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Reviews on Environmental Health

Editor-in-Chief: Carpenter, David O. / Sly, Peter

Editorial Board: Brugge, Doug / Edwards, John W. / Field, R.William / Garbisu, Carlos / Hales, Simon / Horowitz, Michal / Lawrence, Roderick / Maibach, H.I. / Shaw, Susan / Tao, Shu / Tchounwou, Paul B.


IMPACT FACTOR 2017: 1.284

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Volume 33, Issue 3

Issues

Nanostructures: between natural environment and medical practice

Maria Concetta TrovatoORCID iD: http://orcid.org/0000-0001-8518-4918 / Daniele Andronico
  • Istituto Nazionale di Geofisica e Vulcanologia (INGV), Osservatorio Etneo, Sezione di Catania, Piazza Roma 2, Catania, Italy
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/ Salvatore Sciacchitano
  • Department of Clinical and Molecular Medicine, Sapienza University, Policlinico Umberto I, Viale Regina Elena n. 324, Rome, Italy
  • Laboratorio di Ricerca Biomedica, Fondazione Università Niccolò Cusano per la Ricerca Medico Scientifica, Via Don Carlo Gnocchi 3, Rome, Italy
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/ Rosaria Maddalena Ruggeri
  • Department of Clinical and Experimental Medicine, Policlinico Universitario, Consolare Valeria 1, Messina, Italy
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/ Isa Picerno
  • Department of Biomedical and Dental Sciences and Morphofunctional Imaging, Policlinico Universitario, Consolare Valeria 1, Messina, Italy
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/ Angela Di Pietro
  • Department of Biomedical and Dental Sciences and Morphofunctional Imaging, Policlinico Universitario, Consolare Valeria 1, Messina, Italy
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/ Giuseppa Visalli
  • Department of Biomedical and Dental Sciences and Morphofunctional Imaging, Policlinico Universitario, Consolare Valeria 1, Messina, Italy
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Published Online: 2018-09-06 | DOI: https://doi.org/10.1515/reveh-2017-0036

Abstract

Nanoparticles (NPs) are small structures under 100 nm in dimension. Interrelationships among the morphological parameters and toxicity of NPs have been the focus of several investigations that assessed potential health risk in environmentally-exposed subjects and the realistic uses of NPs in medical practice. In the current review, we provide a summary of the cellular mechanisms of membrane-mediated transport, including old and novel molecules that transport nanostructures across cellular membranes. The effects of geochemical exposure to natural NPs are evaluated through epidemiological data and cancerous pathways activated by Fe2+ NPs. Specifically, we discuss screening for papillary thyroid carcinomas in the inhabitants of the Sicilian volcanic area surrounding Mount Etna to compare the incidence of thyroid carcinoma in this population. Lastly, considering the increased production of carbon nanotubes (CNTs), we examine the toxicity and potential use of these engineered NPs in drug delivery of an extensive amount of therapeutic and imaging molecules (theranosis) that can be conjugated to CNTs.

Keywords: nanoparticles; nanotubes; papillary thyroid carcinoma; theranosis; volcanic ash

How to label nanoparticles: new functions for ancient particles

By an approximate definition, all structures with a size ranging between microscopic and molecular dimensions may be defined as nanoparticles (NPs) (1). For an accurate definition of NPs, however, it must focus more specifically on the relationship occurring among sizes and dimensions at the length scale of nanometres (nm). A nanometre corresponds to one billionth of a metre (10−9 m). So far, three main types of NPs have been categorised: nanotextured surfaces (NTS), nanotubes (NT) and spherical NPs (SNPs) (1). For NTS, only one dimension under 100 nm is recognised (1). NT encompass two dimensions: these tubular structures can have a diameter under 100 nm, but a greater length (1). Lastly, SNPs display a nanometre scale on three dimensions, such that the diameter of a single particle under 100 nm is the only spatial dimension (1).

Practically, the main parameters used to define NP classifications are measurements of the morphological dimensions of objects (2). This is the reason that the NP classification launched in the USA and Japan in the 1990s sounded like a new description of old fashioned particles discovered in 1942 through the use of scanning electron microscopy (SEM). In fact, in September 1942, Vladimir Zworykin, better known as a co-inventor of the television, set out the description of the first SEM with a power of resolution of 100 Ångstrom units (0.1 nm) (3). In a scientific paper compiled with James Hillier and Richard Snyder, he summarised the new scanning machine at the Institute of Radio Engineers’ convention in Cleveland, Ohio, and in 1944 acquired a US patent for the use of electron microscopes (US patent no. 2,354,263; 1944) (3). NPs were initially named “ultrafine particles”, and nowadays, the size and distribution of NPs are still analysed using SEM and transmission electron microscopes in diagnostic medical practice (4). The use of electron microscope technology for the isolation of NPs has the triple benefit of individualising the locus where the particles are deposited, in addition to adding morphological features and dimensions of particles (4). By revealing the morphological features of NPs, a large branch of NPs studies have aimed to evaluate the behaviour of NPs in the context of pathological tissues (5). Therefore, for the examination of biological material ex vivo, the use of electron microscopes remains the predominant strategy to recognise at least two spatial dimensions of NPs (4), (5).

