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Am J Physiol Lung Cell Mol Physiol 294: L817-L829, 2008. First published February 8, 2008; doi:10.1152/ajplung.00442.2007
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INVITED REVIEW

Interactions of nanoparticles with pulmonary structures and cellular responses

Christian Mühlfeld, Barbara Rothen-Rutishauser, Fabian Blank, Dimitri Vanhecke, Matthias Ochs, and Peter Gehr

Institute of Anatomy, University of Bern, Bern, Switzerland

Submitted 23 October 2007 ; accepted in final form 5 February 2008


   ABSTRACT
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Combustion-derived and synthetic nano-sized particles (NSP) have gained considerable interest among pulmonary researchers and clinicians for two main reasons. 1) Inhalation exposure to combustion-derived NSP was associated with increased pulmonary and cardiovascular morbidity and mortality as suggested by epidemiological studies. Experimental evidence has provided a mechanistic picture of the adverse health effects associated with inhalation of combustion-derived and synthetic NSP. 2) The toxicological potential of NSP contrasts with the potential application of synthetic NSP in technological as well as medicinal settings, with the latter including the use of NSP as diagnostics or therapeutics. To shed light on this paradox, this article aims to highlight recent findings about the interaction of inhaled NSP with the structures of the respiratory tract including surfactant, alveolar macrophages, and epithelial cells. Cellular responses to NSP exposure include the generation of reactive oxygen species and the induction of an inflammatory response. Furthermore, this review places special emphasis on methodological differences between experimental studies and the caveats associated with the dose metrics and points out ways to overcome inherent methodological problems.

ultrafine particles; engineered nanoparticles; electron tomography; translocation; oxidative stress

THE EPITHELIAL SURFACE of the lungs is the largest surface area of the human body simultaneously in direct contact with the environment. In contrast to the similarly large gut surface, the alveolar surface area is connected in parallel (78), and the structural barrier between air and blood is reduced to a 2.2-µm thin layer (33). The latter consists of surfactant, the underlying watery hypophase, and the epithelial and capillary endothelial cells with the interposed coalesced basal lamina. Although this design is required for efficient gas exchange, every breath also introduces large numbers of airborne particles, toxic gases, and microorganisms that may be deposited, i.e., come into physical contact with pulmonary surface structures. Therefore, the lungs are equipped with defense mechanisms exerting diverse protective strategies including surfactant, epithelium, alveolar macrophages (AM), and dendritic cells, the mucociliary escalator, and secretory immunoglobulins (94).

During the past decades, various aspects of the interaction between airborne particles and pulmonary structures have been investigated. The advent of nanotechnology, however, has provoked great hopes for new inhalative and targeted drug delivery strategies including the application of therapeutics, vaccines, and diagnostics (42). On the other hand, the different physicochemical properties of nano-sized particles (NSP) (compared with larger sized particles of the same components) have raised substantial concerns about the safety of nano-sized material brought into the body with or without intention (61, 76).

In this article we review the current knowledge about interactions between pulmonary structures and NSP as well as the cellular responses. Special emphasis is placed on methodological factors influencing the data reported in the literature.

Definitions

Ambient airborne particulate material (PM) is ubiquitously distributed. The concentration, particle size, and chemical characteristics of particulate material vary widely in space and time (77). A useful expression of particle size is the aerodynamic diameter, which is defined as the diameter of a spherical particle with a density of 1 g/cm3 with the same settling velocity as the particle that is to be characterized (45). PM with an aerodynamic diameter of <10 µm (PM10) is categorized into coarse, fine, and ultrafine particles with aerodynamic diameters between 2.5 and 10 µm, <2.5 µm (PM2.5), and <0.1 µm (UFP, or PM0.1), respectively. The coarse fraction of PM10 includes crystal materials, sea salt, and biological components. Ambient PM2.5 and PM0.1 are predominantly produced by combustion processes and consist primarily of metals, hydrocarbons, and secondary particles generated by chemical reactions with gaseous compounds in the atmosphere (Fig. 1) (26).


