Person:

Demokritou, Philip

In vitro toxicity assessment of engineered nanomaterials (ENM), the most common testing platform for ENM, requires prior ENM dispersion, stabilization, and characterization in cell culture media. Dispersion inefficiencies and active aggregation of particles often result in polydisperse and multimodal particle size distributions. Accurate characterization of important properties of such polydisperse distributions (size distribution, effective density, charge, mobility, aggregation kinetics, etc.) is critical for understanding differences in the effective dose delivered to cells as a function of time and dispersion conditions, as well as for nano–bio interactions. Here we have investigated the utility of tunable nanopore resistive pulse sensing (TRPS) technology for characterization of four industry relevant ENMs (oxidized single-walled carbon nanohorns, carbon black, cerium oxide and nickel nanoparticles) in cell culture media containing serum. Harvard dispersion and dosimetry platform was used for preparing ENM dispersions and estimating delivered dose to cells based on dispersion characterization input from dynamic light scattering (DLS) and TRPS. The slopes of cell death vs administered and delivered ENM dose were then derived and compared. We investigated the impact of serum protein content, ENM concentration, and cell medium on the size distributions. The TRPS technology offers higher resolution and sensitivity compared to DLS and unique insights into ENM size distribution and concentration, as well as particle behavior and morphology in complex media. The in vitro dose–response slopes changed significantly for certain nanomaterials when delivered dose to cells was taken into consideration, highlighting the importance of accurate dispersion and dosimetry in in vitro nanotoxicology.

Background: Photocopiers emit nanoparticles with complex chemical composition. Short-term exposures to modest nanoparticle concentrations triggered upper airway inflammation and oxidative stress in healthy human volunteers in a recent study. To further understand the toxicological properties of copier-emitted nanoparticles, we studied in-vitro their ability to induce cytotoxicity, pro-inflammatory cytokine release, DNA damage, and apoptosis in relevant human cell lines. Methods: Three cell types were used: THP-1, primary human nasal- and small airway epithelial cells. Following collection in a large volume photocopy center, nanoparticles were extracted, dispersed and characterized in the cell culture medium. Cells were doped at 30, 100 and 300 μg/mL administered doses for up to 24 hrs. Estimated dose delivered to cells, was ~10% and 22% of the administered dose at 6 and 24 hrs, respectively. Gene expression analysis of key biomarkers was performed using real time quantitative PCR (RT-qPCR) in THP-1 cells at 5 μg nanoparticles/mL for 6-hr exposure for confirmation purposes. Results: Multiple cytokines, GM-CSF, IL-1β, IL-6, IL-8, IFNγ, MCP-1, TNF-α and VEGF, were significantly elevated in THP-1 cells in a dose-dependent manner. Gene expression analysis confirmed up-regulation of the TNF-α gene in THP-1 cells, consistent with cytokine findings. In both primary epithelial cells, cytokines IL-8, VEGF, EGF, IL-1α, TNF-α, IL-6 and GM-CSF were significantly elevated. Apoptosis was induced in all cell lines in a dose-dependent manner, consistent with the significant up-regulation of key apoptosis-regulating genes P53 and Casp8 in THP-1 cells. No significant DNA damage was found at any concentration with the comet assay. Up-regulation of key DNA damage and repair genes, Ku70 and Rad51, were also observed in THP-1 cells, albeit not statistically significant. Significant up-regulation of the key gene HO1 for oxidative stress, implicates oxidative stress induced by nanoparticles. Conclusions: Copier-emitted nanoparticles induced the release of pro-inflammatory cytokines, apoptosis and modest cytotoxicity but no DNA damage in all three-human cell lines. Taken together with gene expression data in THP-1 cells, we conclude that these nanoparticles are directly responsible for inflammation observed in human volunteers. Further toxicological evaluations of these nanoparticles, including across different toner formulations, are warranted.