Supported by numerous studies that have investigated the ability of NPs to combine with each other inside environmental and biological systems, the definition of NPs has been improved (6), (7), (8), (9). From 1990 to 2015, investigations on NPs have attempted to connect the measure of shape miniaturised with “quantum confinement effects” appearing at the nanometre level (6). These effects are strictly related to the size of the NP, in fact they are inconsistent in macro or micro levels while appearing at nano levels (7). Briefly, when the size of a particle becomes too small to be comparable with the wavelength of the electron, the quantum phenomena drives the nanosystems away from thermal equilibrium by decreasing the wavelength, and subsequently, the emission of blue radiation (8). Quantum phenomena are the basis of different properties acquired by materials reduced to the nanometre scale (8). Some examples include: macroscopic opaque copper becoming transparent; inert platinum converting into catalyst material; solid gold transforming into liquid at room temperature; and stable aluminium becoming combustible while the insulating silicon is converted in conductor material (9).

The scientific discovery of NPs is driven by emerging properties of objects related to the NP’s length. NPs show an increased number of surface area atoms with respect to microparticles or bulk because of the high particle number per unit mass (2). In fact, the relationship between surface area and volume (or mass) for a particle with a diameter of 60 nm turns out to be 1000-times larger than a particle with a diameter of 60 nm, and consequently, the surface area for chemical reactions is greatly enhanced.

These observations have been further improved through evidence that NPs can be manipulated (10), (11). The basis of the nanotechnology definition, proposed by the USA National Nanotechnology Initiative (NNI) and reported in 2015 by the National Science and Technology Council (NSTC), mentions two specific abilities of NP, such as “modelling” and “manipulation”. These two characteristics distinguish fine particles from NPs. Size and shape become features of specific NPs’ abilities appearing only in nanoscale (10), (11).

Toxic and useful characteristics of NPs in medical practice

The threat that NPs may pose to human health have not been scientifically confirmed. The main issue relates to the ability of NPs to enter the biological milieu by crossing cellular membranes, with subsequent internalisation in the cytoplasm or nucleus (12). The mechanisms controlling which NPs can move in and out of cell compartments remains unclear. Multiple biological frameworks are involved in maintaining separation between the cytosol and the extracellular environment (12). Nevertheless, the cellular membrane remains the key cytological parameter for controlling NP flow due to its physiochemical properties (12). Based on evidence that cellular membranes are 10 nm wide, NPs are expected to follow three main pathways to cross the surface lipid bilayer to enter the bilayer interior (12). NPs may move into the internal compartments of cells through passive diffusion in addition to endocytosis (12) (Figure 1).

Nanoparticle pathways to cross cellular membranes.
Figure 1:

Nanoparticle pathways to cross cellular membranes.

Diffusion phenomena of NPs

By diffusion, NPs smaller than 10 nm in dimension should be able to simply slip through the cell membranes. However, this explanation is incomplete due to the diffusivity of NPs evidenced in non-Newtonian biological fluids, which are not only regulated by the dimensions of the NP, but also by the viscosity of the medium and interactions between NPs and macromolecules diluted in a solution (13). Therefore, in order to comprehensively evaluate the diffusion mechanisms of NPs, it must be considered that NPs show different characteristics to traditional chemicals (14). These include their insoluble nature, agglomeration tendencies and non-uniform concentration gradients (14). In addition, the levels of NPs in biological fluids that are able to penetrate the membrane remain ambiguous, and the main factors regulating this movement are still under investigation. In fact, the scattering of NPs is under Brownian motion, and therefore, the NP diffusion coefficient is determined by the ratio of two forces, the chemical potential gradient and the frictional coefficient of the particle related to the solvent (15). However, due to the slight flow in the fluid channel, it has been demonstrated that the frictional coefficient of NPs is increased under liquid motion in respect to steady state (15). Moreover, for NPs with diameters ranging between 3 and 84 nm, such as sodium chloride particles, the temperature must be included among the empirical coefficients influencing the diffusion coefficient of NPs (16). This is because of the correlation between temperature and dimension expressed by the law D~Tα, where α decreases with the increasing particle diameter (16).