Figure 1
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  		Fig. 1. Classification of particulate matter (PM) and sources of particles. Ambient particulate matter can be classified into PM with an aerodynamic diameter <10 µm (PM10), <2.5 µm (PM2.5), and <0.1 µm (PM0.1). The sources of particles within these ranges are denoted. Although manufactured nanoparticles (NP) do not belong to ambient PM, they are included as a source of inhalable particles. [Adapted from Hinds (45)].

 
The terminology of nano-sized materials is variable. In general, laboratory-made, engineered NSP with clear chemical characteristics need to be distinguished from ambient (both natural and anthropogenic) NSP of varying constituents. In accordance with Oberdörster et al. (97), we use the terms NSP for all engineered and ambient materials with a diameter between 1 and 100 nm, nanoparticle (NP) for engineered NSP, and UFP for ambient NSP. Differently shaped NP are directly referred to as nanotubes, nanowires, and nanorods, among others. Finally, the term quantum dot (QD) is used for NSP consisting of a semiconductor core (e.g., CdSe) surrounded by a shell of another semiconductor (e.g., ZnS) with varying surface coatings (8).

Methodological Issues

The analysis of the interactions between NSP and biological systems requires knowledge about several methodological problems and, in most cases, an interdisciplinary approach involving particle chemistry, aerosol physics, and biomedicine.

As recommended in Ref. 98, it is crucial to provide information on size distribution, agglomeration state, shape, crystal structure, chemical composition, surface area, surface chemistry, surface charge, and porosity. The rationale for characterizing NSP rigorously is supported by several studies that have reported an influence of these parameters on the translocation characteristics, cellular uptake, or toxicity (97, 98).

Inhalation of aerosolized NSP is the gold standard for in vivo exposure and can be performed by whole body, head/nose/mouth-only, or lung-only exposure. The quality of whole body exposures depends on an equal distribution of the NSP in the exposure chamber still leaving a relatively high variability in dosage. However, this exposure type is closest to environmental exposure, less stressful for the animals than other exposure types, and can therefore be recommended for chronic exposures. Head/nose/mouth-only exposure guarantees more efficient and controllable doses, and thus allows testing of parameters during exposure, but it introduces stress and cuts the animals off from their food and water supply. The technically demanding lung-only exposures provide the most precise dosage, but the lack of systemic and autonomous nervous system reactions may limit the significance of the results (32, 104, 105). Although artificial, specialized techniques such as intratracheal instillation of NSP containing fluids are frequently used, they should not be regarded as an adequate substitute for inhalation studies (32).

In vitro studies are often based on submerse exposure of cells to NSP suspended in a suitable cell culture medium. Submerse exposure is easy to perform, and dosimetry seems to be simple by measuring the amount added to the cell culture medium. However, the particle dose added does not necessarily reflect the particle dose in contact with the cells, since the main mode of NSP motion is Brownian diffusion, which means that the particles do not sediment on the cells (70). In addition, the decrease in cell viability in a bronchial epithelial cell line upon exposure to NO2 was inversely related to the amount of fluid present on the cells (137). Therefore, exposure of the cell cultures at the air-liquid interface is preferable to submerse exposure. A prerequisite is a biphasic culture system with the cells grown on a porous membrane and the culture medium delivering the nutrients from below (7, 144, 145). The aerosol is guided through a cylinder, which should guarantee an even particle exposure of the whole cell culture surface. The latter depends on the flow characteristics of the aerosol and the size of the particles. Based on the physics of stagnation point flow, in one model the applied dose was calculated from the number and size of the particles in the exhaust measured after the exposure (14, 138). A similarly designed model (6, 7) was used to study the biological activity of cigarette mainstream smoke on A549 cells (115); however, experiences with NSP in this model are not available yet. Other exposure models, e.g., rotating wall cell cultures (82), might prove suitable for NSP exposure if dosimetry is appropriate.

In many studies, monocultures of epithelial cell lines (e.g., A549) or primary cell cultures are used. Coculture systems have been introduced consisting of epithelial cells and endothelial cells (11, 13) or epithelial cells and macrophages (52). We have introduced a triple cell coculture model of the human airway wall, which consists of A549 cells, human blood monocyte-derived macrophages on top, and human blood monocyte-derived dendritic cells at the bottom of the cell culture (15, 117). Using this model, we demonstrated a transepithelial cellular interplay between macrophages and dendritic cells upon exposure to 1-µm fluorescent polystyrene particles (PSP) (16).

