- Email: [email protected]
Carbon nanotubes and central nervous system: Environmental risks, toxicological aspects and future perspectives
Environmental Toxicology and Pharmacology 65 (2019) 23–30
Contents lists available at ScienceDirect
Environmental Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/etap
Carbon nanotubes and central nervous system: Environmental risks, toxicological aspects and future perspectives
T
Alessio Facciolàa, Giuseppa Visallib, Sebastiano La Maestrac, Manuela Ceccarellia, ⁎ Francesco D’Aleoa, Giuseppe Nunnaria, Giovanni Francesco Pellicanòa, Angela Di Pietrob, a
Department of Clinical and Experimental Medicine, Unit of Infectious Diseases, University of Messina, Italy Department of Biomedical and Dental Sciences and Morphofunctional Imaging, University of Messina, Italy c Department of Health Sciences, University of Genoa, 16132, Genoa, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: Carbon nanotubes Environmental exposure Neurotoxicity Drug delivery
Due to their morphological and physicochemical properties, carbon nanotubes (CNTs) enhance the structural properties of several materials and are produced in great volumes. The production and the manufacturing of CNTs-incorporated products can lead to the potential environmental release of CNTs. For these reasons, CNTs can represent a serious concern for human health. Humans are exposed to nanoparticles through inhalation, ingestion and skin uptake. After their entrance, the particles can reach the Central Nervous System (CNS) through three different pathways: the systemic, olfactory and trigeminal pathways. In the first, through systemic blood circulation, nanoparticles cross both the blood-brain and blood-spinal cord barriers, which are highly selective semipermeable barriers that protect the CNS compartments. The second is the step from the nose to brain route and occurs along axons and via nerve bundles that cross the cribriform plate to the olfactory bulb. In the third, the compounds diffuse through the nasal cavity mucosa to reach the branches of the trigeminal nerve in the olfactory and respiratory regions, and they reach brain stem via axonal transport. After their entrance, CNTs reach the CNS where they may cause cytotoxicity of selected neurons in several CNS regions, impairing molecular pathways and contributing to the onset and progression of chronic brain inflammation, microglia activation and white matter abnormalities with an increased risk for autism spectrum disorders, lower IQ in children, neurodegenerative diseases and stroke. The large surface area to mass ratio of CNTs greatly increases surface reactivity. Despite this property considerable contributes to their toxicological profile in biological systems, also makes CNTs very attractive in the medical field, where they can be used as carriers of bioactive molecules, contrast agents, biological platforms and for many other applications in medicine.
1. Introduction Carbon nanotubes (CNTs) are engineered nanoparticles (NPs) formed by graphene (one atom-thick carbon sheet) that can be single rolled sheet, named single-walled (SWCNTs), or concentric multiple sheets (from 2 to 50, holden together by van der Waals interactions) named multi-walled (MWCNTs). The morphological and physicochemical properties of CNTs allow to enhance the structural properties of plastics, rubbers and composite materials (De Volder et al., 2013). Similar to all NPs, the large surface area to mass ratio of CNTs greatly increases surface reactivity. Despite this property considerable contributes to their toxicological profile in biological systems, also makes CNTs very attractive in the medical field, where they can be used as
⁎
carriers of bioactive molecules, contrast agents, biological platforms and for many other applications in medicine (Liopo et al., 2006; Ye & Chen, 2011; Mehra and Palakurthi, 2016). Worldwide CNT production ranges from 350 tons/year to 500 tons/ year and it is assumed that the global CNT market will increase from $2.26 billion in 2015 to $5.64 billion in 2020 (Nowack et al., 2013). The substantial increase in production and the widespread usage of CNTs in industrial applications and consumer products creates the potential for their release, causing an increase of human and environmental exposures to CNTs (Gottschalk and Nowack, 2011) Even if little is known about the fate of CNTs during the full life cycle of polymer/ CNTs nanocomposites, the release has definitely increased (Nowack et al., 2013). During use and after disposal, these materials are exposed
Corresponding author. E-mail address: [email protected] (A. Di Pietro).
https://doi.org/10.1016/j.etap.2018.11.006 Received 27 July 2018; Received in revised form 5 November 2018; Accepted 22 November 2018 Available online 22 November 2018 1382-6689/ © 2018 Published by Elsevier B.V.
