Nanotechnology

In Vitro Immunotoxicology of Quantum Dots and Comparison with Dissolved Cadmium and Tellurium

Health Canada is responsible for the assessment and management of health risks associated with exposure to chemicals and products in the environment.  Nanoparticles are very small particles, between 1 and 100 nanometers in size. Their small size results in novel properties that are useful for many different applications and products. However, these novel properties of nanoparticles, and their increased use, also raise concern about their potential toxicity. One subtype of nanoparticles is quantum dots (QDs), which are used in electronics and imaging. QDs are largely composed of metals (cadmium and tellurium) that can cause damage to animal cells. It is unclear if QD toxicity is caused by the nanoparticles themselves, or by these metals in the nanoparticles that can partially dissolve in water, forming a potentially toxic solution. In this study, Health Canada collaborated with Environment Canada and other organizations to explore whether toxicity was due to the QDs or the solution of metals in partially dissolved QDs.  Toxicity was determined in human blood cells exposed in petri dishes, or cells derived from three species commonly used as models of human and ecological health: mussels, rainbow trout, and mice. The results indicate that mussels are more sensitive to QDs than the dissolved components. However, the intact nanoparticles were not as toxic as their dissolved metal components in the other species. Human and mouse cells were generally more sensitive to the toxic effects of QDs than fish or mussels. Overall, this study recommended that a set of species, as opposed to just one species, be used to determine health risks associated with QDs. Health Canada will be able to use the results of this study to inform the development of tests for assessment of the safety of QDs. Results of this research are published in Environmental Toxicology, 2015, 30(1), 9-25.

Cadmium telluride quantum dot nanoparticle cytotoxicity and effects on model immune responses to Pseudomonas aeruginosa

Health Canada is responsible for the assessment and management of health risks associated with exposure to products and chemicals in the environment. Nanoparticles are very small particles, between 1 and 100 nanometers in size, that are increasingly used in a range of fields including physics, medicine, cosmetics, aeronautics, electronics, mechanics, and the textile industry. Quantum dots (QDs) are nanoparticles that are largely composed of the metals cadmium and tellurium. QDs are used in various electronic and biomedical research applications. While the number of QD applications is growing, there is limited information on their toxicity. In this study, Health Canada examined whether exposure to QDs had toxic effects on human cells grown in the laboratory, and whether QDs altered how the cells responded to bacteria. Cells were exposed to various concentrations of QDs, and analyzed for changes in cell metabolism, cell shape, or changes in the levels of specific molecules involved in immune response. Higher concentrations of QDs caused changes in cell metabolism, cell shape, and in the internal organization of the cells. However, there were no apparent alterations to molecules involved in the immune response. Health Canada then explored whether QD exposure changed the immune responses of the cells towards bacteria. The data showed that cells exposed to QDs, and then exposed to bacteria, exhibited increased toxicity over cells exposed to bacteria alone. In addition, these cells had larger changes in the levels of several molecules involved in immune response. Importantly, these altered responses to bacteria exposure were detectable even if the cells were exposed to lower concentrations of QDs that were previously found to have no discernable effect on cell metabolism or shape. Therefore this study demonstrates that exposure to QDs, even below the concentration at which they are toxic, can alter how cells generate defensive responses against bacteria. Health Canada will use the results of this study to better understand potential health effects associated with exposure to QDs, which is important for safety evaluation of these nanoparticles. This research is published in Nanotoxicology, 2013, 7(2), 202-211.

Cadmium telluride quantum dots cause oxidative stress leading to extrinsic and intrinsic apoptosis in hepatocellular carcinoma HepG2 cells