After exposure, it is essential to analyze the localization of the particles in tissues or single cells. This requires a microscopic approach allowing the identification of single particles unambiguously and the surrounding biological environment with appropriate resolution. For fluorescent NP and QD, confocal laser scanning microscopy with subsequent use of deconvolution algorithms provides valuable information on intra- vs. extracellular localization (46, 120) and, using additional organelle staining, on the intracellular distribution. For many purposes, transmission electron microscopy (TEM) is most appropriate if several caveats are considered (87). Conventional TEM often cannot distinguish NSP from cellular structures or staining precipitations of the same size range and electron density, undoubtedly (102). Thus energy-filtered TEM can be used to analyze the elemental composition of the structure of interest and verify the identity of a NSP (Fig. 2) (37, 55). Electron tomography can be used to visualize NSP in their biological environment in three dimensions (Fig. 3, see Supplemental Fig. 1 and Supplemental Video 1). (Supplemental data for this article is available online at the American Journal of Physiology-Lung Cellular and Molecular Physiology website.) To correlate functional data with particle localization, we recently introduced a stereological method to assess the particle distribution quantitatively (85, 86).


Figure 2
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  		Fig. 2. Titanium detection by electron spectroscopic imaging (ESI) and parallel electron energy loss spectroscopy (PEELS). Nano-sized particles (NSP) may have an appearance similar to subcellular biological structures. ESI and PEELS provide suitable tools to identify the particles unambiguously by analyzing the elemental composition of a structure of interest (101). This analysis is based on the specific loss of electron energy upon contact of the electron beam with the structure of interest. At 464 eV, the PEELS spectrum has a maximum, specific for the L2 orbital of titanium (A–C, F, and G). In this experiment, a triple cell coculture was incubated with titanium dioxide (TiO2) NP. Inside a macrophage, a small agglomerate of NP is shown inside a phagosome (Ph) roughly 1 µm from the plasma membrane and in close proximity to a mitochondrion (D and E). The structure can be clearly identified as a TiO2 NP, based on the identification of titanium (Ti) by analytical electron microscopy. All images are at the same magnification (bar in F, 500 nm), with the exception of D (bar, 1 µm).

 

Figure 3
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  		Fig. 3. Electron tomography of TiO2 NP after uptake by a macrophage. Electron tomography allows the visualization of subcellular structures in 3 dimensions and, hence, of NSP in the biological environment. A stack of images provides X–Y slices at different depths within the specimen either of the whole scene (A–C) or of a structure of interest (D–H) or X–Z slices (I–L). Software-assisted segmentation of the tomogram (65) based on contour drawing obtains an observer interpretation in 3 dimensions that can be viewed from different angles (M and O) and can be used to calculate volumes (V) and surfaces (S) of structures of interest (N). This experiment shows the same agglomerate of TiO2 NP as in Fig. 2 localized within a phagosome inside a macrophage. It helps to get a 3-dimensional picture of the particle localization, structure, and size after active uptake into the cell. GBd, ghosts of Bragg diffractions, indicated by long black arrows; Ph, phagosome; PhM, membrane of the phagosome, indicated by short white arrows; Mi, mitochondrion; Ti, TiO2 NP, indicated by long white arrows. Arrowheads in B provide orientation of the X–Z slices (I–L), and arrowheads in I provide orientation of the X–Y slices (A–C). Bars: A–C, 500 nm; D–L, 100 nm.

 
Analysis of structural alterations upon exposure to a toxicant has a long and successful tradition in pulmonary toxicology (22, 35) but is infrequently used in nanotoxicology. The importance of assessing the structural features of cells and tissues using design-based stereology needs to be emphasized. Recent reviews have highlighted the most important stereological tools for pulmonary research (99, 100, 146).

