In-Vitro Cell Exposure Studies for the Assessment of Nanoparticle Toxicity in the Lung—A Dialog Between Aerosol Science and Biology$

Journal of Aerosol Science 42 (2011) 668–692

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Journal of Aerosol Science

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In-vitro cell exposure studies for the assessment of nanoparticle toxicity in the lung—A dialog between aerosol science and biology$

Hanns-Rudolf Paur a, Flemming R. Cassee b, Justin Teeguarden c, Heinz Fissan d, Silvia Diabate e, Michaela Aufderheide f, Wolfgang G. Kreyling g, Otto Hanninen¨ h, Gerhard Kasper i, Michael Riediker j, Barbara Rothen-Rutishauser k, Otmar Schmid g,n a Institut fur¨ Technische Chemie (ITC-TAB), Karlsruher Institut fur¨ Technologie, Campus Nord, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany b Center for Environmental Health, National Institute for Public Health and the Environment, P.O. Box 1, 3720 MA Bilthoven, The Netherlands c Paciﬁc Northwest National Laboratory, Fundamental and Computational Science Directorate, 902 Battelle Boulevard, Richland, WA 99352, USA d Institute of Energy and Environmental Technologies (IUTA), Duisburg, Germany e Institut fur¨ Toxikologie und Genetik, Karlsruher Institut fur¨ Technologie, Campus Nord, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany f Cultex Laboratories, Feodor-Lynen-Straße 21, 30625 Hannover, Germany g Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum Munchen,¨ Ingolstadter¨ Landstrasse 1, 85764 Neuherberg, Germany h THL National Institute for Health and Welfare, PO Box 95, 70701 Kuopio, Finland i Institut fur¨ Mechanische Verfahrenstechnik und Mechanik—Bereich Gas-Partikel-Systeme, Karlsruher Institut fur¨ Technologie, Campus Sud,¨ Geb. 30.70 Straße am Forum 8, 76131 Karlsruhe, Germany j Universite´ de Lausanne et Geneve, Institute for Work and Health, Rue du Bugnon, 21 1011 Lausanne, Switzerland k Universitat¨ Bern, Pneumologie, Departement klinische Forschung und Inselspital, Murtenstrasse 50, 3010 Bern, Switzerland article info abstract

Article history: The introduction of engineered nanostructured materials into a rapidly increasing Received 12 December 2010 number of industrial and consumer products will result in enhanced exposure to Received in revised form engineered nanoparticles. Workplace exposure has been identiﬁed as the most likely 11 June 2011 source of uncontrolled inhalation of engineered aerosolized nanoparticles, but release Accepted 11 June 2011 of engineered nanoparticles may occur at any stage of the lifecycle of (consumer) Available online 22 June 2011 products. The dynamic development of nanomaterials with possibly unknown toxico- Keywords: logical effects poses a challenge for the assessment of nanoparticle induced toxicity and Nanoparticle safety. Nanotoxicity In this consensus document from a workshop on in-vitro cell systems for nano- Particle toxicity particle toxicity testing1 an overview is given of the main issues concerning exposure to Cell exposure system Air–liquid interface airborne nanoparticles, lung physiology, biological mechanisms of (adverse) action, in- Dose metric vitro cell exposure systems, realistic tissue doses, risk assessment and social aspects of nanotechnology. The workshop participants recognized the large potential of in-vitro cell exposure systems for reliable, high-throughput screening of nanoparticle toxicity. For the investigation of lung toxicity, a strong preference was expressed for air–liquid interface (ALI) cell exposure systems (rather than submerged cell exposure systems) as

$ Based on a workshop sponsored by Gesellschaft fur¨ Aerosolforschung, Karlsruhe, Germany, 5–6 September 2009. n Corresponding author. Tel.: þ49 8931872557; fax: þ49 8931872400. E-mail addresses: [email protected] (H.-R. Paur), fl[email protected] (F.R. Cassee), [email protected] (J. Teeguarden), heinz.fi[email protected] (H. Fissan), [email protected] (S. Diabate), [email protected] (M. Aufderheide), [email protected] (W.G. Kreyling), otto.hanninen@thl.fi (O. Hanninen),¨ [email protected] (G. Kasper), [email protected] (M. Riediker), [email protected] (B. Rothen-Rutishauser), [email protected] (O. Schmid). 1 Workshop on ‘In-Vitro Exposure Studies for Toxicity Testing of Engineered Nanoparticles’ sponsored by the Association for Aerosol Research (GAeF), 5–6 September 2009, Karlsruhe, Germany.

they more closely resemble in-vivo conditions in the lungs and they allow for unaltered and dosimetrically accurate delivery of aerosolized nanoparticles to the cells. An important aspect, which is frequently overlooked, is the comparison of typically used in-vitro dose levels with realistic in-vivo nanoparticle doses in the lung. If we consider average ambient urban exposure and occupational exposure at 5 mg/m3 (maximum level allowed by Occupational Safety and Health Administration (OSHA)) as the boundaries of human exposure, the corresponding upper-limit range of nanoparticle ﬂux delivered to the lung tissue is 3 105–5 10-3 mg/h/cm2 of lung tissue and 2– 300 particles/h/(epithelial) cell. This range can be easily matched and even exceeded by almost all currently available cell exposure systems. The consensus statement includes a set of recommendations for conducting in-vitro cell exposure studies with pulmonary cell systems and identiﬁes urgent needs for future development. As these issues are crucial for the introduction of safe nanomaterials into the marketplace and the living environment, they deserve more attention and more interaction between biologists and aerosol scientists. The members of the workshop believe that further advances in in-vitro cell exposure studies would be greatly facilitated by a more active role of the aerosol scientists. The technical know- how for developing and running ALI in-vitro exposure systems is available in the aerosol community and at the same time biologists/toxicologists are required for proper assessment of the biological impact of nanoparticles. & 2011 Elsevier Ltd. All rights reserved.

1. Introduction

With the advent of nanotechnology, the prospects of manufactured nanomaterials in many applications have progressed rapidly (Gwinn & Vallyathan, 2006). The potential benefits of nanotechnology are undoubted. Currently, numerous types of novel nanoparticles are being produced for widespread application in consumer productions as well as for therapeutic, diagnostic and other novel technological applications. In the wake of this development literally hundreds of thousands ‘new’ types of nanoparticles are already or will be manufactured differing in size, agglomeration stage, shape, surface charge as well as material, surface functionalisation, layered structure and many more properties. Typically, engineered nanoparticles refers to particles which are manufactured (not inadvertently generated) and shorter than 100 nm in at least one dimension. There are currently standardization efforts under way (e.g. ONR CEN ISO/TS 27687:2009-06-01) applying this definition to ‘nanoobjects’, but no generally accepted nomenclature has been established, yet. Material safety regulations traditionally relied on animal testing. For both ethical and financial reasons, animal experiments cannot be performed for each single type of new nanoparticle. Hence, workers’ safety, consumer health and environmental protection concerns require new approaches to toxicity testing of these nanoparticles, which may be exposed to during production, handling, use and recycling of nanoparticles or of materials containing nanoparticles. Toxicology is defined as the study of the adverse effects of substances on living organisms (Timbrell, 1998). One of the fundamental principles of toxicology is that the health ‘risk’ posed by a substance is a function of its potential to cause harm, or ‘hazard’, and the amount of substance a biological system is ‘exposed’ to. This is expressed by the following relation:

Risk ¼ f ðexposure, hazardÞ

Another important principle of toxicology is that all materials are toxic, if exposure occurs in sufficient quantities (Timbrell, 1998). Most commonly this principle is quoted as phrased by Paracelsus (CH, 1493–1541): ‘All things are poison and nothing is without poison, only the dose permits something not to be poisonous’. These fundamental toxicology principles imply that substances with a low hazard generally pose a low risk. If however, there is a high enough exposure of these low hazard substances, then even these substances can be harmful, or even fatal. And of course, the opposite can also occur: high hazard but relative low exposure also results in a low risk. In this paper we focus on hazard identification but we would also like to stress the importance of accurate exposure data. A very important aspect is the identification of the most relevant dose metric for particle toxicity (Schmid et al., 2009; Grass et al., 2010). It is acknowledged that numerous pathways of nanoparticle toxicity (henceforth nanotoxicity) exist including but not limited to oxidative potency, bioactivation of toxic organic compounds (e.g. polyaromatic hydrocarbons), inflammation, binding to biomolecules leading to inactivation or mutations, adjuvant action, and (frustrated) phagocytosis (Donaldson et al., 2005, 2006). Depending on the physicochemical properties of the nanoparticles, one or more of these pathways may be activated. Hence, it is quite obvious that a single dose parameter (e.g. number, mass or surface area) is not likely to adequately describe the toxic effects of any type of nanoparticle. Traditionally mass has been used as dose metric. While this is likely to be a reasonable dose metric for completely soluble or biodegradable nanoparticles, (partially) inert or non-biodegradable nanoparticles reside for an extended period of time in the tissue as particulate matter 670 H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692 interacting with the biological environment via the surface-tissue interface. This is reflected in the mounting evidence that surface area, and not mass or number, best predict the toxic response of numerous types of nanoparticles (Stoeger et al., 2007, 2009; Oberdorster¨ et al., 2007; Donaldson et al., 2008). However it is important to note that while surface area may be a very relevant contributor to the dose metric for insoluble, non-biodegradable particles, there are also other important properties for predicting the toxicity of nanoparticles. E.g. frustrated phagocytosis is encountered for rigid, non-soluble fibers larger than about 10 mm (e.g. asbestos and carbon nanotubes) in the lungs (Poland et al., 2008). A more detailed discussion of the effect of the physico-chemical properties of nanoparticles on the dose definition has recently presented by Grass et al. (2010). Based on the principles of toxicology, nanoparticles toxicity (henceforth referred to as or nanotoxicology), can be defined as ‘the study of the adverse effects of nanoparticles on living organisms’ and further described as a multi- disciplinary science including material science, chemistry, physics and medicine (Donaldson et al., 2004). The field of nanotoxicology focuses upon gaining a thorough understanding of the relationship between the toxicity of nanoparticles depending on their dose levels and properties such as size, shape, reactivity and material composition, i.e. their physicochemical characteristics. One of the main entrance routes into the human organism is the lung due to the constant intake of air (about 20–30 m3/day; ICRP, 1994) and the relatively large surface area 75–150 m2 as compared to the skin (1m2). This large surface allows efficient oxygen/carbon dioxide exchange with the blood (Gehr et al., 1978a, 1978b; Mercer et al., 1994; Wiebe & Laursen, 1995). There actually is a relatively large uncertainty regarding the surface area of the lungs in human populations as the few available studies on lung morphometry (Gehr et al., 1978a, 1978b; Mercer et al., 1994; Wiebe and Laursen, 1995)have examined a very limited number of individuals without even accounting for differences in the body mass or height of the subjects versus the general population or more specific target population groups. While exposure to nanoparticles can also occur through other portals of entry (e.g. skin and gastrointestinal tract), this review focuses in-vitro models to determine toxicity of nanoparticles inhaled into the lungs. The exposure of the lung epithelium to reactive atmospheres (which can contain nanoparticles) has been shown to lead to cellular injury or oxidative stress and is discussed in connection with a variety of diseases like asthma or Chronic Obstructive Pulmonary Disease (COPD) (Barnes, 2000, 2004; Bartal, 2005; Hylkema et al., 2007). Therefore, characterization of the early cellular and molecular reactions of bronchial/alveolar epithelial cells and alveolar macrophages plays an important role in understanding the mechanisms involved in chronic lung diseases. In addition, nanoparticles are also thought to by-pass the lung defense system, reach the systemic circulation and adversely affect the cardiovascular system. Interestingly, engineered nanoparticles are also being used as drug-delivery vehicles (Borm et al., 2006; Borm & Kreyling, 2004). For the assessment and evaluation of the toxic characteristics of inhaled aerosols, the toxicity is usually determined by animal experiments according to the guidelines of the OECD (Organization for Economic Co-operation and Development).