Environmental Toxicology and Pharmacology 65 (2019) 23–30
A. Facciolà et al.
The sidewall covalent functionalization is obtained by some chemical reactions and allows the attachment of organic group to the tips and wall surface of CNT such as the amino groups, added by 1,3-dipolar cycloaddition that keeps unchanged the length and the surface of the CNTs, thus not altering their electronic properties More simply, the addition of carboxylic group is obtained by strong acidic oxidation which also causes a drastic reduction in the length to diameter ratio of the CNT, increasing their biocompatibility (Visalli et al., 2015b). However, carboxylation, requiring strong reaction condition, disrupts the sp2 hybridisation of the carbon atoms and erodes the graphene external layer. In addition to unavoidable surface defects in hexagon ring produced during CNT synthesis, this simple and quick post synthetic process increases surface reactivity, which may affect safety of CNTs (Visalli et al., 2017a, 2017b). Non-covalent functionalization involves van der Waals forces, π-π interactions, electrostatic force and hydrogen bonds between the nanotube surface and hydrophobic/aromatic regions of the amphiphilic organic molecules, forming supramolecular complexes. Non-covalent methods for surface functionalization of CNTs preserve the aromatic structure of CNT and is performed by coating CNTs with several hydrophilic materials such as surfactants, polymers, or, limited to the medical field, biological macromolecules such as protein/peptides and nucleic acids, especially single-stranded (ss) DNA. Anionic, cationic and nonionic surfactants (sodium dodecyl sulphate: SDS, Sodium dodecylbenzene sulphonate: SDBS, sodium cholate: SC, Triton X-100, formed by the hydrophilic polyethylene oxide chains and by an aromatic hydrophobic group) have been applied in the postsynthetic treatment of CNTs and, as shown by electronic transmission microscopy (TEM), surfactants cover CNTs that are maintained uniformly dispersed. However, the surfactants, notoriously able to permeabilize plasma membranes, are cytotoxic not allowing their use in medical fields. More recently, biocompatible surfactants are available. These include the nonionic triblock copolymers named Pluronics that, as a function of the length of their chain, effectively allow CNT dispersion, preserving cell integrity. Several stable nontoxic polymers have been used for the noncovalent functionalization of CNTs. Among others, they include polyethyleneimine: PEI poly (diallyldimethylammonium chloride: PDDC, poly (acrylic acid): PAA, polyoxyethylene sorbitan monooleate: PS-80, poly-m-aminobenzene sulphonic acid: PABS, polyethylene glycol: PEG and, polyvinylpyrrolidone: PPY (Antonelli et al., 2010). Other than to increase water solubility, these nanocomposite materials show a high stability in biological media. Due to the limited cytotoxicity, the water soluble highly hydrated PEG, is the hydrophilic polymer more commonly used. (i.e. PEGylation) to coating CNTs for medical use. Furthermore, depending on the length of the PEG chain, PEGylation increases half-life of CNT in blood vessels, preventing the immunological clearance, due to opsonisation.
