32 research outputs found
High-Throughput Screening Platform for Engineered Nanoparticle-Mediated Genotoxicity Using CometChip Technology
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 (SiO[subscript 2], ZnO, Fe[subscript 2]O[subscript 3], Ag, and CeO[subscript 2]) 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 > Fe[subscript 2]O[subscript 3] > CeO[subscript 2] > SiO[subscript 2] in TK6 cells at 4 h and Ag > Fe[subscript 2]O[subscript 3] > ZnO > CeO[subscript 2] > SiO[subscript 2] 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.National Science Foundation (U.S.) (Grant 1235806)National Institutes of Health (U.S.) (Grant P30ES000002
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Estimating the effective density of engineered nanomaterials for in vitro dosimetry
The need for accurate in vitro dosimetry remains a major obstacle to the development of cost-effective toxicological screening methods for engineered nanomaterials. An important key to accurate in vitro dosimetry is the characterization of sedimentation and diffusion rates of nanoparticles suspended in culture media, which largely depend upon the effective density and diameter of formed agglomerates in suspension. Here we present a rapid and inexpensive method for accurately measuring the effective density of nano-agglomerates in suspension. This novel method is based on the volume of the pellet obtained by bench-top centrifugation of nanomaterial suspensions in a packed cell volume tube, and is validated against gold-standard analytical ultracentrifugation data. This simple and cost-effective method allows nanotoxicologists to correctly model nanoparticle transport, and thus attain accurate dosimetry in cell culture systems, which will greatly advance the development of reliable and efficient methods for toxicological testing and investigation of nano-bio interactions in vitro
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Optimization of a nanotechnology based antimicrobial platform for food safety applications using Engineered Water Nanostructures (EWNS)
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
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Advanced computational modeling for in vitro nanomaterial dosimetry
Background: Accurate and meaningful dose metrics are a basic requirement for in vitro screening to assess potential health risks of engineered nanomaterials (ENMs). Correctly and consistently quantifying what cells “see,” during an in vitro exposure requires standardized preparation of stable ENM suspensions, accurate characterizatoin of agglomerate sizes and effective densities, and predictive modeling of mass transport. Earlier transport models provided a marked improvement over administered concentration or total mass, but included assumptions that could produce sizable inaccuracies, most notably that all particles at the bottom of the well are adsorbed or taken up by cells, which would drive transport downward, resulting in overestimation of deposition. Methods: Here we present development, validation and results of two robust computational transport models. Both three-dimensional computational fluid dynamics (CFD) and a newly-developed one-dimensional Distorted Grid (DG) model were used to estimate delivered dose metrics for industry-relevant metal oxide ENMs suspended in culture media. Both models allow simultaneous modeling of full size distributions for polydisperse ENM suspensions, and provide deposition metrics as well as concentration metrics over the extent of the well. The DG model also emulates the biokinetics at the particle-cell interface using a Langmuir isotherm, governed by a user-defined dissociation constant, KD, and allows modeling of ENM dissolution over time. Results: Dose metrics predicted by the two models were in remarkably close agreement. The DG model was also validated by quantitative analysis of flash-frozen, cryosectioned columns of ENM suspensions. Results of simulations based on agglomerate size distributions differed substantially from those obtained using mean sizes. The effect of cellular adsorption on delivered dose was negligible for KD values consistent with non-specific binding (> 1 nM), whereas smaller values (≤ 1 nM) typical of specific high-affinity binding resulted in faster and eventual complete deposition of material. Conclusions: The advanced models presented provide practical and robust tools for obtaining accurate dose metrics and concentration profiles across the well, for high-throughput screening of ENMs. The DG model allows rapid modeling that accommodates polydispersity, dissolution, and adsorption. Result of adsorption studies suggest that a reflective lower boundary condition is appropriate for modeling most in vitro ENM exposures. Electronic supplementary material The online version of this article (doi:10.1186/s12989-015-0109-1) contains supplementary material, which is available to authorized users
An integrated methodology for assessing the impact of food matrix and gastrointestinal effects on the biokinetics and cellular toxicity of ingested engineered nanomaterials
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 \u3c1% 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
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High-Throughput Screening Platform for Engineered Nanoparticle-Mediated Genotoxicity Using CometChip Technology
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
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Engineering safer-by-design silica-coated ZnO nanorods with reduced DNA damage potential
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