Frequently, these tests provide the LC50 (median lethal concentration), that can be ‘expected to cause death during short term (acute) exposure or within a fixed time after exposure in 50% of animals exposed for a specified time’ (OECD). Various OECD-guidelines for testing acute and chronic toxicity are available, depending on the required information. The determination of repeated dose inhalation toxicity requires long term studies ranging from several days up to two years employing large numbers of test animals, followed by extensive examination of tissue samples (e.g. Pott & Roller, 2003). Furthermore, there are methodological difficulties associated with in-vivo exposures of laboratory animals to nanoparticles (Muhlfeld¨ et al., 2008). Laboratory animals and humans show significant differences in lung physiology and immune system, which are highly relevant for nanotoxicology. Secondly, the lung-delivered nanoparticle dose is typically not well known for in-vivo inhalation experiments, since it cannot be measured directly, but is derived from the (size-resolved) nanoparticle concentration in the inhaled air, the inhaled air volume and empirical equations for the size-dependent nanoparticle deposition in the lungs (Alexander et al., 2008; Mendez et al., 2010). While the former is readily measured that latter two are not. This results in potentially large uncertainties in tissue-delivered dose due to e.g. variability in respiratory parameters and lung morphology (Alexander et al., 2008; Mendez et al., 2010). Nevertheless, animal experiments provide critical information for risk assessment and, for reasons discussed below, at present no in-vitro models are available to replace this type of studies. In the year 2005 10 billion euro were spent on animal experiments worldwide and more than 100 million animals were used (Taylor et al., 2008), about 20% of these for toxicological testing. Although most of these studies are required by legislation, their value is challenged with respect to transferability of the results to humans, the application of extreme doses, false positive correlations of multiple endpoints and low toxicity of most of the tested chemicals (Hartung, 2009). Given the high costs, the ethical concerns and their questionable value presumably false hopes are raised regarding product safety on the basis of animal tests only. Therefore high expectations rest on alternative in-vitro test methods, which apply (human) cell cultures to provide endpoints which are directly associated with specific organs. Especially the use of co-cultures and organ tissue for in-vitro studies in modern exposure systems open new avenues for reliable toxicological studies without animals. Cell-based in-vitro exposure studies might offer a new avenue for toxicity screening of new nanoparticles. This screening should be optimized to identify nanoparticles of relative high toxicity. Only those ‘high-hazard’ nanoparticles should then be assessed by animal testing. Dedicated strategies have been developed to reduce the number of animal experiments for the implementation of the European Guideline REACH (Registration, Evaluation and Authorization of Chemicals) (Lahl, 2005), which regulates the toxicity testing required for all chemicals used in the H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692 671

European community. However, it has to be acknowledged that cell and tissue cultures will not be able to replace animal tests completely, since the complexity of the human organism cannot be represented by cell cultures and since the lifetime of cell cultures is too short to allow for the assessment of chronic toxicity. Despite high hopes in-vitro testing of nanoparticles has not yet replaced the established animal-based procedures of inhalation toxicology. Several challenges are identiﬁed that have to be overcome:

Development of accurate and cost efﬁcient measurement/detection technologies of nanoparticles (relevant for nanotoxicity in general) in biological samples. Establishment of reliable dose metrics for exposure assessment (relevant for nanotoxicity in general). In-vitro dose–response studies using realistic dose ranges guided by exposure assessment. Optimized and standardized sample preparation with appropriate reference material. Optimized and standardized exposure technologies for in-vitro studies using aerosol exposures. Acceptance in risk assessment of data produced by in-vitro models.

Ultimately this will result in linking nanoparticle (physicochemical) properties with toxicological pathways and cellular uptake mechanisms, sufficient and appropriate information for risk assessment and societal acceptance of nanomaterials and products made using nanotechnology. Accordingly this review will present the state of the art on nanoparticle exposure and in-vitro cell exposure systems for nanoparticles and it will offer perspectives for approaching this highly interdisciplinary field of active research. The main chapters cover the topics of nanoparticle exposure, the biological responses and underlying mechanisms, and the available in-vitro exposure methods. Crucial knowledge gaps and urgent research needs with respect to standardization, measurement tools, particle generation, legislation, and toxicological testing are discussed in each of the main chapters. The social and legislative perspectives of nanoparticle toxicity will be addressed in the final chapters of this review.

2. Exposure

2.1. From emission to exposure

Exposure to airborne nanoparticles occurs at the end of an event cascade which is schematically shown in Fig. 1. Nanoparticles are emitted into the environment by primary sources such as natural phenomena, combustion processes or industrial activities (e.g. welding) or release during generation and handling of engineered nanoparticles. As nanoparticles are transported through the environment, they can be physically (size and shape) and chemically modiﬁed due to interactions with sunlight, water and other environmental substances. Among the most relevant processes modifying the properties of the nanoparticles are aggregation, (partial) evaporation, condensation, cloud processing and photochemical transformation. The amount of nanoparticles contained in the inhaled air is typically referred to as the exposure level. However, the biologically more relevant measure is the (biological) dose, i.e. the amount of particles seen by the biological response (effect) system. For inhalation exposures as considered here the dose refers to the amount of particles reaching the lung epithelium. Once the dose is known one can infer the biological effect from toxicological dose–response measurements using either in-vivo (animal) or in-vitro (cell) models. In the lungs airborne particles are mainly deposited onto the lung epithelium due to the combined effects of diffusion, sedimentation and impaction. For the nano-sized particles diffusion is the main deposition mechanism, but for particles larger than about 200 nm sedimentation also starts to play a role. Since diffusion is an isotropic process, the deposition of

Fig. 1. From nanoparticle emission to the induced biological effect. 672 H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692 nanoparticles is spatially quite uniform on the lung tissue for a given lung depth, but its regional deposition efficiency varies significantly depending on particle size, lung morphology and respiratory parameters. For typical breathing patterns, nanoparticles between about 10 and 50 nm deposit very efficiently (maximum of about 50% at 20 nm) in the alveolar region, which represents the potentially most vulnerable site of deposition (very thin tissue barrier towards the circulation; o1 mm, see below) and accounts for more than 90% of the lung surface area. Detailed discussions of the governing parameters of regional lung deposition have been presented elsewhere (ICRP, 1994). As mentioned above, a critical issue is the identification of the most relevant dose metric for exposure and dose determination. The most frequently used concentration measures are particles mass (mass of particles per volume of inhaled air, mg/m3), surface area (mm2/m3), number concentration (1/m3). While particle mass is most suitable for water- soluble material, its predictive power for toxicological effects of non-soluble nanoparticles (as considered here) is very limited (Waters et al., 2009; Stoeger et al., 2006). Similarly, particle number does not appear to provide a relevant dose metric for different types of nanoparticles (Stoeger et al., 2007). Therefore, an increasing number of authors recommends surface area (according to the BET method, if possible) as the most relevant dose metric for nanoparticle toxicity over number or mass concentration (Oberdorster,¨ 1996; Stoeger et al., 2006; Maynard and Kuempel, 2005; Donaldson et al., 2008), while size is mostly relevant to determine the site of action and the mechanism of toxicity (regional deposition efficiency in the lungs, cellular uptake rate, translocation into organs other than the lung, etc., see below). As a caveat we add that in light of the wide range of nanoparticle properties such as shape, size and chemical composition used in modern nanotechnology combined with the complexity of the human organism, it is unlikely that there will ever be a single dose metric suitable for all types of nanoparticles and any kind of biological response. While there are numerous aerosol devices for exposure measurement (Baron & Willeke, 2001), direct dose measurements are impossible, since the detector would have to be placed on the lung epithelium, i.e. inside the human lungs. Consequently, alternative methods have to be developed for inhalation dose measurements. The most commonly used approach is to measure the size-resolved exposure levels using conventional aerosol devices (e.g. a scanning mobility particle sizer) and model the subsequent size-dependent lung deposition with computational models (Ferron et al., 1988; Hofmann, 1996) or with standard lung deposition curves as provided by the ICRP software (ICRP, 1994). More recently, an alternative more direct technology has become available, which infers the lung-deposited nanoparticle surface area (between 10 and 400 nm in diameter) by adjusting the size-resolved response function of the instrument so that it corresponds to the lung-deposited particle surface area (Shin et al., 2007; Asbach et al., 2009; Fissan et al., 2007). In the meantime several instruments of this kind have been made commercially available. They all assume that the particles are non-hygroscopic and that the equivalent mobility diameter corresponds to the equivalent surface area diameter, i.e. they assume spherical, non-hygroscopic particles with smooth surface. While the mean effect of individual lung physiology and respiratory parameters can be incorporated into the response function of these devices via a calibration factor (Asbach et al., 2009), these instruments are not personal samplers, which determine the breath-by-breath lung deposited particle dose, but this parameter can be calculated from the measured lung deposited nanoparticle surface area, when the breathing parameters of a person are known. We note that this is an approximation of the breath-by -reath personal dose, which assumes that the particle lung deposition of an individual can be predicted by the ICRP-Model and the slope of the deposition curve does not change in the covered size range with the kind of person and the activity (Asbach et al., 2009). Several research and development projects have been started to adjust and further develop the existing techniques to measure nanoparticles especially in workplaces (NanoDevice). The developments aim towards simpler, easy to use, on-line personal sampler devices based on assuming spherical particles. Towards agglomerates a first step has been taken. Recently, an instrument has been developed, which not only measures size distributions of spheres but also of agglomerates and determines the number and the size of the primary particles forming the loose agglomerate (UNPA) (Wang et al., 2010). A severe problem in exposure measurement is the always existing background, which is interfering with the measurement of the nanoparticle originating from the workplace activities. The solution thus far is the application of a reference measurement according to recently developed procedures (Kuhlbusch et al., 2008). Alternatively, source apportionment can provide source-attributable exposure estimates that can then be used for studying potential source-specific effects.

2.2. Exposure systems with cells cultured under submerged conditions

In-vitro cell experiments have been widely used to assess the toxicity of nanoparticles. These studies have typically been performed under submerged culture conditions, where the agent to be investigated (here: nanoparticles) is added to the culture medium, which completely covers the cells grown on the bottom of the cell culture dish (Maier et al., 2008; Kim et al., 2005). With this approach a wide variety of biological endpoints and nanoparticles have been investigated including ambient and occupational particles (e.g. soot and welding fume particles) as well as engineered nanoparticles made of e.g. metals, metal oxides, transition metals and organic material either with or without surface modification (Baulig et al., 2009; Jalava et al., 2009; Fritsch et al., 2006; Brunner et al., 2006; Pulskamp et al., 2007; Waters et al., 2009; Limbach et al., 2007a; Rothen-Rutishauser, 2007; Stearns et al., 2001; Becker et al., 2005). While testing under submerged culture conditions is a simple and well-proven approach for soluble molecular toxins, this method has several deficiencies when applied to airborne nanoparticles. First, for primary contact organs such as the lung (or the skin or retina), submerged exposure represents an unrealistic way of exposure for airborne particles, since the in-vivo exposure to airborne nanoparticles occurs at the air–liquid interface (ALI, epithelial cells represent the boundary H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692 673 between ambient air and inner tissue of the organisms) and not under fully immersed (submerged) conditions. Furthermore, unlike dissolved molecular substances, the biological effect of nanoparticles may be altered by surface adsorption of molecules contained in the medium and agglomeration of individual nanoparticles may result in much larger – and hence biologically different – particles than the individual nanoparticles (Limbach et al., 2005; Kreyling et al., 2006). Finally, for reasons discussed below (Section 4.1), it is difficult to infer the cell-deposited nanoparticle dose from the nanoparticle concentration in the cell medium. Consequently, the reliability of dose–response relationships obtained under submerged conditions is limited unless all parameters (particle/agglomerate size, height of the medium above the cells, etc.) are well controlled (Limbach et al., 2005; Teeguarden et al., 2007; Waters et al., 2009).

