Representative cell models for nanotoxicology studies: different approaches and challenges
Nanotechnology is among the fastest growing branches of industry; it provides a wide range of products, from new drugs, to construction materials, to products in daily use such as cosmetics or clothes. Like all new materials, especially those which will be applied in medicine, they should be carefully examined in terms of their impact on human health. Investigations so far show that the toxic potential of engineered nanomaterials (ENMs) is strictly related to their physico-chemical properties, such as shape, size, charge and chemical composition [1-4]. A great number of ENMs should be tested for efficacy (i.e. for desired effects in nanomedicine) and at the same time for safety. Clearly, it is important to produce preferably harmless materials that are in daily use as consumer products or applied in nanomedicine in diagnostics or pharmacology.
Nanotechnology produces a wide range of new materials that require special risk assessment strategies. Those strategies have to be defined for all aspects of risk to evaluate the impact of ENMs on human health and the environment. For evaluation of toxicity, not only fast and high throughput methods are needed but also representative biological models. There is an urgent need to develop well-understood in vitro model cell systems, selected especially for ENM testing and delivering the most reliable information about nano-toxicity. In vitro studies should be focused on cell models originating from representative organs, whose functions would be most affected by ENMs. [5,6]
In vitro toxicology methods allow examination of the toxicity of ENMs and their potential impact on human health in controlled precise experimental conditions, with highly efficient and reliable methods, giving results in a short time, and respecting ethical issues.
This brings us to the question, which cell models from the many available should we choose for nanotoxicology? Which model brings the most relevant information on cellular responses, consistently reflecting the various possible reactions that might occur in organs? And which tissues will be the most affected by ENMs during their “life-cycle” within a living organism? We need to select the most representative organs that will be reached by ENMs and where ENMs will be accumulated. Further, for evaluating ENM toxicity, it is crucial also to define those cells whose function could be most affected by the substances being tested.
Four possible routes of exposure to ENMs have been defined. ENMs can enter the human body via skin (for example from wound dressings, clothing, sun block creams, hair dyes), by inhalation (paints, insulation materials), ingestion (preservatives, toothpastes, water treatment products), or by injection (drugs).
For nano-products, a group of cell models representative for each exposure mechanism should be selected. The chemical analyses (mostly using inductively coupled plasma mass spectroscopy) together with histopathology (by electrons transmission or confocal microscopy) in in vivo models (rats, mice or pigs) are used to define target organs for ENMs and also their translocation and accumulation in animal bodies.
The gastrointestinal tract is the main entrance for ENMs present in food or water. In vivo studies reported damage to epithelial cell microvilli as well as to intestinal glands after exposure to silver nanoparticles. The exposure significantly decreased the weight of exposed animals, perhaps as a result of reduced absorptive capacity of the intestinal epithelium. A significant level of ENMs was found in blood cells, liver, bone marrow, spleen and kidneys. These studies have shown that among target organs only liver exhibited a high level of damaged cells, indicating a significant role of liver in ENM metabolism –acting as a filter for the blood stream. [7-10]
