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Keywords:

  • biodegradation;
  • lung;
  • oxidative stress;
  • single-walled carbon nanotubes

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Role of nanotoxicology in nanomedicine
  5. Pulmonary inflammatory responses to SWCNTs In vivo
  6. SWCNT-induced pulmonary inflammation is accompanied by robust oxidative stress
  7. Redox interactions of SWCNTs with macrophages
  8. Nanoparticles and immunity
  9. Recognition and engulfment of nanotubes by macrophages
  10. Possible carcinogenic/mutagenic responses to carbonaceous nanotubes
  11. Biodegradation of SWCNT by myeloperoxidase of inflammatory cells
  12. Concluding remarks
  13. Conflict of interest statement
  14. Acknowledgements
  15. Disclaimer
  16. References

Nano-sized materials and nano-scaled processes are widely used in many industries. They are being actively introduced as diagnostic and therapeutic in biomedicine and they are found in numerous consumer products. The small size of nanoparticles, comparable with molecular machinery of cells, may affect normal physiological functions of cells and cause cytotoxicity. Their toxic potential cannot be extrapolated from studies of larger particles due to unique physicochemical properties of nanomaterials. Therefore, the use of nanomaterials may pose unknown risks to human health and the environment. This review discusses several important issues relevant to pulmonary toxicity of nanoparticles, especially single-walled carbon nanotubes (SWCNT), their direct cytotoxic effects, their ability to cause an inflammatory response, and induce oxidative stress upon pharyngeal aspiration or inhalation. Further, recognition and engulfment of nanotubes by macrophages as they relate to phagocytosis and bio-distribution of nanotubes in tissues and circulation are discussed. The immunosuppressive effects of CNT and their significance in increased sensitivity of exposed individuals to microbial infections are summarized. Finally, data on biodegradation of SWCNT by oxidative enzymes of inflammatory cells are presented in lieu of their persistence and distribution in the body.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Role of nanotoxicology in nanomedicine
  5. Pulmonary inflammatory responses to SWCNTs In vivo
  6. SWCNT-induced pulmonary inflammation is accompanied by robust oxidative stress
  7. Redox interactions of SWCNTs with macrophages
  8. Nanoparticles and immunity
  9. Recognition and engulfment of nanotubes by macrophages
  10. Possible carcinogenic/mutagenic responses to carbonaceous nanotubes
  11. Biodegradation of SWCNT by myeloperoxidase of inflammatory cells
  12. Concluding remarks
  13. Conflict of interest statement
  14. Acknowledgements
  15. Disclaimer
  16. References

‘As to diseases, make a habit of two things — to help, or at least do no harm’.

– Hippocrates, The Epidemics

In the early 50s, understanding by physicists of the novel unique properties and unusual behaviour of single atomic layer assemblies lead to revolutionary developments in nanotechnology – the field of science producing, studying and using particles that are nanometres in size for industrial applications in the medical, chemical, materials, electronics, sensing and other fields. The discoveries in physics and chemistry of nano-sized materials and developments of nanotechnologies have already revolutionized our lives in different fields through the manufacturing of new types of products from computer chips and energy-saving batteries, to composite construction materials, high effective catalysts, chemical sensors and multiple biomedical diagnostic and therapeutic tools and devices [1]. The advantages and impact of nanotechnology on biomedicine are enormous: from novel approaches to design of artificial organs and tissues for replacement therapies to nano-robotic biosensors, diagnostic devices and tiny vehicles for drug delivery.

Role of nanotoxicology in nanomedicine

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Role of nanotoxicology in nanomedicine
  5. Pulmonary inflammatory responses to SWCNTs In vivo
  6. SWCNT-induced pulmonary inflammation is accompanied by robust oxidative stress
  7. Redox interactions of SWCNTs with macrophages
  8. Nanoparticles and immunity
  9. Recognition and engulfment of nanotubes by macrophages
  10. Possible carcinogenic/mutagenic responses to carbonaceous nanotubes
  11. Biodegradation of SWCNT by myeloperoxidase of inflammatory cells
  12. Concluding remarks
  13. Conflict of interest statement
  14. Acknowledgements
  15. Disclaimer
  16. References

The Hippocratic Oath – one of the most widely known Greek medical texts – is traditionally taken by doctors swearing to ethically practice medicine. The everlasting significance of its ethical principles becomes particularly obvious at the times when new revolutionizing technologies are introduced into clinical practice. It is quite possible that the advent of nanomedicine designates such a groundbreaking moment. Currently, many different types of nanoparticles are being explored for applications in nanomedicine. They can be carbon-based skeletal-type structures, such as carbon nanotubes, carbon nano-capsules and the fullerenes, or spherical lipid-based liposomes, which are already in use for numerous applications in drug delivery and the cosmetic industry. Other examples of carbon-based nanoparticles are chitosan- and alginate- or polymers-based nanoparticles are believed to be effective in oral delivery of proteins, including regulated delivery of insulin.

There is a plethora of examples of successful applications of nanotechnologies in the field of medicine, particularly for the treatment of cancer. The concept of ‘thermal scalpel’ has been developed based on the ability of gold nanoparticles to absorb energy in the near-infrared wavelength spectrum – which is best suited for tissue penetration – to induce hyperthermia and kill tumour cells upon laser irradiation [2]. Accumulation of nanoparticles in neoplastic tissue is achieved by targeting ligands – antibodies and targeted gene therapy vectors – that are also incorporated into the thermal scalpel schema.

