Because of their unique physicochemical properties, engineered nanoparticles have the potential to significantly impact respiratory research and medicine by means of improving imaging capability and drug delivery, among other applications. These same properties, however, present potential safety concerns, and there is accumulating evidence to suggest that nanoparticles may exert adverse effects on pulmonary structure and function. The respiratory system is susceptible to injury resulting from inhalation of gases, aerosols, and particles, and also from systemic delivery of drugs, chemicals, and other compounds to the lungs via direct cardiac output to the pulmonary arteries. As such, it is a prime target for the possible toxic effects of engineered nanoparticles. The purpose of this article is to provide an overview of the potential usefulness of nanoparticles and nanotechnology in respiratory research and medicine and to highlight important issues and recent data pertaining to nanoparticle-related pulmonary toxicity.
- respiratory system
the possibility of nanotechnology dramatically improving the health and quality of life of people throughout the world holds great promise. Predictions of beneficial effects of nanotechnology in numerous industrial, consumer, and medical applications have been promising. By no means an exhaustive list, these applications include those that may lead to more efficient water purification, stronger and lighter building materials, increased computing power and speed, improved generation and conservation of energy, and new tools for the diagnosis and treatment of disease. The optimistic outlook for a future improved by nanotechnology must be tempered, however, by the realization that relatively little is known about the potential adverse effects of nanomaterials on human health and the environment.
The definition of a nanoparticle is generally considered to be a particle with at least one dimension of 100 nm or less. As a result of their small size and unique physicochemical properties, the toxicological profiles of nanoparticles may differ considerably from those of larger particles composed of the same materials (15, 98). Furthermore, nanoparticles of different materials (e.g., gold, silica, titanium, carbon nanotubes, quantum dots) are not expected to interact with and affect biological systems in a similar fashion. As a result, it seems unlikely that the toxic potential and/or mechanisms of nanoparticles can be predicted or explained by any single unifying concept.
The respiratory system represents a unique target for the potential toxicity of nanoparticles due to the fact that in addition to being the portal of entry for inhaled particles, it also receives the entire cardiac output. As such, there is potential for exposure of the lungs to nanoparticles that are introduced to the body via the act of breathing and by any other exposure route that may result in systemic distribution, including dermal and gastrointestinal absorption and direct injection. Interest in the respiratory system as a target for the potential effects, both beneficial and adverse, of nanoparticles is reflected by the steady increase in the number of scientific publications on these subjects during the past decade (Fig. 1).
The purpose of this article is to complement and expand on previous reviews of the pulmonary effects of nanoparticles (11, 14, 34, 35) by providing an overview of potential applications of nanotechnology in pulmonary research and in diagnosis and treatment of disease. In addition, recent advances regarding the potential pulmonary toxicity of nanoparticles as assessed in human, experimental animal, and in vitro studies are discussed. For the purposes of this article, only intentionally engineered nanoparticles are considered; unintentionally generated (e.g., via combustion engines, grilling, welding) and naturally occurring nanoparticles (e.g., via forest fires or volcanic eruptions) are not included in this discussion.
NANOPARTICLES AND THE LUNG
There are myriad nanoparticles to which the respiratory system may be exposed.
There is the potential for the respiratory system to be exposed to a seemingly countless number of unique nanoparticles, essentially none of which has been sufficiently examined for potential toxicity at this time. A substantial number of nanoparticles are already present in the marketplace in consumer products such as sunscreens, cosmetics, and car wax, and many more are sure to follow (a comprehensive list is maintained and updated by the Project on Emerging Nanotechnologies at the Woodrow Wilson International Center for Scholars: http://www.wilsoncenter.org/nano). Although the toxicity of the majority of nanoparticles may prove to be minimal, the fact that there is any potential for adverse effects to result from exposure suggests that prudence is warranted.
Various types of nanoparticles exist including those that are carbon-based (e.g., nanotubes, nanowires, fullerenes) and metal-based (e.g., gold, silver, quantum dots, metal oxides such as titanium dioxide and zinc oxide) and those that are arguably more biological in nature (e.g., liposomes and viruses designed for gene or drug delivery). To demonstrate the complexity of the situation, it is worthwhile to consider the case of carbon nanotubes as an example. Carbon nanotubes can be: 1) produced and/or cleaned using one of several different methods; 2) produced using one of several different metal catalysts; 3) single- or multi-walled; 4) of various lengths; and 5) subjected to numerous surface modifications. The result of these permutations is that a vast number of unique carbon nanotubes can be derived, all of which fall under one broad category, namely the carbon nanotube. Dividing these into single-walled and multi-walled forms reduces the ambiguity only so much, and we are still left with potentially thousands of each type. Furthermore, as has been demonstrated in recent in vitro experiments (37), the potential for nanotube agglomeration or for adhesion of nanotubes to biological molecules and the resultant alteration of their reactivity must be considered. Needless to say, the variations in nanoparticle form and functionality, not only for carbon nanotubes but also for nanoparticles in general, present significant challenges in the assessment of their potential usefulness and toxicity.