These recent investigations on the nature of the diffusion phenomena of NPs have been fundamental for improving our knowledge on the pathogenesis of several infective diseases caused by viruses and bacteria that penetrate mucosal membranes (13). Therefore, investigations regarding the diffusion mechanisms of NPs have crucial implications for potential biomedical applications of NPs in the cure of infective pathologies. To efficiently investigate the mechanisms of the diffusion of NPs through cellular membranes, several tests can be realistically employed in common medical practice (17), (18), (19). Firstly, the membrane of red blood cells is considered to be a paradigmatic model because it is incapable of endocytosis, and thus, NPs should cross the membrane lipid bilayer through passive diffusion only (17). Using fluorescence microscopy, surface-enhanced infrared absorption spectroscopy and electrochemistry approaches in red blood cell membranes, the passive penetration of zwitterionic quantum dots has been demonstrated to be well matched to the pronounced flexibility of the lipid bilayers (17). Interestingly, in this model the membrane structures remained intact and no persistent holes appeared in the bilayers (18). A different method to investigate the diffusion of NPs utilised in vivo cellular models of HeLa cells (19). This analysis, which employed a spinning disk and 4Pi confocal microscopies, explored the penetration of an NP with a 4 nm radius and a D-penicillamine (DPA-QDs) coating into HeLa cells (19). In this examination, a double NPs’ flow was revealed, evidenced by DPA-QD accumulation at the plasma membrane prior to internalisation. Interestingly, the uptake efficiency scaled nonlinearly with NPs concentration, and therefore, a critical threshold density should trigger an internalisation process at a specific time (19). Together, these experimental data point to a mechanism of NPs transport along cellular membranes which is correlated with the membrane topology. In line with this, experimental data of viral diffusion has identified several putative transmembrane regions specialised for the passage of the HIV-1 gp41 fusion protein, including the membrane-proximal external region (20).

Strictly related to the diffusion pathways of NPs, specific cytotoxicities of metallic NPs have been revealed (21), (22), (23). This is because several metals, such as gold, silver and iron oxide NPs, show variable sizes and properties (21), (22), (23). Specifically, gold NPs range between 5 and 40 nm, silver between 5 and 100 nm and iron oxide between 1 and 100 nm (21), (22), (23). The implications of the size variability of NPs are the existence of different cellular internalisation pathways for the same NP. To support this, it has been demonstrated that there is no toxicity when metallic NPs of gold, silver and iron oxide cross cell membranes without the activation of energy-dependent mechanisms such as endocytosis (12). This allows their potential use, even considering the chemical-physical characteristics, in the medical field. In particular, as reported by Long et al. (24), (25), Fe2O3, and Fe3O4-based NPs could be used in magnetic hyperthermia and magnetic resonance imaging (MRI) for cancer treatment and diagnosis. Furthermore, NPs that enter cells by diffusion are mainly localised in the cytosol with no signs of accumulation in lysosomes, which can cause nanotoxicity due to degradation of the nanostructure and in situ release of metal ions (12).

NPs endocytosis machineries

Endocytosis is predominantly a stepwise process by which cells internalise macromolecules and particles through vesicles derived from the plasma membrane (Figure 1) (26). Molecular interactions regulating endocytosis control the immune response, neurotransmission, intercellular communication, signal transduction and the toxicity of particles (26).

The endocytosis machinery of NPs can be used for structures at least ~60 nm in size. Based on the cell type, the endocytosis mechanisms of NPs may include pinocytosis and/or phagocytosis (27) (Figure 1). Almost all cells can internalise NPs by pinocytosis; however, phagocytosis occurs only in specialised cells, namely “professional phagocytes” including macrophages, neutrophils and monocytes. Both endocytosis processes, by which biomolecules can be internalised into cells, are energy-dependent (27). As endocytosis processes involve actin polymerisation, they occur through the stepwise involvement of GTPases (28).

Hight resolution transmission electron microscopy (HRTEM) images of multi walled carbon nanotubes (MWCNT) [pristine (p) and oxidised]. Low and high magnification images are displayed on the left and right side. The bar is equal to 20 nm.
Figure 2:

Hight resolution transmission electron microscopy (HRTEM) images of multi walled carbon nanotubes (MWCNT) [pristine (p) and oxidised].

Low and high magnification images are displayed on the left and right side. The bar is equal to 20 nm.

Pinocytosis

Pinocytic processes represent an area of particular investigation for generating fast trucks for internalising NP drug complexes (26). Cellular internalisation occurring via pinocytosis machinery consists of progressive invaginations of the cellular membranes to generate cytoplasmatic vesicles that contain the internalised NP (26). Four different pinocytosis mechanisms have been described: macropinocytosis, clathrin-mediated endocytosis, caveolae-mediated endocytosis, and mechanisms independent of clathrin and caveolin (26).

First described by Lewis, macropinocytosis is a pinocytic process by which a relatively large amount of the fluid is enveloped by the cellular membrane (29). By macropinocytosis, viruses such as bluetongue virus-1 and the Ebola virus can be absorbed, as well as bacteria and NPs that generate macropinosomes up to 5 μm in dimension (30), (31), (32). In endothelial cells, it has been shown that the macropinocytosis process can internalise chemokines conjugated to NP complexes, as well as multimeric antibodies against the intercellular adhesion molecule (ICAM-1) (33), (34).