A variety of medicines, antioxidants and other substances are currently tested as pay-loads in different schemas of nanoparticle-mediated drug delivery, particularly for cancer therapy [3]. Whilst design of these new generations of nanoparticles-based pharmaceuticals envisions reduced toxicity and side effects of drugs, there is an underappreciated risk associated with the potential ability of carrier systems themselves to cause toxic effects and injury to the patient. It is possible that hazards introduced by carrier-nanoparticles are beyond those posed by conventional chemicals in classical delivery matrices. This may be due, at least in part, to the ability of the very small nanoparticles to more readily cross the various biological barriers within the body. Nanoparticles have become more and more prevalent in reports of novel contrast agents, especially for molecular imaging, and the detection of cellular processes. Nanotechnology has also opened ways for the development of contrast agents and radiopharmaceuticals for specific targeting, which is presently changing research strategies in the field of magnetic resonance imaging, ultrasound imaging and nuclear medicine applications [4, 5]. In the field of public health, potential benefits associated with the employment of nanotechnology are also significant. One of the examples is nanomaterial-based filtration technologies for water purification. Thus, benefits of nanotechnologies in improvements of quality of life are colossal but so are the potential risks. The unique features of nanomaterials may produce unknown effects on health and environment. Therefore, public acceptance remains equivocal and society feels that nanotechnologies should be subject to stringent regulations. It is clear, however, that the regulatory process should be based on solid research, which includes both basic studies of interactions of nanomaterials with cells and components of biofluids as well as applied toxicological assessments of nanomaterials.

Whilst numerous studies aimed at exploiting desirable properties of nanoparticles in biomedical applications have been conducted, there are limited attempts to evaluate potentially toxic and damaging effects of these particles when administered intentionally for medical purposes or after unintentional occupational or environmental exposures [6]. Fast propagation of nanotechnologies into different industries and consumer products is causing exponential growth of nanomaterials production. As a consequence, large amounts of nanomaterials may reach the natural as well as occupational environments thus representing a potential health hazard. Until recently, however, no direct indications of the toxicity of nanoparticles to humans have been reported. A calamitous reminder of possible health hazards associated with the excessive exposure to nanoparticles has been published in September of 2009 [7]. Seven young female workers (aged 18–47 years), exposed to nanoparticles for 5–13 months, all with shortness of breath and pleural effusions were admitted to a hospital in China. Surveys of the workplace confirmed the presence of polyacrylate nanoparticles in the workplace. Pathological examinations of the patients’ lung tissue revealed nonspecific pulmonary inflammation, pulmonary fibrosis and foreign-body granulomas of pleura. Nanoparticles were detected in the cytoplasm and caryoplasm of pulmonary epithelial and mesothelial cells as well as in the chest fluid by electron microscopy. These shocking clinical observations are reminiscent of experimental findings of morphological, biochemical and immunological changes detected after in vivo inhalation exposure of animals to carbonateous nanoparticles [8].

The unusual properties nanoparticles may produce unique biological activities but not enough is known regarding their potential effects on human health and environment. Given the specific smallness of nanomaterials – comparable with the nanoscale of intracellular molecular machinery and organelles – nanotoxicology can be defined as the field of biomedical science that investigates and understands mechanisms and pathways through which nanoparticles or complex nanostructures may interfere with structural and functional organization of biological systems, cause cytotoxicity and affect health [8].

Single Walled Carbon Nanotubes (SWCNT) are amongst the newly developed products and are currently of interest for a variety of applications in electronics, reinforced rods, micro-fabricating conjugated polymer activators, biosensors, enhanced electron/scanning microscopy imaging techniques, etc. As a fibrous structure resembling asbestos in physical properties and health effects, SWCNTs are currently under special attention regarding possible carcinogenicity. US Environmental Protection Agency (US EPA) generally considers SWCNTs to be chemical substances distinct from graphite/other allotropes of carbon listed on the Toxic Substances Control Act (TSCA) Inventory. Many CNTs may therefore be considered as new chemicals under TSCA section 5. On June 24, 2009, EPA issued a direct final rule promulgating significant new use rules (SNURs) for the SWCNTs and multi-walled carbon nanotubes (MWCNTs) for which EPA had received premanufacturing notices and negotiated section 5(e) consent orders [9]. The Federal Register notice states that EPA negotiated the consent orders out of a concern that both the SWCNTs and MWCNTs may cause ‘lung health effects’ and health effects from skin exposure. The potential risks to health from inhalation of SWCNT are due to their small size, as a result of which they could reach distant organs and systems normally inaccessible to larger particles. This includes possibility of crossing cell boundaries and going directly from the lungs into the blood stream reaching other organs in the body, including brain and the immune system. The translocation of nanoparticles is reportedly more effective than that of large particles. The small sized nanoparticles have much higher surface area than the same mass of larger ones; therefore surface area may play a role in vivo adverse outcomes. The small size of nanoparticles has been shown to relate to increased solubility and better bioavailability of materials. Altered chemical and/or physical properties might be expected to be accompanied by modified biological properties, some of which could accelerate cytotoxicity. Respirable fibrous nanoparticles e.g. carbon nanotubes (SWCNT, MWCNT, nanofibres) could cause pulmonary diseases by entering the alveolar region of the lung because their physical dimensions affecting lung mucociliary clearance. It has been shown that some high aspect ratio nanoparticles can have similar morphology (shape) and durability and are therefore likely to persist in the lungs, if inhaled. Carbonaceous materials are highly durable and do not easily dissolve in the lung lining fluids. Hence, they remain in the lungs for a long time, but without causing persistent lung interstitial diseases [10] (see also below about biodegradation of SWCNT).

Interaction of nanoparticles with five organ systems, lung, skin, GI tract, nasal olfactory structures and eyes are most important routes of exposure [11, 12]. However, nanoparticles released from these tissues could enter circulation and translocate to distant organs including the cardiovascular system and brain [13, 14]. The interaction of nanoparticles with the immune system, particularly macrophages, may play a significant role in the translocation process. In this review, we will focus on the effects of nanoparticles on lung toxicity with respect to inflammatory response and oxidative stress; the recognition and engulfment of nanoparticles by lung macrophages. In addition, we will consider the possible effects of nanoparticles on the immune system, as well as their potential genotoxicity. Finally, we will briefly review recent findings on CNT biodegradation pathways.