Nanoparticle accumulation within the lung.
Nanoparticles may reach the lung via inhalation or systemic delivery and do so by incidental/accidental or intentional means. Intentional pulmonary administration is being examined as a means of nanoparticle delivery for imaging and therapeutic purposes and is discussed separately below. Incidental or accidental inhalation exposure to nanoparticles can be envisioned most likely to occur as a result of exposure to occupational aerosols during the production or packaging of nanoparticles or nanostructured materials (89). In addition to pulmonary effects resulting from such exposures, translocation and subsequent systemic exposure and accumulation are also possible and are being investigated. It should be noted that nanoparticles naturally tend to agglomerate into larger particles that can be microns in size, thereby reducing the likelihood of free nanoparticles being respired. However, surface modifications designed to limit particle-particle interactions and protein binding may reduce the tendency for nanoparticle agglomeration and increase the potential for inhalation and deposition within the lungs (131).
Incidental pulmonary exposure as a result of systemic delivery is likely inherent for any nanoparticle that is injected or that might be absorbed following dermal application or ingestion. Although no published human data pertaining to pulmonary accumulation of nanoparticles following systemic exposure were identified, several animal studies have demonstrated pulmonary accumulation of nanoparticles (or of drug-conjugated nanoparticles) by means of determining their quantity in total lung homogenate preparations following their ingestion or intravenous or subcutaneous injection (43, 71, 109, 138, 142, 155). None of these studies investigated whether systemically administered nanoparticles traversed the blood-air barrier to gain access to the interstitium or lung epithelium; however, this is not necessarily a requirement for beneficial (or detrimental) effects to ensue. Although the levels and duration of accumulation appear to vary for the different nanoparticles examined, these data highlight the potential for exposure of the lungs to nanoparticles via the systemic route.
PULMONARY APPLICATIONS OF NANOPARTICLES
Imaging and diagnostic applications.
Many improvements in imaging capabilities that will benefit basic and clinical pulmonary research and disease diagnosis can be envisioned through the application of nanotechnology. Advances that include the delivery of nanoparticle imaging agents to specific cells or tissues of interest, the development of nanoprobes for molecular imaging of disease pathways, and the development of better contrast agents are forthcoming (21, 22, 115). Quantum dots are one type of nanoparticle that is proving to be particularly useful for imaging and diagnostic purposes. These semiconductor nanocrystals have broad absorption spectra and narrow emission spectra, and as their fluorescence is dependent on their chemical composition and size, multiple quantum dots (each with a unique color emission) can be detected simultaneously. Moreover, their relatively large surface area provides the opportunity for attachment of peptides or antibodies that precisely target cell types or tissues for imaging, thereby increasing specificity and decreasing background. In this regard, Akerman et al. (2) demonstrated that quantum dots coated with a peptide that binds to membrane dipeptidase on pulmonary endothelial cells were detected in the lung but not in brain or kidney 5 min after intravenous administration in BALB/c mice. Furthermore, in a study using quantum dots conjugated to monoclonal antibodies, rapid and specific detection of respiratory syncytial virus infection was demonstrated in vitro and in the lungs of BALB/c mice in vivo (137). Quantum dots have also been used to study tumor cell extravasation into lung tissue in C57BL/6 mice (140), highlighting the utility of these nanoparticles in the study of tumor metastasis.
Other nanoprobes for pulmonary imaging and diagnostics are also being examined experimentally. A recent study by le Masne de Chermont et al. (78) demonstrated that inorganic luminescent nanoparticles can be optically excited before injection into mice to provide long-lasting imaging of the lung. This was particularly evident for the positively charged nanoparticles that were studied, as noninvasive external detection revealed significant pulmonary accumulation of these nanoparticles up to 1 h following intravenous injection (78).