2.3. Air–liquid interface (ALI) exposure systems

With the increasing awareness of the inherent problems of using submerged cell cultures for nanotoxicity testing, several in-vitro exposure systems have recently been developed, which allow for controlled nanoparticle exposure of cells cultured at the ALI. The ALI exposure not only represents a more realistic exposure scenario for nanoparticles inhaled into the lung it also provides more control over the effective nanoparticle dose interacting with the cells, which facilitates more reliable dose–response measurements as discussed in Section 4.2. Early versions of modern ALI cell culture techniques were introduced in the 1970s (Voisin et al., 1975, 1977a, 1977b). As seen in Fig. 2 (left panel) by growing the cells on micro-porous membranes the cells can be nutrified from the basal side of the membrane whereas the apical part with the cultivated cells is in direct contact with the test atmosphere consisting of gaseous and/or particulate compounds. The essential aspects for in-vitro exposure devices mimicking realistic inhalation exposure conditions as found in the lung are as follows: (1) complex pulmonary cell systems, which can be cultivated for at least several hours at the ALI (see Sections 3.2.3), (2) direct contact between the cultivated cells and the inhalable substances without interfering medium, (3) uniform exposure of the entire cell layer (4), temperature and humidity conditioning of the air to maintain cell integrity (T37 1C; RH485%) and (5) precise control of the pollutant levels (Rasmussen, 1984; Aufderheide, 2005, 2008) for accurate dosimetry. While issues 1 and 5 will be discussed in more detail below (see Sections 3 and 4, respectively), here we will focus on aspects 2–4. A variety of such ALI cell exposure systems have been described in the literature (Aufderheide and Mohr, 1999, 2000; Aufderheide, 2005; Be´ruBe´ et al., 2009; Fukano et al., 2004; Massey et al., 1998: Bitterle et al., 2006; Mulhopt¨ et al., 2008; Rothen-Rutishauser et al., 2009a, 2009b; Lenz et al., 2009). While most of them rely on diffusion and/or gravitational settling as deposition mechanisms, some exposure systems have been introduced utilizing electrostatic deposition for bipolarly or unipolarly charged particles (Savi et al., 2008; Stevens et al., 2008; de Bruijne et al., 2009). For medical diagnostic (e.g. gold nanoparticles) or therapeutic purposes (nanocarriers for drug delivery via inhalation therapy), nanoparticles may be inhaled as nebulized aqueous suspension. Typically, micron-sized suspension droplets are generated (smaller droplets are difficult to generate, except with electrospray generators, which are not suitable for aqueous suspension) and the droplets are subsequently deposited onto the cells via impaction or sedimentation. One of these systems reported in the literature uses a jet nebulizer combined with an Andersen cascade impactor for inertial droplet deposition on the cells, which are seeded on the impactor stages (Schreier et al., 1998). Other systems spray the aerosol directly onto the cells, where the aerosol deposits via impaction (Knebel et al., 2001; Blank et al., 2007). Recently, an ALI exposure system (ALICE) was introduced, which combines cloud settling (sometimes referred to as bulk motion of aerosol; Hinds, 1999) and single particle sedimentation for fast and efficient droplet deposition onto the cells (Lenz et al., 2009). The most suitable exposure system depends on the specific application. However, typically one of the main criterions is the particle deposition efficiency onto the cells. This issue will be addressed in details below (Section 4.2). Most of the methods described in the literature are in-house systems, but there are also commercially available systems like the CULTEXs exposure technology (Cultex Laboratories GmbH) and the VitroCell system (Mulhopt¨ et al., 2008; Paur et al., 2008).

Fig. 2. Comparison of the protocols for exposing cells to (nano-)particles under submerged (left) and air/liquid interface (ALI) (right) culture conditions to study particle-cell interactions. While the air–liquid interface exposure mimics the interaction of particles with cells in the lungs, the submerged exposure is more realistic for ‘internal’ cells such as in the gastro-intestinal tract. 674 H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692

The CULTEXs glass modules have been used in a wide variety of studies on the biological effects of complex mixtures like diesel exhaust, cigarette smoke, volatile compounds and particles after direct exposure of cells cultivated at the air– liquid interface (ALI) (Aufderheide et al., 2001, 2002, 2003a; Aufderheide, 2005, 2008; Aufderheide and Mohr, 2004; Diabate´ et al., 2008; Fukano et al., 2004; Massey et al., 1998; Pariselli et al., 2006, 2009a, 2009b; Seagrave et al., 2005, 2007; Wolz et al., 2002). A very important feature in ALI exposure systems is the supply of the cell cultures with nutrients. Fluctuations of the medium level beneath the culture inlets change the microclimate above the cells influencing the state of the cells to a great extend. In the recently introduced CULTEXs Radial Flow System (RFS), the medium level is controlled and a static, intermittent or continuous medium supply can be selected. In addition, the circular arrangement of the cell culture inserts around a central gas inlet favors homogenous aerosol distribution throughout the module. As the sampling of nanoparticles for exposure assessment may significantly alter the composition and structure of these materials the exposure of cell cultures to the unaltered aerosol in industry or in ambient conditions is a challenging task. For a rugged environment such as in industrial settings like waste incinerators and biomass combustors a robust yet precise setup for metering complex industrial gas mixtures containing nanoparticles to the exposure system. Such a system is the Karlsruhe Exposure System, which samples the particles from a dilution tunnel by a size selective inlet cutting off particles above 1 mm(Mulhopt¨ et al., 2008; Dreher et al., 2009). In the conditioning reactor the aerosol is humidified and temperature controlled to 39 1C to avoid cell damage due to dry gas flows. Then the air–liquid exposure is carried-out by Vitrocell modules, which include an on-line dose measurement system based on a Quartz-Crystal Microbalance (QCM, see Section 4.2).

3. Biological mechanisms and responses

3.1. The structure of the lung

As nanoparticles get in contact with the skin, the gastrointestinal tract, and the respiratory tract, these biological compartments are ‘designed’ to act as barriers to the passage of nano-sized materials into the organism (Stern & McNeil, 2008). Because the lung is considered by far the most important portal of entry for nanoparticles into the human body this overview will mainly focus on the lung as a potential barrier for inhaled nanoparticles. It should however be noted that evidence has been published that nanoparticles can also deposit on the olfactory epithelium and directly be translocated to the brain (Oberdorster¨ et al., 2009). The respiratory tract can be subdivided into functionally and structurally distinct regions (Ochs & Weibel, 2008). Most proximally is the extra thoracic region, consisting of the nasal cavity, mouth, pharynx and larynx, followed by the tracheobronchiolar region, consisting of trachea, main bronchi, bronchi and bronchioles including terminal bronchioles. The main tasks of the extra thoracic region are air conditioning and air conduction. Ambient air, which is usually of lower temperature and humidity than the air in the lung, is efficiently modified and cleansed of much of the larger particulate material by mucociliary activity (fast particle clearance) before being conducted deeply into the lungs. The tracheobronchiolar region is followed by the proximal part of the alveolar-interstitial region, consisting of the respiratory bronchioli with only a few alveoli apposed, the task of which is air conduction, some gas exchange and slow clearance of particulate material. The following distal part of the alveolar-interstitial region consists of the most peripheral airways, the alveolar ducts with their ‘walls’ completely covered with alveoli, i.e. alveolar entrances, and the alveolar sacs, i.e. alveolar ducts with alveoli closing the end of the terminal ducts, including the interstitial connective tissue. The main task of this region is the exchange of oxygen and carbon dioxide with the blood (Gehr, 1994). However, not only oxygen is inhaled but with every breath we take, millions of particles enter the respiratory system; therefore the airways and the vast area of the alveoli need to be protected from harmful and innocuous particulate material. The respiratory tract has a large internal surface area (alveoli and airways approximately between 75 and 150 m2) and a very thin air–blood tissue barrier, both of which are essential for an optimal gas exchange between air and blood by diffusion. The deposition of particles in the lung is size dependent. Besides the geometry of the airways and the breathing pattern, the particle size is important for deposition and clearance studies in the respiratory tract. Significant amounts of nanoparticles are deposited in the most peripheral region of the lung, in the alveoli, but to a considerable extent also in the extra thoracic airways (Oberdorster¨ et al., 2005). The deeper the particles are deposited in the lung, the longer it takes to clear them and the higher is the probability of adverse health effects due to particle–tissue and particle–cell interactions.

3.1.1. Tissue membranes A series of structural and functional barriers protects the respiratory system against harmful and innocuous particulate material (Nicod, 2005). Most of the clearing structures are part of the barrier components of the lungs (Rothen-Rutishauser et al., 2007). They include the surfactant ﬁlm (Gehr et al., 1990; Gil & Weibel, 1971; Schurch¨ et al., 1990), the aqueous surface lining layer including the mucociliary escalator (Kilburn, 1968), a population of macrophages (professional phagocytes) in the airways and in the alveoli (Brain, 1988; Lehnert, 1992), the epithelial cellular layer endowed with tight junctions and adherens junctions between the cells (Godfrey, 1997; Schneeberger & Lynch, 1984), a network of dendritic cells inside and underneath the epithelium (Holt & Schon-Hegrad, 1987; McWilliam et al., 2000). H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692 675

Besides the lung epithelium the basement membrane (Maina & West, 2005), the connective tissue (Dunsmore & Rannels, 1996), and the capillary endothelium (Dudek & Garcia, 2001; Schneeberger, 1977) serve as structural barriers against inhaled particulate material. Deposited inhaled particles first encounter the surfactant film which is located at the ALI of the liquid lining layer covering continuously the internal surface of the lung. Surfactant facilitates the displacement of the particles towards the cells lying beneath (Gehr et al., 1990; Schurch¨ et al., 1990), for example macrophages (professional phagocytes), which play important roles in particle clearance. Displaced particles are either transported out of the lung by the mucociliary escalator or come into close association with macrophages, the epithelium and dendritic cells which are located inside or beneath the epithelium. Of particular interest is therefore how dendritic cells come into contact with inhaled particles. Dendritic cells realize, as sentinels and most competent antigen-presenting cells, a surveillance network in the pulmonary tissues (Holt et al., 1990; McWilliam et al., 2000; Nicod, 1997). Recently, it has shown that dendritic cells and macrophages collaborate as sentinels against fine particles by building a transepithelial interdigitating network of cell processes (Blank et al., 2007). In spite of this intricate defense system, it has been shown that various kinds of different particulate matter are able to overcome these barriers and enter the human body. Also the fact that respiratory diseases are frequent and of increasing prevalence (Peters et al., 1997; Schulz et al., 2005; Wichmann et al., 2000) has directed substantial interest on this topic and a lot of research needs to be done to gain more knowledge on how and when particles evade those barriers.

3.1.2. Cellular membranes After displacement of nanoparticles into the hypophase the particles come into close contact with the membranes of the cells (Rothen-Rutishauser, 2007). All cell membranes have a common structure: they consist of a very thin ﬁlm of lipid and protein molecules, held together mainly by non-covalent interactions (Kendall, 2007; Singer & Nicolson, 1972). The lipid molecules are arranged as an about 5 nm thick continuous double layer in the cell membranes. This lipid bilayer serves as a relatively impermeable barrier to the passage of most water-soluble molecules. The transmembrane protein molecules mediate speciﬁc functions as transporting proteins across the bilayer or catalyzing membrane-associated reactions. Some proteins serve as structural links that connect the cytoskeleton through the lipid bilayer to the extracellular matrix or an adjacent cell by integrins and cadherins, while others serve as receptors to detect and transducer chemical signals into the cells environment (Eisenberg et al., 1984). All eukaryotic cells (such as lung cells) contain functionally distinct, membrane-enclosed compartments. The main types are the nucleus and the organelles which include mitochondria, endoplasmic reticulum, Golgi apparatus, peroxisomes, lysosomes and endosomes. Nucleus and organelles are enclosed by a lipid bilayer containing distinct proteins (Warren & Wickner, 1996). Nanoparticles can cross the membranes of organelles since they have been localized in lysosomes, mitochondria and the nucleus, the mechanisms of internalization are, however, not known so far (Rothen- Rutishauser et al., 2009a, 2009b).