Background: Tobacco use is the single most preventable cause of morbidity and mortality in humans. Limited data exist regarding the extent of the problem among Cyprus youth. We use the Global Youth Tobacco Survey to assess the prevalence of cigarette smoking among middle and high school students as well as the social environment in which this is taking place. Methods: The survey was conducted by the Cyprus International Institute for the Environment and Public Health in association with Harvard School of Public Health. A two-stage cluster sample design was used to select a representative sample of students from middle and high schools registered with the Republic of Cyprus in 2005–2006. The study questionnaire consisted of 99 questions and participation in the survey was voluntary. Statistical analyses were performed taking into consideration the specific design of the study and the sample weights associated with each completed questionnaire. Results: The prevalence of current smoking, defined as having smoked cigarettes on one or more days of the past 30 days, is 13% among boys and 7% among girls in middle schools, and 36% among boys and 23% among girls in high schools. Furthermore, 16% of middle school students and more than 24% of high school students that had never smoked indicated that they are likely to initiate smoking within the next year. Exposure to environmental tobacco smoke is also very high with 91% of students reporting being exposed to smoke in places outside home. In addition, more than 95% of current smokers reported that they had bought cigarettes in a store during the past month and were not refused cigarettes because of their age. Conclusion: Smoking prevalence among Cyprus middle and high school students is high and there are indications of an increase in the prevalence of smoking among girls over the last few years. Susceptibility rates, exposure to second-hand smoke, and access to and availability of cigarettes to youth are also high and concerning. The present survey indicates that the problem of cigarette smoking among youth in Cyprus is significant and requires collective action immediately.

A novel system for generation of engineered nanomaterials (ENMs) suitable for in situ toxicological characterization within biological matrices was developed. This Versatile Engineered Nanomaterial Generation System (VENGES) is based on industry-relevant, flame spray pyrolysis aerosol reactors that can scaleably produce ENMs with controlled primary and aggregate particle size, crystallinity, and morphology. ENMs are produced continuously in the gas phase, allowing their continuous transfer to inhalation chambers, without altering their state of agglomeration. Freshly generated ENMs are also collected on Teflon filters for subsequent physicochemical and morphological characterization and for in vitro toxicological studies. The ability of the VENGES system to generate families of ENMs of pure and selected mixtures of iron oxide, silica, and nanosilver with controlled physicochemical properties was demonstrated using a range of state-of-the-art-techniques. Specific surface area was measured by nitrogen adsorption using the Brunauer-Emmett-Teller method, and crystallinity was characterized by X-ray diffraction. Particle morphology and size were evaluated by scanning and transmission electron microscopy. The suitability of the VENGES system for toxicological studies was also shown in both in vivo and in vitro studies involving Sprague-Dawley rats and human alveolar-like monocyte derived macrophages, respectively. We demonstrated linkage between physicochemical ENM properties and potential toxicity.

The likelihood of intentional and unintentional engineered nanoparticle (ENP) exposure has dramatically increased due to the use of nanoenabled products. Indeed, ENPs have been incorporated in many useful products and have enhanced our way of life. However, there are many unanswered questions about the consequences of nanoparticle exposures, in particular, with regard to their potential to damage the genome and thus potentially promote cancer. In this study, we present a high-throughput screening assay based upon the recently developed CometChip technology, which enables evaluation of single-stranded DNA breaks, abasic sites, and alkali-sensitive sites in cells exposed to ENPs. The strategic microfabricated, 96-well design and automated processing improves efficiency, reduces processing time, and suppresses user bias in comparison to the standard comet assay. We evaluated the versatility of this assay by screening five industrially relevant ENP exposures (SiO2, ZnO, Fe2O3, Ag, and CeO2) on both suspension human lymphoblastoid (TK6) and adherent Chinese hamster ovary (H9T3) cell lines. MTT and CyQuant NF assays were employed to assess cellular viability and proliferation after ENP exposure. Exposure to ENPs at a dose range of 5, 10, and 20 μg/mL induced dose-dependent increases in DNA damage and cytotoxicity. Genotoxicity profiles of ZnO > Ag > Fe2O3 > CeO2 > SiO2 in TK6 cells at 4 h and Ag > Fe2O3 > ZnO > CeO2 > SiO2 in H9T3 cells at 24 h were observed. The presented CometChip platform enabled efficient and reliable measurement of ENP-mediated DNA damage, therefore demonstrating the efficacy of this powerful tool in nanogenotoxicity studies.