The potential therapeutic applications of nanoparticles in respiratory and systemic diseases are numerous (20, 21, 112, 115, 133). A considerable thrust of recent research has been focused on determining the suitability of nanoparticles of various types to serve as vectors for the pulmonary delivery of drugs or genes via inhalation or systemic administration, whereas other efforts have been directed toward developing and delivering nano-sized drug particles to the lung (Table 1). The majority of the studies reported to date have focused on the utility of these strategies for the treatment of pulmonary infection. As an example, gene transfer using intranasal administration of chitosan-DNA nanospheres was shown to prophylactically inhibit respiratory syncytial virus infection and to reduce allergic airway inflammation in mice when given prophylactically or therapeutically (74, 75). Moreover, nanoparticle-mediated intranasal delivery of short interfering RNA (siRNA) targeted against a specific viral gene, NS1, has also been shown to inhibit respiratory syncytial virus infection in mice and rats (72, 161).
The usefulness of nano-sized drug particles as treatment modalities in models of pulmonary infection has also been investigated. Inhalation of aerosolized nano-sized itraconazole resulted in significantly higher lung concentrations in mice than did oral administration (138) and was found to prophylactically inhibit invasive pulmonary aspergillosis and reduce infection-related deaths in mice, whereas oral drug administration did not (4, 59). In addition, Pandey et al. (110) demonstrated that a single inhalation of aerosolized poly (DL-lactide-co-glycolide) nanoparticles loaded with antitubercular drugs (isoniazid, rifampicin, or pyrazinamide) resulted in therapeutic plasma drug levels for up to 6 days in guinea pigs and found that repeated inhalations were as effective as more frequent oral administrations of free drug in treating experimental tuberculosis. A subsequent study revealed that a single subcutaneous injection of these antitubercular drug-containing nanoparticles in mice resulted in therapeutic plasma drug levels for up to 32 days and was more effective at reducing bacterial counts in the lungs and spleen than was daily oral administration of free drug (109). Finally, Zahoor et al. (158) reported that the same antitubercular drugs were more effective than free oral drugs when they were encapsulated in alginate nanoparticles and administered via inhalation to guinea pigs.
Other studies relevant to the potential utility of nano-sized drugs in disease treatment have examined siRNA-mediated suppression of target mRNA levels following intranasal administration of chitosan-based nanoparticles in mice (61) and the pharmacokinetics of lipid-coated nanoparticles of 5-fluorouracil in hamsters (58). Moreover, allergic airway inflammation in mice has been shown to be reduced by intravenous administration of polymer nanoparticles coated with a P-selectin inhibitor (67) and by intranasal administration of chitosan nanoparticles carrying theophylline (79). Importantly, Dames et al. (30) recently reported on the ability to externally direct inhaled magnetically charged iron oxide nanoparticles to specific areas of the lungs of mice without adversely affecting respiratory mechanics, demonstrating for the first time that targeted aerosol delivery to the lungs is achievable. Such an approach could prove to be beneficial in the treatment of localized lung infections or tumors.
Although the majority of the toxicity studies that are discussed below focused on nonbiodegradable nanoparticles such as metals and carbon nanotubes, nanoparticles designed for clinical pulmonary drug delivery will likely be biodegradable (133). In this regard, Dailey et al. (29) reported that intratracheal administration of biodegradable polymeric nanoparticles to BALB/c mice did not induce pulmonary inflammation (measured as bronchoalveolar lavage fluid neutrophil influx, protein content, and lactate dehydrogenase activity), whereas nonbiodegradable polystyrene nanoparticles did. In addition to the treatment of lung diseases, the inhalation route is being explored for the systemic delivery of drugs to treat a variety of nonpulmonary ailments. This is due in part to the large surface area of the lungs and the relatively high bioavailability of many small molecules when administered by this route (113). As discussed below, human studies have not demonstrated systemic translocation of nanoparticles following inhalation, although some animal studies suggest that it is possible. Indeed, experimental animal data demonstrating achievement of therapeutic plasma drug levels following inhalation of nanoparticle-encapsulated antitubercular drugs (109, 110, 158) indicate that this approach may be feasible. Efforts to develop safe and effective nanoparticles for aerosol delivery are ongoing (33, 41, 52, 53, 124, 130) and will undoubtedly lead to significant advances in the treatment of respiratory and systemic diseases.
