Concept and Project Objective(s)
NanoTOES has 6 major scientific objectives, which are indicated in the following list. The numbers refer to the projects contributing to each objective, which are described in section B.1.3.
- Identify mechanisms by which nanomaterials induce cellular stress and immune activation (1,2,3,4,5,7,9).
- Correlate size, shape, composition, coating of nanomaterials with defined cellular responses (1,2,3,4,5,6,8,13)
- Distinguish cell-specific from general cellular responses for cells from selected tissues (2,3,4,5,8,10,13)
- Identify the relevance of bystander substances and contaminants for nanotoxicity (1,2,6,7,9,12)
- Analyse the influence of biological compounds and entities on engineered NP (4,9,10,11,12,13)
- Develop and modify laboratory methods to allow their application in the work-place and in the environment (1,3,6,7,10,11)
Furthermore, all projects contribute to the training objective, which is to facilitate training of highly skilled experts in nanotoxicology based on an interdisciplinary and intersectoral training programme as outlined in section B.1.3.
The biological effects of engineered nanomaterials are currently a topic of considerable debate. Concerns relate to work-place safety at sites of production and application of nanomaterials, to consumer safety, and to environmental impact following discarding or accidental release of nanoparticles (NP). Intentional application of nanomaterials during diagnostic or therapeutic applications constitutes another area of interest because existing procedures may not be sufficient to estimate their desired biological effects and, even more importantly, any undesired side effects of such treatments. This emerging field requires genuinely interdisciplinary activities and strong intersectorial links between academia and industry. There are many national and international activities in this area, which are producing a lot of data right now, so we can look forward to a surge of publications.
However, a lot of data is not the same as full understanding. One reason for this caveat is that many different types of assays are being used to investigate questions of nanotoxicology: some are relatively standard methods with greater or lesser degrees of relevant validation, others may be modified or developed more specifically for nanomaterials. In any event there are for many nanotoxicological assessments presently no methods which can be defined as “gold standards”, and it will be some time before a consensus is reached on which methods are relevant, properly-validated and useful in common practice. In the meantime the literature is confused, and comparative testing across laboratories presents considerable methodological challenges. Indeed, it is difficult to compare data from different laboratories when neither materials nor methods are fully standardised and documented: nanomaterials may be insufficiently characterised, and assays may be selected because they are available, rather than because they are appropriate. The opportunity for other groups using somewhat different materials, methods or endpoints, to arrive at different or opposite conclusions is unsurprising and indeed inevitable. Whilst this situation may be irritating for researchers, it is deeply unsatisfactory for consumers and regulatory agencies, which depend on scientifically based advice for their decisions.
In order to improve this situation NanoTOES shall pursue the following work packages.
See the Project Work Packages.
Standardised methods for the synthesis of nanomaterials as well as their physicochemical characterisation and biological evaluation are urgently needed to enable the generation and validation of reliable data describing interactions between living systems and NP. Recent investments by the EU into nanotoxicology should result in an initial portfolio of standardised protocols designed for the evaluation of NP. To complement and extend that protocol development, and most importantly to train those who will implement and disseminate these activities, NanoTOES has set itself the goal of training young researchers in the synthesis, characterisation and biological evaluation of NP, using laboratories operating best practice in the field. The ITN NanoTOES will link ongoing projects concerned with nanosafety (e.g., partners UCD, NERC, NILU and IST are coordinating relevant FP7-NMP and FP7-Health projects) while providing a specific focus on the training of future experts. Partners ACS, Grimm and Bayer represent the relevant industries and will help to coordinate the training activities of the ITN with the needs of the private sector.
A focus will be to define nanotoxicological methods in such a way that their values, limits and restrictions are well documented, and reasons for possible errors are understood. One of the accomplishments expected from NanoTOES is a textbook on methods in nanotoxicology, which will outline procedures and pitfalls. Such a “cookbook” will contribute towards the development of generally accepted standards, which are now largely missing.
Appropriateness of research methodology. As the reference nanomaterial, which will be used across all projects in order to generate a significant body of data, as well as facilitating training of the ESR and ER in nanomaterial handling and testing, we haven chosen SILVER nanoparticles for the following reasons:
- Ag NP have biological activity by themselves. They are used as disinfectants and pesticides, have a well-known bactericidal activity, and toxicity to eukaryotic cells has been reported as well.
- Ag NP are metallic. They can be thus representative for other metallic NP like Au or Pt, and they get resonantly heated or enhance the Raman spectroscopy.
- Ag NP slowly oxidize their surface. They can be thus also representative of oxide NP and act as catalysts. This property also makes them suitable for considering aging, and could impact on their interaction with and impact on cellular biomolecules.
- There are reproducible recipes for the size and shape control. It is possible to produce spheres, cubes, rods etc. from a few nm to hundreds of nm sizes.
- Their strong Surface Plasmon Resonance (SPR) allows to monitor NP fate (SPR is sensitive to the NP size and shape) and the NP environment (SPR is highly sensitive to the surroundings of the NP).
- They are commercially available and important in the market. Silver NP are present in many products from colloids to clean vegetables to washing machines and fridges. In addition to using materials produced by NanoTOES partners 9 and 12 it is thus feasible to study commercial preparations as well.
- They can be corroded and release Ag cations which are toxic for bacteria and have a health impact for humans as well. Silver NP and cations released thereof are also of concern for the environment, and will be studied in parallel.
- They can be functionalized. There are many published reproducible strategies for the functionalization of the Ag NP surfaces, e.g., via thiols.