Interaction With Pulmonary Structures

The anatomy, breathing pattern, and particle size are the principle factors determining the pulmonary compartment to which a particle gains access (18, 19). For NSP, the main transport mechanism is diffusion with only minimal contributions of gravitation or inertia (124). Therefore, NSP are not retained in the tracheobronchiolar region but reach the alveolar region (44), where they are retained for a long period of time and are effectively deposited (95). The possible interactions with the pulmonary structures, which are discussed below, are summarized in a flow chart (Fig. 4) that provides an overview of hypothetical interactions currently under investigation.


Figure 4
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  		Fig. 4. Summary of possible interactions of NSP with pulmonary structures. Upon inhalation, NSP reach the gas-exchanging region of the alveoli, where they interact with surfactant and subsequently are displaced to the hypophase. There, they may interact with surfactant proteins A and D (SP-A, SP-D) or enter different cell types, including alveolar epithelial type II (AE2) or type I (AEI) cells, alveolar macrophages (AM), or alveolar dendritic cells (DC). Entering of the cells may lead to interference with specific cellular functions (surfactant metabolism in AEII) or result in secretion of proinflammatory cytokines, such as tumor necrosis factor-{alpha} (TNF-{alpha}). Clearance of intracellular particles occurs via the mucociliary escalator for AM or migration to lymph nodes for DC. Inflammatory proteins may open up the way for a paracellular route of the NSP, which in the healthy lung does not seem to contribute significantly to particle translocation. Most likely, translocation occurs via AE1 to the interstitium and, eventually, to the blood circulation over endothelial cells.

 
In the alveoli, upon contact with surfactant, larger sized particles are displaced from the airspace to the hypophase due to wetting forces (34, 39, 125), a phenomenon that probably also occurs with NSP. In the hypophase, the particles may interact with proteins (e.g., surfactant proteins A and D or glycoproteins) (30) or may be taken up by AM (36). Recent evidence suggests that UFP interfere with normal lipid and macrophage metabolism by increasing lipid peroxidation and decreasing killing of bacteria (75). Surfactant lipids and proteins adsorb to the surface of PM2.5 particles and carbon nanotubes, thereby modulating the function of pulmonary surfactant (56, 121). Similarly, gold NP sequester lung surfactant and may interfere with its normal function (9).

With respect to AM, only 20% of the retained iridium NSP radiolabeled with 192Ir were accessible to lavage in the rat lung, with ~90% of these particles being associated with AM (66). In contrast, the lavageable fraction of 0.5-, 2-, and 10-µm PSP amounted to ~80% of retained particles in the rat (96). This indicates that NSP are not phagocytized as effectively by AM as larger sized particles and that a large fraction of the NSP is not accessible to lavage, possibly by binding to epithelial surface proteins, uptake of epithelial cells, or translocation over the alveolar epithelium. This conclusion is supported by a study on rat lung AM after inhalation exposure to titanium dioxide (TiO2) NP, which showed that only a small fraction of the deposited NP are taken up by the AM (38). Importantly, aggregates of ultrafine carbon particles reduced the phagocytic activity of human AM collected by bronchoalveolar lavage from healthy volunteers (73) and decreased the phagocytosis of microorganisms by AM (74).

The fate of the NSP not taken up by AM is a subject of controversial discussion. After a period of 12 wk of whole body exposure, a large fraction of the retained TiO2 NP were localized in the interstitial space (95). Interstitial localization of NSP after inhalation was confirmed in the rat lung after inhalation of TiO2 NP (37, 86) and iridium NP radiolabeled with 192Ir (126).

For the translocation mechanism of NSP over the alveolar epithelium, different mechanisms are discussed (Table 1). There is little evidence for paracellular transport of NSP in the healthy lung, but it may be present when permeability is increased, e.g., due to hydrogen peroxide, histamine (79), or additive ozone inhalation (12). Endocytosis as the main mode of particle uptake has been shown by several authors (58, 129, 131, 132, 135). Inhaled gold particles were observed within membrane-bound vesicles in AM and alveolar type I cells of rat lungs (135). In vitro, similar results were obtained for the uptake of gold NP by macrophage cells, with the particles localized in pinocytotic vesicles and lysosomes (129), as well as for the uptake of TiO2 NP by A549 cells, with the NP localized inside vesicular structures including multivesicular and lamellar bodies (131, 132). Low temperature and metabolic inhibitors inhibited the uptake of silica-coated NP, suggesting an energy-dependent mechanism (58). In macrophages, certain receptors are involved in the endocytotic processes, such as scavenger receptors (5, 54, 112). According to our own experiments, caveolae-dependent uptake seems to be important in human monocyte-derived macrophages, since the cholesterol-extracting agent methyl-β-cyclodextrin inhibited the uptake of fluorescent ultrafine PSP but not fine PSP (unpublished data). Furthermore, as suggested by Supplemental Fig. 1, clathrin-coated transport vesicles also contribute to active NP uptake.