Clathrin-mediated pinocytosis internalises vesicles 150 nm in diameter (32). Clathrin is a triskeleton construction consisting of three heterodimers, each with one heavy and one light chain (35). By linking adaptor proteins, the clathrin structure assembles around the vesicles and mediates pinoytosis through membrane invagination (35). Ultrastructural investigations have shown that the internalisation of NPs by clathrin-mediated pinocytosis is independent of cell type and tissue of origin. This is because both lung fibroblast and liver cell types have been demonstrated to be able to absorb and internalise gold NPs by the clathrin pinocytosis pathway (36).

Caveolin and the caveolar pinocytic processes appear because of the caveolin protein and the ability of its assembly products to bind directly to membrane cholesterol (37), (38). There are three subtypes of caveolin proteins: caveolin-1, caveolin-2 and caveolin-3 (37), (38). The former two proteins are responsible for caveolae formation in non-muscle cells, and the latter in muscle cells; however, the function of individual caveolin proteins may be diverse (37), (38). To support this hypothesis, different investigations have demonstrated a probable tissue-specific function of caveolins, as several mammalian cell lines, such as human colorectal adenocarcinoma cells or blood cell lineages, do not have detectable levels of these proteins (39).

Since the early 1990s, cellular uptake independent of clathrin and caveolin machineries have been investigated. These have utilised different approaches from ultrastructural evaluations to molecular examinations (40). NPs’ vesicles originating from clathrin- and caveolae-independent pinocytosis are about 50 nm in size, and there is clear evidence in animal systems that clathrin-dependent and -independent endocytosis forms may coexist in the same cell (32), (41). By the late 1990s, the idea of clathrin-independent endocytosis was working toward a definition of target organelles for NPs’ pinocitosis machineries. The intracellular organelles that are likely targets of NP include mitochondria and lysosomes (42), (43). Ultrastructural studies in Nicotiana tabacum L. pollen tubes have simultaneously visualised multiple endocytic cargo for gold NPs internalisation (44). A remarkable diversity of endocytic processes has been revealed because of diverse gold NPs’ internalisation sites located in the subapical plasma membrane that recycle NPs through the Golgi apparatus (44). However, these results were not confirmed in human prostate cancer cells in regard to new therapeutic approaches by drugs with targeted toxic effects in cellular organelles (45), (46).

Phagocytosis

Large particles preferentially enter the cellular cytoplasm by phagocytosis, and the ingestion of NPs larger than 500 nm in size generally trigger this process (47). NPs that enter the cell in this way need to be firstly recognised by immunoglobulins (IgG and IgM), complement component (C3, C4, and C5) or blood serum proteins to build NP-protein complexes (48).

Due to the vesicle morphology and requirement for Rho-GTPases, the phagocytosis process can be classified as: (i) Ig receptor type, FcR-mediated termed “zipper-like”; or (ii) CR3 type phagocytosis, termed “trigger-like” (28). Related to Rac1 and Cdc42 activities, in “zipper-like” phagocytosis, tyrosine kinase immunoreceptors are activated with subsequent actin rearrangement, membrane remodelling and intrusion of receptor ligand complex in a bowl with a zipper-lock arrangement (28), (47). Conversely, dependent on RhoA, “trigger-like” phagocytosis is triggered by treatment with phorbol esters, and NPs are dispersed in a large cup (28), (47).

Fate of NPs after internalisation

Following the binding of NP complexes to the cell surface and interaction with the receptor, membrane invagination is induced and a vesicle enclosing the NP is formed (48). The vesicles are internalised and subsequently fuse with lysosomes by forming phagosomes with a diameter of 0.5–10 μm (49). Lastly, by acidification and enzymolysis of lysosomes, the cargo containing phagosomes are destroyed, thereby producing the so-called “lysosome-enhanced Trojan horse effect” (50). This is because the protective intracellular mechanisms working to degrade foreign bodies turn out to be the cause of their toxicity (50). In fact, the release of intracellular ions elicited by the acidic conditions of the lysosomes trigger intracellular toxicity through the dispersion of NPs throughout the internal compartments of cells (50). In experiments using chelating and lysosomotropic agents, the “Trojan horse effect” has been demonstrated for different NPs such as metallic, metal oxide and semiconductor NPs (50).

NPs and cancer: morphological features

The interrelationship among NPs’ morphological parameters (i.e. size and shape) with the cancerous effects of nanostructures has been the focus of several investigations attempting to elucidate the mechanisms of NP toxicity (51). This issue may be approached by two different methods: firstly, by reporting the cancerous effects of natural exposure to NPs such as volcanic ash in restricted areas; and secondly, by exploring the outcomes on cancer pathways in cellular systems exposed to fibrous silicate minerals in the NP form.