to a wide range of environmental agents, such as ultraviolet radiation, ozone and microbial agents that could cause the release of CNTs from the polymer matrices. The potential mechanisms of release during the polymer/CNTs nanocomposite lifecycle include biodegradation, mechanical damage (abrasion, scratching and sanding), washing, diffusion, matrix degradation (photo-, thermo- and hydrolytic) and incineration. For these reasons, CNTs can represent a serious concern for environmental and human health (Pacurari et al., 2016). Novack et al. recognises different settings of CNT release and subsequent human exposure distinguished in professional, consumer and environmental exposures. For example, the professional exposure is unlikely for injection moulding but very likely for manufacturing. The consumer exposure is unlikely for sports equipment and electronics but very likely for tires and textiles. The environmental exposure is unlikely or very unlikely for injection moulding, manufacturing, sports equipment and electronics but very likely for tires and textiles. In general, it can be concluded that the expected release of CNTs from products and articles is unlikely, except for in the manufacturing and subsequent processing of tires and textiles and in recycling operations (2013). However, except for high energy machining processes, which affect production workers only that can also be accidentally exposed to high CNTs concentrations, most likely the resulting exposure for the general population will be low. 1.1. Synthesis and postsynthetic treatment of CNTs SWCNTs and MWCNTs have similar lengths that range from a few hundred nanometres to several tens of micrometres, while the diameter is considerably different and equal to 1–3 nm and 10–200 nm for SWCNTs and MWCNTs, respectively. The extremely high length to diameter ratio (i.e., aspect ratio) awards to these engineered NPs distinctive characteristics. Referring to more specific reviews (Karimi et al., 2015), we briefly report that CNTs are synthesized by several methods developed over time and with variable yield and degree of purity. These include: i) electric arc discharge, in an inert gas atmosphere formed by Ar or He, ii) laser ablation and, iii) chemical vapour deposition (CVD) using hydrocarbons as carbon source (acetylene, propylene, ethylene or methane) and metal catalysts such as iron, nickel and cobalt or rare-earth metals. In turn, CVD methods include several synthesis processes such as hot filament CVD (HFCVD), plasma enhanced CVD (PECVD), radio frequency plasma-enhanced CVD (RF-PECVD), microwave plasma-enhanced CVD (MPECVD), water-assisted CVD (WA-CVD). Whatever is the process of synthesis, one must consider that major batch-to-batch variations in length, width and number of walls exist between different CNT-preparations since it is difficult to control all the parameters (De Volder et al., 2013). A prerequisite for their application in almost all fields and, above all, in medicine, is given by surface engineering of CNTs, i.e. functionalization, that, modifying the morphological and physicochemical characteristics of CNTs, also changes their impact on health and has a dual purpose. Due to the van der Waals forces occurring at the surface, pristine CNTs tend to agglomerate in aqueous environment. On the one hand, the functionalization mitigates the strong hydrophobicity, enhancing their dispersibility in water solutions and, on the other, it allows the bond of the graphene surface to form composite materials. Functional groups are attached to the CNT surface by covalent bonds or non-covalent interactions. The strong chemical bonds between carbon atoms in the hexagonal ring, conferring an outstanding chemical stability, allow several functionalization treatments that, generally, maintain unabridged the honeycomb structure of the CNT walls. The covalent insertion of surface charged organic functional groups, attached onto the CNT backbone, create electrostatic repulsive forces between the single tubes, promoting dispersibility. As assessed by zeta potential measurement, the electrostatic repulsion between particles stabilize aqueous colloidal systems, hindering the particle aggregation.
1.2. Cellular uptake of CNTs Due to their shape, CNTs can enter through the skin or lungs and reach other sites. In general, these NPs can penetrate into cells through two different ways: the endocytic pathway and/or passive diffusion (Fig. 1) and then translocate into different subcellular compartments. In comparison to pristine, functionalized CNTs, obtained by adding hydrophilic groups are preferentially singly dispersed and cross cell membranes by a passive diffusion pathway. Unlike the endocytosismediated internalisation of CNT tangles, this mechanism reduces cytotoxicity not causing lysosomial content leakage by overload endosomes (Visalli et al., 2017a, 2017b). Moreover, functionalization improves interfacial adhesion, promoting CNTs-cell membranes interactions and it is essential to regulate biological half-life, biodistribution and pharmacokinetics of CNTs (Mu et al., 2009) In the first mechanism, CNTs are internalised in endosomes that 24