Health Canada is responsible for the assessment and management of health risks associated with exposure to products and chemicals in the environment. Nanoparticles are very small particles, between 1 and 100 nanometers in size, that are increasingly used in a range of fields including physics, medicine, cosmetics, aeronautics, electronics, mechanics, and the textile industry.  Quantum dots (QDs) are very small particles that are largely composed of the metals cadmium and tellurium. QDs are used in a range of products including solar cells, light emitting devices (LEDs), quantum computing and applications, and in biological imaging. There is growing evidence that QDs may cause harmful health effects, although the mechanisms underlying this are not well understood. For example, it is unclear whether the effects are due to the QDs directly, or due to cadmium from the particles dissolving in the surrounding solution and then causing health effects. In this study Health Canada, in collaboration with scientists from Carleton University, sought to determine the mechanisms by which exposure to QDs results in health effects. Human liver cells grown in the laboratory were exposed to either QDs, or an equivalent amount of cadmium dissolved in solution. Cells were then analyzed for changes in structure, or changes in various functions. The results showed that QD exposure was harmful to the cells by causing oxidative stress, which led to cell death. Oxidative stress is an umbrella term that refers to various types of cellular damage caused by chemically-reactive molecules that contain oxygen. The health effects caused by QDs were greater than that caused by exposure to cadmium dissolved in solution, suggesting that the dissolved cadmium cannot explain all of the impacts associated with QDs. Health Canada will be able to use the results of this study to better understand the health impacts of exposure to QDs, which is important for safety evaluation of these nanoparticles.  Results of this research are published in Toxicology, 2013, 306, 114-123.

Comparison of toxicity of uncoated and coated silver nanoparticles

Health Canada is responsible for the assessment of health risks associated with exposure to products and chemicals in the environment.  Nanoparticles are very small particles, between 1 and 100 nanometers in size. Silver-containing nanoparticles (Ag-NP) are used in a wide array of applications and consumer products due to their antimicrobial, electrical, and optical properties. However, there is growing evidence that Ag-NPs may be harmful, causing alterations in the immune system, increases in oxidative stress and cell damage. Oxidative stress refers to various types of cellular damage caused by chemically-reactive molecules containing oxygen. The exact mechanisms by which Ag-NP cause these effects, and the role that size and chemical modifications of the particles play, remain unclear. In this study Health Canada, in collaboration with scientists from the University of Ottawa, used different sizes of Ag-NPs with or without two types of chemical coating (citrate or polyvinylpyrrolidone) to explore the effects of these properties measures of health. Mouse and human cells in petri dishes in the laboratory were exposed to various types of Ag-NP for 24 hours and tests were conducted to determine effects. Uncoated Ag-NPs reduced cell survival, with larger effects induced by smaller particles. Uncoated particles also increased oxidative stress and reduced levels of molecules involved in activating the immune system. Coated Ag-NPs reduced survival, but only at higher concentrations, with greater effects associated with polyvinylpyrrolidone-coated than citrate-coated particles. Coated Ag-NPs increased levels of the same immune system molecules as uncoated. These results show that the degree of health impact, and the underlying mechanisms, may be dependent on the size and coating of the Ag-NPs. The study will help Health Canada better understand safe application ofAg-NP. This research is published in the

2013, 429, 012025, doi: 10.108811742-6596/429/1/012025.

Critical Experimental Parameters Related to the Cytotoxicity of Zinc Oxide Nanoparticles

Health Canada is responsible for the assessment and management of health risks to Canadians associated with exposure to products and chemicals in the environment. Materials composed of zinc oxide nanoparticles (ZnO-NPs) are used and sold commercially for cosmetic, food, medical and industrial applications. Given this widespread use of ZnO-NPs, Health Canada has a need to understand their chemical and physical characteristics and any associated toxicity in order to better assess the potential health risks. There is a scarcity of literature and data on ZnO-NPs, whose characteristics and biological effects may be completely different from their non-nano forms. To address this lack of data, Health Canada conducted a study that first compared ZnO-NPs with their larger ZnO (non-nano) counterparts for size, shape and elemental content, and then evaluated their biological effects in several mouse and human cell systems. The results revealed that both nano and non-nano forms were highly clumped and capable of causing cell damage that was proportional to the dose applied. Some cell types were more sensitive than others, and only some tests could detect a difference in the toxicity of the various types of ZnO materials. These data demonstrate the importance of applying a variety of different assays to evaluate the potential toxicity of various nanoparticles. The study will be used by Health Canada for the design of future assays for the evaluation of nanomaterials. Results of this research are published in the Journal of Nanoparticle Research, 2014, 16(6), 2440.