PULMONARY TOXICITY OF NANOPARTICLES
As a number of physicochemical factors can influence the potential biological interactions and toxicity of nanoparticles, it is important to consider the extent to which the physicochemical properties of nanoparticles have been characterized in any given study. Without sufficient characterization, it is extremely challenging to interpret the results of individual studies and virtually impossible to compare the results of different studies, even in cases where the same nanoparticle has been investigated. As a result, the ability to identify parameters that might influence toxicity is hampered. Although there is not yet a universally accepted standard set of parameters that is deemed necessary for nanoparticle characterization, recent reports have highlighted several key physicochemical elements for which it is strongly recommended that data be reported (103, 143). These include method of synthesis, size, size distribution, shape, composition, crystal structure, aggregation and agglomeration status, dissolution, purity, surface area, and other surface characteristics. Characterization of nanoparticles in the context of the experimental exposure media (cell culture media, dosing solution, aerosol, etc.) is also of considerable importance as some physicochemical parameters are likely to differ depending on whether they are determined in the experimental media or in the bulk (i.e., “as received”) state. Unfortunately, the inclusion of all these parameters in publications describing nanoparticle toxicity studies appears to be rare. However, using these parameters as a checklist and applying the guidance outlined in recent opinions on the matter (103, 143), the in vivo animal studies and in vitro cell culture studies summarized in Tables 2–4 have been categorized according to our assessment of whether or not they contain “sufficient” or “insufficient” particle characterization data. The discussions of these studies in the sections that follow focus primarily on those considered to contain sufficient information pertaining to nanoparticle characterization, although studies that contain insufficient characterization data but nonetheless highlight important concepts are also discussed.
Studies in humans.
As summarized elsewhere (7, 107), inhaled particles of different sizes exhibit different fractional depositions within the human respiratory tract. Although inhaled ultrafine particles (<100 nm) deposit in all regions, tracheobronchial deposition is highest for particles <10 nm in size, whereas alveolar deposition is highest for particles approximately 10–20 nm in size (7, 107). Particles <20 nm in size also efficiently deposit in the nasopharyngeal-laryngeal region. Human studies of potential adverse pulmonary effects resulting from exposure to engineered nanoparticles appear to be limited, although a number of investigations into pulmonary deposition patterns of inhaled nanoparticles in the healthy and diseased lung have been conducted (5, 24, 28, 93). Computational models predict increased deposition of inhaled nanoparticles in diseased or constricted airways (44), and, consistent with this prediction, obstructive lung disease and asthma have both been demonstrated to increase their pulmonary retention (5, 24). Nonetheless, Pietropaoli et al. (114) did not observe differences between healthy and asthmatic subjects in respiratory parameters assessed up to 45 h after a 2-h inhalation of ultrafine carbon particles (up to 25 μg/m2), nor was airway inflammation observed in either group (measured as exhaled nitric oxide). Moreover, the same study reported that exposure of healthy subjects to a higher concentration of ultrafine carbon particles (50 μg/m2 for 2 h) resulted in decreased midexpiratory flow rate and carbon monoxide diffusing capacity 21 h after exposure, albeit still in the absence of airway inflammation (114). Thus nanoparticles may influence respiratory function and gas exchange without a concomitant induction of inflammation.
Several studies have also examined the potential for inhaled manufactured ultrafine particles (i.e., 99mtechnetium-labeled carbon nanoparticles) to translocate from the lungs to the systemic circulation in humans. This is an important issue to consider as inhaled engineered nanoparticles may exert adverse cardiovascular effects, similar to the proposed mechanism for the nanoparticulate fraction of urban air pollution (15, 40). All but one of the studies reported to date indicate that inhaled 99mtechnetium-labeled carbon nanoparticles are not detected outside of the lungs in appreciable quantities after inhalation (17, 91, 93, 100, 150, 151). However, as alluded to by Mills et al. (91), these findings do not indicate that other nanoparticles will behave in the same manner, nor do they rule out the possibility that nanoparticles may interact with and influence the vasculature. Moreover, the studies conducted to date have used a single inhalation exposure protocol, and it is possible that repeated exposures may result in greater pulmonary accumulation and translocation of significant quantities of nanoparticles to the circulation.
Studies in experimental animals.
Pulmonary effects resulting from airway administration of nanoparticles have been examined in a number of experimental animal studies, a summary of which is presented in Table 2. Although the primary outcomes of interest in the majority of these studies have been pulmonary inflammation and fibrosis, several have investigated distribution patterns within the lung and the potential translocation and systemic distribution of nanoparticles following pulmonary administration; these are summarized in Table 3. In addition to the endpoints listed in Tables 2 and 3, carcinogenic effects of inhaled nanoparticles (ultrafine particles) have, in some instances, been found to be more severe than those of larger size analogs. This is thought to result primarily from lung particle overload due to the inability of alveolar macrophages to recognize and/or clear particles of this size, leading to particle build up, chronic inflammation, fibrosis, and tumorigenesis. These effects are discussed in detail elsewhere (14, 101) and will not be covered here.