NPs present in volcanic ash and thyroid cancer

The cells of organisms have always been unknowingly exposed to NPs (52). However, some natural processes, such as volcanic ash, create environments that may influence the occurrence of inflammatory diseases or the onset of cancers (53). Therefore, geochemical studies on volcanic ash can be considered natural examples of inflammatory and cancerous effects mediated by human exposure to NPs (52), (53), (54). During explosive eruptions, magma is discharged at temperatures of 1000–1300°C, including solid, liquid and gas particles (54). Volcanic ash consists of solid particles with dimensions less than or equal to 2 μm. Mafic magma (enriched with MgO) typically produce coarse ash, while silicic magmas (enriched with SiO2) are associated with eruptions, thereby forming fine ash (54). The shape of the ash particles may vary from irregular to blocky to sub-rounded to elongated based on the magma composition and the eruptive style and process by which they were erupted (54). As visualised by SEM, the superficial faces appear to be arranged in vesicular structures with numerous cavities (55). Many NP metals, such as Pb, Hg, Cu, Zn and Cd, are present in volcanic ash, as well as surface transition metals like Fe2+ (53). Limited studies have evaluated the mechanisms involved in the cellular inflammatory responses to volcanic ash, and despite numerous epidemiological reports that show a significant association between exposed areas under volcanoes and increased cancer incidence, the intracellular cancerous pathways activated by exposure to ash remain unclear (53), (56), (57).

To investigate the in vitro effects of volcanic ash on the inflammatory response, the morphological and chemical features of ash derived from the 2010 eruptions of the Icelandic volcano, Eyjafjallajökull, was investigated (58). The cellular responses to ash were evaluated on the epithelial and macrophage systems of rats and humans, and the results of this study demonstrated an effect of ash from Eyjafjallajökull on alteration of the inflammatory response in macrophages (58).

It has been demonstrated that volcanic ash with a high content of Fe2+ NP may induce oxidative DNA damage in human peripheral blood mononuclear cells (53). This is because the Fe2+ mineral is able to produce the deleterious hydroxyl radical when in contact with hydrogen peroxide (H2O2) (53). In addition to the direct genotoxic effect of OH, the signal role of reactive oxygen species (ROS) in triggering the inflammatory cascade must be considered since redox imbalance chronic inflammation-induced play a pivotal role in cancer onset. The amount of Fe2+ in volcanic ash is specific to each volcano. Mount Etna, located in the eastern part of Sicily (Italy), is the highest and most active volcano in Europe, and produces a solid ash with a high content of Fe2+ NP during eruption (59). During the 2011 Etna eruption, FeO particles were among the top three components of ash fragments, accounting for 10.13% of the weight of ash samples (57) (Table 1). The thyroid gland is the only endocrine organ that uses H2O2, which is required for thyroid hormone synthesis (60). Cultured thyroid cells naturally contain H2O2 as well as antioxidants that protect cells from H2O2-mediated oxidative damage (61). In rats that have the ability to remove H2O2, the loss of this balance results in thyroid cell dysfunction and thyroid diseases (61). Notably, papillary thyroid carcinoma (PTC) is closely linked to an intracellular increase in H2O2 following a change in the disposal mechanisms that regulate H2O2 production, such as reactive oxygen species (ROS) (60). These experimental investigations seem to confirm the epidemiological data on PTC distribution in Sicilian volcanic areas (56), (62). In fact, PTC is the most common form of thyroid cancer, appearing more frequently in the population living in the south-east area of Etna (56), (62), (63). Noteworthy, prevailing winds above the volcano blow toward this area of Etna (64), which affects both the air and ground composition due to huge volcanic ash falls, which have been frequent over the past 20 years (57), (65). In addition, Etna is one of the highest gas and aerosol emitters in the world during “quiescent periods” (66), (67). After being transported, such material may strongly impact the lower flanks of the volcano, posing a potential hazard for the health of local people, particularly in the most inhabited south-east areas of Etna (57). Inhabitants of this area of the Catania province have a significant increase in PTC rates relative to the rates of PTC in other Sicilian provinces, therefore, volcanic ash cannot be excluded as a risk factor for PTC (56), (62). Interestingly, PTC from patients of the Etna volcanic area carry the BRAF V600E gene, a marker of disease aggressiveness, more frequently than the tumours of patients that live elsewhere in Sicily (62). These data led us to investigate in greater depth the natural toxic qualities of NPs that favour the onset of human cancers.

Table 1:

Chemical analyses of volcanic ash collected from 2011 Etna eruption.a

Engineered nanoparticles: carbon nanotubes (CNTs)

In addition to natural NPs, exposure to anthropogenic NPs, such as the by product of combustion processes, can also impact human health. Recently, another cause of NP exposure was identified to be the increased production of several synthesised metallic and non-metallic NPs. Due to their physicochemical properties, CNTs are highly versatile, and therefore, produced in great volumes, improving the structural properties of plastics, rubbers, electronics and composite materials (68). The quantities produced globally are estimated to range between 11 and 1000 t/year (69).