3.2. Cell culture systems

3.2.1. Indicators of nanoparticle toxicity As mentioned above inhaled nanoparticles can deposit on the lung surface and subsequently overcome the tissue barrier as well as the cellular membranes (Rothen-Rutishauser et al., 2009a, 2009b). Once located inside the cell, certain nanoparticles have been shown to be cytotoxic (Borm et al., 2006; Nel et al., 2006; Oberdorster¨ et al., 2005). In addition, nanoparticles are also known to cause several biological responses including the generation of reactive oxygen species (Gonzalez-Flecha, 2004), alter cell signaling (Brown et al., 2004; Clift et al., 2010), as well as cause an enhanced expression of pro-inflammatory cytokines (Muller et al., 2005) without causing cytotoxicity. Furthermore, associations between oxidative stress and inflammatory responses to nanoparticles have also been described within the literature (Donaldson et al., 2005; Rahman & MacNee, 2000). In the presence of environmental stressors, the production of reactive oxygen species within cells increases, overwhelming the cellular antioxidant defense system. Interaction of reactive oxygen species with DNA can cause DNA damage, resulting in DNA strand breaks and covalent modifications of DNA (Oberdorster¨ et al., 2005). Oxidative stress, regardless of the reason of initiation, can cause an inflammatory reaction in cells. The onset of oxidative stress can cause altered cell signaling pathways which can, in turn, activate pro-inflammatory mediators. It has been shown that these mediators are then responsible for inflammatory responses observed (Donaldson et al., 2006). Different cytokines play a role as inflammatory mediators such as Interleukin-8 and the pro-inflammatory chemokine tumor necrosis factor-a. Also cell death can be attributed to numerous cell cascades, resulting in different cell death processes. There are however, two specific forms of cell death which have received increased attention in relation to NP exposure to cells (Kanduc et al., 2002). The first of these processes is apoptosis, also known as controlled cell death. The second, necrosis, is a pathological process, as it has been shown to occur in response to externally induced toxicity, including inflammation. For instance silver nanoparticles have been shown to produce apoptosis via oxidative stress and altered gene expression, however, the possible mechanism for silver induced apoptosis is not clear yet (Ahamed et al., 2010). Cancer is known to occur as a result of a sequence of events that include genetic alterations. DNA damage is considered to be an important aspect of carcinogenesis, and therefore genotoxicity assays have been introduced to assess the cancer 676 H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692 risks of poorly soluble particles (Schins & Knaapen, 2007). Two principal modes of genotoxic action have been introduced for particles (Knaapen et al., 2004; Schins & Knaapen, 2007). Primary genotoxicity is defined as genetic damage elicited by particles in the absence of pulmonary inflammation, whereas secondary genotoxicity implies a pathway of genetic damage resulting from the oxidative DNA attack by reactive oxygen species. At this moment, however, little is understood regarding the potential for nanoparticles to cause genotoxicity, mutations and subsequently, cancer. Despite this, a number of animal studies have shown genotoxic effects in cells following exposure to nanoparticles. In addition, recent studies with carbon nanotubes have shown that certain types of these fibrous nanoparticles can cause secondary genotoxicity in the lungs of mice (Jacobsen et al., 2009), as well as induce granulomas (Poland et al., 2008) and mesothelioma in the peritoneal cavity in-vivo (Takagi et al., 2008). These asbestos- like symptoms point to the importance of the length, diameter and solubility of nanoparticles for toxicity considerations. Although it is essential that further investigation is given to all nanoparticles types, it is imperative that increased attention is dedicated to the carcinogenic potential of carbon nanotubes due to their similarity to asbestos, as well as their potential advantageous characteristics within the technological and engineering industries.

3.2.2. Nanoparticle uptake mechanisms The plasma membrane of the cells is a dynamic structure and segregates the chemically distinct intracellular milieu (the cytoplasm) from the extracellular environment by coordinating the entry and exit of small and large molecules. While essentially small molecules are able to traverse the plasma membrane through the action of integral membrane protein pumps or channels, macromolecules must be carried into the cells in membrane bound vesicles derived from the invagination and pinching-off of pieces of the plasma membrane to form endocytic vesicles. This process is termed endocytosis and two types of endocytosis are distinguished on the basis of the size of the endocytic vesicles formed: pinocytosis (‘cellular drinking’) involves the ingestion of fluid and molecules via small vesicles (o0.15 mm in diameter) whereas phagocytosis involves the ingestion of large particles such as microorganisms and cell debris, at the formation of large vesicles called phagosomes (generally 40.25 mm in diameter). Whereas all eukaryotic cells are continuously ingesting fluid and molecules by pinocytosis, large particles are ingested mainly by specialized phagocytic cells such as macrophages (Conner & Schmid, 2003). The different mechanisms of cellular entering and intracellular trafficking described so far for nanoparticles have been discussed in detail by various reviews (Muhlfeld¨ et al., 2008; Rothen-Rutishauser et al., 2009a, 2009b; Unfried et al., 2007) and are shown in Fig. 3. The kinetics of all known processes are considered to depend largely on nanoparticle surface properties as well as on in in-vivo surface modifications, e.g. by interactions with endogenous proteins (Oberdorster¨ et al., 2005).

Fig. 3. Cellular uptake mechanisms of NPs and related intracellular trafficking: (A) Phagocytosis, an actin-based mechanism occurring primarily in professional phagocytes, leading to phagosomes (AI) and phago-lysosomes (L). (B) Macropinocytosis, also an actin-based pathway, engulfing NPs with poor selectivity, leading to macropinosomes (BI) which might be exocytosed or fuse with lysosomes (L). (C) Clathrin-mediated endocytosis, associated with the formation of a clathrin lattice and depending on the GTPase dynamin, forming primary endosomes (CI) and late endosomes (CII) including multivesicular bodies (CIII). (D) Clathrin and caveolae independent endocytotic pathways. (E) Caveolae-mediated endocytosis, with typical flask-shaped invaginations made of caveolin dimers, also dynamin-dependent and forming caveosomes (EI), which fuse with the ER (EII) or translocate through the cell (EIII). (F) Particle diffusion/transport through the apical plasma membrane, resulting in particles located freely in the cytosol. Reproduced from Brandenberger et al. (2010) with permission for the publisher. H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692 677

Phagocytosis involves the uptake of large particles (40.25 mm) into cells. It is an actin-based, mostly receptor mediated mechanism (Aderem and Underhill, 1999). Particle uptake by professional phagocytes (e.g. macrophages) serves as defense mechanism to remove foreign material from the body. In contrast to ﬁne particles (1 mm in size), the internalization of nanoparticles (0.078 mm) after cytochalasin D treatment could not be blocked completely in cultured macrophages indicating cellular uptake of nanoparticles does not occur via any actin-based mechanism (Geiser et al., 2005). Caveolae-mediated endocytosis is described as ﬂask-shaped invaginations of the plasma membrane of a diameter of 50–100 nm. Whether or not this process is important for the endocytosis of nanoparticles needs further research. Clathrin mediated endocytosis is very well studied and constitutively occurs in all mammalian cells. It is, like most pinocytic pathways, a receptor mediated endocytosis. Again, whether or not clathrin mediated endocytosis is important for nanoparticles needs to be studied in more detail. All of the previously presented endocytic pathways have in common, that the internalized particle is ultimately located in an intracellular vesicle. However, there are studies which reported the intracellular localization of NP of different materials, which were not membrane bound, indicating alternative pathways for particles to enter the cells (Geiser et al., 2005; Lesniak et al., 2005; Rothen-Rutishauser et al., 2006). Besides other possible mechanisms passive and active (receptor mediated) diffusion through membrane pores and passive uptake by van der Waals or steric interactions (subsumed as adhesive interactions) (Rimai et al., 2000) are suggested by the authors of these studies. However, it remains to be determined which chemical and physical properties of membranes and particles are responsible for the translocation of nanoparticles into cells, the nucleus and organelles both in-vitro and in-vivo.

3.2.3. 3D cell culture model and applications The complex nature of the lung architecture which includes very distinct anatomical regions presupposes the use of different in-vitro models. In a nanoparticle exposure scenario an in-vitro model should highlight the most important characteristics of its corresponding region in the respiratory tract. Most of the studies done in the nanotoxicity field have been done using in-vitro models which consist of human cell lines derived from the epithelium of the airways and alveolar (gas-exchange) region (Rothen-Rutishauser et al., 2008a). The majority of studies are performed with mono-cultures; however, recent publications have shown that co-cultures using a combination of different cell types have an influence on the observed cellular response (Lehmann et al., 2009; Muller et al., 2009). Recently, we have developed a triple cell co-culture in-vitro model of the human airway wall to study the cellular interplay and the cellular response of epithelial cells, human blood monocytes derived macrophages and dendritic cells to particles (see Fig. 4)(Blank et al., 2007; Rothen-Rutishauser, 2007; Rothen-Rutishauser et al., 2005, 2008a). In this model, monolayers of two different epithelial cell lines, A549 (Lieber et al., 1976) and 16HBE14o epithelia (Forbes, 2000), were grown on a micro-porous membrane in a two-chamber system. After isolation and differentiation of human blood derived monocytes into macrophages and dendritic cells (see also paragraph ‘limitations of cell culture models’), they were added at the apical side and at the basal side of the epithelium, respectively. After the triple cell co- culture was established, cell densities of macrophages and dendritic cells within the culture were quantified using the specific surface markers CD14 and CD86 for the labeling of macrophages and dendritic cells, respectively, and the quantitative occurrence of macrophages and dendritic cells resembled very closely the in-vivo situation (Blank et al., 2007). After its thorough evaluation, this model was exposed to particles (either airborne or suspended in medium) of different materials (polystyrene, titanium dioxide and gold) and of different sizes (r1 mm) (Blank et al., 2007; Rothen- Rutishauser, 2007; Rothen-Rutishauser et al., 2008b; Brandenberger et al., 2010). Translocation and cellular localization of particles were studied as well as the effects of particles on cellular interplay and signaling. Recently, we have shown that dendritic cells and macrophages collaborate as sentinels against fine polystyrene particles (1 mm in diameter) by building a transepithelial interdigitating network of cell processes (Blank et al., 2007). The translocation of nanoparticles into the different cell types however, is different compared to their larger particle counterparts (Rothen-Rutishauser et al., 2007). This triple cell co-culture system might be used for other epithelial models, for example, the gastrointestinal tract or the skin, by replacing the lung epithelial cells by any other epithelial cell type.

Fig. 4. Laser scanning microscopy images of the triple cell co-culture model. Epithelial cells (dark gray, volume rendering), macrophages (white, surface rendering; black arrows), and dendritic cells (dark gray, surface rendering; white arrow) are shown. The same data set is shown from top (A), from bottom (B), and without epithelial cells from top (C). Reproduced and adapted with permission from ALTEX (Rothen-Rutishauser et al., 2008b). 678 H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692

Building on the broad experience with ambient particulates and ultrafine particulates, nanotoxicology has largely focused in pulmonary effects, with a particular emphasis on inflammation, oxidative stress, cytotoxicity and genotoxicity (Doak et al., 2009; Donaldson et al., 2009; Jones and Grainger, 2009; Kroll et al., 2009). Each of these are likely mechanisms of action, and logical endpoints for assaying and ranking the hazards of nanomaterials (Nel et al., 2006), but a critical question remains: do these assays or mechanisms of action sufficiently represent the breadth of possible mechanisms and responses? Largely unexplored is the potential for nanomaterials to impact critical cellular functions, which lead to neither overt toxicity nor inflammation, but can affect the health of an organism. For example, the liver Kupffer cells play a primary role in the systemic clearance of bacteria escaping the gastrointestinal tract, proving for protection against system infection. Consequently, Kupffer cells are a major site of accumulation of nanomaterials reaching the blood circulation (Odegaard et al., 1978; Moghimi & Hunter, 2001; Krasinskas et al., 2004; Sadauskas et al., 2009), and nanomaterials can remain in these cells for decades. Very little, however, is understood about the potential for nanoparticle accumulation in Kupffer cells to disrupt their normal bacterial clearance function following low, chronic environmental exposures. Pulmonary macrophages provide protection against inhaled bacteria, and although ambient particulates are known to reduce pulmonary bacterial clearance (Zhou & Kobzik, 2007), the impact of nanoparticle exposure on pulmonary bacterial clearance has not been explored. There is a need to expand the field of nanotoxicology to include the broad exploration of potential mechanisms of action in sites remote to the lung, where tissue exposure has been shown following relevant routes of exposure.

