Like two sides of the same coin, nanotechnology can be both boon and bane for respiratory medicine. Nanomaterials open new ways in diagnostics and treatment of lung diseases. Nanoparticle based drug delivery systems can help against diseases such as lung cancer, tuberculosis, and pulmonary fibrosis. Moreover, nanoparticles can be loaded with DNA and act as vectors for gene therapy in diseases like cystic fibrosis. Even lung diagnostics with computer tomography (CT) or magnetic resonance imaging (MRI) profits from new nanoparticle based contrast agents. However, the risks of nanotechnology also have to be taken into consideration as engineered nanomaterials resemble natural fine dusts and fibers, which are known to be harmful for the respiratory system in many cases. Recent studies have shown that nanoparticles in the respiratory tract can influence the immune system, can create oxidative stress and even cause genotoxicity. Another important aspect to assess the safety of nanotechnology based products is the absorption of nanoparticles. It was demonstrated that the amount of pulmonary nanoparticle uptake not only depends on physical and chemical nanoparticle characteristics but also on the health status of the organism. The huge diversity in nanotechnology could revolutionize medicine but makes safety assessment a challenging task.
Over the past years nanomaterials have found their way into more and more areas of life. Examples are new coatings and pigments, electronic devices as well as cosmetic products like sunscreens and toothpastes. On top of that, much effort is done to adopt nanotechnology for the treatment of human diseases. The term “Nano” refers to structures in the range of 1 to 100 nm. In contrast to nanoparticles, which have to measure between 1 and 100 nm in all dimensions, nanomaterials may consist of elements bigger than 100 nm but need to be structured in the nanoscale and exhibit characteristic features associated with their nanostructure . In this context, the International Organization for Standardization defined the term nano-object as a material with one, two or three external dimensions in the nanoscale  (Fig. 1). Nanomaterials have an extremely high surface area to volume ratio. Therefore, some of them are very reactive or catalytically active. Moreover, in the nanoworld quantum effects become visible and lead to some of the unique properties of nanoparticles. Like viruses and cellular structures, some nanoparticles are able to self-assemble to more complex structures . This makes them interesting candidates for novel drugs. On the other hand it is necessary to redefine toxicology because of nanotechnology. Unlike classical toxicology, where dose and composition matter, in nanotoxicology the focus has to be set on properties like morphology, size, size distribution, surface charge, and agglomeration state as well. Nanotechnology is important for respiratory medicine for several reasons. Firstly, it offers new approaches to treat diseases of the respiratory tract. However, as nanotechnology usage in consumer products, cosmetics, and medicine is continuously increasing, it is also pivotal to understand potentially adverse effects of nanomaterials on the respiratory system. Additionally, studying respiratory effects of manufactured nanomaterials helps to understand the impact of combustion exhaust and ultra-fine dusts on human health. On top of that, the lung is probably the most important gateway of nanoparticles to the human organism. For the assessment of safety in nanotechnology it is therefore also important to elucidate which nanoparticle properties determine pulmonary resorption and biodistribution (Fig. 2).
Although clinical application of nanotechnology in therapeutics and diagnostics is still rare, there are multiple promising candidates for future use in the field of respiratory medicine.
Nanoparticles can act as vessels for drugs because they are small enough to reach almost any region of the human organism. Drugs can be bound chemically to the nanoparticles by a multitude of different linker molecules or by encapsulation. This allows better control of toxicokinetics. However, the main advantage is the capability of targeted drug delivery. The targeting can be active or passive. In case of tumor diseases, the leaky and immature vasculature of fast growing tumors can be taken advantage of in order to achieve passive targeting of chemotherapeutic loaded nanoparticles. This is called the enhanced permeability and retention (EPR) effect . The first generation nano drug delivery systems rely entirely on the EPR effect. One example is Genoxol-PM, a polymeric paclitaxel loaded poly(lactic acid)-block-poly(ethylene glycol) micelle-formulation . This nanocarrier has recently been tested in a phase II trial in patients with advanced non-small cell lung cancer (NSCLC). 