Environmental Toxicology and Pharmacology 65 (2019) 23–30
A. Facciolà et al.
perivascular microglia survey the systemic blood environment for adverse stimuli, such as inflammatory cytokines and soluble/particulate pollutants that reach the systemic circulation via respiratory tract penetration. Small lipophilic molecules easily enter into the blood and cross the BBB compared to high molecular weight and hydrophilic molecules (Kafa et al., 2016). Several “in vitro” studies highlighted a similar capability of CNTs to cross the BBB. Kafa et al., using a BBB co-culture model comprised of primary porcine brain endothelial cells and primary rat astrocytes, revealed that functionalised MWCNTs (f-MWCNTs) were quickly internalised via endocytosis and then released from endocytic vesicles near the abluminal side of the cells within 24–48 h. This process did not damage the cell membranes and did not involve the tight junctions during translocation, suggesting a transcellular route to cross the BBB. The same group investigated the role played by the diameter of CNTs on BBB translocation. Particularly, they showed that “wide” MWCNTs (∼35.9 nm diameter) translocated in a higher percentage than the “thin” MWCNTs (∼9.2 nm diameter) (Kafa et al., 2016). Moreover, other studies demonstrated the capacity of CNTs to penetrate biological membranes without perturbing the membrane integrity (Wang et al., 2016). Similarly, “in vivo” studies provided the evidence of the capacity of CNTs to reach the brain tissues beyond the BBB. In particular, although in vitro data suggested that wide f-MWCNTs are more efficient in crossing the BBB, the available in vivo data suggests that uptake in healthy brain tissues after systemic injection is favoured by f-MWNTs with smaller diameters. In any case, wider MWCNTs exhibit better brain retention, and it was demonstrated that the conjugation of CNTs with angiopeptin-1 (ANG) enhances brain uptake of wider MWCNTs but not of thinner ones. ANG modified f-MWCNTs of wider diameter seem to be the most suitable candidates among the ones studied for BBB and brain tumour double targeting (Costa et al., 2016). Wang et al. have studied the brain accumulation of f-MWCNTs using single photon emission computed tomography/computed tomography (SPECT/CT) imaging and autoradiography after systemic intravenous administration of radiolabelled f-MWCNTs. The accumulation was evident, especially in the mid-brain region, at the early time points between 0.5 h and 4 h (2016).
Fig. 1. Different mechanisms of CNTs cellular uptake.
then merge with lysosomes to form endolysosomes. For all types of CNTs (SWCNTS and MWCNTs), the process is energy-dependent and clathrin-dependent (Mu et al., 2009). Instead, passive diffusion, also called “needle-like penetration”, is an energy-independent process that occurs after the interaction between CNTs and the plasma membrane surface, which is promoted by the high hydrophobicity of the CNTs. By crossing the phospholipid bilayer, CNTs penetrate into the cytoplasm where, similarly to all foreign particles and damaged cellular components, they can be subjected to autophagocytic processes over time. Particularly, clusters of CNTs are internalised through endocytic processes, whereas single CNTs mainly use passive membrane diffusion (Costa et al., 2016). The internalisation mechanism of CNTs is dependent not only on their physicochemical properties but also on the phagocytic nature of cells. In professional phagocytic cells, CNTs penetrate mainly through phagocytosis, and they accumulate in the lysosomal compartment, even if the stoppage of this pathway still allows the uptake of CNTs by passive diffusion. Conversely, it was established that SWCNTs penetrate the cell membrane of non-phagocytic cells by passive diffusion and accumulate in the mitochondria (Costa et al., 2016). Antonelli et al. investigated the differential uptake of CNTs into human monocyte-derived macrophages of fluorescently labelled nanotubes as a function of their length (2010). The results showed that CNTs > 400 nm in length were mainly localised in endosomes, while the fluorescent signal from shorter CNTs was diffuse throughout the cytosol, supporting their extravesicular localisation. This suggests that the shortening of CNTs allows for their uptake by passive diffusion, even in phagocytic cells (Antonelli et al., 2010).