Pulmonary inflammatory responses to SWCNTs In vivo

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Role of nanotoxicology in nanomedicine
  5. Pulmonary inflammatory responses to SWCNTs In vivo
  6. SWCNT-induced pulmonary inflammation is accompanied by robust oxidative stress
  7. Redox interactions of SWCNTs with macrophages
  8. Nanoparticles and immunity
  9. Recognition and engulfment of nanotubes by macrophages
  10. Possible carcinogenic/mutagenic responses to carbonaceous nanotubes
  11. Biodegradation of SWCNT by myeloperoxidase of inflammatory cells
  12. Concluding remarks
  13. Conflict of interest statement
  14. Acknowledgements
  15. Disclaimer
  16. References

Three major responses are induced by exposure of animals to SWCNTs delivered through pharyngeal aspiration: (i) direct damage of cells, particularly of type II epithelial cells, (ii) encapsulation of agglomerated forms of SWCNTs and formation of granulomas and (iii) nongranulomatous inflammatory reaction to dispersed SWCNTs and resultant interstitial fibrosis. Our previous work has detailed these specific features of inflammatory response to aspired SWCNTs and established that SWCNT caused unusual pulmonary effects in C57BL/6 mice that combined a robust acute inflammation with early onset of progressive fibrosis and granulomas formation. Appearance and accumulation of typical biomarkers of cell damage such as increase in total protein, lactate dehydrogenase (LDH) and gamma-glutamyl transferase activities in bronchoalveolar lavage were accompanied by augmented levels of 4-hydroxynonenal (a characteristic secondary product of lipid peroxidation with profibrotic properties [15] as well as depletion of glutathione and other low-molecular weight thiols (the major water-soluble antioxidants) in lungs. At the cellular level, an accelerated inflammatory response was evidenced by an early arrival and accumulation of polymorphonuclear neutrophils (PMNs), followed by influxes of lymphocytes and macrophages [15]. This was accompanied by a rapid induction of proinflammatory cytokines, including TNF-α and IL-1β, followed by the production of pro-fibrogenic transforming growth factor (TGF)-β1. A early onset of progressive fibrosis in mice exhibited two distinct morphologies: (i) SWCNT-induced granulomas mainly associated with hypertrophied epithelioid cells surrounding SWCNT agglomerates and (ii) diffuse interstitial fibrosis and alveolar wall thickening likely associated with deposition of dispersed SWCNT. Comparison with well-documented responses to standard particles equal doses of ultrafine carbon black particles or fine crystalline silica (SiO2) – demonstrated that they were remarkably less effective than SWCNTs in inducing granulomas or alveolar wall thickening and caused a significantly weaker pulmonary inflammation and damage. Not surprisingly, the exuberant inflammatory response induced by SWCNTs resulted in functional respiratory deficiencies and decreased bacterial clearance (Listeria monocytogenes). Combined with other reports in the literature [16–18], our results suggest that occupational exposures to respirable SWCNT particles at the current permissible exposure limit (PEL) (for graphite particles) may represent a significant risk of developing some lung lesions.

Single exposure to bulk of CNT may be associated with potential artifactual effects due to instillation/agglomeration of nanotubes. These complications can be avoided by employment of inhalation exposures using stable and uniform SWCNT dispersions. Therefore, we developed a new aerosolization technique and conducted a study in which the inhalation of unpurified SWCNT (iron content of 17.7% by weight) at 5 mg m−3, 5 h day−1 for 4 days was compared with pharyngeal aspiration of varying doses (5–20 μg per mouse) of the same SWCNT [10]. The chain of pathological events in both exposure routes was similar both showing early inflammatory response and oxidative stress culminating in the development of multifocal granulomatous pneumonia and interstitial fibrosis. SWCNT inhalation was more effective than aspiration in causing inflammatory response, oxidative stress, collagen deposition and fibrosis as well as mutations of K-ras gene locus in the lung of C57BL/6 mice.

SWCNT-induced pulmonary inflammation is accompanied by robust oxidative stress

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Role of nanotoxicology in nanomedicine
  5. Pulmonary inflammatory responses to SWCNTs In vivo
  6. SWCNT-induced pulmonary inflammation is accompanied by robust oxidative stress
  7. Redox interactions of SWCNTs with macrophages
  8. Nanoparticles and immunity
  9. Recognition and engulfment of nanotubes by macrophages
  10. Possible carcinogenic/mutagenic responses to carbonaceous nanotubes
  11. Biodegradation of SWCNT by myeloperoxidase of inflammatory cells
  12. Concluding remarks
  13. Conflict of interest statement
  14. Acknowledgements
  15. Disclaimer
  16. References

Massive cell injury culminating in the demise has been universally associated with the initiation and propagation of free radical oxidation reactions and excessive accumulation of their products [19]. Whether oxidative stress is causative to cellular destruction or is a trivial consequence of the damaged cell’s inability to effectively control and maintain redox balance is an important but frequently unknown issue. Excessive oxidative stress has been proposed as a common paradigm for the toxicities of different nanoparticles, including CNT [20, 21].