Besides silver NP, other types of NP will be investigated as well, but silver NP will serve as reference material which is investigated by all partners, which will produce an exhaustive and novel set of data characterizing features of the reference materials with a wide range of methods. It is in fact necessary to include other particle types, in order to allow studies on the full range of NP effects and interactions.
For example, silver is susceptible to oxidation, especially the surface of the NP, therefore we will also study NP produced from other noble metals such as Au or Pt and other oxides as Fe2O3 or CeO2 to further our understanding of interactions between inorganic NP and biological entities. Silver and other particles to be investigated will be specifically produced by the experienced partners 9 (ICN) and 12 (Bayer), but will also be obtained from commercial sources. This will allow the ESR and ER to study both novel, highly characterized and functionalized materials, and materials arriving in bulk qualities on the market. It will thus be possible throughout this project to correlate biological effects with specific characteristics of the NP used, in particular, composition, size, shape, purity and aggregation behaviour. For the choice of other NP types, a high priority will be given to NP intended for medical applications. Nanomedicine has to deal with potential side effects of bringing high amounts of NP directly into the body, so tests for nanotoxicity are extremely important. Assays allowing the quick identification of toxic effects may substantially speed up product development. From the point of view of nanosafety, medical applications are the occasion where the highest exposure levels arise, so it has to be verified whether such high concentrations may elicit specific effects.
The investigation of physical, chemical and biological factors regarding their influence on the cell-biological effects of NP requires more sensitive in vitro methods which are able to detect the effects on cell assemblies on the single cell level. To detect the effect of NP on cells, it is common to examine the response of a cellular population, such as a cell monolayer or tissue. Such methods therefore only provide information on the average response of the cells within the sample. This might be a disadvantage; especially when using low concentrations of NP, since some particles can interact with single cells and induce a response, whereas most other cells are not affected. Responses of a few cells only will not be detectable when combining the results of the complete cell population. This approach will establish an innovative method for determining NP-induced effects at the single cell level in dependency of the concentration of NP by using adhering reporter cells on a microfabricated chip with miniaturized cell culture chambers. The chip-supported microscopic technique (provided by partner 3) allows easy detection of the response of each individual cell in a well. Using this method, the effect of treated NP on the IL-8-promoter induction and viability of stably reporter cells (generated by partner 1) is determined, whereby green fluorescence protein (GFP) is used as reporter gene. This chip based system presents a new non-invasive in vitro alternative test method to assess toxicity of nanomaterials with a very significant potential for commercial applications. For example, this assay format will make it possible to study effects induced by NP over time in single, undisturbed cells, greatly facilitating kinetic studies for toxicology as well as for assessment of NP intended for medical or diagnostic use.
The fate of nanomaterials post-production will be followed in an interdisciplinary approach towards a full analysis of their behaviour before, during and after use. Products containing NP may be stored for prolonged periods and under various conditions. We will analyse changes in NP during realistic storage times and conditions to reflect shelf life of the products. It will also be considered that dry particles may get wet and particles in liquid form may dry.
The behaviour of NP upon contact to biological compounds is not sufficiently understood. We will thus use the novel approach to characterise parameters of nanomaterials during exposure to biological agents. Methods will include observations on single NP to allow a full time- resolved study on the fate of individual particles. Changes in NP properties may be related to coating, chemical reactions affecting surface characteristics, aggregation, fragmentation or even dissociation of particles. NanoTOES will contribute to an improved understanding of the behaviour of NP in real-life situations, taking into account degradation, clustering, association with biological substances and other factors which will not only affect the behaviour of NP in living systems and in the environment, but which may also increase or reduce the potential toxicity of the NP.
Based on thorough understanding of NP properties as they have developed during various ageing conditions, we will establish toxicity profiles for these products in a range of relevant systems. In addition to conventional readouts related to toxicity we will also analyse the behaviour of individual cells. This will allow us to establish what percentage of cells shows reactions towards NP and whether individual characteristics of cells, like cell cycle stage, are relevant for their responsiveness. Considering that tumours, allergies or inflammatory reactions may be initiated by single cells, these studies are important to complement information derived from whole cell cultures.
Environmental studies will consider both aqueous and soil environments, documenting the distribution of particles in these media, association with abiotic and biological entities and resulting toxicity according to established ecotoxicological test systems.
Cell-based analysis potential provides the means of testing, in a relatively convenient way, with medium-throughput, the effect of nanostructured materials on human cells. In so doing, this testing should, to be useful, predict the effect of those materials in/on the human body. The predictive value of information obtained by cell-based analysis depends, however, fundamentally on the quality of cells being used. Cell lines that have been in culture for many years may have lost the memory of their tissue of origin; therefore, their predictive value may be low. Primary cells recently isolated from human tissue generally retain their in vivo characteristics for longer than the duration of a cell-based assay; their predictive value is likely to be high. Primary cells offer challenges in terms of limited supply, and the culture requirements to retain functionality. AvantiCell’s culture methodology allows primary cell population expansion, allowing extensive analysis from a single isolate. Additionally, proprietary materials can be used to enhance cell microenvironment and meet the cells’ fastidious requirements that mitigate loss of in vivo-like function.These primary cells’ repertoire of functions is not easily captured by conventional single or dual fluorescence-based assay readouts. And added avantage of AvantiCell’s approach is the inclusion of optional cell-proteomic analysis, with medium throughput, to obtain comprehensive data on the cell function and enable the identification of subtle changes in cell phenotype resulting from NP exposure.