1.3.2. Olfactory pathway The nasal cavity provides an alternative transport pathway for direct brain delivery of some inhaled particles and airborne pollutants that are strongly related to cognitive impairment and neurodegenerative diseases. The nose to brain pathway involves both the olfactory nerve and trigeminal nerve (Selvaraj et al., 2017). The nasal cavity consists of three regions: vestibule, respiratory and olfactory regions with a surface area of approximately 160 cm2 in humans. The first is the anterior external region that opens to the outside and is not involved in absorptive functions. The second is formed by different cellular types (ciliated and non-ciliated columnar cells, mucus secreting goblet cells and basal cells) and is mainly involved in the absorption of pollutants/ drugs The third region is the olfactory region and includes the olfactory receptor and basal and sustentacular cells with a surface area of 10 cm2. The olfactory system originates with specialised bipolar neurons, named olfactory neurons, found within the olfactory epithelium that lines this portion of the nasal cavity. Projections from the olfactory neurons form the olfactory nerve (cranial nerve I), which ultimately terminates in the olfactory bulb after the nerve fibres pass through the skull. Molecules reach the olfactory receptor neurons by a paracellular or transcellular mechanism (Selvaraj et al., 2017). The integrity of nasal epithelium along with the tight junctions, desmosomes, adherent junctions and spaces between the epithelial cells only partially prevent the entry of compounds by paracellular transport. The olfactory pathway is the determining step of the nose to brain route and occurs along axons and via nerve bundles that cross the cribriform plate to the olfactory bulb. From the olfactory nerve fibres, the substances can reach the cerebrospinal fluid (CSF) and olfactory bulb (Dhuria et al., 2009).
1.3. CNT uptake in central nervous system Humans are exposed to NPs through inhalation, ingestion and skin uptake. After their entrance, the particles can reach the central nervous system (CNS) through three different pathways: the systemic, olfactory and trigeminal pathways (Selvaraj et al., 2017) (Fig. 2). 1.3.1. Systemic pathway Though the systemic blood circulation, NPs cross the blood- brain barrier (BBB) and blood-spinal cord barrier (BSCB), which are the highly selective semipermeable barriers that protect the compartments of the CNS. These structures limit the entry to small lipophilic molecules or those for which an active and specific transporter is expressed (e.g., glucose, leptin, ghrelin or transferrin-bound iron). Different cell types compose these structures: endothelial cells, pericytes and astrocytes. Age, diseases, pollutants and toxins can compromise the protective role of the BBB, which normally hinders the entry of macromolecules, toxins and small organic drugs (Selvaraj et al., 2017). Some brain regions have a more fenestrated BBB; in these areas the 25
Environmental Toxicology and Pharmacology 65 (2019) 23–30
A. Facciolà et al.
Fig. 2. Different pathways of CNTs neurological uptake.
pollutants escape the metabolic barrier of the olfactory epithelium, reaching the brain with neurotoxic effects. Oberdörster et al. demonstrated that the olfactory bulb was a site of NP accumulation after its exposure via inhalation (2002).