Synthesis and Physicochemical Characterization of Mesoporous Nanoparticles

Nano-sized particles are very small (one billionth of one metre in at least one dimension) and can exhibit specific properties, which in many cases are different from larger particles of the same material. These properties vary with nanoparticle size and surface chemistry and it is these unique properties of nanomaterials that have made them attractive in the production/manipulation of consumer products to impart novel characteristics. Silica is a form of silicon that when prepared in the nano-scale can be used in various technologies, such as catalytic supports for chemical processing, electronics, and in various advanced biomedical applications including medical imaging of tumours. Although silica nanoparticles become very useful at the nano-scale, the properties that make them very useful, may also affect the way they interact with biological systems to change their toxicity profiles. A particular challenge for studying potential toxicity is the lack of well characterized silica nanoparticles at different sizes and various corresponding surface modified forms. In this study, Health Canada scientists synthesized and characterized a library of silica nanoparticles with approximate particle sizes of 25, 70, 100, 170, and 600 nm, thus producing a fully characterized library of materials whose toxicological properties can be studied in future investigations. Results of this study are published in the Journal of Nanomaterials, 2014, Article ID 176015. doi:10.1155/2014/176015.

Nanosilver Cytotoxicity in Rainbow Trout (Oncorhynchus mykiss) Erythrocytes and Hepatocytes

Health Canada helps protect and promote health by using existing legislative and regulatory frameworks to mitigate the potential health risks of nanomaterials and to help realize their health benefits. Silver nanoparticles (AgNPs) are used in many consumer products, such as bandages, ointments and fabrics, because of their antimicrobial activity. Although relatively little information is available on the human health and environmental impacts of AgNPs exposure, there is the potential for such particles to accumulate in environments such as natural waters. In this study, which was led by the University of Ottawa, researchers examined the toxicity of AgNPs to liver and blood cells that were isolated from freshwater trout, a model species for these effects. In order to understand the cellular effects, inhibitors and promoters that influence the clumping of the AgNPs or reduce cellular enzyme activity were used. Health Canada helped to characterise the AgNPs, assess particle uptake and the resulting changes in cellular structure and morphology. These data demonstrated that AgNPs caused toxicity in both liver and blood cells and identified the cellular mechanisms that limit damage. The results help to clarify the difference between ionic silver and AgNP toxicity and will ultimately lead to a better understanding of the biological processes that are affected by nanoparticles. Health Canada will use the results of this research to better inform future risk assessments on the human health impacts posed by these materials. Results of this research are published in the journal of Comparative Biochemistry and Physiology - C Toxicology and Pharmacology, 2013, 159(1), 10-21.

Carbon Black Nanoparticle Instillation Induces Sustained Inflammation and Genotoxicity in Mouse Lung and Liver

Nanomaterials (NMs) are increasingly being used in the marketplace in a wide range of products and substances that Health Canada is responsible for regulating. While there are a great many new applications and benefits, there is inadequate information on risks associated with NMs at this time. Nanoparticles (NPs) are extremely small (1-100 nanometers in at least one dimension) particles that when inhaled, either in ambient air or occupational settings, may penetrate deep into the lungs and move into the blood and peripheral organs. To better understand the potential health risks in the workplace and from environmental exposures, Health Canada undertook a study to determine what NP exposure levels cause toxicity, the precise mechanisms of toxicity, and the duration of toxicity in the lung and other affected tissues. In this study, signs of damaged DNA and inflammation were measured in mice that were exposed to the model NP, carbon black. It was found that damage to DNA occurred in both the lungs and liver of the mice. The damage to DNA persisted long after exposure and was found to correlate strongly with inflammation, suggesting inflammation likely played an important role in the DNA damage. This is important because damage to DNA can lead to cancer. These results will help Health Canada determine what health effects may occur in humans from exposure to NMs and aid in setting exposure limits. This study was done in collaboration with the Danish National Research Centre for the Working Environment and the University of Copenhagen and was published in Particle and Fibre Toxicology (2012 Feb 2), 9(1):5-18.