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  		Table 1. Summary of selected in vivo and in vitro studies on translocation and cellular entering of NSP

 
Apart from endocytosis, other mechanisms of NP entering into the cells are possible. The localization of TiO2 NP inside red blood cells after inhalation exposure in rats (37), of fluorescent ultrafine PSP in red blood cell culture (118), of ambient UFP in mitochondria of macrophage and epithelial cell culture (69), of C60 fullerenes inside the nucleus and the free cytoplasm of human monocyte-derived macrophages (108), and of 1.4-nm gold particles bound to the nuclear DNA of 11 different normal or cancer cell lines (141) provides evidence that other mechanisms apart from the usual endocytotic pathways are involved in the internalization of NSP into cells. Adhesive interactions due to electrostatic forces, Van der Waals, or steric interactions are suspected to be involved in these processes (41, 114, 119). It may be concluded that the physicochemical properties of the NSP, the experimental conditions, and the characteristics of the target cells influence the extent and characteristics of the NSP uptake (142).

It is very likely that size, composition, and surface characteristics influence the entering mechanism and the intracellular trafficking of particles (103, 111, 120, 152). Especially, protein adsorption may determine the mechanism of NSP trafficking, as evidenced in rat brain endothelial cells (57). The coating of NP surface by specific peptides was recently described as a potential mechanism to control the translocation behavior of NP. Insertion of the peptide into the membrane with subsequent adhesion of the NP to the membrane may open a tight membrane pore, allowing particle translocation (72). Studies addressing the translocation mechanism therefore need to characterize the applied NSP and its adsorption of endogenous molecules exhaustively.

In a rat model, a small fraction of the inhaled TiO2 NP crossed the blood-air barrier and reached the circulation, where they also gained access to the cytoplasm of erythrocytes (37, 86). Such intracellular localization of fluorescent ultrafine PSP in a cultured erythrocyte is shown in Fig. 5. Translocation to the circulation was also reported for instilled diesel exhaust particles in a hamster model (93) but not for instilled fluorescent ultrafine PSP in an isolated rabbit lung model (92). In the human lung, however, there is only one study that describes a rapid and significant translocation of inhaled carbonaceous NP to the systemic circulation and extrapulmonary organs (91), whereas most other studies only detected a low degree of translocation for iridium (66) or carbon NSP (81, 147). The strong translocation of 99mTc-labeled particles in Ref. 91 was convincingly related to a greater translocation of soluble 99mTc-pertechnetate, which was cleaved from the carbon particles (81). It is therefore currently accepted that a very small fraction of inhaled NSP translocates to the circulation; however, knowledge about the significance of this translocation is lacking so far.


Figure 5
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  		Fig. 5. Laser scanning microscope micrograph of fluorescent polystyrene NP attached to or within an erythrocyte. After incubation of human erythrocytes with fluorescent polystyrene NP, particles were found inside or attached to the erythrocyte. Since erythrocytes are nonendocytotic cells, the entering of the NP into red blood cells (RBC) suggests that mechanisms other than endocytosis are also involved in the entering of NP into cells. Autofluorescence of the cells is shown in red; particles are green. With the use of a surpass algorithm (RBC red, transparent mode) it can be shown that many particles are inside the cells.