One of the major factors for the potential involvement of oxidative reactions in pathogenic mechanisms is the presence of catalysts, most frequently transition metals. Participation of transition metals such as iron (Fe), copper (Cu), cobalt (Co) and nickel (Ni) in catalysis of redox oxidation reactions is essential for effective induction of oxidative stress. One should bear in mind that the metals in CNT samples are present almost exclusively in elemental rather than ionic form that is not readily available for the catalytic purposes. Most commonly used methodologies to manufacture SWCNTs include employment of transition metal catalysts, such as Fe, Co and Ni. Inflammation involves production of reactive oxygen species (ROS), mostly superoxide radicals and hydrogen peroxide, and reactive nitrogen species (nitric oxide and resulting peroxynitrite) by immune cells PMNs and macrophages [22]. Combination of transition metals with oxygen radicals is known to synergistically enhance each other to cause disproportionally high inflammatory response and oxidative damage. Therefore, it is not surprising that SWCNTs-induced inflammatory response is characterized by accumulation of typical biomarkers of oxidative stress. Amongst those depletion of total antioxidant reserves (decreased levels of glutathione and other low-molecular weight thiols as well as protein SH-groups), consumption of the major lipid-soluble antioxidant of membranes (vitamin E), and accumulation of a typical biomarker of lipid peroxidation (4-hydroxy-nonenal) were all documented in mice exposed to SWCNTs by pharyngeal aspiration (see above). If the mutual enhancement of transition metal-catalysed oxidative stress and inflammatory response are indeed major contributors, then SWCNTs containing metals may induce greater lung damage than purified SWCNT where the metal catalysts have been removed. This indeed has been demonstrated by using SWCNT with different content of iron [10, 15].

The activated NADPH oxidase of recruited phagocytes is readily identifiable as a source of oxidative stress during inflammation. In addition to direct transition metal-dependent pathways through which CNT can trigger redox cascades, activation of NADPH oxidase may play a critical role in oxidative stress associated with inflammatory response. Further, timely elimination of PMNs through apoptosis and their subsequent clearance by macrophages is a necessary stage in the resolution of pulmonary inflammation whereby NADPH oxidase contributes to control of apoptotic cell death and clearance of PMNs. NADPH oxidase may be an important regulator of the transition from the acute inflammation to the chronic fibrotic stage in response to SWCNT. Indeed, we found that NADPH oxidase-deficient mice (which lacked the gp91(phox) subunit of the enzymatic complex) responded to SWCNT exposure with a marked accumulation of PMNs and elevated levels of apoptotic cells in the lungs, production of pro-inflammatory cytokines, decreased production of the anti-inflammatory and pro-fibrotic cytokine, TGF-β and significantly lower levels of collagen deposition, as compared with C57BL/6 control mice. This indicates that reactive oxygen species produced by NADPH oxidase of inflammatory cells may affect and determine the course of pulmonary response to SWCNT [23].

Oxidative stress commonly accompanies cytotoxic effects originating from exposure of cells and animals to engineered nanomaterials, including carbon nanotubes. However, whether this is a correlation or a causative effect remains to be elucidated. One of experimental approached to address the issue is the possible protective effect of antioxidants or exacerbation of damage in antioxidant deficiency. It is logical to expect that animals with compromised antioxidant defences would be more sensitive and elicit an enhanced inflammatory response. To test this experimentally, we employed C57BL6 mice with dietary vitamin E-deficiency. This was achieved by maintenance of the animals on vitamin E-insufficient diet [24]. In these animals, vitamin E-deficiency also caused decreased levels of other antioxidants (vitamin C and GSH) and elevated levels of lipid peroxidation products. Most notably, these animals displayed very high levels of both acute and chronic inflammatory responses. This was evidenced by higher amounts of inflammatory cells (PMNs, macrophages), higher levels of released cytokines (TNF-α, IL-1 β, IL-6, TGF-β, IL-10), as well as more pronounced deposition of collagen in the lung [24].

Redox interactions of SWCNTs with macrophages

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Role of nanotoxicology in nanomedicine
  5. Pulmonary inflammatory responses to SWCNTs In vivo
  6. SWCNT-induced pulmonary inflammation is accompanied by robust oxidative stress
  7. Redox interactions of SWCNTs with macrophages
  8. Nanoparticles and immunity
  9. Recognition and engulfment of nanotubes by macrophages
  10. Possible carcinogenic/mutagenic responses to carbonaceous nanotubes
  11. Biodegradation of SWCNT by myeloperoxidase of inflammatory cells
  12. Concluding remarks
  13. Conflict of interest statement
  14. Acknowledgements
  15. Disclaimer
  16. References

Macrophages are the primary responders to different particles that initiate and propagate inflammatory reactions and oxidative stress [25, 26]. Recognition, tethering, engulfment and digestion of nanoparticles by macrophages may be important for regulation of inflammatory response as well as for the fate of nanopartciles, their distribution and potential biodegradation. Interaction of particles with macrophages commonly results in the activation of their NADPH oxidase system leading to the production and release of ROS, mainly superoxide radicals [27]. The latter dismutates (spontaneously or via SOD-catalysed pathways) to yield hydrogen peroxide. Elimination of superoxide is important to prevent its very effective interaction with nitric oxide, which results in the formation of peroxynitrite (ONOO-), a potent oxidant causing massive nitration of protein tyrosine resides. Accumulation of nitrotyrosines has been associated with oxidative/nitrosative stress and tissue damage [28, 29]. Alternatively, the product of superoxide radical dismutation, H2O2, can be decomposed, in the presence of transition metals, to yield another very potent oxidant, hydroxyl radical (OH·) via a well known Haber–Weiss mechanism. As OH· radicals can indiscriminately attack essentially any biomolecule and cause its oxidative modification, excessive generation of hydroxyl radicals has been also linked to oxidative damage [30, 31]. In this pathway, the presence of catalytically active transition metals is critical to the fate of superoxide/H2O2 produced by inflammatory cells (macrophages). The presence of transition metals (Fe, Co, Ni) as obligatory decorating metals in the manufacturing of SWCNTs may have a very substantial impact on the induction of macrophage-driven reactions. Using iron-rich (unpurified) SWCNT (26 wt% of iron) and iron-stripped (purified) SWCNT (0.23 wt% of iron) we reported that iron-rich SWCNT were more effective in generating hydroxyl radicals spin-trapped with 5,5-dimethyl-I-pyrroline-N-oxide (DMPO), than purified SWCNT. Similarly, EPR spin-trapping experiments in the presence of zymosan-stimulated RAW 264.7 macrophages showed that nonpurified SWCNT more effectively converted superoxide radicals generated by xanthine oxidase/xanthine into hydroxyl radicals as compared to purified SWCNT. After stimulation of RAW 264.7 macrophages (with zymosan or PMA), iron-rich SWCNT caused significant loss of low-molecular weight thiols and accumulation of lipid hydroperoxides. Thus, transition metal contaminations in SWCNT substantially affect redox-dependent responses of macrophages [22]. These results are concordant with in vitro studies with cell-free systems in which SWCNT with high iron content displayed high redox activity in a cell-free model system and generated higher levels of EPR-detectable ascorbate radicals resulting from ascorbate oxidation.