3.3. Considerations for selecting appropriate cell systems

There are a large number of in-vitro test systems ranging from single cell line cultures, primary cells and 3D and co- culture models to tissue slices. Roughly 7% of the alveolar surface in the human lung is covered by alveolar type II cells, which make up 16% of the total alveolar cells (Crapo et al., 1982). Capillary endothelial cells and cells in the interstitial space comprise up to 30% and 37% of all lung cells, respectively. The number of alveolar macrophages shows great variability (1–20%), likely due to highly variable exposures to exogenous compounds including inhaled particles, smoke and bacteria The correct choice of a cell system for in-vitro testing should be based on a working hypothesis. In order to compare the toxicity of various nanoparticle types it might be sufficient to choose submersed cultures, but in such kind of studies different cell types need to be included, because macrophages would have a different cellular response (i.e. secretion of cytokines/chemokines) than epithelial cells. However, to study to what extend insoluble particles can be transported from the alveoli to the blood, co-cultures of epithelial and endothelial cells cultured on a membrane can be exposed to nanoparticles at the ALI with ALI exposure systems as described above (Hermanns et al., 2010). For even more realistic in-vitro models of the in-vivo exposure conditions in the lungs, it is possible to add layers of lining fluid as well as mucus or surfactant onto epithelial cells (cultured at the ALI) from the bronchial or alveolar regime or use the alveolar type II cell line A549 which releases surfactant upon culture at the ALI (Blank et al., 2006). Another consideration is the most likely site of lung deposition for a given particle upon inhalation. As a rule of thumb, the smaller the size of a particle, the deeper it will penetrate into the lung. When focussing on supermicron particles, bronchial epithelial cells such as the BEAS-2 can be considered whereas particles below 100 nm are likely to reach the alveoli and hence alveolar type I and II epithelial cells like the A549 cells are more favorable, but it has to be taken into consideration that A549 cells are derived from adenocarcinoma. However, A549 cells are an accepted model for nanotoxicity studies (Duffin et al., 2007; Limbach et al., 2007a, 2007b; Stearns et al., 2001) (see also Table 2). It is important to note that the cellular response of epithelial cells strongly depends on their interaction with other types of pulmonary cells. This has also been demonstrated in-vitro by comparing the response of mono- and co-culture cell systems after particle challenge (Alfaro-Moreno et al., 2008). The addition of immune cells to the culture system is of great importance to study inflammatory effects upon particle exposure. It has been shown that epithelial cells and immune cells such as dendritic cells as well as macrophages continuously cross-talk in vivo through intercellular signaling in order to maintain homeostasis and to coordinate immune response (Roggen et al., 2006). Dendritic cells and macrophages collaborate as sentinels against foreign particulate antigens by building a transepithelial interacting cellular network through cytoplasmic processes between epithelial cells across the epithelium, or alternatively, can transmigrate through the epithelium in-vivo and in-vitro to capture particles present at the epithelial surface (Holt, 2005; Blank et al., 2007). Another study has shown that human bronchial epithelial cells exposed to diesel exhaust particles can induce dendritic cell maturation (Bleck et al., 2006) which might explain the rise in allergic disorders. Therefore, appropriate cell culture systems need to be developed and used to study possible adverse effects of inhaled nanoparticles on epithelial cells combined with immune cells. Hence, it is recommended to use cell models resembling in-vivo conditions as closely as possible. For inhalation scenarios this implies a strong preference for co-culture systems (Aufderheide et al., 2003b; Savi et al., 2008; Diabate´ et al., 2008; Ning et al., 2008; Brandenberger et al., 2010) or even slices of lung tissue (Bion et al., 2002) cultured at the ALI. ALI exposures are also considered more useful for obtaining dose–response relationships (see below), whereas the technically more simple submerged culture systems are particularly useful for initial screening for nanoparticle toxicity. The interpretation of the cellular response relies heavily on the characterization of the exposures (dose, particle properties, etc.), which is at present something that deserves more attention and more cross talk between biologists and aerosol scientists. In general, in-vitro systems can be very useful to elucidate biological mechanisms, but they are not very well H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692 679 accepted for risk assessment, since they do not adequately resemble the response of the entire organism of humans. In any case, there is a wealth of publications on in-vitro test systems and it is advised to study the literature carefully prior to selecting a cell system for toxicity testing. Table 1 shows the key parameters that should be considered for testing nano- sized particles in in-vitro systems

3.4. Limitations of cell culture models

The mechanisms governing the biological response of an organism to nanoparticles exposure are extremely complex and are influenced by the interaction of different cell types, cytokines and the microenvironment in the vicinity of the nanoparticles. Such a complex situation cannot be completely reproduced by an in-vitro system. Even animal models can only approximate the conditions during human exposure due to physiological differences between man and animals. The presently available cell models illustrate the great variability with respect to morphological, biochemical and/or functional features of a cell system. Therefore, an in-vitro cell model should be selected and characterized carefully with respect to the requirements needed for a certain study. Some of the most commonly used cell lines for pulmonary toxicity testing have been shown in Table 2. It is important to note that the respiratory tract contains more than 40 different types in total and each cell type has its specific function such as for example barrier structure (epithelial cells) or immune defense (antigen presenting cells). There are many studies published comparing the toxicity of nanoparticles in different cellular models such as cell lines, i.e. immortalized cells, primary cells (taken from humans via e.g. bronchoalveolar lavage or biopsy) and tissue slices Each of these different cellular models has its advantages and disadvantages. For instance, immortalized cell lines are relatively simple to culture and tend to show highly reproducible response, while primary cells tend to behave more realistically, but are more difficult to acquire, are more cumbersome to deal with and are frequently more heterogeneous in response. In addition, polarity of epithelial cells, tight junctions, and other characteristics of differentiated epithelial cells will likely contribute to differences seen in biological/toxicological responses. Since all of these issues are relevant for the biological response of the cells, it is difficult to compare the results obtained with different cell types. It is of course of great importance that the chosen cell system is adequate for scientific question to be addressed For instance for studying tissue penetration and cellular trafficking of nanoparticles, the epithelial barrier should be present and possibly one other cell type. Ideally, one also includes the liquid lining fluids, i.e. the mucus layer and alveolar lining fluid covering bronchial and alveolar epithelial cells. Apart from the selected biological response variables and the route and type of exposure the duration of exposure is a critical feature for in-vitro test systems. In general cell exposures can be performed for up to 2–3 days (typically exposures have only be performed for a few hours), but longer exposure durations are very difficult to maintain due to the limited lifetime of the cells or due to an overgrowth of the cells forming multiple layers instead of a monolayer, if cultured at the ALI. This limits the hazard screening of nanoparticles with in-vitro cell cultures to short-term (acute) exposures and the in- vitro cell systems provide very little if any information on longer (chronic) exposure scenarios as frequently encountered

Table 1 Recommended biological parameters to assess nanoparticle responses. It is important to note that cytotoxicity should always be measured for quality control, since all other response parameters may be severely mitigated (biased), if the cells are not viable anymore.

Cytotoxicity (but work below cytotoxic levels) Oxidative stress (or oxidative capacity in abiotic systems) Mediators that affect immune system including inﬂammatory mediators Genotoxicity Translocation of nanoparticles and effect on cell skeleton

Table 2 Human cell culture models of the airway or alveolar epithelium used for nanotoxicological studies.

Cell lines References

Airway epithelial cells Calu-3 Bivas-Benita et al. (2004), Grenha et al. (2007) and Rotoli et al. (2008) 16HBE14o- Brzoska et al. (2004) and Holder et al. (2008) BEAS-2B Herzog et al. (2007), Jang et al. (2006), Park et al. (2007) and Veranth et al. (2007)

Alveolar epithelial cells A549 Dufﬁn et al. (2007), Park et al. (2007) and Stearns et al. (2001) Immortalized human alveolar type 2 cells with Kemp et al. (2008) alveolar type 1 phenotype

3D cultures Triple cell co-culture model (epithelial cells, Rothen-Rutishauser et al. (2007), Rothen-Rutishauser et al. (2008a) and Rothen- macrophages and dendritic cells) Rutishauser et al. (2008b) 680 H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692 under real life conditions. It has to be considered, that the maximum survival for cells in incubators might become a limiting factor, and, in addition, the survival of the cells is very much dependent on the cell type. Currently, a 3D in-vitro model of the human airway epithelium composed of primary bronchial epithelial cells (i.e. the MucilAirTM system from Epithelix (www.epithelix.com)) exists, which has been used for chronic exposures to chemicals, however, the potential of this system for chronic nanoparticle exposures needs to be evaluated. Another issue is the time between an exposure and the measurement of the subsequent biological effects. Both rapid responses (such as induction of chemokines/cytokines) and effects occurring on a slow time scale (such as induction of genotoxicity) can only be measured by post-exposure incubation of the challenged cells for different durations. The maximum survival for cells in incubators may therefore become a limiting factor. Finally, in-vitro effects of particles are known to be difﬁcult, but not impossible, to relate to their in-vivo equivalents as will be discussed below (see Section 4.3). Considering these limitations when working with cell culture models, in-vitro cell systems are expected to become an essential tool for pre-screening of the toxicity of nanoparticles, since they allow for high-throughput toxicity testing using biological entities (cells), which is much more meaningful than pure physico-chemical characterization of nanoparticles. Furthermore, in-vitro cell culture models are a valuable tool to perform basic research on cellular response mechanisms without interference from systemic feedback loops.

4. Dosimetry

The dose–response paradigm of toxicology is predicated on the principle that response is proportional or at least most closely related to the dose of the affector (here: nanoparticles) at the site of action (Hardman & Limbird, 2001). The use of target tissue dose (here amount of nanoparticles reaching the cells), rather than less speciﬁc dosimetric measures such as exposure concentration (e.g. amount of nanoparticles per volume cell culture medium containing the cells) or administered dose (e.g. amount of nanoparticles added to the cell culture medium), has been shown to improve correlations between dose and response for drugs, chemicals, inhaled gases and particles (Treinen-Moslen, 2001; Witschi & Last, 2001; Brown et al., 2005; Schroeter et al., 2006). There is then a history of high conﬁdence in the use of target tissue dose for in-vivo dose–response assessment, and target tissue dose has become the standard for dose–response assessment in pharmaceutical safety assessment and chemical risk assessment (NRC, 1994).On the other hand, target tissue dosimetry is also an important, but largely ignored aspect of the in-vitro dose–response paradigm for industrial chemicals and pharmaceuticals. The target tissue/site in in-vitro cell culture systems is the cell, or cellular targets such as receptors (Teeguarden & Barton, 2004), rather than a tissue remote from the site of administration. In this section we will discuss if and how the target tissue dose can (or cannot) be determined with in-vitro cell exposure systems cultured under submerged or at air–liquid interface (ALI) conditions.

