thereby reducing suspension consumption and (2) assessing AgNPs release. Monitoring measurements were carried out at two locations: inside and outside the spray chamber (far field). We calculated the deposition efficiency (E), finding it to be enhanced by increasing the spray pressure from 1 to 1.5 bar, but to be lowered when the number of operating sprays was increased, demonstrating the multiple spray system to be less efficient than a single spray. Far-field AgNPs emission showed a particle concentration increase of less than 10% as compared to the background level. This finding suggests that under our experimental conditions, our spray-coating process is not a critical source of worker exposure.

Polyvinyl alcohol/silver electrospun nanofibers: Biocidal filter media capturing virus-size particles.

In response to the nowadays battle against SARS-CoV-2, we designed a new class of high performant filter media suitable to advance the facemask technology and provide new efficient widespread solutions against virus propagation. By means of the electrospinning technology we developed filter media based on polyvinyl alcohol (PVA) nanofibers doped with AgNPs combining three main performance requirements: high air filtration efficiency to capture nanometer-size particles, low airflow resistance essential to ensure breathability and antimicrobial activity to inactivate aerosolized microorganisms. PVA/AgNPs electrospun nanofibers were produced by electrospinning the dispersion of colloidal silver into the PVA water solution. A widespread physicochemical characterization was addressed to the Ag colloidal suspension. The key functional performances of the electrospun nanofibers were proven by water stability, antibacterial activity, and filtration efficiency and pressure drop measurements performed under conditions representative of facemasks. We assessed a total bacterial depletion associated to a filtering efficiency towards nano-aerosolized particles of 97.7% higher than required by the EN149 standard and a pressure drop in line with FFP1 and FFP2 masks, even at the highest filtration velocity. Such results pave the way to the application of PVA/AgNPs electrospun nanofibers in facemasks as advanced filtering media for protecting against airborne microorganisms.

TiO2@BSA nano-composites investigated through orthogonal multi-techniques characterization platform.

Biocompatible coating based on bovine serum albumin (BSA) was applied on two different TiO2 nanoparticles (aeroxide P25 and food grade E171) to investigate properties and stability of resulting TiO2@BSA composites, under the final perspective to create a "Safe-by-Design" coating, able to uniform, level off and mitigate surface chemistry related phenomena, as naturally occurring when nano-phases come in touch with proteins enriched biological fluids. The first step towards validating the proposed approach is a detailed characterization of surface chemistry with the quantification of amount and stability of BSA coating deposited on nanoparticles' surfaces. At this purpose, we implemented an orthogonal multi-techniques characterization platform, providing important information on colloidal behavior, particle size distribution and BSA-coating structure of investigated TiO2 systems. Specifically, the proposed orthogonal approach enabled the quantitative determination of bound and free (not adsorbed) BSA, a key aspect for the design of intentionally BSA coated nano-structures, in nanomedicine and, overall, for the control of nano-surface reactivity. In fact, the BSA-coating strategy developed and the orthogonal characterisation performed can be extended to different designed nanomaterials in order to further investigate the protein-corona formation and promote the implementation of BSA engineered coating as a strategy to harmonize the surface reactivity and minimize the biological impact.

Photocatalytic Oxidation of HMF under Solar Irradiation: Coupling of Microemulsion and Lyophilization to Obtain Innovative TiO2-Based Materials.

The photocatalytic oxidation of biomass-derived building blocks such as 5-hydroxymethylfurfural (HMF) is a promising reaction for obtaining valuable chemicals and the efficient long-term storage of solar radiation. In this work, we developed innovative TiO2-based materials capable of base-free HMF photo-oxidation in water using simulated solar irradiation. The materials were prepared by combining microemulsion and spray-freeze drying (SFD), resulting in highly porous systems with a large surface area. The effect of titania/silica composition and the presence of gold-copper alloy nanoparticles on the properties of materials as well as photocatalytic performance were evaluated. Among the lab-synthesized photocatalysts, Ti15Si85 SFD and Au3Cu1/Ti15Si85 SFD achieved the higher conversions, while the best selectivity was observed for Au3Cu1/Ti15Si85 SFD. The tests with radical scavengers for both TiO2-m and Au3Cu1/Ti15Si85 SFD suggested that primary species responsible for the selective photo-oxidation of HMF are photo-generated electrons and/or superoxide radicals.

Use of Cotton Textiles Coated by Ir(III) Tetrazole Complexes within Ceramic Silica Nanophases for Photo-Induced Self-Marker and Antibacterial Application.