Although it is recognized that the pathogenic mechanisms underlying animal models of lung fibrosis and human lung fibrosis are not necessarily the same, increased collagen deposition and structural changes to the lungs that can result in altered respiratory mechanics are common features of both. As such, for the purpose of this review, fibrosis in animal models is considered to be defined by increased collagen content and/or histopathological evidence of structural alterations to the lung that are consistent with fibrosis. With regard to inflammation and fibrosis, studies have focused primarily on the effects of carbon nanotubes, carbon black, fullerenes, silica, and metal-based nanoparticles including titanium dioxide, silver, and nickel. The reader is referred to recent reviews for more detailed descriptions of some of the available data for carbon nanotubes (34, 56, 76). In brief, single- and multi-walled carbon nanotubes have been shown to induce inflammatory and fibrotic responses in mice and rats following administration by intratracheal instillation or oropharyngeal aspiration. However, differences in the type and extent of injury induced by the two forms of nanotubes have been observed, and some reports indicate a lack of inflammation due to carbon nanotube instillation or inhalation (88, 92). Of note, when administered at equal doses (on a milligrams per kilogram body weight basis) to mice by intratracheal instillation, one type of single-walled carbon nanotube was found to be more toxic than silica quartz (77), which itself poses a serious occupational health hazard in chronic inhalation exposure scenarios. However, the authors of this study were unable to rule out the possibility of nickel contamination in the nanotube suspensions as contributing to the observed toxicity (77).
Although not the most physiologically relevant exposure routes, instillation and aspiration have dominated pulmonary toxicity studies of carbon nanotubes, likely due in part to their relative ease and cost efficiency compared with inhalation exposure protocols. However, concerns including the potential formation of nanotube aggregates and how responses to these aggregates might differ from responses to more dispersed species necessitate caution in interpreting the available data. Indeed, Mercer et al. (90) recently reported that the deposition pattern and inflammatory/fibrotic response to aspirated single-walled carbon nanotubes in mice depended on whether the nanotubes were dispersed before instillation. Specifically, dispersion was found to result in incorporation of the single-walled carbon nanotubes into the alveolar interstitium and an increase in collagen accumulation following aspiration administration (90), whereas the same authors had previously demonstrated that nondispersed single-walled carbon nanotubes caused a primarily granulomatous response associated with deposition of nanotube agglomerates (127). To date, very few inhalation exposure studies with carbon nanotubes have been reported (80, 92). No biochemical analyses were performed in the study by Li et al. (80), but the histopathological responses of the lung to instilled multi-walled carbon nanotubes (0.05 mg/mouse) were compared in a nonquantitative manner to those following inhalation of aerosols of multi-walled carbon nanotubes for up to 15 days over the course of a 24-day period (resulting in estimated lung burdens of 0.07, 0.14, or 0.21 mg/mouse). The authors observed considerable differences in nanotube deposition pattern and bronchial and alveolar responses between the two administration methods. In particular, aggregated clumps of nanotubes were evident following instillation and were associated with areas of bronchial and alveolar inflammation and destruction of alveoli, whereas the aerosol inhalation exposure resulted in much less severe inflammation and a general thickening of alveolar walls. Similarly, Mitchell and colleagues (92) did not observe an inflammatory response in the lungs of mice following 7 or 14 days of inhalation exposure to multi-walled carbon nanotubes, nor did they observe any evidence of fibrosis. Two important differences between exposure via aerosol inhalation and exposure via instillation or aspiration include the dose rate and dose distribution. Both of these factors are likely to significantly impact the nanoparticle agglomerate state in the lungs following exposure, the responses of the lung cells to the deposited material, and the rate of translocation (if it occurs at all). Thus the observed dissimilar findings in toxicity outcomes reported to date highlight the importance of considering the method of administration when assessing lung inflammatory and fibrotic responses to carbon nanotubes and likely to other nanoparticles as well. This is particularly relevant given that human exposures are likely to occur via inhalation of nanoparticle-containing aerosols and not by bolus instillation or aspiration of nanoparticle-containing solutions.