4.1. Submerged cell exposure systems

The prevailing dosimetric assumption for in-vitro systems with cells cultured under submerged culture conditions is that the nominal concentration of a test material in the culture media is proportional to the cellular dose and is therefore a good measure of dose at the target site (cell culture). This assumption is reasonably accurate for soluble chemicals where saturable processes such as metabolism and active transport do not influence cellular concentrations. Dose–response differences between two test chemicals can therefore be attributed to factors other than target tissue/site dose such as metabolism, partitioning, potency, efficacy or the characteristics of its binding to a receptor. This paradigm has been widely and successfully used to assess the relative potency of drug candidates and the relative toxicity of industrial and environmental chemicals (Padron et al., 2000; Eisenbrand, et al., 2002; Allen, et al. 2005; Bakand et al., 2005). It has also been used to assess the relative potency of particulate matter, which is the subject of this study (Bakand et al., 2005). The well accepted principles of target cell dosimetry developed for chemicals and particles in-vivo can be extended to provide an equivalent definition of dose for nanomaterials in-vitro: amount, duration of dosing, and material identity. However, in contrast to chemicals, the material identity dimension for nanomaterials is relatively complex. Nanomaterials do not have a single stable molecular identity; rather, they are defined by the materials comprising the core particle, any intentional or acquired surface modifications, as well as their macro scale properties, e.g. size, shape, agglomeration state. In addition, like the paradigm for chemicals, dose for nanoparticles in-vitro can be defined at various levels of specificity with regards to the site of action and mode of action, reflecting administered dose at the most nonspecific level, apparent exposure at a more specific level, or cellular dose at the most specific level (Teeguarden et al., 2007). There are important implications to the use of any one of these metrics of dose. For in-vitro studies, a clear distinction should be made between exposure and dose metrics for nanomaterials. Nominal media concentrations (e.g. mass, surface area or number per volume of cell culture medium in terms of mg/ml, cm2/ml, ml1, respectively) and their derivatives most commonly used in in-vitro studies, are NOT measures of dose, they are measures of exposure, and do not accurately reflect the amount of material coming in contact with the cells. Processes such as diffusion, sedimentation, and perhaps convection, all of which are dependent on the size, shape and density of the nanoparticles, influence the relationship between the nanoparticles in solution above cells and nanoparticles reaching the cells (Limbach et al., 2005; Teeguarden et al., 2007), the latter being a measure of nanoparticles dose. The general comfort H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692 681 in using these exposure metrics, rather than dose metrics, is misplaced because there is no justifiable expectation that in- vitro exposure and dose metrics consistently have a direct relationship. The key point for nanotoxicologists is that for submerged in-vitro cell exposure systems with standard media heights of one or more millimeters above the cell layer, the theoretical transport rates by diffusion and sedimentation are sufficiently different between particles of different sizes and different densities, and between materials with different agglomeration states, that large differences in the amount of particles reaching cells in a given time period (cellular dose) are likely and there is no simple relationship between particle characteristics and the amount of particles reaching the cells. That is, the exposure–dose relationship is complex. For example, despite concluding that nominal media concentrations were an appropriate metric of dose in-vitro Lison et al. (2008) showed no clear relationship between nominal media concentration and cellular dose for 35 nm amorphous silica. More important, perhaps, is the fact that measures of exposure (media concentrations), may misrepresent dose to the cell by many orders of magnitude when viewed across particle size (Limbach et al., 2005; Teeguarden et al., 2007). This can clearly be established based on principles of particle motion in liquids that have been well understood for more than 100 years (Einstein, 1905; Dusenberry, 2009). Thus, there appears to be no theoretical, computational, or experimental support for the adequacy of conventional measures of dose for in-vitro nanotoxicology studies—nominal media concentrations (NMC) on a mass, surface area or particle number basis. Surprisingly, this fact has received little attention in the literature, where the focus seems to be predominately on the suitability of assays, cell types, and methods of characterization (Doak et al., 2009; Donaldson et al., 2009; Kroll et al., 2009; Marquis et al., 2009; Park et al., 2009; Stone et al., 2009) A successful dosimetry paradigm for nanotoxicology or nanobiology should parallel the widely accepted and applied paradigm for particles in-vivo, and should reflect certain core attributes; a focus on dose to the cell, adequate characterization of the particles, and represent the mode of action under study. These core attributes ensure that the operating paradigm for nanomaterial dosimetry support accuracy, scalability and interpretability of all in-vitro nanomaterial research. Some advances have been achieved in the recent years to address the dosimetric drawbacks in submerged exposure scenarios. The cellular dose has been determined by removing the cell culture medium and quantifying the amount of nanoparticles associated with the cells by elemental analysis. The dose applied to A549 cells after exposure to a silver nanoparticle suspension has been determined by atomic absorption spectroscopy (AAS) (Foldbjerg et al., 2010). Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the cellular content of platinum nanoparticles in HUVEC cells (Limbach et al., 2005; Elder et al., 2007; Lison et al., 2008). However, these methods are time-consuming and cost-intensive and they do not allow a distinction between nanoparticles taken up by the cells and those merely attached to the cell membrane.

4.2. Air–liquid interface (ALI) cell exposure systems

As mentioned above one of the main advantages of ALI over submerged cell exposure systems is that the airborne nanoparticle are passed over the apical side of the cells from where they can deposit directly onto the cells without first having to penetrate a thick layer of cell culture media as under submerged exposure conditions. Thus, the nanoparticle dose delivered to the target tissue (here cell layer) can be calculated from the nanoparticle concentration in the air entering the exposure system and the deposition efficiency of the nanoparticles onto the cells, i.e. the cell-deposited fraction of the particles entering the exposure system. Both nanoparticle concentration and deposition efficiency can be measured with standard aerosol devices (Bitterle et al., 2006). For ALI in-vitro cell exposure systems relying on diffusion and/or sedimentation alone as particle deposition mechanisms, the deposition efficiency is typically fairly low. For instance, the deposition efficiency of 200 nm particles is about 0.7% in the CULTEXs glass modules (Desantes et al., 2006). Using a stagnation point flow system to direct the aerosol flow to the cells enhances the deposition efficiency to 1–2% (Tippe et al., 2002; Bitterle et al., 2006), which is still low, but almost constant over a broad size range from about 50 to 500 nm due to the compensating effects of diffusive and gravitational deposition (Desantes et al., 2006). For particles smaller than 50 nm or larger than 500 nm in (mobility) diameter, particle deposition is enhanced due to more pronounced diffusional and sedimentational deposition, respectively (Desantes et al., 2006). The newest generation of in-vitro cell exposure systems adopts two main technological improvements: enhancement of the nanoparticle deposition efficiency and real-time monitoring of the cell-deposited nanoparticle dose (target tissue dose). These technological advances are expected to substantially improve in-vitro nanotoxicity testing, since efficient use of substances is critical for potentially limited (expensive) types of nanoparticles and real-time dose determination improves the reliability of measured dose–response relationships. Electrostatic precipitation is one of the main mechanisms to improve the deposition efficiencies. For bipolarly charged 50-600 nm (airborne) particles, deposition efficiencies can be enhanced to 15-35% under the influence of an alternating electrostatic field (Savi et al., 2007). Larger deposition efficiencies are not possible with typically used diffusion chargers, since bipolar diffusion charging leaves the majority of submicron-sized particles uncharged (Baron and Willeke, 2001). The so-called EPDExS system selects a monodisperse subfraction of nanoparticles from a bipolarly charged polydisperse sample aerosol with a differential mobility particle sizer (DMA) and deposits the selected unipolar (positive or negative) subfraction with an efficiency of near unity onto a cell-covered substrate using a constant electrical field (Stevens et al., 2008). 682 H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692

At first glance the reported deposition efficiency of 100% may seem as the ultimate step towards optimal use of limited supplies of nanoparticles. However, as mentioned above in the EPDExS system the nanoparticles are passed through a DMA, which typically does not select more than about 1% (at best) of the total particle population due to limited charging efficiency and narrow width of the transfer function of the DMA. Thus, no more than about 1% of the nanoparticles entering the mobility analyzer are actually depositing onto the cells. The obvious next step of technological development is the use of a unipolar charger in combination with a high efficiency electrostatic precipitator. Since commercially available unipolar chargers have a charging efficiency of near 100% for large enough particles (430 nm), electrostatic precipitators should be a useful tool for spatially uniform and near 100% efficient deposition of airborne polydisperse nanoparticles onto cells. Such a system has recently been introduced, but the deposition efficiency was not reported (Gaschen et al., 2010). de Bruijne et al. (2009) have modified a commercially available electrostatic precipitator by placing lung cell cultures in the region of electrostatic particle deposition. A deposition efficiency of 90% was reported for the particles between 19 and 882 nm. Cellular effects due to the electric field were found negligible compared to the effect due to the deposition of diesel particulates. There are currently two ALI cell exposure systems, which allow for (quasi) real-time dose measurements utilizing a quartz crystal microbalance (Mulhopt¨ et al., 2009; Lenz et al., 2009). The system by Lenz et al. will be discussed below. The stagnation point flow system described by Mulhopt¨ et al. (2009) consists of six identical cell exposure units operated in parallel. By replacing the cell-covered membrane of one of the cell exposure units with a quartz crystal microbalance real- time measurement of the cell-deposited nanoparticles mass can be performed. While this system allows for accurate dose measurement, its diffusion-driven deposition efficiency is currently limited to a few percent for the most interesting size range between about 150 and 500 nm (Tippe et al., 2002; Desantes et al., 2006). Typically this results in a maximum cell- deposited dose of the order of 0.1 mg/(cm2h) and 450 nanoparticles/cell/h, if maximum aerosol exposure concentrations of about 5 mg/cm3 and 5 106 nanoparticles/cm3, respectively (Mulhopt¨ et al., 2008; Bitterle et al., 2006), are applied. This mass deposition rate is four orders of magnitude larger than the estimated upper limit in the lungs (at the ‘hot spots’) for realistic ambient exposure conditions (3 10–5 mg/cm2 h) and still a factor of 20 larger than the deposition rate in the lungs for the worst case exposure scenario (5 10–3 mg/cm2 h) (see Section 4.3 and Table 3). Considering that the lifetime dose for ambient exposure conditions is about 0.66 mg/cm2 (or 6.6 mg/cm2 at local hot spots; see below) these diffusion- based cell exposure systems are even suitable for depositing lifetime doses of nanoparticles onto cells cultured at the ALI within a matter of days. This issue will be discussed in more detail below. As mentioned above electrostatic precipitation will help to enhance these deposition efficiencies even further, if required. For nanoparticles, which are generated, stored and/or used in liquid suspensions, nanoparticle-containing droplets can be inhaled by workers or consumers. To simulate this exposure scenario the nanoparticle suspension can be nebulized into micron-sized droplets and subsequently deposited onto the cells at the ALI. Previously described methods for deposition of micron-sized droplets onto cells have relied on inertial impaction and sedimentation as the deposition mechanism (Fiegel et al., 2003; Grainger et al., 2009; Schreier et al., 1998; Knebel et al., 2001; Blank et al., 2006; Bur et al., 2009). The use of impactorsorimpingers(Fiegel et al., 2003; Grainger et al., 2009; Schreier et al., 1998; Bur et al., 2009) requires high flow rates for effective particle deposition, which may impair cell viability (Bur et al., 2009). The newest generation of droplet-based cell exposure systems is expected to focus on the flowing three issues: minimization of the destruction of potentially delicate nanoparticles during the nebulisation process (e.g. functionalized nanoparticles or gene transfer complexes), optimization of the accuracy and repeatability of the deposition efficiency as well as minimization of the stress exerted on the cell systems during the exposure procedure. The recently introduced ALICE system (Lenz et al., 2009) addresses these issues by combining cloud settling (sometimes referred to as bulk motion of aerosol) and single particle sedimentation as deposition mechanism for fast (10 min; entire exposure), low-stress and efficient delivery of the nanoparticles to the cells. The vibrating membrane nebulizer (investigational eFlow, Pari, Germany; MMD¼4.5-5.4 mm) used in the ALICE has been shown to maintain the functional integrity of potentially delicate therapeutic agents, such as biopharmaceuticals, since – unlike jet nebulizers or ultrasonic generators – vibrating membrane nebulizers avoid both high shear forces and heating of the nebulized liquid during the generation process (Kesser and Geller, 2009; Wagner et al., 2006). The cell deposited dose can be controlled with a quartz crystal microbalance over a wide dynamic range 0.02–200 mg/cm2 (for 1 ml of sprayed suspension) by changing the nanoparticle (mass) concentration in the suspension from 0.0012% to 12%. For commercially available transwell plates, the cell-specific deposition efficiency of the ALICE is currently limited to 7.2%, but a deposition efficiency of at least up to 57% is possible, if the entire bottom plate of the exposure chamber could be covered by cells (Lenz et al., 2009).