The molecules are distributed from the CSF to the brain by mixing with interstitial fluid in the cerebral parenchyma (Selvaraj et al., 2017). Olfactory transport involves two different pathways: one intracellular and the other extracellular. The intracellular mechanism involves the internalisation of materials by an olfactory neuron in an endocytic vesicle that reaches the neuron's projection site, and, finally, it is released via exocytosis. In the extracellular pathway, the materials cross the nasal epithelium and reach the lamina propria, before being transported externally along the length of the neuronal axon. The intracellular transport requires hours to days to reach different regions of the brain. Instead, the extracellular, involving transport through perineural channels, is very fast and takes only a few minutes to directly reach the brain (Crowe et al., 2018). Transport of airborne xenobiotics along the olfactory nerve is a route to reach the CNS that bypasses the protective BBB. Various metals (e.g., manganese, iron, cadmium, thallium, mercury, cobalt and zinc) as well as carbon particles appear to move into the brain following inhalation or intranasal/tracheal instillation exposure. However, not all studies confirm these transport routes, and they highlighted how interspecies differences are important. In humans, the relative surface area of the nasal olfactory mucosa is much smaller than that of rodents (5% vs. 50%), making humans less susceptible to airborne pollutants. In nasal mucosa, there is a “metabolic” barrier present and phase II enzymes, such as UFP-glucuronosyltransferase, epoxide hydrolase, sulfotransferases and glutathione S-transferases, detoxify reactive metabolites originating following phase 1 reactions, or, in any case, they conjugate harmful pollutants after the contact between mucosal cells and xenobiotics (Zhang et al., 2012). This barrier provides a first line of defence for the brain, limiting axonal transport of exogenous material from the olfactory nerve cells to the olfactory bulb. However, several airborne
1.3.3. Trigeminal pathway The trigeminal nerve (i.e., cranial nerve V) is formed by three branches—ophthalmic, maxillary and mandibular, respectively. The ophthalmic and maxillary branches play an important role in nose to brain transport that can occur by intracellular transport (endocytosis) or by axonal transport. Neurons from these branches reach the nasal mucosa and some segments end in the olfactory bulb. Once the compounds diffuse through the nasal cavity mucosa, they reach the branches of the trigeminal nerve in the olfactory and respiratory regions; and, via axonal transport, they reach the brain stem. This pathway was demonstrated by Thorne et al. who reported that the intranasal administration of insulin-like growth factor-I (IGF-I) rapidly reached the brain via the trigeminal neuronal pathway (2004). After going through the mucus and bypassing mucociliary clearance, there are several mechanisms involved in transport through the mucosa: paracellular, transcellular, carrier-mediated transport, receptor-mediated transport and transcytosis. While the paracellular route occurs between adjacent cells through intercellular spaces, the transcellular route refers to transport across cells and may occur by carrier-mediated transport or by endocytosis. 1.4. Toxicological effects of CNTs in biological systems The high hydrophobicity combined with the high aspect ratio enables CNTs to efficiently cross biological membranes, and their uptake, 26
Environmental Toxicology and Pharmacology 65 (2019) 23–30
A. Facciolà et al.
by the activation of the NF-κB or AP-1 transcription factors (Palomäki et al., 2011). The microglia activation, due to prolonged release of proinflammatory cytokines, has a pathogenic role in neurodegenerative diseases leading to a loss of neuronal cells. A two-fold faster rate of decline in AD patients is associated with systemic inflammation as determined via increases in serum TNF-α. The mechanism is confirmed by McAlpine et al. who showed the inhibition of TNF-α signalling significantly reduced amyloid beta protein deposition in the brain in a mouse model of AD (2009). Bardi et al. investigated the neurotoxicity of different types of CNTs through cortical stereotactic injection in mouse brain. Particularly, oxMWCNTs-NH3+ caused cytokine and glial cell activation, suggesting that oxidation can contribute to a sustained inflammatory reaction in healthy brain, while MWCNTs-NH3+ was better tolerated, causing only a local and transient inflammatory response. This different effect was observed