Nanoparticles and immunity

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Role of nanotoxicology in nanomedicine
  5. Pulmonary inflammatory responses to SWCNTs In vivo
  6. SWCNT-induced pulmonary inflammation is accompanied by robust oxidative stress
  7. Redox interactions of SWCNTs with macrophages
  8. Nanoparticles and immunity
  9. Recognition and engulfment of nanotubes by macrophages
  10. Possible carcinogenic/mutagenic responses to carbonaceous nanotubes
  11. Biodegradation of SWCNT by myeloperoxidase of inflammatory cells
  12. Concluding remarks
  13. Conflict of interest statement
  14. Acknowledgements
  15. Disclaimer
  16. References

Lately, several in vivo investigations were conducted in which effects of CNT on the immune responses were studied. According to Park et al. [32], both local pulmonary and systemic immune responses can be triggered by exposure of mice to CNT. Dose-dependent increases in the content of several pro-inflammatory cytokines – IL-1β, TNF-α, IL-6, IL-4, IL-5, IL-10, IL-12 and IFN-γ– were documented in BAL fluid and blood of exposed animals. Accordingly, increased total IgE production was found in blood of CNT exposed mice. Based on these observations, the authors suggested that pulmonary exposure to CNT caused activation of alveolar macrophages, recruitment of immune cells into the lung, and facilitated differentiation of CD4+ T cell into Th1 cells and Th2 cells. Overall, these responses may be in charge of the allergic pulmonary outcomes observed in mice treated with CNT. The adjuvant capacity and allergic immune responses to carbonaceous nanomaterials are dependent on the particle size and surface area [33]. Assessment of acute effects on the innate immune system revealed that SWCNT and MWCNT given to mice along with OVA increased serum levels of OVA-specific IgE, eosinophils counts in BAL and secretion of Th2 cytokines measured in cell supernatants obtained from mediastinal lymph nodes. However, exposures to ultrafine particles plus OVA increased IgG2a, PMN numbers, TNF-α and MCP1. Thus, specific features of activation of the innate immune system by particles depend on their size. Ryman-Rasmussen et al. [34], demonstrated that inhaled MWCNT caused pulmonary fibrosis in mice with allergic asthma. The authors suggested that fibrogenic response found in mouse airways after combined ovalbumin sensitization and MWCNT inhalation required platelet derived growth factor (PDGF), a potent fibroblast mitogen, and TGF-β1. Combined ovalbumin sensitization and MWCNT inhalation also synergistically increased IL-5 mRNA levels, further contributing to airway fibrosis. These data indicate that inhaled MWCNT require pre-existing inflammation to cause airway fibrosis. Thus, population with pre-existing allergic inflammation may be susceptible to airway fibrosis elicited by respirable MWCNT.

It has been shown that inhalation of MWCNT suppressed systemic immunity assessed by proliferative assay of spleen cells [35]. Notably, activity of constitutive cyclooxygenase (COX) in spleen of mice was affected by inhalation of MWCNT. Based on experiments with COX knockout mice, the authors suggested that COX is involved in immuno-suppression triggered by inhalation of MWCNT. It is possible that CNT induced weakened immunity is associated with increased sensitivity to infections. This may be particularly important in the context of realistic exposures to CNT occurring in conjunction with other pathogenic impacts such as microbial infections. Our studies of interactions between pharyngeal aspiration of SWCNT and bacterial pulmonary infection of C57BL/6 mice with Listeria monocytogenes (LM) showed that sequential exposure to SWCNT/LM amplified lung inflammation and collagen formation. Despite this robust inflammatory response, SWCNT pre-exposure significantly decreased the pulmonary clearance of LM-exposed mice. Decreased bacterial clearance in SWCNT-pre-exposed mice was associated with decreased phagocytosis of bacteria by macrophages and a decrease in nitric oxide production by these phagocytes. Pre-incubation of naïve alveolar macrophages with SWCNT in vitro also resulted in decreased nitric oxide generation and suppressed phagocytizing activity toward LM. Failure of SWCNT-exposed mice to clear LM led to a continued elevation in nearly all major chemokines and acute phase cytokines into the later course of infection. In SWCNT/LM-exposed mice, bronchoalveolar lavage neutrophils, alveolar macrophages and lymphocytes, as well as lactate dehydrogenase level, were increased compared with mice exposed to SWCNT or LM alone. These data suggest that enhanced acute inflammation and pulmonary injury after SWCNT associated with delayed bacterial clearance exposure may lead to increased susceptibility to lung infection in exposed populations [23].