4.3. Estimated in-vivo nanoparticle dose deposited on the human alveolar epithelium

One of the main caveats of currently performed in-vitro toxicological studies is the unrealistically high dose which is typically used in these studies reaching values of 65 mg/cm2 or more (Volckens et al., 2009). To put these dose levels into perspective, we estimate a realistic and a worst case dose level delivered to the lungs during a single day and over the entire lifetime. The following discussion focuses on the alveolar regime, since – as discussed above – the alveoli represent more than 90% of the lung surface area and they are considered the most vulnerable pulmonary region due to the ineffective nanoparticle clearance mechanism (phagocytosis), the very thin protective liquid layer on the tissue (o0.1 mm lining ﬂuid and surfactant) and an extremely thin tissue barrier (o1 mm) separating the inhaled air from the blood vessels (Pinkerton et al., 1992; ICRP, 1994; Geiser & Kreyling, 2010). H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692 683

Table 3 Cell-deposited nanoparticle dose for in-vitro exposure systems and for human (in-vivo) inhalation exposure. It is evident that all of the exposure systems (ALI, namely CULTEXs glass modules, Vitrocell and ALICE, as well as submerged systems) can reproduce the range of target tissue dose fluxes occurring in the lungs. The CULTEX/Vitrocell and ALICE represent the low and high end of the typically found target tissue dose fluxes in air–liquid interface exposure systems, respectively. In fact all in-vitro exposure systems allow for about 10–107-fold higher dose fluxes, where the CULTEXs/Vitrocell and the submerged exposure systems are at the low and high end, respectively.

System/exposure scenario Exposure concentration Tissue Target tissue dose ﬂux deposition efﬁciency (%) Mass Number Mass per surface Number per cell concentration concentration areaa (lg/cm2/h) (cell1 h1) (lg/m3) (cm3)

Human inhalation (realistic ambient exposure)b 10 3 104 30c 3 105 2 Human inhalation (worst case exposure)b 5000 5 106 30c 5 103 300 Submerged exposure (maximum dose) 100 mg/mld 1014/mld 0–100e 0–5 104f 0–2 108f CULTEXs/Vitrocellg 10–5000h 3 104–5 106h 1.5i 2 104–0.1j 3–450k ALICEg 0–100 mg/mll 0–1014/mll 56l 0–140l 0–5 105l

a The mass flux is defined as the mass of particles depositing on the target tissue (or cells) per surface area of the tissue and per time. b The details of the so-called realistic and worst case exposure scenario can be found in Section 4.3. The targeted tissue dose flux was calculated for the alveolar regime, since nanoparticle deposition occurs predominantly in this region. The dose values include a safety factor of 10 in order to guarantee that these values can be considered upper limits. c The lung deposited particle fraction depends on various parameters including respiratory parameters and particle properties. In general alveolar deposition ranges from about 50% to 10% for particles with 10–400 nm mobility diameter. Hence, as an estimate for lung deposition of nanoparticles we are using the mean value of 30%. d In contrast to air–liquid interface exposure systems, the concentration values for suspended exposure is given in terms of nanoparticles mass (or number) per volume liquid covering the cells. Volume concentrations of 40% are possible for stabilized nanoparticle suspensions (Studart et al., 2007). Hence, we conservatively estimate 10% (100 mg/g or 100 mg/ml) as a realistic maximum nanoparticle mass concentration in the aqueous suspension. The corresponding particle number concentration depends on particle size and density. Here we arbitrarily assume a particle diameter and density of 100 nm and 2 g/cm3 (to get an upper limit for the particle number we are using a material with low density: carbon). e The exact deposition efficiency is frequently not known, since it depends on various parameters such as nanoparticle (monomer) size, agglomeration state, density and exposure time. At least some of these parameters are typically not all known. For more details, please refer to Section 4.1. f 3 Here we assumed that 1 ml of a nanoparticle suspension (dp ¼100 nm; density¼2 g/cm ) is given into one well of a standard 24-well plate, which has a 2 cm2 growth area for the cells i.e. 0.5 cm of suspension above the cells and a cell density of about 3 105 cm2. g These cell exposure systems are described in Section 2.3. h The concentration range is derived from the values estimated for the realistic and worst case human inhalation exposure scenario (see rows above). i The deposition efficiency is relatively independent of particle size in the range between 50 and 500 nm (diameter) due to compensating effects of diffusion and sedimentation. j Typically, the CULTEX/VitroCell system is operated at a flow rate of 100 ml/min and the cell-covered surface area is 4.5 cm2 with a cell density of 2.2 105 cm2. k Here we assume that the cell number per surface area was 2.2 105 cm2. l In contrast to the CULTEXs and VitroCell system, the ALICE was designed to allow for much larger dose rates. Typically 1 ml of a nanoparticle suspension is nebulized and deposited onto the cells. As volume concentrations of 40% are possible for stabilized nanoparticle suspensions (Studart et al., 2007), we conservatively estimate 10% (100 mg/g or 100 mg/ml) as a realistic upper limit for the nanoparticle mass concentration in the suspension. For nanoparticles with relatively low density (e.g. carbon: 2 g/cm3 to get an upper limit for the particle number) and large diameter (here 100 nm), this corresponds to a nanoparticle number concentration of 1014 ml1. The 1 ml of suspension is deposited with an efficiency of 56% over an area of 400 cm2. The cell density is 3 105 cm2. The entire exposure takes about 10–15 min. Here we are assuming that only one exposure is performed per 1 h.

For a healthy, moderately active adult, we may assume a daily inhaled air volume of 25 m3 (ICRP, 1994), an alveolar lung surface area of 100 m2, an alveolar deposition efficiency of about 30% (10–50% for 10–100 nm particles with its maximum at about 20 nm) (ICRP, 1994). The realistic average dose level in an urban environment is estimated assuming an ultrafine particle mass concentration of 10 mg/m3 (particles with mobility diameter o100 nm). This corresponds to an average daily tissue dose of 7.5 10–5 mg/cm2 (particle mass per cm2 lung epithelium; 24 h clearance from the alveolar region is negligible; ICRP, 1994) or 7.5 10–4 mm2/cm2 (assuming a specific BET surface area of 10 m2/g for ultrafine ambient particles) (see Table 3). Locally we estimate that these values could be up to about a factor of 10 larger in ‘hot spots’ (7.5 104 mg/cm2 and 7.5 103 mm2/cm2) occurring due to spatially non-uniform deposition (e.g. secondary flows at bifurcations; higher particle concentration near the mouth or nose), spatially non-uniform clearance from the lung tissue (Bala´sha´zy et al., 2003; Donaldson et al., 2008) and underestimation of the particle lung deposition efficiency (e.g. due to dealing with particles near 20 nm, where the maximum of alveolar deposition efficiency is; electrostatic charge on the particles). Since the most pronounced biological effects are expected to occur at these hot spots, the following discussion will include this safety factor of 10 (Bala´sha´zy et al., 2003). Consequently, the lifetime dose (at the hot spots) under realistic ambient conditions is 6.6 mg/cm2 (70% long-term clearance of nanoparticles from alveolar region is assumed (Kreyling and Scheuch, 2000)) or 66 mm2/cm2 (lifetime: 80 years). It is worth noting that the lifetime burden of biopersistent fraction of the ultrafine ambient particles is about a factor of 5 lower than the values reported here, since about 80% of ambient particulate matter is either dissolved in the lung fluids or metabolized. For the worst case exposure 684 H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692 scenario, we assume that the individual is working under highly polluted conditions. As an upper limit for workplace nanoparticles mass concentrations we use 5 mg/m3, the currently recommended Occupational Safety and Health Administration (OSHA) standard for respirable nuisance dust (averaged over an 8 h work shift). Consequently, for an eight hour shift the OSHA standard corresponds to daily alveolar mass dose of 0.13 mg/cm2 (clearance is neglected; 100% biopersistance) or 6.30 mm2/cm2 (specific BET surface area of ZnO: 50 m2/g) at the hot spots. Hence, the maximum lifetime dose accumulated by a worker during work hours is up to 420 mg/cm2 or 2.1 104 mm2/cm2 (5 workdays per week for 50 weeks per year over 45 years; with 70% clearance) with an additional small (o10%) contribution of 29 mg/cm2 or 1400 mm2/cm2 from urban air during off-working hours (assumed 50 mg/m3 as worst case scenario for long-term ambient particle exposure) accumulated over the entire lifetime of 80 years. This yields an overall worst case lifetime dose of 450 mg/cm2 or 2.3 104 mm2/cm2. This calculation indicates that for highly polluted occupational conditions the particle burden in the lungs may be dominated by occupational particles, even if the allowed occupational exposure limits are not exceeded. It is also instructive to consider dose levels in terms of nanoparticles per cell. For a number concentration of 3 104 cm3 in ambient air, whereas almost 100% (80–90%) can be considered nanoparticles (mobility diameter smaller than 100 nm (Kreyling et al., 2003)) about 2.3 1011 nanoparticles are deposited onto the alveolar epithelium per day. Under highly polluted conditions such as in some occupational settings, the particle concentrations can be significantly higher. However, due to rapid coagulation of nanoparticles it is typically not possible to maintain number concentrations above 5 106 cm3 for more than several minutes, which is consistent with the exposure levels observed by Demou et al. (2009) at a nanoparticles production site. Thus, even under worst case conditions no more than 3.8 1013 nanoparticles/ day can be deposited in the lungs (100% of them are assumed to be biopersistent). Assuming that the alveolar epithelium consists of 2 1010 epithelial type I cells, 3 1010 epithelial type II cells and 6 109 alveolar macrophages (Stone et al., 1992), 40 nanoparticles per cell will be deposited daily (3.5 105 nanoparticles/cell for 80 years lifetime dose with 70% clearance), where again we have included the safety factor of 10 to provide a maximum estimate. Correspondingly, under worst case conditions (5 106 cm3) an alveolar surface cell will accumulate up to 6700 nanoparticles per day or 5.9 107 nanoparticles/cell over an 80 years lifetime. Normalized to lung surface area (100 m2) the corresponding nanoparticle dose is 2.3 106 cm2 (lifetime: 2.0 1010 cm2) for (realistic) urban conditions and 3.8 108 cm2 (lifetime: 3.3 1012 cm2) for the (unrealistic) worst case scenario. Most of the available in-vitro cell exposure systems can match the range of target tissues dose fluxes occurring in the lungs. As seen from Table 3 the range of mass and number flux delivered to the lung tissue is 3 10–5–5 10–3 mg/cm2 h) and 2–300 particles per cell per hour, respectively, depending on the exposure scenario. This range can be produced by both submerged and ALI exposure systems quite easily. In fact all in-vitro exposure systems allow for larger dose fluxes. For ALI systems, about 10-fold higher mass dose fluxes (mg/cm2/h) can be accomplished with the CULTEXs glass modules and Vitrocell systems and even up to about 105-fold enhanced mass dose fluxes can be accomplished with the ALICE (Table 3). If the number dose flux (cell1 h1) is considered, the CULTEXs glass modules and Vitrocell systems provide an almost identical range (3–4500 nanoparticles/cell/h) as seen in the lungs (2–300 cell1 h1), while the ALICE system covers the range from 0 to 5 105 cell1 h1 depending on the nanoparticle concentration in the nebulized suspension (Table 3). These values are still significantly lower than the up to 107-fold higher than the worst case exposure doses, which can be realized with submerged exposure systems. Ideally, in-vitro nanotoxicity studies with cells from the peripheral lungs should be performed with realistic dose levels. Since cell systems cannot be cultured for more than a few days, the daily dose levels of ultrafine or nanoparticles for realistic ambient exposure (7.5 10–4 mg/cm2 (or 3.1 10–5 mg/cm2/h), 7.5 10–3 mm2/cm2, 2.3 106/cm2, 40/cell) or worst case occupational exposure (0.13 mg/cm2 (5.2 10–3 mg/cm2/h), 6.3 mm2/cm2, 3.8 108/cm2, 6700 cell1) can be used as a guideline, where the safety factor of 10 guarantees that these values are upper limits for the different scenarios. It has been shown, that the threshold value for pro-inflammatory effects observed under in-vitro cell conditions (submerged) is about 1 cm2/cm2 (surface area of the nanoparticles per exposed cell surface area) for low-solubility, low-toxicity particles (e.g. TiO2, BaSO4) using interleukin-8 (IL-8) gene expression of A549 cells as biological endpoint (Donaldson et al., 2008). This in-vitro threshold dose is about a factor of 15 higher than the estimated daily dose possibly deposited at hot spots of the lung under worst case conditions (6.3 mm2/cm2) and it is even similar to the entire lifetime dose of ultrafine ambient particles (66 mm2/cm2 for 10 mg/m3 exposure level). This is confirmed by numerous mass-based studies showing no statistically significant in-vitro cellular response below about 1–10 mg/cm2 (e.g. Bitterle et al., 2006; Volckens et al., 2009, Lenz et al., 2009; Holder et al., 2008), which is in the range of the entire lifetime dose of ultrafine ambient particles (6.6 mg/cm2 for 10 mg/m3 exposure level). Hence, during in-vitro testing almost an entire lifetime dose has to be ‘dumped’ onto the cells within a few hours or less to obtain measurable cellular responses. It is unclear, if the mechanistic studies performed under these highly unrealistic dose conditions provides meaningful insights into the cellular pathways of nanoparticle toxicity, but it is important to note, that otherwise no mechanistic data would be available at all. Recently, Donaldson and colleagues have shown that this in-vitro toxicity threshold of 1 cm2/cm2 was consistent with the in-vivo inflammatory response in rats characterized by the influx of inflammatory neutrophil granulocyte cells into the lungs (Donaldson et al., 2008), which is considered the ‘gold standard’ for inflammatory response. The concordance between in- vitro and in-vivo inflammatory response suggests that in-vitro cell cultures may not only be used as screening tool for nanoparticle toxicity testing, but may even allow under carefully controlled conditions an approximate estimate of relevant threshold dose levels for risk assessment and regulatory measures of nanoparticles exposure. As a caveat we add, H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692 685 that the in-vitro dose–response relationship is strongly dependent not only on the type of in-vitro cell system (cell type, single- or multi-cell co-culture) (Alfaro-Moreno et al., 2008), but also on the type of exposure (submerged or ALI) (Volckens et al., 2009; Lenz et al., 2009; Bitterle et al., 2006). Thus, it is crucial to identify the most relevant in-vitro cell system for a given risk–exposure scenario.