despite the uptake levels in different brain tissue cells (astrocytes, microglia and neurons) being similar for the two engineered NPs. Compared to MWCNTs-NH3+, the release of the proinflammatory cytokines TNF-α and IL-1β and microglia activation were significantly higher after injection of ox-MWCNTs-NH3+ (2013). A recent follow-up study revealed that microglia are involved in the cytotoxic response generated by f-MWCNTs in brain tissue. This study showed that exposure to various types of f-MWCNTs (ox-MWCNTs, oxMWCNTs-NH3+ and ox-MWCNTs-am-NH3+) resulted in different effects in base on cellular types. At doses up to 100 μg/mL, they did not induce any damage in neuronal cultures isolated from the two different brain regions studied (frontal cortex (FCO) and striatum (ST) within 24 h. In contrast, the assayed f-MWCNTs caused cytotoxicity in microglia-containing cultures. The authors concluded that the observed brain region-specific sensitivity to MWCNTs exposure most likely was related to the number of microglial cells in different brain regions (Bussy et al., 2015). CNT-induced cytotoxicity was observed even in neuronal cell cultures. The toxic effect, reported by Bussy et al. in human neuroblastoma cells (SH-SY5Y) incubated with co-polymer (Pluronic F127)-coated nanotubes, was dependent on the dispersibility, concentration, incubation time (up to 2 weeks) and contaminant content of CNTs. Particularly, concerning the impurities, CNTs with high iron catalyst content decreased cell viability of pheochromocytoma cells and disrupted the cytoskeleton (Bussy et al., 2015). Visalli et al., using differentiated SH-SY5Y, highlighted the neurotoxic and proinflammatory effects of pristine (p) and functionalised (f) MWCNTs (i.e., submitted to strong acid oxidation to allow the covalent insertion of terminal carboxyl groups into CNTs). The authors observed significant oxidative damage in the neuronal-like cells confirmed by the analysis of DNA damage using the comet assay. Moreover, they highlighted that MWCNT-induced ROS overproduction activated a proinflammatory response as shown by transcript levels of IL-1β, IL-6 and TNF-α, confirmed by the cytokine levels detected in the cell supernatants. The authors hypothesised that the superimposable pro-oxidant activity of both CNTs was imputable to excessive lengths with regard to the p-MWCNTs (10–20 μm vs. 200–1000 nm) and to higher surface reactivity with regard to the f-MWCNTs (2017). Even if the presence of carboxyl groups enhances water dispersibility and causes a reduction in the length to diameter ratio of CNTs, making them more biocompatible, these effects are nullified by the erosion acid-induced by the graphene external layers. This increases the surface reactivity and, consequently, the cellular toxicity (Visalli et al., 2015b). CNT-induced neurotoxic effects include a decrease in cell activity, as shown by Chen et al. that demonstrated the inhibition effect on CA1 glutamatergic synaptic transmission in rat hippocampal slices in vitro (Chen et al., 2014). Gao et al. explored the in vivo relationship between the autophagic flux and synaptic plasticity damage caused by MWCNTs, observing autophagy enhancement and synaptic plasticity damage in the CA1 area (2015). The same authors demonstrated that exposure to MWCNTs
as described above, has been observed in several mammalian cell lines (Pantarotto et al., 2004; Visalli et al., 2015a, 2015b). After their internalisation and according to their physicochemical properties and functionalisation, CNTs accumulate in various subcellular compartments, such as the cytosol, endosomes, perinuclear region, mitochondria and nucleus (Pantarotto et al., 2004; Mu et al., 2009). Whereas lightweight nanosized CNTs aerosolise their unintentional inhalation is highly probable and has raised considerable concern about the possible impact on human health (Pacurari et al., 2016). In the first years, toxicological studies focused almost exclusively on the effects of CNTs in the respiratory system. This is attributable to the morphological similarity of CNTs, especially MWCNTs, to asbestos amphiboles (Palomäki et al., 2011; Donaldson et al., 2013). Like asbestos fibres, CNTs active several pathways, causing apoptosis, fibrosis, genotoxicity, tumourigenesis and inflammation in the lungs as highlighted in a previous review (Trovato et al., 2018). Briefly, following inhalation, epithelial cells and macrophages of the respiratory system are exposed to CNTs which are phagocytized by macrophages, triggering the response of other immune cells. The macrophages also release the inflammasome