NanoTEST in a Nutshell

There is a huge effort underway in nanotechnology with the development of nanomaterials which have better properties and are more effective than their parent bulk materials, and also are safe to use. In nanomedicine safety aspects can largely be addressed with the same methodologies and tools as are needed for testing efficacy. The impact of medical nanomaterials is also easier to investigate because human exposure is already known. Since in nanomedicine, nanoparticles (NPs) enter the body, their interaction with cells and tissue is inevitable and must be investigated. The main aim of the FP7 NanoTEST project (www.nanotest-fp7.eu, Dusinska et al., 2009) was to study the biological impact of NPs in nanomedicine as a basis for understanding molecular and cellular pathways that can lead to toxic effects, together with the development of appropriate methods to test them. This supplement consists of 13 scientific papers including a commentary, all of them from the NanoTEST project, showing the approach towards development of testing strategies and high throughput methods for hazard assessment of nanomaterials used in nanomedicine. All in vitro studies were harmonized with NPs from the same batch with identical dispersion protocols, exposure time, concentration range, culture conditions and time-courses. The first paper addresses critical aspects of assessing the safety of nanomaterials in medicine: the balance between risks and benefits; and the major challenges encountered when studying biological impact, biocompatibility, distribution in the human body, biodegradation and excretion routes, and dispersion in the environment (Juillerat et al., 2015). A final goal of NanoTEST was the development and validation of tools for evaluating the biological impact of NPs. This could only be achieved by addressing interactions of NPs with cells, living tissues and their possible effects in the human (and animal) body. For hazard assessment of NPs, specific characteristics related to size and surface properties, that might influence their behaviour and adverse effects, must be taken into consideration. In NanoTEST titanium dioxide (TiO2) NPs of nominal size 20 nm, iron oxide (8 nm) both uncoated (U-Fe3O4) and oleic acid coated (OC-Fe3O4), rhodamine-labelled 25 nm (Fl-25 SiO2) and 50 nm (Fl-50 SiO) amorphous silica and polylactic glycolic acid polyethylene oxide polymeric (PLGA-PEO) NPs, were investigated. The main characteristics of these NPs are described in Guadagnini et al. (2015a). The major goal of this paper was to evaluate the available toxicity tests and to investigate possible interference with tested nanomaterials. The panel of NP suspensions used in this project showed that many NP characteristics (composition, size, coatings and agglomeration) interfere with a range of in vitro cytotoxicity assays. The paper also proposes how to avoid interference of NPs with testing systems as the first step of a screening strategy for biomedical NPs. The information and recommendations provided by the authors are also valuable for NP safety assessment generally. NanoTEST addressed the main toxicity endpoints – cytotoxicity, oxidative stress, immunotoxicity and genotoxicity – using various in vitro cell culture models representing eight different organs. Results from vascular system, placenta, brain, kidney, gastrointestinal system and (partially) blood have already been published elsewhere (Aranda et al., 2013; Cartwright et al., 2011; Halamoda Kenzaoui et al., 2012a,b,c, 2013a,b; Kazimirova et al., 2012; Magdolenova et al., 2012a,b). One of the main routes of exposure to NPs is through the lungs. Lung epithelial cells are the first target cells after inhalation but also secondary targets after injection of NPs due to the small distance between the epithelial cells and the blood capillaries. Several nanomaterials are already used for lung therapeutics and diagnostic purposes. Guadagnini et al. (2015b) studied possible adverse pulmonary responses by evaluating cytotoxicity, reactive oxygen species (ROS) production and pro-inflammatory responses. The effects of PLGA, silica, iron oxide and TiO2 NPs were studied using human bronchial (16HBE) and alveolar epithelial cells (A549) with different sensitivity depending on cell type, toxicity endpoint and NPs used. PLGA NPs were proposed as good candidates for negative control NPs and SiO2 NPs were revealed to be the best benchmark NPs. The authors concluded that measurement of oxidative stress does not systematically allow the prediction of cellular responses and proposed that a battery of assays and cell lines are necessary to evaluate the pulmonary effects of NPs. Blood is the main route for biodistribution of therapeutic NPs, and NPs that pass through the lungs or gastrointestinal tract are also distributed to other organs through the blood circulation. NPs were therefore studied in vitro in a blood cell model, using both stable cell lines as well as primary human blood cells (Magdolenova et al., 2015; Tulinska et al., 2015). A human blood cell model was used for immunotoxicity and genotoxicity testing to measure the response to PLGA-PEO NPs in fresh peripheral whole blood cultures and in isolated peripheral blood mononuclear cell cultures from 13 human volunteers, showing that primary blood cells are suitable for detecting the response to NPs (Tulinska et al., 2015). Using several immunotoxicity tests, proliferative activity of T-lymphocytes and T-dependent B-cell response in cultures stimulated with mitogens, cytotoxicity of natural killer cells, phagocytic activity of granulocytes and Correspondence: Maria Dusinska, E-mail: maria.dusinska@nilu.no N an ot ox ic ol og y D ow nl oa de d fr om in fo rm ah ea lth ca re .c om b y 95 .1 02 .1 79 .3 9 on 0 4/ 29 /1 5