 
Cellular and Molecular Responses

Three main cellular responses to particle exposure have been described for a variety of NSP, including the generation of reactive oxygen (ROS) and nitrogen species (RNS), the release of proinflammatory, inflammation-associated proteins, and injury of nuclear DNA. Information on morphological alterations associated with these responses, including emphysema and granulomatous and fibrotic lesions, is based on single reports and warrants systematic stereological confirmation (23, 130). The key questions arising from these observations are, do NSP have a higher toxicological potential than larger sized particles of the same material? If so, what is the appropriate parameter to evaluate the concentration of NSP in relation to their toxicological potential? In other words, which particle characteristic is most closely correlated with the biological reactions to particle exposure? Do particles have to enter cells or even particular organelles to induce cellular changes?

The higher toxicological potential of NSP compared with larger sized particles of the same material was deduced from the exposure of cell cultures or mice to the same mass of fine particles (25, 113). At a given mass, the number and total surface area of the NSP exceed those of the fine particles by far. A spherical particle 1 µm in diameter equals the volume/mass of roughly 8,000 spherical particles 0.05 µm in diameter presuming they have the same density as the larger particle. The total surface of the mass-equivalent number of NSP would exceed the surface of the 1-µm particle 20 times. In a recent study from our laboratory, the ability of PSP to generate tumor necrosis factor-{alpha} (TNF-{alpha}) in a triple cell culture model was compared between 1- and 0.05-µm particles at the same number concentration. In this case, only the fine particles increased TNF-{alpha}, whereas the NP had no significant effect (120). Also, the comparison between different micro- and nano-sized metal oxides with soil dust did not support the concept of a generally higher toxicity of the NSP (143). Which particle characteristics then are most closely correlated with the biological responses? The first factors that come to mind are the larger physical surface area and number of the NSP per unit mass. The second factor relates to the chemical composition of the particles, including the insoluble particle core and soluble or insoluble molecules attached to the surface of the particle. For example, combustion-derived UFP usually consist of a carbon core that carries a number of adsorbed transition metals and (polyaromatic) hydrocarbons.

In the mouse lung, three different inflammatory parameters measured in bronchoalveolar lavage fluid were most closely correlated with particle surface after exposure to six different instilled carbon particles (133), but the search for the best dose metric has not finished yet. It also has been speculated that the toxicological potential of NSP is related to their ability to enter cells via pathways different from usual endocytic uptake and thus gain access to mitochondria or the nucleus (69, 108, 141). Evidence for a causal relationship between particle localization and a specific cellular response, however, is lacking so far. In addition, particle toxicity may also be driven by the internalization of organic hydrocarbons attached to the particle surface without any entering of the particles themselves (106).

There is consensus that the induction of ROS and RNS by NSP is a key event in the toxicological cellular responses to NSP exposure (27, 142). A direct particle surface effect was implicated by the finding that nonenzymatically generated ROS appear in cell free systems after addition of carbon black NP (148). Since UFP, particularly combustion-derived UFP, contain transition metals and organic compounds, their release from the particle surface was found to generate ROS by Fenton-type reactions or redox cycling, respectively (67, 148, 150). A direct interaction of the NSP with subcellular structures significantly involved in redox reactions, i.e., plasma membrane, cytosol, mitochondria, or endoplasmic reticulum (24), may increase the physiological generation of ROS. In this regard, the localization of UFP inside mitochondria of macrophage and epithelial cell culture was hypothesized to be causative for the observed ROS generation (69). The interaction of NSP with mitochondrial and endoplasmic reticulum membranes may also lead to a loss of membrane reactivity, thus leading to a dysregulation of Ca2+ homeostasis (151) with subsequent activation of nitric oxide synthases and the generation of RNS (63). The literature on this topic provides evidence that various NSP, including UFP (reviewed in Ref. 40) and combustion-derived UFP (reviewed in Ref. 27), as well as manufactured NP (71, 110, 127), have the potential of inducing oxidative stress in pulmonary cells but also that other NP may be useful as oxygen radical scavengers (53, 123, 139).