43 patients were treated with four 3-week cycles of Genexol-PM at 230 mg/m2 on day 1 combined with gemcitabine 1000 mg/m2 on day 1 and day 8. With a response rate of 46.5 %, the therapy showed favorable antitumor activity. Moreover, emetogenicity was low. However, frequent grade 3/4 adverse events like neutropenia and pneumonia were observed . The second generation nanoparticle drug delivery systems possess targeting ligands. These can be antibodies, aptamers, small molecules and proteins (Fig. 3). The attached ligands actively guide the nanoparticles and therefore the drugs to the tumor cells. Tumor specific monoclonal antibodies are already widely used in cancer therapy. Those antibodies can be attached to nanoparticles for active targeting. In a recent study polyglycolic acid nanoparticles, that were conjugated with cetuximab antibodies for targeting and loaded with the drug paclitaxel palmitate, were administered intravenously to mice with A549-luc-C8 lung tumors. The survival rate of these mice increased significantly compared to the control group . Another approach involves aptamers as targeting agents. Aptamers are synthetic oligonucleotides that are capable of binding specific target structures. Their small size, their simple synthesis, and their lack of immunogenicity make them promising ligands for nanoparticles. Moreover, small molecules such as folate can be used for targeting tumor cells that express a high density of folate receptors. In addition, tumors often overexpress receptors for several proteins. Proteins like transferrin therefore are common targeting ligands . These second generation nanocarriers are already used clinically against lung cancer with substances like Aurimmune Cyt-6091 and Bind-014. Aurimmune Cyt-6091 is a drug delivery system based on gold nanoparticles functionalized with polyethylene glycol (PEG) and tumor necrosis factor alpha (TNF-α). It has been used against adenocarcinoma of the lung in a phase I clinical trial. The TNF-α serves both as targeting and therapeutic agent in this case . A phase II clinical trial for non-small cell lung cancer patients has been planned . The nano drug delivery system Bind-014 is currently tested in a phase II clinical trial as second-line therapy for patients with non-small cell lung cancer . Bind-014 nanoparticles consist of a polylactic acid (PLA) core, in which the anti-tumor drug docetaxel is physically entrapped. The particles are surface-decorated with PEG to reduce elimination from the immune system and contain ligands against prostate-specific membrane antigen (PSMA) for targeting. PSMA is expressed in prostate cancer cells and in the neovasculature of nonprostate solid tumors, such as NSCLC . Preliminary data demonstrates, that Bind-014 is clinically active and well tolerated. It also showed promising effects on patients with KRAS mutations, where ordinary anti-tumor agents usually fail. Additionally, adverse effects like anemia, neutropenia and neuropathy were significantly reduced compared to solvent based docetaxel .
Nanoparticle based drug delivery also offers potential in other fields of respiratory medicine. In experiments with tuberculosis infected guinea pigs, it was demonstrated that inhaled alginate nanoparticles encapsulating isoniazid, rifampicin, and pyrazinamide showed better bioavailability and higher efficiency than oral drug medication . Similar results were presented by Pandey et al. with the three antitubercular drugs encapsulated in poly (DL-lactide-co-glycolide) nanoparticles. Moreover, another study demonstrated that pirfenidone loaded nanoparticles have higher anti-fibrotic efficacy in the treatment of mice with bleomycin-induced pulmonary fibrosis than dissolved pirfenidone .
Nanoparticle induced hyperthermia can be used to locally destroy tumor cells. Heat generation is usually achieved by two approaches, magnetic and photothermal hyperthermia. In magnetic hyperthermia, an extracorporeal coil creates an alternating magnetic field that heats magnetic nanoparticles inside a tumor. This increases the temperature in the tumor without affecting healthy tissue. A recent study assessed the effect of inhalable superparamagnetic iron oxide nanoparticles in a mouse model of NSCLC. Compared to the non-targeted nanoparticles, the epidermal growth factor receptor (EGFR) targeted nanoparticles showed significantly more effective tumor shrinkage after magnetic hyperthermia treatment . The other approach, photothermal therapy uses laser radiation in the visible or near infrared spectrum and photosensitizing nanoparticles such as gold or graphene. A commercial product called auroshell is available for tumor therapy. Auroshell nanoparticles consist of a silica core surrounded by a thin layer of gold. The gold nanoshells are administered intravenously and accumulate in the tumor due to the EPR effect. Upon exposure of the tumor to a near infrared laser, the laser energy is efficiently converted to heat by the gold nanoshells . This therapy, which is called AuroLase, is currently undergoing clinical trial in patients with primary and/or metastatic lung tumors  (Fig. 4).
Like viruses, nanoparticles can be used as vectors for genes. But in contrast to viruses, they are less immunogenic and have higher DNA transport capacity. In a study, DNA loaded polyethylenimine nanoparticles were used in order to treat lipopolysaccharide induced acute lung injury in mice. After intravenous injection of the nanoparticles, the beta2-Adrenic Receptor genes in the nanoparticles led to a short lived transgene expression in alveolar epithelia cells. As a result the 5-day survival rate improved from 28 % to 64 %. The severity of the symptoms measured by alveolar fluid clearance, lung water content, histopathology, bronchioalveolar lavage cellularity, protein concentration, and inflammatory cytokines was also significantly attenuated . DNA loaded nanoparticles are also promising candidates in the treatment of cystic fibrosis. It was shown in a clinical trial that nasal application of DNA nanoparticles is safe and evidently leads to vector gene transfer . One major problem in this context is to overcome the mucus barrier. In a recent study, it was demonstrated that densely PEG-coated DNA nanoparticles can rapidly penetrate extracorporeal human cystic fibrosis and extracorporeal mouse airway mucus. In addition, those particles exhibited better gene transfer after intranasal administration to mice than conventional carriers .