5. Social aspects and risk assessment

Nanotechnologies carry a high potential for improved material efficiency and solving countless technical problems in durability, sustainability, precise control, manipulation and construction of materials. Simultaneously unforeseen concerns are raised by the very same properties that make them promising. As Maynard et al. (2006) stated, the pursuit of responsible nanotechnologies can be tackled only through a series of grand challenges. The third article of the United Nations Declaration of Human Rights (UN, 1948), stating that ‘Everyone has the right to life, liberty and security of person’, highlights the role of the society in protecting the safety of the citizens. There is no doubt about the importance of this role in context of nanotechnologies. However, it is also obvious that the regulation and management of the risks is also in the greatest interests of enterprises, too. Individual enterprises that take on the task of developing new (nano-)products and (nano-)materials can hardly cover all associated risks from human exposure assessment to environmental fate and life cycle analysis. Nevertheless, enterprises could later be made liable for potentially induced damage. Hence, proper management of these risks is not only in the best interest of the society, but also in the best interest of the enterprises. Efficient management of the risks has to be based on balanced division of roles between scientific research, regulation, reasonable application of the precautionary principle and instigator responsibility. Acceptable (ethical) regulation must be based on science and appropriate handling of the inherent uncertainties in our understanding of the nature of the effects and their probabilities. Regulation must consider the probability (here mainly related to exposure levels) and magnitude of the risks (hazard) of individual nanotechnologies and balance the precautionary actions well with the risks; failing to balance these factors leads to either violation of the public safety or an excessive load of unnecessary protection. Application of the strictest forms of the precautionary principle (Rio Summit, 1992; Wingspread Conference, 1998) indeed could easily cripple nanotechnology as any new technologies either non-functional or economically infeasible. The major pathway to reduce the uncertainties surrounding the risks of nanomaterials is the careful evaluation of exposure–dose–response relationships (Oberdorster¨ et al., 2008; Seaton, 2006). Exposure assessment is relatively straightforward using both existing monitoring techniques as well as large variety of models. There is abundant toxicological and epidemiological data on ambient particles that seem to be more or less harmful regardless of their source or composition. New epidemiological data is emerging concerning also the nano-sized ambient particles (e.g. Stolzel¨ et al., 2007; Peters et al., 2009; Breitner et al., 2009). Due to the lack of more targeted data concerning a specific nanomaterial it seems a reasonable application of the precautionary principle to assume that the unknown new materials are as safe or as harmful as the widely spread ambient particles. This first approximation of toxicity can then be refined in more detailed studies. Nevertheless, for the years to come toxicology will be the main discipline providing us with data on the toxicity of specific nanomaterials. Developments of toxicological methods are tightly integrated with using laboratory animals and in-vitro methods and balancing between these have also strong societal implications. Nanotechnology is most likely facing a public debate similar to those which have confronted the nuclear, chemical and biotechnology industries, and the economical success as well as realization of the technological promises can proceed only via gaining the public acceptance—thus public communication and engagement are essential for its successful development (Donaldson, 2009). Maintaining societal support for the development of nanotechnology will not be simple. It will require making decisions in the face of both large uncertainties and a wide range of choices and possible risk scenarios. While this is the case with all new technologies, some believe that the challenges with nanotechnology will be greater than ever before (Drexler et al., 1991). But we will only be able to tackle this task by accepting it as a well-worth scientific, environmental and societal challenge.

6. Conclusions

Societal acceptance of nanoproducts, which is vital for the dynamic development of the nanotechnology, depends largely on the risk-benefit ratio associated with nanotechnology. This requires realistic assessment of nanoparticle exposure based on studies on the mechanisms of nanotoxicity, the determination of dose–response relationships and the assessment of realistic target tissue doses. In all of these areas considerably more work is required. For pulmonary toxicity of nanoparticles, particle surface area is emerging as one of the most relevant dose metrics for insoluble and non-biodegradable particles. Consequently, the development and refinement of devices for the measurement of nanoparticle exposure, especially in terms of lung-deposited particle surface area, are desirable. However, surface area alone will not be adequate to predict the toxicity. Especially for (partially) soluble, (partially) biodegradable and fiber- like particles other metrics appear to be more adequate. Significant effort is undertaken by many research groups to determine the critical determinants of nanoparticles that play a role in the development of adverse health effects, e.g. oxidative potential, particle number concentrations, three dimensional aspects etc. On top of the physicochemical properties, the development of fast (in-vitro) screening methods and models to determine nanoparticles toxicity and the 686 H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692 underlying mechanisms of action and biological pathways is important to cope with the potential threat posed by the vast number of new nanoparticle types introduced into the marketplace today. In the interest of limiting animal experiments to a minimum, comprehensive toxicity screening should be performed not with animals, but with in-vitro cell systems (or slices of organ tissue). New developments in the isolation and cultivation of pulmonary and respiratory tract cell/tissue systems (single- and multi-cell cultures) should be combined with optimized methods of nanoparticle exposure. Although the technically simple submerged culture system has been widely used for nanotoxicity screening, the workshop participants expressed a strong preference for the use of air–liquid interface (ALI) cell exposure systems for the following reasons:

(1) ALI exposure systems resemble in-vivo exposure conditions of the lungs more realistically than submerged cell systems. (2) Cell systems (single- or multi-cell cultures, tissue slices) cultured at the ALI can be covered with a mucus layer and/or surfactant lining ﬂuid mimicking the lung epithelium. This is not possible under submerged culture conditions. (3) In submerged cell systems, the interaction of nanoparticles with the cell culture medium may alter the physicochemical and hence the toxicological properties of the nanoparticles. (4) The cell-delivered nanoparticle dose is much more readily measured and controlled in ALI cell systems and real-time methods for the determination of the cell-deposited nanoparticle dose are available for ALI exposure systems only. In addition, an ALI system also has the advantage of controlling the dose rate, which is not possible for submerged cultures.

It is acknowledged that as long as there are no ALI cell exposure systems for high-throughput measurements available, the technically more simple submerged culture system is particularly useful for initial, high-throughput screening for nanoparticle toxicity. Hence, there is an urgent need for developing in-vitro cell exposure systems at the air–liquid interface, which are (1) suitable for high-throughput screening, (2) provide accurate dosimetry with real-time monitoring techniques, (3) allow for simple handling by technical personnel without extensive expertise in aerosol technology and (4) incorporate real-time monitoring of the biological response of the cell systems. The aerosol community is well equipped to make a substantial contribution to at least the former three issues. The interpretation of the cellular dose–response relies heavily on the well-controlled and well-characterized exposure conditions (target tissue dose, particle properties, etc.). The already existing guideline of the OECD (2010) for the characterization of nanoparticle properties for toxicity assessment includes the parameters such as particle size distribution, solubility, chemical composition, etc. Yet, there is also an urgent need for the demonstration of the superiority of ALI in-vitro models above in vivo studies in terms of their predictive value for human hazards. To avoid unnecessary concern towards the hazard of nanoparticles no-effect levels should also be reported and the typically observed threshold doses for toxicological responses should be put into perspective of realistic exposure doses. If we consider average ambient urban exposure and occupational exposure at the currently recommended Occupational Safety and Health Administration (OSHA) standard for respirable nuisance dust as the boundaries of human exposure, the corresponding range of upper-limit mass and number flux delivered to the lung tissue is 3 105 103 mg/cm2/h and 2–300 particles/cell/h, respectively. This range can be easily matched and even exceeded by almost all currently available cell exposure systems reaching an up to 107-fold higher dose flux with submerged exposure systems. This implies that the frequently observed surface threshold dose for in-vitro cell response of low-solubility, low-toxicity particles of about 1cm2 nanoparticle surface area deposited on 1 cm2 of tissue is about equal to the lifetime dose of ambient ultrafine particles accumulating in the lungs under realistic urban conditions. This raises concerns regarding the scientific relevance of in-vitro studies delivering the entire lifetime dose within a few hours. Hence, while in-vitro toxicity studies are certainly valuable for mechanistic studies and toxicity screening, their relevance for risk assessment should be considered with great caution. Another limitation of in-vitro cell systems is the fact that toxicological response and/or the susceptibility or disease status of a complex mammalian organism cannot be mimicked in its entirety by an in-vitro system. Hence, even the most sophisticated in-vitro cell exposure systems will not be able to completely substitute animal testing. The members of this workshop content that it is in the interest of public health, environmental safety and sustainable economical growth to work towards providing a sound basis for risk assessment of nanoproducts. This will eventually result in the issuance of relevant guidelines by the OECD and/or national regulatory bodies to ensure that safe nanoproducts enter the marketplace.

Acknowledgment

We thank the Gesellschaft fur¨ Aerosolforschung e.V. for funding of the workshop ‘In-Vitro Exposure Studies for Toxicity Testing of Engineered Nanoparticles—A dialog between Aerosol Science and Biology’, 5–6 September 2009, Karlsruhe, Germany. The excellent organization of this Workshop by Dr. Gunthard Metzig and Ms. Sybille Mann is acknowledged gratefully. Finally we thank Sonja Mulhopt¨ for her critical review of the manuscript and Isil Bal for support in editing the document. H.-R. Paur et al. / Journal of Aerosol Science 42 (2011) 668–692 687

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