RAMAN LABEL-FREE VISUALISATION OF TITANIUM DIOXIDE NANOPARTICLES UPTAKE IN BJ CELL LINES

Titanium dioxide nanoparticles represent one of the most frequently applied nanomaterials. Due to its advantageous physicochemical properties affecting the final products, use of this nanomaterial in daily used products is increasing. Beside the addition into glaze or enamels, titanium dioxide nanoparticles are found in UV protective cosmetic products applied on skin. According to the studies confirming the potential carcinogenic effect of titanium dioxide nanoparticles application of such nanomaterial may cause health risk. Cellular uptake of nanoparticles and their distribution in cell environment may play an important role in nanoparticles toxicological effect. Thus, evaluation of cellular uptake of nanoparticles is the additional step for evaluation of nanoparticles toxicology. The main objective of this study was to confirm the assumption of the cellular uptake of tested TiO2 nanoparticles using human fibroblasts BJ cell lines and confocal Raman microscopy as a new, promising, label-free imaging technique for studying the distribution of exogenous substances in cells. The results of this study confirm that tested TiO2 nanoparticles are uptaken by cells and distributed in intracellular environment, where form aggregates, possibly during their transport via endocytosis

THE EFFECT OF ACETYLSALICYLIC ACID ON ANGIOGENESIS IN VITRO

Angiogenesis, the formation of new blood vessels, is an essential aspect of, among others, embryonic development, wound healing and the female reproductive cycle. It is also necessary for the expansion of tumour masses beyond a minute volume. Acetylsalicylic acid (ASA) is a non-steroidal anti-inflammatory drug with additional antitumour activity. We tested ASA for its ability to inhibit angiogenesis in a simplified angiogenesis model, hASC+HUVEC co cultured in vitro, using immunocytochemical staining with fluorescence-marked antibodies and observation of tubulelike structures and their branching under a fluorescence microscope. We confirmed that ASA is an efficient and useful angiogenesis inhibitor and deserves further attention. We intend using the designed angiogenesis model and the methods described for observing changes in angiogenesis after anti tumour photodynamic therapy (PDT), and also for enhancing PDT efficiency by addition of angiogenesis inhibitors

Behaviour of silver nanoparticles in simulated saliva and gastrointestinal fluids

Continuously increasing application of silver nanoparticles (AgNPs) requires information on their safety and performance under biological conditions. Assessment of AgNPs in biological systems is also related to availability of robust toxicological methods for evaluation of toxic potential of AgNPs and information on their physicochemical state. Silver nanoparticles were subjected to action of simulated saliva, gastric and intestinal fluids, appropriately supplemented with digestive enzymes pepsin or pancreatin. The behaviour of AgNPs was determined using dynamic light scattering and transmission electron microscopy, and their toxicity as well as capability to induce inflammatory reactions were assessed using reconstructed human tissue models (EpiOral, EpiGingival, EpiIntestinal). The study revealed that during exposure to the fluids, AgNPs size and morphology changed and depended on composition and pH of the respective fluid. If present, the change in terms of growth of AgNPs size occurred immediately after contact of AgNPs with the respective fluid and continued with prolonged time of contact. A pilot study on reconstituted human tissue models revealed low toxicity and inflammatory effects of AgNPs and confirmed the suitability of 3-D models for toxicological studies including bioavailability. © 201717-05095S, GACR, Grantová Agentura České RepublikyCzech Science Foundation [17-05095S]; Ministry of Health, Czech Republic conceptual development of research organization (National Institute of Public Health - NIPH) [75010330]; TBU in Zlin; funds of specific academic research [IGA/CPS/2017/001

Characteristics of silver nanoparticles in vehicles for biological applications

Silver nanoparticles (AgNPs) have been used for decades as anti-bacterial agents in various industrial fields such as cosmetics, health industry, food storage, textile coatings and environmental applications, although their toxicity is not fully recognized yet. Antimicrobial and catalytic activity of AgNPs depends on their size as well as structure, shape, size distribution, and physico-chemical environment. The unique properties of AgNPs require novel or modified toxicological methods for evaluation of their toxic potential combined with robust analytical methods for characterization of nanoparticles applied in relevant vehicles, e.g., culture medium with/without serum and phosphate buffered saline. © 2015 Elsevier B.V. All rights reserved.Internal Grant Agency of the Ministry of Health of the Czech Republic [NT 14060-3/2013]; TBU in Zlin [IGA/FT/2014/004

Sensitization potential of medical devices detected by in vitro and in vivo methods

Medical devices must be tested before marketing in accordance with ISO EN 10993-10 in order to avoid skin sensi­tization. This standard predominantly refers to the in vivo test but does not exclude the use of in vitro methods that have been sufficiently technically and scientifically validated for medical device testing. It is foreseen that, due to the complexity of the sensitization endpoint, a combination of several methods will be needed to address all key events occurring in the sensitization process. The objective of this pilot study was to evaluate the sensitization potential of selected medical devices using a combination of in chemico (DPRA, OECD TG 442C) and in vitro (LuSens, OECD TG 442D) methods in comparison with the in vivo (LLNA DA, OECD TG 442A) method and to suggest a possible testing strategy for the safety assessment of medical device extracts. Overall, one of the 42 tested samples exhibited positive results in all employed test methods, while 33 samples were predicted as non-sensitizing in all three performed methods. This study demonstrated good agreement between in vitro and in vivo results regarding non-sensitizing samples; however, some discrepancies in positive classification were recorded. A testing strategy is suggested in which negative results are accepted and any positive results in the in chemico or in vitro tests are followed up with a third in vitro test and evaluated in accordance with the “2 out of 3 approach”. This strategy may reduce and replace animal use for testing the sensitization potential of medical devices.publishedVersionPeer reviewe

The Rise of Three Rs Centres and Platforms in Europe.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general