Nanoparticles have the potential to improve pulmonary x-ray diagnostics. Folic acid-modified dendrimer-entrapped gold nanoparticles were utilized as imaging probes for targeted CT imaging. In in-vitro and in-vivo tests, the nanoparticles were trapped in the lysosomes of folic acid receptor expressing lung adenocarcinoma cells (SPC-A1). It was possible to detect the tumor cells by micro-CT imaging after nanoparticle uptake. In addition, it was also shown that the particles possess good biocompatibility, with no impact on cell morphology, viability, cell cycle, and apoptosis . Nanoparticles can also be used to enhance MR diagnostics of lung tissue. In experiments with intratracheal administration of Gadolinium-DOTA nanoparticles in mice, signal enhancements in several organs including the lung were measured with ultrashort-echo-time-proton-MRI. The signal change over time in the different organs demonstrated the passage of the nanoparticles from the lung to the blood, then to the kidneys, and finally to the bladder .
Toxic effects of nanoparticles are a major concern in pulmonary medicine. Especially ultrafine particles of low soluble, low toxic materials like titanium dioxide, carbon black, and polystyrene are overall more toxic and inflammatory than fine particles of the same material. This applies to both synthesized nanoparticles and natural dusts . For nano related toxicity multiple mechanisms seem to be important. In the following, the interaction with the immune system, the creation of oxidative stress, and toxic effects on the genome are taken a closer look at. In order to correlate toxic effects with nanoparticle properties, it is necessary to thoroughly characterize the selected nanoparticles prior to administration.
The most commonly used methods to characterize nanoparticles for toxicology studies are transmission electron microscopy (TEM) for size, morphology, and agglomeration, dynamic light scattering (DLS) for the size distribution of the particles, zeta potential measurement for nanoparticle surface charge, and x-ray diffraction (XRD) for the particles’ crystal structure. In some cases such as gold and silver nanoparticles, UV-vis spectroscopy can be used to determine size and size distribution due to a special size dependent optical activity . Ideally, nanoparticle characterization is repeated after administration as changes of the nanoparticles during the application process are possible. In in-vitro experiments, nanoparticles are usually applied by mixing with cell culture medium. The dissolved components of the medium, especially the ions, lead to agglomeration and precipitation of many nanoparticles, causing significant changes in their physicochemical properties. Similar effects are to be expected when nanoparticles come into contact with surfactant or other biological fluids. It was shown, that some nanoparticles tend to form protein coronae in biological systems .
Many nanoparticles possess properties that give them the potential to influence the immune system. In this context, nanoparticles’ ability to penetrate cellular boundaries, to escape phagocytation by macrophages, to act as haptens, and even to disturb the Th1/Th2 balance might be essential . For carbon black nanoparticles, a recent study investigated the effects of inhalative exposure on mice with bleomycin-induced pulmonary fibrosis. The analysis of histology as well as cytokine expression suggested that the nanoparticles triggered an inhalation exacerbated lung inflammation. The author concluded that especially for people with pulmonary preconditions inhalation of nanoparticles can lead to serious health problems . In this context, another study found out that PEGylated cationic shell-cross-linked knedel-like (cSCK) nanoparticles produced significantly less airway inflammation than non-PEGylated ones. This was explained by a change in endocytosis. In contrast to the clathrin-dependent endocytosis of non-PEGylated particles, the PEGylated cSCK nanoparticles showed a clathrin-independent route . On the other hand, some nanomaterials exhibit impressive immune modulating activity. As an example, [Gd@C82(OH)22]n, a fullerene derivate with a gadolinium atom inside showed anticancer activity without being cytotoxic (Fig. 5). In vitro studies demonstrated that [Gd@C82(OH)22]n activated dendritic cells (DCs) and even induced phenotypic maturation of those cells. Moreover, the [Gd@C82(OH)22]n treated DCs also stimulated allogenic T cells in a Th1 characteristic. The effect of [Gd@C82(OH)22]n was comparable, probably even stronger than the effect of lipopolysaccharide (LPS) on DCs. The study also verified that the nanoparticles were free of LPS contamination. In-vivo experiments on ovalbumin (OVA) immunized mice showed enhanced immune responses comparable to the adjuvant effect of Alum on OVA mice. However, whereas Alum lead to a Th2 response pattern with IL-4, IL-5 and IL-10 upregulation, [Gd@C82(OH)22]n caused a Th1 pattern with upregulation of IFNγ . Similar results were demonstrated in another study using a murine asthma model. OVA