Recognition and engulfment of nanotubes by macrophages

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Role of nanotoxicology in nanomedicine
  5. Pulmonary inflammatory responses to SWCNTs In vivo
  6. SWCNT-induced pulmonary inflammation is accompanied by robust oxidative stress
  7. Redox interactions of SWCNTs with macrophages
  8. Nanoparticles and immunity
  9. Recognition and engulfment of nanotubes by macrophages
  10. Possible carcinogenic/mutagenic responses to carbonaceous nanotubes
  11. Biodegradation of SWCNT by myeloperoxidase of inflammatory cells
  12. Concluding remarks
  13. Conflict of interest statement
  14. Acknowledgements
  15. Disclaimer
  16. References

Redox effects of SWCNTs and their transition metal content can be realized during the immediate contact of particles with cells or at a distance through ROS and other reactive intermediates generated by transition metals. As the lifetime of free radical intermediates is short, it is likely that remote interactions are less effective than direct particle/cell contacts. It is therefore important to assess the extent to which SWCNTs are recognized and engulfed by macrophages. Previous work has established that functionalized SWCNTs, particularly those carrying negative charge on their surface, are readily ingested by macrophages [36, 37]. By contrast, nonfunctionalized SWCNTs are poorly recognized by macrophages and do not effectively induce typical macrophage activation responses, such as superoxide production by NADPH oxidase or NO· production by iNOS [22].

Significant literature indicates that recognition of apoptotic cells by macrophages is largely and universally dependent on the appearance on the cell surface of an acidic phospholipid, phosphatidylserine (PS), which is normally confined to the cytosolic leaflet of plasma membrane [38]. In other words, externalization of PS during apoptosis generates an ‘eat-me’ signal for macrophages. Further, nonapoptotic cells with externalized PS are well recognized by macrophages resulting in the suppression of their ROS and reactive nitrogen species (RNS) production [39, 40]. This suggests that coating of SWCNTs with PS could interface them with macrophages and stimulate the recognition, tethering, and engulfment. This may be important for regulation of the inflammatory response to SWCNT. Moreover, this approach can be utilized for targeted delivery of specialized cargos – regulators, inhibitors – into macrophages aimed at control of their functions. Indeed, SWCNT coating with a phospholipid ‘eat-me’ signal, PS, makes them recognizable in vitro by professional phagocytes such as murine RAW264.7 macrophages, primary monocyte-derived human macrophages. Macrophage uptake of PS-coated nanotubes was suppressed by the PS-binding protein, Annexin V and endocytosis inhibitors, and changed the pattern of pro- and anti-inflammatory cytokine secretion. Loading of PS-coated SWCNT with pro-apoptotic cargo (cytochrome c) allowed for the targeted killing of RAW264.7 macrophages. In vivo aspiration of PS-coated SWCNT stimulated their uptake by lung alveolar macrophages in mice [41]. These studies thus demonstrate that noncovalent modification of SWCNTs with specific phospholipid molecules can be employed for targeted delivery and regulation of professional phagocytes [41].

Possible carcinogenic/mutagenic responses to carbonaceous nanotubes

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Role of nanotoxicology in nanomedicine
  5. Pulmonary inflammatory responses to SWCNTs In vivo
  6. SWCNT-induced pulmonary inflammation is accompanied by robust oxidative stress
  7. Redox interactions of SWCNTs with macrophages
  8. Nanoparticles and immunity
  9. Recognition and engulfment of nanotubes by macrophages
  10. Possible carcinogenic/mutagenic responses to carbonaceous nanotubes
  11. Biodegradation of SWCNT by myeloperoxidase of inflammatory cells
  12. Concluding remarks
  13. Conflict of interest statement
  14. Acknowledgements
  15. Disclaimer
  16. References

As CNT exhibit a high dimensional aspect ratio, questions concerning asbestos-like pulmonary outcomes of exposures have been raised. Recent studies of the responses of mice to intra-abdominal injection of MWCNT have received significant attention. Takagi et al. [42], reported that mesotheliomas were observed 6 months after intraperitoneal injection (i/p) in both MWCNT-exposed and the asbestos-exposed mice, but not in the fullerene-exposed mice. In addition, the death rate for the MWCNT-exposed group was significantly greater than for the asbestos-exposed group. Of note, MWCNT were administered to mice heterozygous for the tumour suppressor gene, p53; these animals are known to be particularly sensitive to asbestos-induced mesothelioma [43]. Although the findings of Takagi et al. are striking, the study has been criticized for the use of an extraordinarily high (3 mg per mouse) exposure dose [44, 45]. This high exposure dose may have resulted in extensive abdominal fibrosis causing death by constriction ileus. Recent findings of Poland et al. [46], documented an acute response to intra-abdominal instillation of a significantly lower dose (50 μg per mouse) of MWCNT. These investigators found that long MWCNT but not short MWCNT caused inflammation of the abdominal wall, with the formation of so-called foreign body giant cells, and an increase in polymorphonuclear leukocytes harvested by peritoneal lavage at seven days postexposure. In this study too, responses to long MWCNT were similar in magnitude to those caused by asbestos. Whilst these data are undoubtedly important, it still remains to be elucidated whether the acute inflammatory response would persist and progress to mesothelioma. There is a lag-time of up to 30 years for mesothelioma formation in humans exposed to pathogenic asbestos fibres. Whether inhaled MWCNT would be able to migrate to the intrapleural space and affect the mesothelium – these questions have yet to be addressed. Notably, Muller et al. [47], did not find a carcinogenic response 2 year posttreatment of rats with MWCN by dosing them into peritoneal cavity. In contrast to data published by Poland et al. [46], shorter and tangled MWCNT bundles given to mice by i/p did not elicit inflammatory responses whilst longer fibres were effective in causing inflammation [47]. In the study with rats i/p injected with MWCNT aggregates similar to the tangled material used by Poland et al. [46], no mesotelioma formation was documented [47]. Thus, there are still uncertainties with regards to whether longer fibres used by Poland et al. [47], will cause lung cancer, mesothelioma, or obstructive pulmonary diseases during/after exposure to carbonaceous nanomaterials.