Carbon black, fullerenes, silica, and metal-based nanoparticles have also been studied for their ability to induce inflammatory and fibrotic responses in the lungs of experimental animals following delivery via instillation, aspiration, and/or inhalation (Table 2). Increased lung inflammation resulting from exposure to nano-sized particles compared with that resulting from an equivalent mass of micron-sized particles has been demonstrated in some studies (16, 45, 49, 54, 82, 118, 160), whereas others have found this not to be the case (9, 120, 135, 149). Potential factors in the increased inflammatory profile observed for nanoscale materials in some studies include their size, increased number, and higher surface area per unit mass compared with that of larger particles of the same material (15, 98, 104). Titanium dioxide is a good example of how both the size and form of a nanoparticle can influence its pulmonary toxicity, as a nanoscale anatase form of titanium dioxide was found to induce greater lung inflammatory responses than those resulting from a nanoscale rutile form and from a micron-sized anatase form following intratracheal administration in rats (148). The increased ratio of surface area to mass for nanoparticles means that a greater percentage of the atoms or molecules of a given particle are present on the surface of the particle, thereby providing an increased number of potential reactive groups at the particle surface that may influence toxicity. Although this appears to be a useful metric for assessing the toxic potential of some nanoparticles, there is consensus among experts in the field that no single dose metric (i.e., particle number, size, surface area, or other) has emerged to be useful for assessment of the reactivity and potential toxicity of nanoparticles in general (89, 144). Rather, it is likely that the most appropriate means of expressing dose-related toxicity for nanoparticles of interest will continue to be determined on an individual basis.
Although the majority of studies have examined the pulmonary effects of nanoparticles administered alone, some have incorporated nanoparticle exposure as a potential modifying factor in established models of airway inflammation (Table 2). Carbon black nanoparticles of 14-nm size, but not of 56-nm size, were found to exacerbate endotoxin-induced airway inflammation in mice when given concomitantly by intratracheal administration (62). Similarly, both 14- and 56-nm carbon black nanoparticles promoted allergen-induced inflammation in mice that was associated with increased levels of eotaxin, TARC, MIP-1α, GM-CSF, and IL-2, -5, -6, -10, and -13 protein in lung tissue (63, 64), although the exacerbating effect was more prominent for the smaller sized particles. Enhancement of allergic airway inflammation in mice by the intranasal administration of carbon black nanoparticles at the time of allergen sensitization (32) or by inhalation 24 h before allergen challenge (3) has also been reported. Finally, Shvedova and coworkers (126) recently demonstrated that intratracheal administration of single-walled carbon nanotubes to mice increased the inflammatory response to a subsequent infection with Listeria monocytogenes and that this effect was associated with decreased bacterial clearance and increased airway levels of several acute phase cytokines and chemokines. Although only two of the six studies described above contained sufficient nanoparticle characterization data (3, 126), the cumulative data from all six studies suggest a detrimental effect of nanoparticle exposure on airway inflammation induced by other agents.
As discussed earlier, studies in humans generally indicate that inhaled manufactured ultrafine particles (i.e., 99mtechnetium-labeled carbon nanoparticles) do not translocate from the lungs to the systemic circulation (17, 91, 93, 150, 151). A number of studies in experimental animals have also addressed the possibility of nanoparticle translocation from the lungs to the circulation and extrapulmonary tissues (Table 3), some of which indicate that certain nanoparticles may have the capacity to do so. For example, Semmler et al. (123) observed detectable albeit very low levels of insoluble iridium nanoparticles in the liver, spleen, brain, and kidney of rats following a single inhalation exposure, whereas Ji et al. (65) reported that the content of silver in the liver of male rats increased in a concentration-dependent manner following inhalation of silver nanoparticles for 5 days/wk for 4 wk. Detection of nanoparticles in the liver following pulmonary delivery may not reflect a direct translocation from the lungs into the bloodstream, however, as the possibility of mucociliary transport and subsequent swallowing of the nanoparticles, leading to gastrointestinal absorption and detectable liver accumulation, must be considered. Also of significant interest is the potential translocation of inhaled nanoparticles to the brain via the olfactory nerve, as has been demonstrated in rats following exposure to inhaled nanoparticles of elemental carbon (36 nm), manganese oxide (30 nm), and silver (12–15 nm) (39, 65, 105). Whether such translocation to the brain has neurological or other consequences remains to be determined.