Pulmonary Response to Surface-Coated Nanotitanium Dioxide Particles Includes Induction of Acute Phase Response Genes, Inflammatory Cascades, and Changes in Micrornas: A Toxicogenomic Study

Nanomaterials (NMs) are very small in size (1 to 100 nanometers in at least one dimension) and are increasingly being used in products that Health Canada is responsible for regulating. Due to their small size, many NMs exhibit different properties compared to the larger-scale materials with the same chemical composition. Nano-sized titanium dioxide is widely used in a variety of consumer products, and this work was undertaken to help Health Canada and its international partners develop a greater understanding of the biological impacts of this material. Using genomics approaches, which investigate the responses of all the genes within an organism to a stressor, this study aimed to understand the potential toxicity and to identify the underlying molecular mechanisms of toxicity of surface modified nano-sized titanium dioxide in a mouse model. The results suggested that inhalation of a dose level of surface-modified nano-sized titanium dioxide, which is comparable to what people could be exposed to occupationally, elicits lung inflammation in mice. The use of genomics approaches further revealed specific biological mechanisms of the inflammation process that were activated. The study provided greater understanding of the detailed mechanisms behind lung responses to the inhalation of nano-sized particles. Such studies will be useful in identifying some biological markers of exposure or effects of exposure to NMs that could be used as early identifiers or predictors of potential adverse outcomes in humans. This study, a collaboration with the Danish National Research Centre for the Working Environment, was published in Environmental and Molecular Mutagenesis (2011 Jul), 52(6):425-439.

Poly(Ethylene Imine) Nanocarriers do not Induce Mutations nor Oxidative DNA Damage In Vitro in Mutamouse Fe1 Cells

Poly(ethylene imine) or PEI has been widely used as a polymer to carry genetic material (i.e., DNA) due to its ability to form stable nanoscale (i.e., 1 to 100 nanometers in at least one dimension) complexes. This ability has stimulated research on the use of PEI polymers to carry genetic material into diseased tissue (e.g., cancerous tumours) for treatment purposes. These polymers are desirable because they provide an alternative to using virus particles; however, there is concern regarding their ability to generate reactive oxygen species (ROS) that can induce undesirable genetic damage. Several other nanoscale materials, including carbon black, zinc oxide (ZnO), and titanium dioxide (TiO₂), have been highlighted for their ability to induce toxic effects via the generation of ROS and inflammation. Health Canada conducted this study to increase the understanding of the effects of nanoparticles and to better characterize the genetic toxicity of a material with potential therapeutic use. A mouse cell culture model was employed to assess the ability of several PEI-based polymers to induce genetic damage and mutations. In addition, oxidative damage was assessed in exposed cells. The results showed that none of the PEI-based polymers investigated could induce toxic effects, oxidative DNA damage or genetic mutations. In contrast, the reference compound, benzo[a]pyrene induced significant levels of DNA damage and genetic mutations. The absence of genotoxic activity for the PEI polymers examined suggests that they may be applicable in a clinical setting, information which may be useful for product assessment purposes. This study was a collaboration with the German Research Center for Environmental Health and Philipps-Universität Marburg and was published in Molecular Pharmaceutics (2011 Jun 6), 8(3):976-981.

Mutation Spectrum in Fe1-Muta™ Mouse Lung Epithelial Cells Exposed to Nanoparticulate Carbon Black

Carbon black is a major industrial nanoscale (1 to 100 nanometers in one dimension) chemical produced by incomplete combustion of petroleum products. It is used in inks, paints, rubber, and plastics, with global production exceeding 10 million tonnes in 2005. Since carbon black has established human health effects, it is widely used as a model in research investigations to increase the understanding of the potential health effects caused by nanoparticles (NPs). In an earlier study, it was shown that exposure of cultured mouse cells to NPs of carbon black from a standard known as Printex 90 induced DNA damage and genetic mutations. The present study was conducted by Health Canada to provide more detailed information on the types and locations (spectrum) of the genetic mutations in order to determine how carbon black causes mutations. Cells exposed to the NPs of carbon black showed a substantially different spectrum of mutations in comparison to the unexposed control cells. Analysis of the results showed a highly significant difference between the exposed cells and the control cells with respect to the location of the DNA mutation, as well as the type of mutations. Overall, the results obtained provide support for the contention that the effect of carbon black exposure on the genetic material of these cultured cells is caused by the generation of highly reactive oxygen molecules. This reactive oxygen damages the DNA and elicits permanent changes in genetic coding (i.e., mutations). Developing an understanding of how NPs of carbon black cause mutations will provide insight into the nature and extent of potential human health risks attributable to nanomaterials, which will be useful in risk assessment. This study was conducted in collaboration with the Danish National Research Centre for the Working Environment and the University of Copenhagen and was published in Environmental and Molecular Mutagenesis (2011 May), 52(4):331-337.