NSP-induced ROS generation may either affect the integrity and functionality of proteins and membrane lipids or DNA by oxidation directly or may induce cellular responses indirectly via redox-sensitive signaling pathways. Direct events include the potential dysfunction of cytoskeletal proteins, enzymes, etc., lipid peroxidation, and loss of cellular integrity, as well as DNA mutations. The signaling pathways induced by ROS generation link NSP to other toxicants like asbestos and cigarette smoke (reviewed in Ref. 84). So far, there is convincing evidence that diesel exhaust (including both particulates and volatile compounds) activates the transcription factors nuclear factor-{kappa}B (NF-{kappa}B) and activating protein 1 (AP-1) (109), with AP-1 being more important for the release of UFP-associated interleukin-8 (IL-8) (60). The activation of NF-{kappa}B has been shown to be regulated by intracellular Ca2+ through interaction with Ca2+-binding proteins and protein kinase enzymes (47), which was recently shown to be important in macrophages exposed to UFP (20, 21). Both NF-{kappa}B and AP-1 can be activated by upstream mitogen-activated protein kinase (MAPK) cascades, which can be started upon activation of receptor tyrosine kinases (84). Apart from its ligand, the receptor tyrosine kinase epidermal growth factor receptor (EGFR) can be activated by oxidative stress (1), linking the UFP-induced proliferation to the tyrosine kinase activity of EGFR (134, 136). In addition, the expression of several EGFR ligands is increased by particle exposure. The activation of NF-{kappa}B and AP-1 form a converging point that regulates the expression of genes involved in inflammation, cell proliferation, apoptosis, and differentiation (128, 149). The enhanced expression of proinflammatory proteins such as TNF-{alpha} and interleukins is a common end point investigated in studies on the toxicity of NSP with differential secretion by specific cell types. To name a few, increased inflammatory markers were demonstrated for gold NP in vitro (120), for carbon NP in ovalbumin-sensitized mice (4) and AM (10), for Teflon fume particles in rats (51), for multiwall carbon nanotubes both in vivo and in vitro (28, 88), and for TiO2 NP in A549 cells (131). In contrast, it needs to be mentioned that other studies have only observed modest or no increased inflammatory responses to gold NP exposure of a macrophage cell culture (129) or to concentrated ambient PM exposure in spontaneously hypertensive rats (64). The signaling pathways described above are summarized in a flow chart (Fig. 6) to provide a short visual orientation of the current knowledge.


Figure 6
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  		Fig. 6. Proposed cellular responses. The toxicological key response to NSP exposure is the generation of reactive oxygen (ROS) and nitrogen species (RNS), for which both intrinsic and extrinsic NSP characteristics are responsible (122). Besides direct effects on cellular molecules, ROS are known to activate the transcription factors nuclear factor-{kappa}B (NF-{kappa}B) or activating protein 1 (AP-1). Activation of NF-{kappa}B and AP-1 may also be triggered by mitogen-activated protein kinase (MAPK) pathways and upstream by epidermal growth factor receptor (EGFR) activation, either directly by NSP or by NSP-triggered expression of EGFR ligands. Penetration of the nucleus enables direct interaction of NSP with nuclear DNA. All cellular reactions mentioned above may alter the expression of genes involved in inflammation, cell proliferation, and differentiation and apoptosis.

 
In summary, there is a growing body of literature suggesting the potential of inhaled NSP to activate pathophysiological pathways in the lung that are known to be involved in a range of important human diseases, such as chronic obstructive pulmonary disease (COPD), asthma, and carcinoma, among others (3, 17, 62). Besides the exact molecular mechanisms involved in NSP toxicology, it will be necessary to define the toxicological significance for normal human health, susceptible human individuals (such as young, old, or diseased), and preferentially exposed individuals, e.g., at the workplace.

NSP in Medicine

The preceding chapters were devoted to the current knowledge on the interactions between inhaled NSP and pulmonary structures stressing the toxicological side. However, "to extrapolate toxicity from nano-sized diesel exhaust particles to similarly sized protein particles is clearly nonsense" states a recent commentary (29). In fact, the applications of nanoparticles in medicine range from contrast agents in medical imaging to carriers for drug or gene delivery to specific cells (89).