There are a number of important factors that must be considered when assessing data purporting to support direct translocation of a nanoparticle from the lungs to the systemic circulation and secondary organs. These include solubility, potential leaching of a radiolabel, inflammation or injury induced by the nanoparticle, and others. Results of rat studies with inhaled insoluble iridium and gold nanoparticles and soluble cadmium oxide nanoparticles offer some insight into some of these factors. Data from studies of inhaled iridium and gold nanoparticles, in which pulmonary inflammation was not induced, indicate that translocation of these insoluble particles from the lower respiratory tract to secondary organs accounts for only a minute fraction of the administered dose (73, 123, 134); in the case of iridium, the vast majority was found to be cleared via the thoracic airways to the larynx, gastrointestinal tract, and eventually the feces (73, 123). For soluble cadmium oxide, lung injury was reported to be the underlying cause of translocation and accumulation in the liver following inhalation as this phenomenon only occurred at a high dose at which injury was induced and not at a low dose in the absence of injury (135). Thus translocation to the systemic circulation can be low even for a relatively soluble nanoparticle, indicating that other mechanisms such as the affinity of binding to cell membranes or proteins may be important. These and other studies reinforce the necessity to consider a variety of factors that can influence the deposition, retention, clearance, and translocation of nanoparticles within and from the lung.
Studies in cell culture.
Studies performed to assess the in vitro toxicity of nanoparticles can be used as part of a screening strategy to identify potentially hazardous substances and to elucidate underlying mechanisms of toxicities observed in vivo. A considerable amount of information from in vitro (i.e., cell culture) analyses of the pulmonary toxicity of nanoparticles has been published (Table 4). Related to the in vivo reports of inflammation and fibrosis induced by carbon nanotubes discussed earlier, in vitro assessments indicate that nanotubes may have the ability to induce oxidative stress and cytotoxicity in pulmonary cells of animal and human origin, although it should be noted that not all studies are in agreement on this matter. Indeed, metal contaminants in nanotube preparations have been found to be causative of some of the adverse outcomes observed in certain in vitro investigations (68, 116), whereas anti-inflammatory properties of nanotubes have been reported in others (37). In addition, an important consideration in studies of nanotube cytotoxicity is the means by which cell injury or death is quantified, as nanotubes have been found to interact with some commonly used dye-based assays and to lead to false-positive indications of cytotoxicity (31, 153). Nonetheless, the cumulative data suggest that carbon nanotubes may have the capacity to induce oxidative stress and cytotoxicity in lung cells.
Studies have also been performed to assess the potential toxic effects of nano-sized metals and metal oxides in a variety of lung cell types in vitro (Table 4). In general, these materials also appear to have the capacity to induce oxidative stress and cytotoxicity, although, not surprisingly, there are apparent differences in the extent of these effects among the different nanoparticles studied. Furthermore, the concentrations required to induce such effects are often relatively high (i.e., in the micrograms per milliliter range) wherein potential effects of agglomeration should be, but are not always, considered. As with any in vitro toxicity study, the range of concentrations tested in nanoparticle toxicity studies should be broad so as to allow for assessment of potentially different responses (and underlying mechanisms) at the low and high ends of the concentration-response curve. Moreover, similar to the interference of cytotoxicity assays by carbon nanotubes, nano-sized metals and metal oxides may interfere with cytokine assays commonly used to assess oxidative stress and inflammation (139), thus necessitating caution in interpreting data derived using such assays. Titanium dioxide is a commercially important nanoparticle that has been shown to induce oxidative stress in, among others, A549 and BEAS-2B lung cell lines (51, 84, 94). Of note, the form of nanoscale titanium dioxide appears to be an important determinant of its cytotoxicity, as the anatase form has been found to be more potent than the rutile form in murine (RAW 267.9) and human (A549) lung cell lines (121, 129). These in vitro data documenting differences in cytotoxicity due to different forms of nanoscale titanium dioxide are generally consistent with findings in recent in vivo pulmonary toxicity studies in rats (148).
Overall impressions from toxicity data.