This study was aimed at the production and characterization of coated cotton textiles with luminescent ceramic nanophases doped with cationic Ir(III) tetrazole complexes. We confirmed that SiO2 nanoparticles (NPs) do not affect the phosphorescent properties of the complexes that maintain their emission (610 and 490 nm). For the first time we transferred the luminescence feature from nanosol to textile surface, highlighting the advantages of using nanosilica as an encapsulating and stabilizing matrix. The optimized Ir@SiO2 suspensions were homogenously applied onto the cotton surface by dip-pad-dry-cure technique, as proved by the 2p-fluorescence microscope analysis. Once we verified the self-marker properties of the Ir(III) complex, we observed an excellent washing fastness of the coating with a very limited release. SiO2 in the washing water was quantified at maximum around 1.5 wt% and Ir below the inductively coupled plasma optical emission spectrometry (ICP-OES) detection limit of 1 ppm. A Franz cell test was used to evaluate any possible ex-vivo uptake of Ir@SiO2 nanoparticles across human skin tissues, showing that epidermis and dermis stop over 99% of Ir, implying a reduced impact on human health. The light-induced antimicrobial potential of the Ir@SiO2 were assessed toward both Gram(-) and Gram(+) bacteria. The results encouraged further developments of such functional textiles coated by self-markers and antibacterial active nanophases.

Length-dependent toxicity of TiO2 nanofibers: mitigation via shortening.

Length and aspect ratio represent important toxicity determinants of fibrous nanomaterials. We have previously shown that anatase TiO2 nanofibers (TiO2 NF) cause a dose-dependent decrease of cell viability as well as the loss of epithelial barrier integrity in polarized airway cell monolayers. Herein we have investigated the impact of fiber shortening, obtained by ball-milling, on the biological effects of TiO2 NF of industrial origin. Long TiO2 NF (L-TiO2 NF) were more cytotoxic than their shortened counterparts (S-TiO2 NF) toward alveolar A549 cells and bronchial 16HBE cells. Moreover, L-TiO2 NF increased the permeability of 16HBE monolayers and perturbed the distribution of tight-junction proteins, an effect also mitigated by fiber shortening. Raw264.7 macrophages efficiently internalized shortened but not long NF, which caused cell stretching and deformation. Compared with L-TiO2 NF, S-TiO2 NF triggered a more evident macrophage activation, an effect suppressed by the phagocytosis inhibitor cytochalasin B. Conversely, a significant increase of inflammatory markers was detected in either the lungs or the peritoneal cavity of mice exposed to L-TiO2 NF but not to S-TiO2 NF, suggesting that short-term macrophage activation in vitro may not be always a reliable indicator of persistent inflammation in vivo. It is concluded that fiber shortening mitigates NF detrimental effects on cell viability and epithelial barrier competence in vitro as well as inflammation development in vivo. These data suggest that fiber shortening may represent an effective safe-by-design strategy for mitigating TiO2 NF toxic effects.

In Vitro Toxicity of TiO2:SiO2 Nanocomposites with Different Photocatalytic Properties.

The enormous technological relevance of titanium dioxide (TiO2) nanoparticles (NPs) and the consequent concerns regarding potentially hazardous effects that exposure during production, use, and disposal can generate, encourage material scientists to develop and validate intrinsically safe design solution (safe-by-design). Under this perspective, the encapsulation in a silica dioxide (SiO2) matrix could be an effective strategy to improve TiO2 NPs safety, preserving photocatalytic and antibacterial properties. In this work, A549 cells were used to investigate the toxic effects of silica-encapsulated TiO2 having different ratios of TiO2 and SiO2 (1:1, 1:3, and 3:1). NPs were characterized by electron microscopy and dynamic light scattering, and cell viability, oxidative stress, morphological changes, and cell cycle alteration were evaluated. Resulting data demonstrated that NPs with lower content of SiO2 are able to induce cytotoxic effects, triggered by oxidative stress and resulting in cell necrosis and cell cycle alteration. The physicochemical properties of NPs are responsible for their toxicity. Particles with small size and high stability interact with pulmonary cells more effectively, and the different ratio among silica and titania plays a crucial role in the induced cytotoxicity. These results strengthen the need to take into account a safe(r)-by-design approach in the development of new nanomaterials for research and manufacturing.

NanoTiO2@DNA complex: a novel eco, durable, fire retardant design strategy for cotton textiles.

A strategy for the design of cotton flame retardant coatings is described, exploiting the natural intumescent formulation of nucleic acids, the intrinsic thermal inertia of TiO2 ceramic phase and the strong affinity that TiO2 nanoparticles shows for amphiphilic biomolecules (e.g. proteins, nucleic acids) and hydrophilic substrates. Two different self-assembled TiO2@DNA systems are obtained by mixing two opposite charged TiO2 nanoparticles suspensions with DNA solution. The colloidal behavior of separate and mixed phases was investigated, as well as the morphological, chemical structure and thermal properties of resulting coatings, in order to make some hypothesis on the observed synergic effects. Scanning electron microscopy (SEM) and Attenuated Total Reflectance (ATR) spectroscopy demonstrated the homogeneous distribution of nano-TiO2@DNA based coatings on the treated fabrics. The washing fastness tests evidenced the highly improved performance of hybrid coatings if compared with DNA alone. The flammability and cone calorimetry test results confirmed this positive synergy that resulted quite effective in slowing down (or even blocking) the propagation of a flame within the treated substrates and in improving the resistance of the fabrics to the applied heat flux, these latter leaving increased residues at the end of the test.