The respiratory tract is one of the main routes of exposure for both ENMs and environmental nanoparticles. Inhaled ENMs can be deposited in one of the target zones of the respiratory tract (nasopharyngeal, tracheobronchial and alveolar), and translocated with intra- or extracellular fluids or by clearance. ENMs deposited in the upper part of the respiratory tract can be removed by a physical process. Smaller ENMs, accumulated in the alveolar area, can cross the blood barrier, where they are phagocytised by macrophages or accumulated in lymphocytes and platelets. With the blood stream, ENMs are translocated to secondary organs such as the heart, bone narrow, reproductive organs, brain, spleen or liver. However, the concentration of inhaled ENMs in these tissues is usually not higher than 1% of the deposited dose. The low level of accumulated ENMs, compared with exposure doses, highlights another problem with designing an in vitro study: to get reliable information comparable with the environmental situation, for cells that represent secondary organs, lower doses should be considered. [7, 11-13]
Toxicology studies based on interaction of ENMs with skin are mostly focused on titanium oxide as a main component of sun block creams. Previous studies reported controversial results. On the one hand, researchers proved that titanium oxide stays on the skin surface and cannot penetrate through the lower layer of living skin.On the other hand, some experiments proved that after prolonged (30 day) exposure, TiO2 can penetrate skin and be located in the deep layer of the epidermis, reaching different tissues, and accumulating in several organs, (liver, brain, heart and spleen) after 60 days exposure. [7, 14-16]
Because of the great potential of ENMs for use in medicine (drug delivery, cancer therapy, diagnostics), another focus is on their interaction with the cardiovascular system. In vivo studies reported high levels of ENMs in blood cells, mostly in macrophages, lymphocytes, and platelets rather in erythrocytes. However, it is most likely that liver and kidneys are the most affected by ENMs, as they play important roles as blood filters. [7,16,17]
After defining target organs for ENMs we should consider which cell models will be most reliable for each tissue. Definitely, macrophages demand special attention because of their role in phagocytosis and ENM uptake. Primary cells harvested from human donors or immortal cell lines such as RAW or THP-1 are frequently used. Endothelial cells and also epithelia play important roles as they are the first contact barrier for ENMs in all exposure routes, and they are often selected as representative models for in vitro studies because a similar phenotype is maintained to that of cells in vivo (with a mucin coating). For evaluation of the impact of ENMs on liver, studies should concentrate especially on hepatocytes, which are the most abundant cell type, responsible for most of the liver’s functions. In general, in vivo assays are based on immortalised cell lines, or on primary cells harvested from tissue. The former are highly repeatable and easy to handle compared with the latter. However, a consequence of immortalization is loss of many of the unique properties of the source primary organs, and so these systems can deliver unreliable information. Additionally, different cell lines representative of the same tissues can react by different mechanisms and with different sensitivity to the same ENMs. Furthermore, the stable cell lines, specially isolated from cancer cells, are characterized by a faster cell cycle compared to primary cells. This can strongly affect ENM testing because uptake, bio distribution in the cells and toxicity of ENMs can all be affected by proliferation rate. Primary cells require human donors or animal sacrifices, which increase experimental costs and raise ethical issues. On the other hand, they can be more representative as models for nanotoxicology and can be used to validate stable cell lines. This comparison was already made with liver cell lines to select the most representative cell models. 
Summing up, nanotoxicology research should focus not only on correlations between ENM properties and biological response, or optimizing high throughput assays, but also on defining standard cell models representative for one main exposure route. Studies should focus on a comparison between immortal cell lines and primary cells and also on the correlation between in vitro and in vivo studies to establish a relevant approach for risk assessment for ENMs. This comparison will allow us to determine the best cell lines for nanotoxicology and also the ideal biological conditions to maintain a reliable in vitro model.
 Wentong Lu, Dulal Senapati, Shuguang Wang, Oleg Tovmachenko, Anant Kumar Singh, Hongtao Yu, Paresh Chandra Ray: Effect of surface coating on the toxicity of silver nanomaterials on human skin keratinocytes. Chemical Physics Letters 2010, 487: 92–96