These engineered NPs are allotropes of carbon, named CNTs for their cylindrical shape. They are formed by single or concentric multiple layers of one atom-thick carbon walls (graphene), designated as single-walled CNT (SWCNT, Ø 1–3 nm) and multi-walled CNT (MWCNT, Ø 10–200 nm), respectively (70). For both types of CNTs, the length ranges from a few hundred nanometres to several tens of micrometres. This allows an extremely high length-to-diameter ratio, far superior to any other material, allowing these engineered NPs (ENPs) to have peculiar features (Figure 2).

Starting with graphite, CNTs are produced by electric arc discharge, laser ablation, high-pressure carbon monoxide disproportionation or by chemical vapour deposition (CVD) using metal catalysts such as iron, nickel and cobalt (71), (72). As with all NPs, CNTs have a large surface area to mass ratio, associated with increased surface reactivity. This property, which greatly contributes to their toxicological profile in biological systems, also makes CNTs very attractive in the medical field due to their ability to be used as carriers of bioactive molecules (73), (74), (75).

Several CNT-based delivery systems have been designed due to high nanotube hydrophobicity that, in addition to the high length-to-diameter ratio, enables them to efficiently penetrate biological membranes and accumulate in intracellular compartments (76). Uptake of CNTs into mammalian cells has been confirmed in several cell lines (74), (77). As reported above, the uptake mechanisms for CNTs also include an endocytic pathway, where the CNTs are included in endosomes which subsequently merge with the lysosomes in the perinuclear compartment (74), and a passive diffusion pathway through cellular membranes, which does not consume energy (78). Mu et al. (79) reported that endocytosis-mediated internalisation preferentially occurs for CNT tangles while single CNTs are internalised by passive penetration, also named needle-like crossing.

Regardless of the internalisation mechanism, CNTs can be considered efficient carriers that are capable of targeting drugs to specific cells, thereby reducing the dosages and any side effects (80). Their large surface area, ranging from 50 to 1315 m2/g, allows their conjugation to a variety of therapeutic and imaging molecules (theranosis), and also enables real-time monitoring of the effectiveness of the treatment (81). In addition, the inner cavity of CNTs can be filled with drugs or probe molecules (82). The loading of bioactive molecules into CNTs could overcome several administration problems including insolubility, poor biodistribution and the inability of certain therapeutic or diagnostic molecules to cross cellular barriers (73).

In addition, CNTs are able to improve cell adhesion, dendrite elongation, signal transfer between neurons and network activity (83), making them able to be used in regenerative medicine to restore normal function by tissue engineering approaches (84).

In each case, the use of CNTs in the medical field, together with other applications, involves their functionalisation by post-synthesis surface modification, such as by adding hydrophilic molecules that can bind bioactive molecules for drug delivery. Functionalisation enhances water dispersibility and improves interfacial adhesion. Functional groups are attached to the CNT surface by covalent bonds or non-covalent interactions (van der Waals forces and π-π interactions) between the nanotube surface and hydrophobic/aromatic regions of the amphiphilic molecules. The covalent insertion of surface functional groups (hydroxyl, carboxylic and carbonyl), obtained by submitting pristine CNT to strong acidic conditions, causes a drastic reduction in the length to diameter ratio of the CNT. On the one hand, this reduces toxicity, but on the other hand, it disrupts the sp2 hybridisation of the carbon atoms and erodes the graphitised external layer. This causes a huge increase in surface reactivity, which may have an impact on the safety of CNTs (85).

Regardless of the use of CNTs as nanodelivery systems, for which the biosecurity must be ensured before product approval, the pathological consequences due to unintentional CNT exposure have caused serious concerns in the scientific community (86). However, in vivo and in vitro studies, performed to analyse the health impacts of CNTs, have led to contradictory results. These may be attributed to the different methods of production, the presence of contaminants (i.e. amorphous coal and bioavailable metal catalyst residues), the intrinsic physicochemical features of CNTs and, last but not least, the cell models used to assess the toxicological effects. In addition, due to the high hydrophobicity, CNTs easily aggregate into bundles in the physiological aqueous environment, thereby altering their bioavailability, which makes risk assessment more difficult (87).

Considering the ease with which lightweight nanosized MWCNTs aerosolise and their steadily increased production, inhalation of MWCNTs cannot be excluded. Different to the occupational risk of workplace exposure to pristine and functionalised CNTs, the general population is exposed almost exclusively to functionalised CNTs that are released by composite materials increasingly used in everyday life.

The respiratory system is highly susceptible to CNTs and, due to their morphological similarity to asbestos amphiboles, MWCNTs in particular, the pathological consequences including cytotoxicity, apoptosis, fibrosis, genotoxicity, tumorigenesis and inflammation have been highlighted (88). MWCNTs and asbestos fibres share a needle-like shape, pro-oxidant capability and biopersistence (77), (89). Like asbestos fibres, MWCNTs have the capacity to cause toxic effects by activating several pathways involved in cytotoxic and genotoxic effects. In addition to oxidative stress, mitochondrial impairment, cell cycle arrest, cell death, DNA and chromosomal aberrations, demonstrated by our research group and by several other authors (77), (85), (90), (91), (92), (93), acute and chronic inflammation has been widely studied both in vitro and in vivo (94), (95), (96).