Inflammation and fibrosis have been considered as significant risk factors in pulmonary carcinogenesis [48]. Amongst the mutated genes implicated in pulmonary tumorigenesis, K-ras oncogene is frequently found in lung tumours of mice exposed to chemicals [13, 49]. Several studies have shown an association between lung fibrosis and an increased risk of lung cancer [48, 50, 51]. It was proposed that fibrosis might be involved in the carcinogenesis by the occurrence of atypical or dysplastic epithelial changes, which progressed to invasive malignancy [50]. Shvedova et al. [10], demonstrated that exposure to respirable SWCNT caused K-ras mutations found in mouse lungs. Interestingly, one of the mutations found in the lung of SWCNT-treated mice (at day 28 post-SWCNT inhalation) consisted of a double mutation occurring at codons 12 and 8 [GGT to GAT and GTG to ATG (valine to methionine), respectively]. The role of this double mutation is unknown and may be specific for SWCNT exposure. However, the involvement of K-ras mutations in fibrosis-associated lung cancer is not understood and merits further investigation. The detected mutations frequency of 62.5% (10/16) in mice after SWCNT inhalation is significantly higher than that in the untreated group of mice 26.7%. Further studies involving a bigger sample size may help in determining the usefulness of this gene mutation as a biomarker of exposure to SWCNT. Furthermore, the potential role in lung carcinogenesis requires further investigation by comparing C57BL/6 mice with other mouse strains susceptible to lung tumour formation.

Exposure to respirable SWCNT caused bronchiolar epithelial cell hypertrophy with both hypertrophy and hyperplasia, and the inflammation being consistently granulomatous (day 28 post exposure). Anuclear macrophages along with other mitotic changes in macrophage cells with anaphase bridges indicating possible spindle aberrations were seen in lung sections of exposed mice at 7–28 days post inhalation (Fig. 1). An atypical mitotic figure was observed in mouse at 7 days post inhalation and material consistent with SWCNT nanoropes intertwined with the abnormal mitotic figure (Fig. 1). This certainly suggests the potential for SWCNTs to interfere with the mitotic spindle. Recently, it has been reported that exposure of cells to SWCNT caused disruption of the mitotic spindle [52]. SWCNT bundles are similar to the size of microtubules that form the mitotic spindle that may be incorporated into the mitotic spindle apparatus. Therefore, exposure to SWCNT could disrupt the mitotic spindle apparatus thus induce abnormal chromosome number well associated with a greater risk of developing cancer. The extraordinary level of chromosomal abnormalities following SWCNT exposure underscores the importance of the SWCNT-induced damage to the mitotic spindle, and the importance of additional studies to uncover the mechanism of damage. Mitotic spindle damage and aneuploidy have also been observed following in vitro treatment with the potent occupational carcinogen, chrysotile asbestos [53]. Chrysotile asbestos has been seen in DNA and in the bridge of cytokinesis; however, association with the centrosome/centrosome damage and/or mixing with the mitotic spindle has not been documented with asbestos. The SWCNT bundles are very similar to the size of the microtubules [54]. Centrosome fragmentation, mitotic spindle disruption and aneuploidy are well-known characteristics of cancer cells [55, 56]. However, long-term in vivo studies are required to evaluate whether pulmonary exposure to SWCNT would result in lung cancer. Accordingly, further research is necessary to fully address mechanism(s) of chromosomal damage and persistence of mitotic spindle aberrations induced by SWCNT in lung tissues.

image

Figure 1.  Histopathology of lung section from the SWCNT inhalation study (5 mg m−3, 5 h day−1, 4 days): Seven days after the last SWCNT exposure, anuclear macrophages (solid arrow) along with an anaphase bridge (open arrow) in a dividing macrophage containing SWCNT.

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Biodegradation of SWCNT by myeloperoxidase of inflammatory cells

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Role of nanotoxicology in nanomedicine
  5. Pulmonary inflammatory responses to SWCNTs In vivo
  6. SWCNT-induced pulmonary inflammation is accompanied by robust oxidative stress
  7. Redox interactions of SWCNTs with macrophages
  8. Nanoparticles and immunity
  9. Recognition and engulfment of nanotubes by macrophages
  10. Possible carcinogenic/mutagenic responses to carbonaceous nanotubes
  11. Biodegradation of SWCNT by myeloperoxidase of inflammatory cells
  12. Concluding remarks
  13. Conflict of interest statement
  14. Acknowledgements
  15. Disclaimer
  16. References