AIM: As engineered nanoparticles (ENPs) increasingly enter consumer products, humans become increasingly exposed. The first line of defense against ENPs is the epithelium, the integrity of which can be compromised by wounds induced by trauma, infection, or surgery, but the implications of ENPs on wound healing are poorly understood. MATERIALS & METHODS: Herein, we developed an in vitro assay to assess the impact of ENPs on the wound healing of cells from human cornea. RESULTS & DISCUSSION: We show that industrially relevant ENPs impeded wound healing and cellular migration in a manner dependent on the composition, dose and size of the ENPs as well as cell type. CuO and ZnO ENPs impeded both viability and wound healing for both fibroblasts and epithelial cells. Carboxylated polystyrene ENPs retarded wound healing of corneal fibroblasts without affecting viability. CONCLUSION: Our results highlight the impact of ENPs on cellular wound healing and provide useful tools for studying the physiological impact of ENPs.

Zinc oxide (ZnO) nanoparticles absorb UV light efficiently while remaining transparent in the visible light spectrum rendering them attractive in cosmetics and polymer films. Their broad use, however, raises concerns regarding potential environmental health risks and it has been shown that ZnO nanoparticles can induce significant DNA damage and cytotoxicity. Even though research on ZnO nanoparticle synthesis has made great progress, efforts on developing safer ZnO nanoparticles that can maintain their inherent optoelectronic properties while exhibiting minimal toxicity are limited. Here, a safer-by-design concept was pursued by hermetically encapsulating ZnO nanorods in a biologically inert, nanothin amorphous SiO2 coating during their gas-phase synthesis. It is demonstrated that the SiO2 nanothin layer hermetically encapsulates the core ZnO nanorods without altering their optoelectronic properties. Furthermore, the effect of SiO2 on the toxicological profile of the core ZnO nanorods was assessed using the Nano-Cometchip assay by monitoring DNA damage at a cellular level using human lymphoblastoid cells (TK6). Results indicate significantly lower DNA damage (>3 times) for the SiO2-coated ZnO nanorods compared to uncoated ones. Such an industry-relevant, scalable, safer-by-design formulation of nanostructured materials can liberate their employment in nano-enabled products and minimize risks to the environment and human health.

A chemical free, nanotechnology-based, antimicrobial platform using Engineered Water Nanostructures (EWNS) was recently developed. EWNS have high surface charge, are loaded with reactive oxygen species (ROS), and can interact-with, and inactivate an array of microorganisms, including foodborne pathogens. Here, it was demonstrated that their properties during synthesis can be fine tuned and optimized to further enhance their antimicrobial potential. A lab based EWNS platform was developed to enable fine-tuning of EWNS properties by modifying synthesis parameters. Characterization of EWNS properties (charge, size and ROS content) was performed using state-of-the art analytical methods. Further their microbial inactivation potential was evaluated with food related microorganisms such as Escherichia coli, Salmonella enterica, Listeria innocua, Mycobacterium parafortuitum, and Saccharomyces cerevisiae inoculated onto the surface of organic grape tomatoes. The results presented here indicate that EWNS properties can be fine-tuned during synthesis resulting in a multifold increase of the inactivation efficacy. More specifically, the surface charge quadrupled and the ROS content increased. Microbial removal rates were microorganism dependent and ranged between 1.0 to 3.8 logs after 45 mins of exposure to an EWNS aerosol dose of 40,000 #/cm3.