Selective Oxidation of HMF via Catalytic and Photocatalytic Processes Using Metal-Supported Catalysts.

In this study, 5-hydroxymethylfurfural (HMF) oxidation was carried out via both the catalytic and the photocatalytic approach. Special attention was devoted to the preparation of the TiO2-based catalysts, since this oxide has been widely used for catalytic and photocatalytic application in alcohol oxidation reactions. Thus, in the catalytic process, the colloidal heterocoagulation of very stable sols, followed by the spray-freeze-drying (SFD) approach, was successfully applied for the preparation of nanostructured porous TiO2-SiO2 mixed-oxides with high surface areas. The versatility of the process made it possible to encapsulate Pt particles and use this material in the liquid-phase oxidation of HMF. The photocatalytic activity of a commercial titania and a homemade oxide prepared with the microemulsion technique was then compared. The influence of gold, base addition, and oxygen content on product distribution in the photocatalytic process was evaluated.

Dip coating of air purifier ceramic honeycombs with photocatalytic TiO2 nanoparticles: A case study for occupational exposure.

Nanoscale TiO2 (nTiO2) is manufactured in high volumes and is of potential concern in occupational health. Here, we measured workers exposure levels while ceramic honeycombs were dip coated with liquid photoactive nanoparticle suspension and dried with an air blade. The measured nTiO2 concentration levels were used to assess process specific emission rates using a convolution theorem and to calculate inhalation dose rates of deposited nTiO2 particles. Dip coating did not result in detectable release of particles but air blade drying released fine-sized TiO2 and nTiO2 particles. nTiO2 was found in pure nTiO2 agglomerates and as individual particles deposited onto background particles. Total particle emission rates were 420×109min-1, 1.33×109µm2min-1, and 3.5mgmin-1 respirable mass. During a continued repeated process, the average exposure level was 2.5×104cm-3, 30.3µm2cm-3, <116µgm-3 for particulate matter. The TiO2 average exposure level was 4.2µgm-3, which is well below the maximum recommended exposure limit of 300µgm-3 for nTiO2 proposed by the US National Institute for Occupational Safety and Health. During an 8-hour exposure, the observed concentrations would result in a lung deposited surface area of 4.3×10-3cm2g-1 of lung tissue and 13µg of TiO2 to the trachea-bronchi, and alveolar regions. The dose levels were well below the one hundredth of the no observed effect level (NOEL1/100) of 0.11cm2g-1 for granular biodurable particles and a daily no significant risk dose level of 44µgday-1. These emission rates can be used in a mass flow model to predict the impact of process emissions on personal and environmental exposure levels.

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 'purpose-specific active applicability window' 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.

Toxicity of surface-modified copper oxide nanoparticles in a mouse macrophage cell line: Interplay of particles, surface coating and particle dissolution.

The rapid dissolution of copper oxide (CuO) nanoparticles (NPs) with release of ions is thought to be one of the main factors modulating their toxicity. Here we assessed the cytotoxicity of a panel of CuO NPs (12 nm ±â€¯4 nm) with different surface modifications, i.e., anionic sodium citrate (CIT) and sodium ascorbate (ASC), neutral polyvinylpyrrolidone (PVP), and cationic polyethylenimine (PEI), versus the pristine (uncoated) NPs, using a murine macrophage cell line (RAW264.7). Cytotoxicity, reactive oxygen species (ROS) production, and cellular uptake were assessed. The cytotoxicity results were analyzed by the benchmark dose (BMD) method and the NPs were ranked based on BMD20 values. The PEI-coated NPs were found to be the most cytotoxic. Despite the different properties of the coating agents, NP dissolution in cell medium was only marginally affected by surface modification. Furthermore, CuCl2 (used as an ion control) elicited significantly less cytotoxicity when compared to the CuO NPs. We also observed that the antioxidant, N-acetylcysteine, failed to protect against the cytotoxicity of the uncoated CuO NPs. Indeed, the toxicity of the surface-modified CuO NPs was not directly linked to particle dissolution and subsequent Cu burden in cells, nor to cellular ROS production, although CuO-ASC NPs, which were found to be the least cytotoxic, yielded lower levels of ROS in comparison to pristine NPs. Hierarchical cluster analysis suggested, instead, that the toxicity in the current in vitro model could be explained by synergistic interactions between the NPs, their dissolution, and the toxicity of the coating agents.