Degradability is an important factor in the assessment of toxicity of nanomaterials. Nondegradable nanomaterials can accumulate in organs and also intracellularly where they can exert detrimental effects to the cells (Fig. 2). For instance, long-term accumulation of medicinal gold salts (nanoparticles) in the body reportedly caused adverse or toxic effects in patients [57]. SWCNTs are known to be bio-persistent and may remain inside macrophages in spleen and liver for prolonged periods of time following parenteral administration [58]; carbon nanotubes have also been observed in the lungs of exposed mice up to 1 year after pharyngeal administration (unpublished information). On the other hand, biodegradable nanomaterials could also yield unpredictable toxic responses due to toxic degradation products [59]. For instance, leaching of toxic core components such as cadmium from quantum dots with induction of oxidative stress has been suggested as a mechanism of in vivo toxicity of these nanomaterials [59]. Controlled biodegradation of nanomaterials thus represents one of the important challenges not only in the field of nanotoxicology but also in the field of nanomedicine, as the safe implementation of nanomaterials for biomedical purposes is contingent on the biodegradation and/or clearance of the exogenous nanomaterials. In a recent proof-of-concept study, Park et al. [60], reported that multi-functional porous silicon nanoparticles self-destructed in a mouse model into renally cleared components – likely orthosilicic acid – in a matter of weeks with no evidence of toxicity in animal tissues. Moreover, we and others have recently demonstrated the enzymatic degradation of SWCNT by incubating CNTs in a cell-free system with horseradish peroxidase (HRP) and low amounts of H2O2 [61]. These results mark a promising possibility for carbon nanotubes to be degraded by HRP in environmentally relevant settings. More recently, we have demonstrated biodegradation of carbon nanotubes by myeloperoxidasse of phagocytic cells (neutrophils) in physiologically relevant environments (Kagan et al., submitted for publication, 2009). Importantly, SWCNTs fully biodegraded by myeloperoxidase were inefficient in inducing typical inflammatory and oxidative stress responses characteristic of naïve nanotubes after pharyngeal aspiration in mice [15]. These results open new opportunities for controlled regulation of CNT content in the lung via directed manipulations of intensity of inflammatory responses (e.g. by using selective chemokines).

image

Figure 2.  Depicts the major pulmonary responses and their pathogenic consequences as well as biodegradation pathways triggered after animal exposure to SWCNT via pharyngeal aspiration or inhalation.

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Concluding remarks

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Role of nanotoxicology in nanomedicine
  5. Pulmonary inflammatory responses to SWCNTs In vivo
  6. SWCNT-induced pulmonary inflammation is accompanied by robust oxidative stress
  7. Redox interactions of SWCNTs with macrophages
  8. Nanoparticles and immunity
  9. Recognition and engulfment of nanotubes by macrophages
  10. Possible carcinogenic/mutagenic responses to carbonaceous nanotubes
  11. Biodegradation of SWCNT by myeloperoxidase of inflammatory cells
  12. Concluding remarks
  13. Conflict of interest statement
  14. Acknowledgements
  15. Disclaimer
  16. References

A plethora of diversified man-made nanostructured materials, such as fullerenes, nanoparticles, nanopowders, nanotubes, nanowires, nanorods, nanofibres, quantum dots, dendrimers, nanoclusters, nanocrystals and nanocomposites, are globally produced in large quantities due to their wide potential applications [12]. This implies that human exposure to nano-sized materials is essentially inevitable; they can enter the body through the lungs or other organs via food, drink and medicine and affect many different organs and tissues. Huge diversity of nanostructured materials defines the broad spectrum of their potential injurious effects that will be largely defined by their unique properties, including size/surface area, concentration, solubility, chemical and biological properties and stability. The toxicity of nanostructured materials could be reduced by chemical approaches, such as surface treatment, functionalization and composite formation.

Whilst toxic effects of CNTs have not been sufficiently studied, existing data indicate that they may be harmful to living organisms [12]. As summarized on Fig. 1, in the lung, their toxic effects may be associated with robust and unusual inflammatory response that includes early onset of fibrosis. A significant component of CNT’s toxicity is not only due to markedly enhanced oxidative stress dependent on the presence in CNT of transition metals but also due to their activation of NADPH oxidase triggering massive production and release of oxygen radicals. The ability of CNT to cause immuno-suppression may lead to elevated sensitivity to microbial infections in exposed individuals. Finally, the complex synergistic nature of inflammatory/oxidative stress responses to CNT includes mutagenicity possibly leading to carcinogenesis. Whilst significantly more detailed studies are necessary to more completely characterize health hazards associated with exposures to CNT, stimulation of pathways leading to their biodegradation seems to be one promising approach. In this regard, identification of myeloperoxidase of inflammatory cells as an oxidation based catalytic biodegradation mechanism seems particularly important.

Obviously, further rigorous and extensive in vitro and in vivo nanotoxicological studies are necessary to determine their mechanisms of toxicity, biodegradation pathways, pulmonary distribution and translocation, before risk assessment can be conducted and appropriate prevention strategies can be developed and implemented to assure the safe production and use of carbon nanotubes.

Acknowledgements

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Role of nanotoxicology in nanomedicine
  5. Pulmonary inflammatory responses to SWCNTs In vivo
  6. SWCNT-induced pulmonary inflammation is accompanied by robust oxidative stress
  7. Redox interactions of SWCNTs with macrophages
  8. Nanoparticles and immunity
  9. Recognition and engulfment of nanotubes by macrophages
  10. Possible carcinogenic/mutagenic responses to carbonaceous nanotubes
  11. Biodegradation of SWCNT by myeloperoxidase of inflammatory cells
  12. Concluding remarks
  13. Conflict of interest statement
  14. Acknowledgements
  15. Disclaimer
  16. References

This study was supported by NIOSH (Grant No. OH008282), National Occupational Research Agenda (NORA) (Grant No. 92700Y), National Institutes of Health (NIH) and the 7th Framework Programme of the European Commission (EC-FP7-NANOMMUNE-Grant Agreement No. 214281). The authors are thankful to Dr A. Hubbs and Mrs K. Clough-Thomas for assisting in Fig. 1 and Fig. 2, respectively.

References

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Role of nanotoxicology in nanomedicine
  5. Pulmonary inflammatory responses to SWCNTs In vivo
  6. SWCNT-induced pulmonary inflammation is accompanied by robust oxidative stress
  7. Redox interactions of SWCNTs with macrophages
  8. Nanoparticles and immunity
  9. Recognition and engulfment of nanotubes by macrophages
  10. Possible carcinogenic/mutagenic responses to carbonaceous nanotubes
  11. Biodegradation of SWCNT by myeloperoxidase of inflammatory cells
  12. Concluding remarks
  13. Conflict of interest statement
  14. Acknowledgements
  15. Disclaimer
  16. References