Quantum dots are NSP that emit strong fluorescent light with wavelengths that depend on particle size and are relatively stable against photobleaching. These features make QD attractive tools in optical imaging. Attachment of specific molecules to the surface of QD made it possible to map lymph nodes below the skin of mice and pigs up to a depth of 1 cm (59) or to direct QD to endothelial cells of pulmonary blood vessels in mice (2). With the use of tumor-specific surface coatings, QD can be targeted specifically to tumor cells, allowing site-specific in vivo imaging (31).

Superparamagnetic iron oxide NP differentially enhance the contrast of liver, lymph nodes, and bone marrow in MRI (83). Once again, coating of these NP with specific molecules may target the particles to tumors (48) or metastases (43) that would otherwise not be detectable by MRI. Similarly, labeling of polymer NP targeting the intercellular adhesion molecule 1 with 64Cu allows the investigation of the biodistribution of the NP by PET (116).

Strategies of drug and gene delivery employing NP hold great potential for medicine by helping to overcome typical problems of drug delivery such as systemic side effects, water insolubility, or inefficient transport across the blood-brain barrier. For example, the water-insoluble drug paclitaxel could be loaded into NP of a natural polymer, albumin, thus reducing the side effects of other formulations (80) and increasing drug dose within the tumor (49). In the lung, two studies have shown that the inflammatory response of allergic asthma mice was significantly reduced by a liposome-based NP system (50). Furthermore, application of a polymer-drug conjugate, chitosan/interferon-{gamma} pDNA nanoparticles, reduced the allergen-induced airway inflammation in mice by augmenting the expression of interferon-{gamma} (68). Potential applications of NP in pulmonary medicine include gene delivery in cystic fibrosis, aerosol therapy of infectious diseases, vaccination strategies (107), cancer (140), and tuberculosis (90). For more detailed reviews of the potentials of nanomedicine, the reader may refer to some excellent reviews on this topic (89, 107).

Future Perspectives

Unintentional exposure to UFP due to air pollution is a subject of concern. Nanotechnology is going to provide inhalative tools for diagnostics and therapy, but unintentional inhalation of NSP may occur at the workplace or due to application of NSP-containing consumer products. Therefore, it is justified to put significant effort into assessing the interactions between the lungs and inhaled combustion-derived and engineered NSP. In several European countries and the U.S., this awareness has led to the development of governmental action plans pointing out the most relevant issues associated with the exposure of NSP.

The present review article has highlighted some of the questions currently under debate in NSP research. However, it has become clear that research into the biological effects of NSP will greatly benefit from interdisciplinary approaches (involving chemistry, physics, toxicology, morphology, and molecular biology) and strict guidelines, because the interpretability of the current literature is hampered by the diverse methodology or the lack of important methodological information. We are convinced that the combination of cellular responses with appropriate visualization and quantitative analysis of NSP localization will greatly promote the understanding of the effects of NSP on the lungs and other organs.

Studies addressing the acute toxicological or therapeutic potential of NSP will need to be complemented by systematic investigations using realistic subchronic or chronic in vivo exposure models to NSP. This will need to include dose-response relationships.

Finally, it should be noted that the decision whether nanoparticles are "good" or "bad" is not a black-and-white decision. Future investigations need to define rigorously which particle characteristic is responsible for a certain effect. If this can be achieved in a matter-of-fact way and based on scientific evidence rather than on political interests, the greatest benefit for society will be achieved: application of safe nanomaterials in technology and medicine but also regulatory measures to decrease the risks of ambient UFP and harmful NP.


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This work was supported by Swiss National Science Foundation Grants 326000-113159, 3100A0-116417 (to M. Ochs) and 3100A0-118420 (to B. Rothen-Rutishauser), the Swiss Agency for the Environment, the Doerenkamp-Zbinden Foundation, and the Johanna Duermueller-Bol Foundation.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the expert technical assistance of Sandra Frank, Beat Haenni, Claudia Haller, Andrea Stokes, and Barbara Tschirren. We thank Loretta Müller for carefully proofreading the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Mühlfeld, Institute of Anatomy, Univ. of Bern, Baltzerstrasse 2, CH-3000 Bern 9, Switzerland (e-mail: muehlfeld{at}ana.unibe.ch)


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Proceedings of the ATS, August 15, 2009; 6(5): 398 - 402.
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