sensitized mice that were additionally treated with the nanomaterial graphene oxide during allergen sensitization had stronger airway remodeling and hyperresponsiveness than mice that have only been treated with OVA. The graphene oxide lead to a downregulation of Th2 dependent markers such as IL-4, IL-5, IL-13 IgE and IgG1 but increased Th1-associated IgG2a. Moreover, the graphene oxide increased the macrophage production of mammalian chinitases, chitinase-3-like protein 1 (CHI3L1), and AMCase, which could be the reason for the overall augmentation in airway remodeling and hyperresponsiveness . However, this kind of immune modulation can also be utilized for therapeutic purposes. In a recent study a nanoparticle-based vaccine has been used to treat dust mite allergies in mice. The immune-modulating carriers were generated by loading dust mite allergen Der p2 and the potent Th1 adjuvant unmethylated cytosine-phosphate-guanine (CpG) into biodegradable poly(lactic-co-glycolic acid) (PLGA) polymer particles. Mice treated with those nanoparticles showed significantly lower airway hyperresponsiveness as well as lower IgE antibody levels after a 10 day intranasal Der p2 instillation compared to the control group. The authors conclude that this biodegradable nanoparticle-based vaccination strategy has significant potential for treating HDM allergies .
Oxidative stress is often brought in context with nanotoxicology. It can be measured directly with dichlorofluorecein or indirectly by the upregulation of reactive oxygen species (ROS) eliminating enzymes like superoxide dismutase . Another approach involves tests whether the nanoparticle dependent toxicity can be reduced by the application of an antioxidant. Widely used semiconductor materials such as lead sulfide nanoparticles may have the potential to generate oxidative stress in the lung. A recent study tested the toxicity of intratracheally applied 30 nm and 60 nm lead sulfide nanoparticles on rats. Oxidative damage was evaluated based on superoxide dismutase, total antioxidant capacity, and concentration of malondialdehyde. In addition to inflammatory responses, both 30 nm and 60 nm groups showed increased oxidative damage compared to control groups. The effect was significantly stronger for the 30 nm lead sulfide compared to the 60 nm nanoparticles . Another nanomaterial which is associated with oxidative stress is nanosized titanium dioxide. Li et al. induced pulmonary injury in mice by daily intranasal instillation of suspended 294 nm TiO2 nanoparticles for 90 days, demonstrating that the rate of reactive oxygen species (ROS) generation increased with increasing TiO2 doses. Moreover lipid, protein and DNA peroxidation products were identified in elevated doses, which suggests that ROS dependent lung damage was significant in the nanoparticle treated animals . Furthermore, in vitro tests on BEAS-2B and A549 lung cell lines demonstrated that the commonly used nanoparticles ZnO and Fe2O3 are very different in terms of creating oxidative stress. The Fe2O3nanoparticles with an average diameter of 39 nm were distributed in the cytoplasm, whereas the 63 nm ZnO nanoparticles were trapped in organelles such as the endosome. In contrast to the Fe2O3 nanoparticles the ZnO nanoparticles caused reactive oxygen species production as well as cell cycle arrest, cell apoptosis, mitochondrial dysfunction and glucose metabolism perturbation (Table 1).
Another important type of toxicity caused by nanoparticles is genotoxicity. A common method to quantify genotoxicity is the comet assay, which uses electrophoresis to detect DNA strand breaks. This assay was used in a recent study to check whether intratracheal instilled fullerene C60nanoparticles induced DNA damage in male rats. However, despite inflammatory responses and hemorrhages in the alveoli of the C60 treated rats, there was no significant increase in fractured DNA in their lung cells. Therefore, it was concluded that even at inflammation inducing doses, fullerene C60 nanoparticles have no potential for DNA damage in the lung cells of rats . Similarly, another study demonstrated that intratracheal instillation of anatase TiO2 nanoparticles on rats did not result in genotoxicity. None of the TiO2 groups showed an increase in fractured DNA while the positive control with ethyl methanesulfonate exhibited significant increases . In contrast to those results, Kyjovska ZO et al. found that even in low doses, where no inflammation occurs, Printex 90 carbon black nanoparticles induce genotoxicity in mice. There was no inflammation, cell damage and acute phase response, which means that the increased DNA strand breaks are related to direct DNA damage caused by the nanoparticles . On the other hand, a recent study suggests that CeO2 nanoparticles may be even used as antioxidant and anti-genotoxic agents in the lung. After treatment with the oxidative stress-inducing agent KBrO3, BEAS-2B cells pretreated with the CeO2 nanoparticles showed significantly less intracellular ROS as well as a reduction in DNA damage compared to non-pretreated cells .