Silica modification of titania nanoparticles enhances photocatalytic production of reactive oxygen species without increasing toxicity potential in vitro.

Titania (TiO2) nanoparticles were surface modified using silica and citrate to implement a 'safe-by-design' approach for managing potential toxicity of titania nanoparticles by controlling surface redox reactivity. DLS and zeta-potential analyses confirmed the surface modification, and electron microscopy and surface area measurements demonstrated nanoscale dimensions of the particles. Electron paramagnetic resonance (EPR) was used to determine the exogenous generation of reactive oxygen species (ROS). All the produced spray dried nanotitania lowered levels of ROS when compared to the corresponding dispersed nanotitania, suggesting that the spray drying process is an appropriate design strategy for the control of nano TiO2 ROS reactivity. The modification of nanotitania with silica and with citrate resulted in increased levels of ROS generation in exogenous measurements, including photoexcitation for 60 minutes. The dichlorodihydrofluorescein (DCFH) assay of dose-dependent production of oxidative stress, generated by pristine and modified nanotitania in macrophages and alveolar epithelial cells, found no significant change in toxicity originating from the generation of reactive oxygen species. Our findings show that there is no direct correlation between the photocatalytic activity of nanotitania and its oxidative stress-mediated potential toxicity, and it is possible to improve the former, for example adding silica as a modifying agent, without altering the cell redox equilibrium.

Lipopolysaccharide Adsorbed to the Bio-Corona of TiO2 Nanoparticles Powerfully Activates Selected Pro-inflammatory Transduction Pathways.

It is known that the adsorption of bioactive molecules provides engineered nanoparticles (NPs) with novel biological activities. However, the biological effects of the adsorbed molecules may also be modified by the interaction with NP. Bacterial lipopolysaccharide (LPS), a powerful pro-inflammatory compound, is a common environmental contaminant and is present in several body compartments such as the gut. We recently observed that the co-incubation of LPS with TiO2 NPs markedly potentiates its pro-inflammatory effects on murine macrophages, suggesting that, when included in a NP bio-corona, LPS activity is enhanced. To distinguish the effects of adsorbed LPS from those of the free endotoxin, a pellet fraction, denominated P25/LPS, was isolated by centrifugation from a mixture of P25 TiO2 NP (128 µg/ml) and LPS (10 ng/ml) in the presence of fetal bovine serum. Western blot analysis of the pellet eluate indicated that the P25/LPS fraction contained, besides proteins, also LPS, pointing to the presence of LPS-doped NP. The effects of adsorbed or free LPS were then compared in Raw264.7 murine macrophages. RT-PCR was used to evaluate the induction of cytokine genes, whereas active, phosphorylated isoforms of proteins involved in signaling pathways were assessed with western blot. At a nominal LPS concentration of 40 pg/ml, P25/LPS induced the expression of both NF-κB and IRF3-dependent cytokines at levels comparable with those observed with free LPS (10 ng/ml), although with different time courses. Moreover, compared to free LPS, P25/LPS caused a more sustained phosphorylation of p38 MAPK and a more prolonged induction of STAT1-dependent genes. Cytochalasin B partially inhibited the induction of Tnfa by P25/LPS, but not by free LPS, and suppressed the induction of IRF3-dependent genes by either P25/LPS or free LPS. These data suggest that, when included in the bio-corona of TiO2 NP, LPS exhibits enhanced and time-shifted pro-inflammatory effects. Thus, in assessing the hazard of NP in real life, the enhanced effects of adsorbed bioactive molecules should be taken into account.

Multiple endpoints to evaluate pristine and remediated titanium dioxide nanoparticles genotoxicity in lung epithelial A549 cells.

Titanium dioxide nanoparticles (TiO2 NP) are broadly used in a wide range of applications. Several studies have reported that TiO2 NP possess cytotoxic and genotoxic properties that could induce adverse health effects in humans. The FP7 Sanowork project was aimed to minimize occupational hazard and exposure to engineered nanomaterials (ENM), including TiO2 NP, through the surface modification in order to avoid possible adverse toxic effects for humans. In this study we investigated cytotoxicity, genotoxicity and epigenetic properties of TiO2 NP uncoated and coated with silica or citrate, as well as of the benchmark material P25. We used a panel of in vitro assays in the human lung epithelial cell line A549, in order to better understand if the remediation strategy adopted was able to counteract possible toxic effects of uncoated TiO2 NP. Our results showed that the uncoated TiO2 NP were both cytotoxic and genotoxic, and the remediation strategy adopted did not reduce the adverse effects of uncoated TiO2 NP. In particular, the presence of citrate was able to increase their cytotoxicity and genotoxicity, exerting also epigenotoxic effects, as evaluated by the marked reduction of LINE-1 methylation levels.