 Tiago Morais, Maria Elisa Soares, José Alberto Duarte, Leonor Soares, Sílvia Maia, Paula Gomes, Eulália Pereira, Sónia Fraga, Helena Carmo, Maria de Lourdes Bastos: Effect of surface coating on the biodistribution profile of gold nanoparticles in the rat. European Journal of Pharmaceutics and Biopharmaceutics 2012, 80: 185–193
 Chunbai He, Yiping Hu, Lichen Yin, Cui Tang, Chunhua Yin: Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials 2010, 31: 3657–3666
 Syed K Sohaebuddin, Paul T Thevenot, David Baker, John W Eaton, Liping Tang:
Nanomaterial cytotoxicity is composition, size, and cell type dependent. Particle and Fibre Toxicology 2010, 7:22
 Vicky Stone, Helinor Jahnston, Roel P. F. Schins: Development of in vitro systems for nanotoxicology: methodological considerations. Critical Reviews in Toxicology 2009, 39:613-626
 Clinton F. Jones, David W. Grainger: In vitro assessments of nanomaterial toxicity Advanced Drug Delivery Reviews 2009, 61:438-456
 Yuliang Zhao, Bing Wang, Weiyue Feng, Chunli Bai: Nanotoxicology: Toxicological and biological activities of nanomaterials. Nanoscience and nanotechnologies
 Brigesh Shahare, madhu Yashpal, Gajendra Singh: Toxic effects of repeated oral exposure of silver nanoparticles on small intestine mucosa of mice. Toxicology Mechanisms and Methods 2013, 23:161-167
 Meike van der Zande, Rob J. Vandebriel, Elke Van Doren, Evelien Kramer, Zahira Herrera Rivera, Cecilia S. Serrano-Rojero, Eric R. Gremmer, Jan Mast, Ruud J. B. Peters, Peter C. H. Hollman, Peter J. M. Hendriksen, Hans J. P. Marvin, Ad A. C. M. Peijnenburg, and Hans Bouwmeester: Distribution, Elimination, and Toxicity of Silver Nanoparticles and Silver Ions in Rats after 28-Day Oral Exposure. ACSNano 2012, 8: 7427-42
 Tae-Keun Hong, Nirmalya Tripathy, Hyun-Jin Son, Ki-Tae Ha, Han-Sol Jeong, Yoon-Bong Hahn: A comprehensive in vitro and in vivo study of ZnO nanoparticles toxicity. Journal of Materials Chemistry B 2013, 1: 2985-2992
 Yang Liu, Yuxi Gao, Lili Zhang, Tiancheng Wang, Jiangxue Wang, Fang Jiao, Wei Li, Ying Liu, Yufeng Li, Bai Li, Zhifang Chai, Gang Wu, and Chunying Chen: Potential health impact on mice after nasal instillation of nano-sized copper particles and their translocation in mice. Journal of Nanoscience and Nanotechnology 2009, 9: 1-9
 Jae Hyuck Sung, Jun Ho Ji, Jung Duck Park, Jin Uk Yoon, Dae Sung Kim, Ki Soo Jeon, Moon Yong Song, Jayoung Jeong, Beom Seok Han, Jeong Hee Han, Yong Hyun Chung, Hee Kyung Chang, Ji Hyun Lee, Myung Haing Cho, Bruce J Kelman, Il Je Yu: Subchronic inhalation toxicity of silver nanoparticles. Toxicological Sciences 2008, 2: 452-61
 Jun Ho Ji, Jae Hee Jung, Sang Soo Kim, Jin-Uk Yoon, Jung Duck Park, Byung Sun Choi, Yong Hyun Chung, II Hoon Kwon, Jayoung Jeong, Beom Seok Han, JAe Hyeg Shin, Jae Hyuck Sung, Kyung Seuk Song, II Je Yu: Twenty-Eight-Day Inhalation Toxicity Study of Silver Nanoparticles in Sprague-Dawley Rats. Inhalation Toxciology 2007, 19:745-751
 Nakissa Sadrieh, Anna M. Wokovich, Neera V. Gopee, Jiwen Zheng, Diana Haines, David Parmiter, Paul H. Siitonen, Christy R. Cozart, Anil K. Patri, Scott E. McNeil, Paul C. Howard, William H. Doub, Lucinda F. Buhse: Lack of Significant Dermal Penetration of Titanium Dioxide from Sunscreen Formulations Containing Nano- and Submicron-Size TiO2 Particles. Toxicological Sciences 2010, 1:156-166
 Matteo Crosera, Massimo Bovenzi, Giovanni Maina, Gianpiero Adami, Caterina Zanette, Chiara Florio, Francesca Filon Larese: Nanoparticle dermal absorption and toxicity: a review of the literature. International Archives of Occupational and Environmental Health 2009, 9: 1043-1055
 Jianhong Wu, Wei Liu, Chenbing Xue, Shunchang Zhou, Fengli Lan, Lei Bi, Huibi Xu, Xiangliang Yang, Fan-Dian Zeng: Toxicity and penetration of TiO2 nanoparticles in hairless mice and porcine skin after subchronic dermal exposure. Toxicology Letters 2009, 1:1-8
 Zhongjun Du, Dali Zhao, Li Jing, Guanqun Cui, Minghua Jin, Yang Li, Xiaomei Liu, Ying Liu, Haiying Du, Caixia Guo, Xianqing Zhou, Zhiwei Sun: Cardiovascular Toxicity of Different Sizes Amorphous Silica Nanoparticles in Rats After Intratracheal Instillation. Cardiovascular Toxicology 2013, 3:194-207
 Helinor J. Johnston, Manuela Semmler-Behnke, David M. Brown, Wolfgang Kreyling,
Lang Tran, Vicki Stone: Evaluating the uptake and intracellular fate of polystyrene nanoparticles by primary and hepatocyte cell lines in vitro. Toxicology and Applied Pharmacology 2010, 242:66-78