Research on the biodistribution of nanoparticles requires tracking of the applied nanoparticles in the test animal. Conventional light microscopy is not able to detect nanoparticles because of Abbe’s law. Therefore, electron microscopic imaging is often required. However, light microscopy can be used to describe the nanoparticle induced changes in the cell morphology without being able to see the nanoparticles themselves. Additionally, nanoparticles can be indirectly made detectable in light microscopy by a method called autometallography. This is a silver staining that can be used to increase the size of several types of nanoparticles like gold, silver, and some metal sulfides and selenides in the histological section . This technique was used to detect silver nanoparticles in the olfactory bulb and lateral brain ventricles of mice that had been intranasally treated with 25 nm silver nanoparticles .
Most research about biodistribution of nanoparticles in organisms focuses on intravenous injection. However, nanoparticles were shown to be able to pass the blood air barrier of the lung. Whether or not nanoparticles can travel through the lung into the body seems to be size dependent. This was evaluated by injecting neutron activated radioactive gold nanoparticles of 1.4 nm and 18 nm intratracheally to rats. The bigger nanoparticles almost completely retained in the lung while significant amounts of the smaller 1.4 nm particles were found in blood, liver, skin and carcass 24 h after instillation . Choi H. S. et al. applied nanoparticles of different size and charge to mice. The nanoparticles were tracked in different organs through fluorescence labeling. It was demonstrated that nanoparticles rapidly translocated to the mediastinal lymph nodes if they possess a hydrodynamic diameter of 34 nm or less and a neutral or anionic surface. Bigger and positively charged nanoparticles exhibited no significant uptake  (Fig. 6). In addition to physical parameters of the applied nanoparticles the health status of the exposed organism also seems to play an important role. A recent study showed that the distribution of oropharyngeal instilled 40 nm gold nanoparticles is influenced by additional LPS treatment. The gold content of organs was measured with inductively coupled plasma mass spectroscopy. BALB/C mice that had been oropharyngeal treated with LPS 24 h prior to the nanoparticle administration exhibited less gold content in their lungs than untreated mice. In both groups gold was detected in different organs. High concentrations were found in heart and thymus in the non LPS group, while the LPS treated mice accumulated most of the gold in the spleen. The author concluded that nanoparticle uptake may depend on medical preconditions .
Over the last decade, major breakthroughs in nanotechnology have been achieved. It is only a matter of time before new nano based drugs reach respiratory medicine. Especially the fields of targeted drug delivery, gene therapy, and hyperthermia offer great potential for modern drugs. On the other hand the increased use of nanomaterials in all fields of life also bears the risk of exposure through inhalation. It is therefore essential to understand pulmonary toxicology of nanomaterials in all its facets. However, it is still very unclear why the toxic effects of nanoparticles in the respiratory tract are so inhomogeneous and not well predictable. In this context, not only local reactions of lung and airways but also nanoparticle uptake and distribution in the organism are important factors and therefore fields of current research. As only few nanoparticle compositions have been tested, it is questionable whether those results can be easily adapted to other nanoparticles. Because of the continuously increasing diversity of engineered nanoparticles, toxicology can hardly keep pace with the safety assessment of future products. Therefore, more attention should be set on this wide field of research.
A Phase 2 Study to Determine the Safety and Efficacy of BIND-014 (Docetaxel Nanoparticles for Injectable Suspension) as Second-line Therapy to Patients With Non-Small Cell Lung Cancer. [Accessed March 30, 2015]. ClinicalTrials.gov . 23-1-2015. Ref Type: Electronic Citation.
Pulmonary applications and toxicity of engineered nanoparticles.
Card JW1, Zeldin DC, Bonner JC, Nestmann ER.
Am J Physiol Lung Cell Mol Physiol. 2008 Sep;295(3):L400-11.
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.
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).
he 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.
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.
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.
A simplified depiction of potential factors that may influence the effects of engineered nanoparticles on the respiratory system.
Fig. 2. From: Pulmonary applications and toxicity of engineered nanoparticles.
Jeffrey W. Card, et al. Am J Physiol Lung Cell Mol Physiol. 2008 September;295(3):L400-L411.
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.
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 ​and3,3, 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.
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.