Particle–cell interactions in physiological media are important in determining the fate and transport of nanoparticles and biological responses to them. In this work, these interactions are assessed in real time using a novel atomic force microscopy (AFM) based platform. Industry-relevant CeO2 and Fe2O3 engineered nanoparticles (ENPs) of two primary particle sizes were synthesized by the flame spray pyrolysis (FSP) based Harvard Versatile Engineering Nanomaterials Generation System (Harvard VENGES) and used in this study. The ENPs were attached on AFM tips, and the atomic force between the tip and lung epithelia cells (A549), adhered on a substrate, was measured in biological media, with and without the presence of serum proteins. Two metrics were used to assess the nanoparticle cell: the detachment force required to separate the ENP from the cell and the number of bonds formed between the cell and the ENPs. The results indicate that these atomic level ENP–cell interaction forces strongly depend on the physiological media. The presence of serum proteins reduced both the detachment force and the number of bonds by approximately 50% indicating the important role of the protein corona on the particle cell interactions. Additionally, it was shown that particle to cell interactions were size and material dependent.

Background: Engineered nanomaterials (ENMs) are increasingly added to foods to improve their quality, sensory appeal, safety and shelf-life. Human exposure to these ingested ENMs (iENMS) is inevitable, yet little is known of their hazards. To assess potential hazards, efficient in vitro methodologies are needed to evaluate particle biokinetics and toxicity. These methodologies must account for interactions and transformations of iENMs in foods (food matrix effect) and in the gastrointestinal tract (GIT) that are likely to determine nano-biointeractions. Here we report the development and application of an integrated methodology consisting of three interconnected stages: 1) assessment of iENM-food interactions (food matrix effect) using model foods; 2) assessment of gastrointestinal transformations of the nano-enabled model foods using a three-stage GIT simulator; 3) assessment of iENMs biokinetics and cellular toxicity after exposure to simulated GIT conditions using a triculture cell model. As a case study, a model food (corn oil-in-water emulsion) was infused with Fe2O3 (Iron(III) oxide or ferric oxide) ENMs and processed using this three-stage integrated platform to study the impact of food matrix and GIT effects on nanoparticle biokinetics and cytotoxicity . Methods: A corn oil in phosphate buffer emulsion was prepared using a high speed blender and high pressure homogenizer. Iron oxide ENM was dispersed in water by sonication and combined with the food model. The resulting nano-enabled food was passed through a three stage (mouth, stomach and small intestine) GIT simulator. Size distributions of nano-enabled food model and digestae at each stage were analyzed by DLS and laser diffraction. TEM and confocal imaging were used to assess morphology of digestae at each phase. Dissolution of Fe2O3 ENM along the GIT was assessed by ICP-MS analysis of supernatants and pellets following centrifugation of digestae. An in vitro transwell triculture epithelial model was used to assess biokinetics and toxicity of ingested Fe2O3 ENM. Translocation of Fe2O3 ENM was determined by ICP-MS analysis of cell lysates and basolateral compartment fluid over time. Results: It was demonstrated that the interactions of iENMs with food and GIT components influenced nanoparticle fate and transport, biokinetics and toxicological profile. Large differences in particle size, charge, and morphology were observed in the model food with and without Fe2O3 and among digestae from different stages of the simulated GIT (mouth, stomach, and small intestine). Immunoflorescence and TEM imaging of the cell culture model revealed markers and morphology of small intestinal epithelium including enterocytes, goblet cells and M cells. Fe2O3 was not toxic at concentrations tested in the digesta. In biokinetics studies, translocation of Fe2O3 after 4 h was <1% and ~2% for digesta with and without serum, respectively, suggesting that use of serum proteins alters iENMs biokinetics and raises concerns about commonly-used approaches that neglect iENM – food-GIT interactions or dilute digestae in serum-containing media. Conclusions: We present a simple integrated methodology for studying the biokinetics and toxicology of iENMs, which takes into consideration nanoparticle-food-GIT interactions. The importance of food matrix and GIT effects on biointeractions was demonstrated, as well as the incorporation of these critical factors into a cellular toxicity screening model. Standardized food models still need to be developed and used to assess the effect of the food matrix effects on the fate and bioactivity of iENMs since commercial foods vary considerably in their compositions and structures. Electronic supplementary material The online version of this article (10.1186/s12989-017-0221-5) contains supplementary material, which is available to authorized users.