Carbon nanotubes stimulate the production and secretion of inflammatory cytokines and chemokines such as tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6 and IL-8 (97), (98). The known mechanisms of CNT-induced inflammation include both direct induction of IL-1 gene expression, activation of proinflammatory genes such as TNFα, or indirectly by activating the transcription of nuclear factor kappa β (NF-κβ).

Following MWCNT inhalation, epithelial cells and macrophages are exposed. Macrophages phagocytise this foreign matter (99) and induce the response of other immune cells (100). They also release the NLRP3 inflammasome, a multiprotein complex which controls the activation and maturation of the IL-1β cytokine (99). IL-1β, together with IL-1R and IL-18, belong to the IL-1 cytokine family which are important mediators of inflammatory responses (101). In turn, NLRP3 activation is triggered by many different signals including pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Long, needle-like MWCNTs activate NLRP3 by ROS overproduction, inducing IL-1β production (101).

Similar to asbestos (102), the induction of tumorigenesis by CNTs may be mechanistically related to chronic inflammation and a prolonged release of proinflammatory cytokines (103). However, the International Agency for Research on Cancer (IARC) recently classified only MWCNT-7 as being potentially carcinogenic to humans (Group 2B), while all other types of CNTs (SWCNTs and MWCNTs) were inserted in Group 3, i.e. not classified as carcinogens for humans (104). MWCNT-7 (1–19 μm in length; 40–170 nm diameter) has been reported to cause mesotheliomas in male and female rats (105) and bronchioloalveolar adenoma and carcinoma in male mice (106).

Many in vivo and in vitro studies have examined the toxicological and proinflammatory effects of MWCNTs on the lung system (i.e. alveolitis, pulmonary fibrosis, chronic obstructive pulmonary disease, i.e. COPD, and granuloma) which is primarily exposed to these airborne ENPs. Oxidative stress is a common mechanism involved in the lung cell toxicity of CNTs (107), causing lipid peroxidation, mitochondrial impairment and DNA damage. In particular, mitochondrial dysfunction, which may be also related to CNT-induced lipid peroxidation, causes metabolic impairment due to the shift from oxidative phosphorylation to anaerobic glycolysis (108). The increased residence time of electrons in complexes I and III of the respiratory chain (109) causes a decrease in transmembrane mitochondrial potential (Δψm) that can further amplify the CNT-induced redox imbalance through the production of endogenous ROS. Moreover, mitochondrial damage caused by the release of caspase-activating proteins can trigger the intrinsic apoptotic pathway, as observed in lung epithelial cells exposed to asbestos (110) and to oil fly ash (111), (112).

Despite the pathogenic role of redox imbalance, the mechanical damage induced by CNTs should not to be underestimated. This includes genomic instability and dysfunction of the endocytic compartment due to the internalisation of CNTs. In the former, needle-shape CNTs cause disruption of the mitotic spindle or kinetochore proteins (100), while changes in lysosomal permeability lead to the release of hydrolytic enzymes into the cytoplasm, triggering apoptosis. Moreover, as observed by our research group in a human alveolar cell line (77), membrane damage of the acidic compartment causes release of intralysosomal low-molecular-mass iron. This redox-active iron, produced during the autophagocytic degradation of metalloproteins such as cytochromes (113), causes redox imbalance regardless of the presence of bioavailable metal catalyst residues in CNTs (85).

However, inhaled CNTs are also translocated to extrapulmonary organs (114). Owing to their hydrophobicity, these internalised ENPs may cross the blood-brain barrier (BBB) to reach the central nervous system (CNS) where they may cause cytotoxicity of selected neurons in several CNS regions, impairing molecular pathways and contributing to the onset and progression of neurodegenerative diseases (80), (115), (116), (117). The BBB is a complex network comprising brain endothelial cells, astrocytes and pericytes that control the influx and efflux of nutrients and other molecules to the brain parenchyma. The CNTs circumvent the physical and biochemical blockage of the BBB by both energy-dependent pathways (transcytosis) and passive energy-independent mechanisms (i.e. needle-like crossing) (80).

A further potential CNS exposure to CNTs is via olfactory neurons, as was recently highlighted for several airborne pollutants that are strongly related to cognitive impairment and neurodegenerative diseases (118), (119). Direct “nose-to-brain” transport into the olfactory bulbs and other brain regions by anterograde pathways has been demonstrated for ultrafine particulate matters (PMs) deposited on the olfactory epithelium (120), (121). This pathogenic mechanism is plausible, especially considering that the early symptoms of many types of dementia frequently include alterations in smell and anosmia (122).