Several important points related to the potential pulmonary toxicity of engineered nanoparticles are evident in the preceding discussion of human, experimental animal, and cell culture studies. First, as there are currently no published human toxicity data, the potential adverse pulmonary effects of engineered nanoparticles in humans remain unknown. Although it is reasonable to assume that nanoparticles such as carbon nanotubes and certain metal oxides may be deleterious to the respiratory system (based on their asbestos-like shape and the recognized toxicities of their larger sized counterparts, respectively), the lack of reliable predictive data indicates that established workplace safety measures and good judgment are the best measures of protection for those most likely to encounter high level exposures. Second, and related to the first point, appropriate nanoparticle characterization is key to generating meaningful data. Without a thorough understanding of the physicochemical properties of the nanoparticle under investigation, particularly in the relevant exposure media, the results of any toxicity study are descriptive at best and provide no sound basis for comparison to other studies or for predicting potential toxicities in other cellular or animal studies or in humans. Although less than half of the studies summarized in Tables 2–4 provide what we feel are sufficient characterization data, a trend toward improved nanoparticle characterization is apparent in the more recent literature and will hopefully continue as existing testing methods are improved, new testing methods are validated, and all become more readily available. Third, even with thorough nanoparticle characterization, in vitro toxicities observed in cell culture are not always predictive of in vivo pulmonary toxicities. Good examples that support this concept are two recent studies by Sayes and colleagues (119, 120) that reveal very little correlation between in vitro and in vivo pulmonary toxicities of nanoscale zinc oxide and fullerenes. These and other studies reinforce the fact that additional time and effort will need to be invested before reliable in vitro approaches to predict in vivo toxicities of nanoparticles are established. Finally, based on the preceding three points, it is suggested that attempts to extrapolate the doses and concentrations examined in animal models and cell culture experiments to corresponding potential human exposure levels and toxicities are generally ineffectual at the present time. Although it is obviously an important goal of nanoparticle toxicity testing, the uncertainties and inter-study inconsistencies that currently plague nanoparticle characterization, dose metrics, and endpoint assessments make predictions of effects in humans extremely challenging.
The studies summarized in this section highlight the extensive research that is being undertaken to understand the potential pulmonary toxicity of engineered nanoparticles, all with the aim to identify adverse pulmonary effects that might occur in humans and/or to reveal the underlying mechanisms of such effects. Although significant progress has been made, there is clearly much that remains to be done. The continued evolution of appropriate testing strategies and the cooperation and exchange of information among various interested parties (academia, industry, regulatory agencies, nongovernmental organizations, etc.) will be crucial to realizing successful outcomes in these important areas of research.
This overview was undertaken to summarize the potential beneficial and detrimental effects of engineered nanoparticles in the lung. As depicted in Fig. 2, a number of factors can influence the effects of nanoparticles in the lung, most of which have been examined to varying degrees in the studies presented in this article. There are currently no established guidelines for determining the potential toxicity of engineered nanomaterials in the lung or any other organ. As such, it is not surprising that data from studies using a variety of methods, cell types, animal models, and endpoints have been reported in the literature, nor is it surprising that some studies are clearly more scientifically sound than others. We chose to present representative studies from the cumulative body of literature pertaining to nanoparticle effects in the lung to highlight the various avenues of research that are being explored in this rapidly expanding field. However, in discussing toxicity data, we attempted to maintain the focus on data that have been generated in studies that provided sufficient detail on nanoparticle characterization, due to the fact that such characterization is a crucial element in the assessment of potential adverse effects of nanoparticles.
Improvements in the diagnosis and treatment of respiratory diseases as a result of the application of nanotechnology are anticipated, and experimental evidence indicates that engineered nanoparticles have unique properties that may render them beneficial in visualizing disease processes earlier and in delivering therapeutics to the lung, possibly even to specific areas within the lung. Using the lungs as a portal of entry for nanoparticles in the treatment of systemic diseases is also being explored and holds tremendous promise. However, nanotechnology is not without its limitations, and of foremost concern is the current lack of knowledge regarding the potential toxicity of engineered nanoparticles. As has been summarized here, a considerable amount of data from in vitro and in vivo studies indicates that nanoparticles have the capacity to exert adverse pulmonary effects, although not all nanoparticles are equivalent in this regard. In addition, in vitro toxicities are not always predictive of in vivo effects or potencies and vice versa, underscoring the need for the continued development and refinement of a suitable testing strategy for assessing the pulmonary effects of nanoparticles. It is anticipated that continued investigation into the mechanisms underlying the adverse in vitro and in vivo effects summarized in this review and their relevance to human lung physiology and disease will lead to a better understanding of the potential hazards associated with nanoparticle exposure and to the development of safe and effective respiratory medical applications and therapeutics based on nanotechnology.
D. C. Zeldin is supported by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Environmental Health Sciences. J. C. Bonner is supported by the College of Agricultural and Life Sciences at North Carolina State University and by NIH Grant R21 ES015801-01.
We are grateful to Drs. Michael Fessler and Steve Kleeberger for helpful comments during preparation of this manuscript.
- Copyright © 2008 the American Physiological Society