In particular, this alternative transport pathway through the cribriform plate could allow the direct distribution of ultralight airborne CNTs to the hippocampus, hypothalamus, amygdala, orbitofrontal cortex, thalamus and entorhinal cortex. However, while olfactory epithelium allows foreign matter, which are potential causes of neurodegenerative diseases, to reach specific brain regions, it also opens unimagined opportunities for their treatment, which could include a rapid non-invasive method of administration. While the foreign compounds that are deposited into the respiratory region of the nasal cavity can reach the brain by the systemic circulatory system, the compounds present in the olfactory mucosa reach the circulatory system much quicker. Unlike the former pathway that requires crossing of the BBB, this direct pathway is only restricted by the reduced surface of the olfactory mucosa. Despite this, intranasal administration of drugs for brain-targeted drug delivery increases the delivery efficiency of biocompatible ENPs, which could represent a novel and exciting strategy for the treatment of several neurodegenerative disorders and brain gliomas due to its ability to bypass the BBB.

Regardless of its potential use in brain nanodelivery, the role of CNTs in the pathogenic pathways of neurodegenerative disorders is plausible, but still not sufficiently assessed. Similar to the natural and anthropogenic NPs (120) deposited on the olfactory epithelium, ultralight CNTs could also exploit the direct “nose-to-brain” transport.

In addition to aggregation and deposits of misfolded proteins, impairment of the degradation pathways for damaged cell components, as well as synapse dysfunction and epigenetic deregulation of gene expression, the causes of brain tissue damage include oxidative stress, mitochondrial impairment, DNA damage and neuroinflammation (119). The latter, which cause the onset and progression of neurodegenerative diseases, were observed by our research group in the in vitro experiments, performed in neuronal-like cells exposed to MWCNTs (123). In particular, the high lipid content of nervous tissue, together with its elevated aerobic metabolic activity, makes the brain exceptionally vulnerable to redox imbalance (119). In addition to direct ROS-induced damage of all biological molecules, by products of lipid peroxidation, such as reactive aldehydes (malondialdehyde, MDA, and 4-hydroxynonenal, HNE), play a pivotal role in neurodegeneration, causing genotoxicity and protein damage.

Moreover, also in the CNS, CNT-induced oxidative stress seems to activate NF-κB or AP-1 transcription factors, thereby upregulating the expression of cytokine genes (101). It is well known that chronic inflammation and the prolonged release of proinflammatory cytokines play a key role in neurodegenerative diseases due to microglia activation, which amplifies the oxidative damage leading to a loss of neuronal cells.

Conclusion

Since 1942, NPs have been identified by electron microscopes as “ultrafine particles”, and since 1990, these particles have been the target of emerging technologies, named nanotechnologies, because they involve particles that appear uniquely within the nanometre scale.

Nanoparticles may induce toxicity through cellular mechanisms by which cells internalise nanostructures. Further, Fe2+ NPs have a role in activation on cancerous pathways implied in PTC carcinogenesis. This has been demonstrated in thyroid porcine membrane lipids showing a specific susceptibility to ferrous ion in dependent manner on Fe2+ concentration (124). These data suggest a role of geochemical exposure to volcanic ash in PTC because of high content of Fe2+ NP in volcanic ash of Mount Etna.

Lastly, due to the large surface area and the presence of an inner cavity that can be filled with drugs or probe molecules, an extensive amount of therapeutic and imaging molecules (theranosis) can be conjugated to CNTs. The loading of bioactive molecules into CNTs could overcome several problems related to administration including insolubility, poor biodistribution and the inability of therapeutic or diagnostic molecules to cross cellular barriers.

Acknowledgements

M.C.T., A.D. and G.V. wrote the first draft of the manuscript. D.A. and S.S. contributed to the writing of the manuscript. M.C.T., G.V., A.D., D.A., S.S., R.M.R. and I.P. agree with the manuscript results and conclusions. M.C.T., G.V. and A.D. jointly developed the structure and arguments for the paper. S.S., D.A, R.M.R. and I.P. made critical revisions and approved final version. All authors reviewed and approved of the final manuscript.

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About the article

Corresponding author: Maria Concetta Trovato, MD, PhD, Department of Clinical and Experimental Medicine, University of Messina, Policlinico Universitario, Consolare Valeria 1, 98125 Messina, Italy, Tel.: +39-090-2212590, Fax: +39-090-2212523


Received: 2017-10-10

Accepted: 2018-06-20

Published Online: 2018-09-06

Published in Print: 2018-09-25


Research funding: Authors state no funding involved.

Conflict of interest: Authors state no conflict of interest.

Informed consent: Informed consent is not applicable.

Ethical approval: The conducted research is not related to either human or animal use.


Citation Information: Reviews on Environmental Health, Volume 33, Issue 3, Pages 295–307, ISSN (Online) 2191-0308, ISSN (Print) 0048-7554, DOI: https://doi.org/10.1515/reveh-2017-0036.

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