NLRP3, a multiprotein complex whose activation is caused by many different signals, including pathogenassociated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) (Palomäki et al., 2011). Long, needle-like CNTs activate NLRP3 by reactive oxygen species (ROS) overproduction. The inflammatory cascade continues and NLRP3, in turn, induces the expression of IL-1 gene production of IL-1β and cytokines (Palomäki et al., 2011; Arnoldussen et al., 2015). Also lung epithelial cells actively contribute to the inflammatory response since CNT exposure causes the activation of the nuclear factor kappa β (NF-κβ) (Arnoldussen et al., 2015). In addition to proinflammatory effects, a common mechanism of CNT toxicity is the oxidative damage due to redox imbalance that causes in exposed cells, other than DNA oxidative damage, metabolic impairment and, due to mitochondrial djsfunction, apoptosis, as observed in alveolar cells exposed to asbestos (Hartz et al., 2008; Palomäki et al., 2011; Donaldson et al., 2013) and to oil fly ash (Di Pietro et al., 2011; Visalli et al., 2015a, 2015b). 1.5. Toxicological effects of CNTs on CNS Recent studies indicate that airborne pollutants alter BBB function. Following exposure, endothelial cell damage in the cerebral vasculature, is confirmed by the increases of both intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression. Moreover, in vitro studies using whole brain rat capillaries have shown that treatment with particulate matter (also formed by NPs) causes production of proinflammatory cytokines and ROS and decreases expression of various tight junction proteins (Hartz et al., 2008). Owing to their high hydrophobicity, CNTs may cross the BBB to reach several CNS regions where they may damage selected neurons. CNTs impair molecular pathways and contribute to the onset and progression of chronic brain inflammation, microglia activation and white matter abnormalities with an increased risk for autism spectrum disorders, lower IQ in children, neurodegenerative diseases (Parkinson’s disease (PD) Alzheimer’s disease (AD) and multiple sclerosis) and stroke (Bussy et al., 2015; Migliore et al., 2015; Costa et al., 2016; Kafa et al., 2016). The pathological effects CNT-induced of brain tissue are due to ROS overproduction, mitochondrial dysfunction, genotoxicity and neuroinflammation (Bussy et al., 2015). Considering the high lipid levels and the elevated aerobic metabolic activity of nervous tissue in addition to the proportionally lower antioxidant content, the brain is mostly susceptible to redox imbalance (Migliore et al., 2015). The byproducts of lipid peroxidation, such as reactive aldehydes (malondialdehyde (MDA) and 4-hydroxynonenal (HNE), contribute to neurodegeneration caused by the ROS-induced damage of all biological macromolecules. Moreover, also in the CNS, CNTs trigger the inflammatory response 27
Environmental Toxicology and Pharmacology 65 (2019) 23–30
A. Facciolà et al.
surface area, ranging from 50 to 1315 m2/g, coupled to very low weight makes CNTs efficient drug delivery platform considering that also the inner cavity of CNTs can be filled with drugs or probe molecules. CNTs may also provide protection from enzymatic degradation or clearance, thus increasing the half-life of the drug within the biological fluids. As reported above, by the endocytic and/or passive diffusion pathways CNTs are internalised in cell compartments. When the CNTs are finely dispersed, the uptake happens preferentially by passive penetration while the endocytosis-mediated internalisation happens for CNT tangles (Mu et al., 2009). Regardless of the uptake pathway inside the cells, the loaded bioactive molecules are released as a result of the low pH and the lysosomal enzymes present in endolysosomes and autophagosomes, respectively. Then, free drug will be distributed in the interested cell compartment as shown by Iannazzo et al. for the intercalating drug doxorubicin (2017), which causes cancer cell death and could allow the treatment of gliomas and other brain cancer types. The possibility of attacking many molecules of different types, including targeting ligands that are recognised only by the cells to be treated (as antibodies, peptides or proteins, vitamins, etc.) allow to reduce drug dosages and, consequently, any side effects, maximizing the therapeutic effect (Mehra and Palakurthi, 2016). Monoclonal antibodies, disialoganglioside GD2, vitamins and all other compounds recognised by receptors expressed on the phospholipid bilayer of nerve cells can be used as targeting ligands to recognise specific cells in the CNS (Iannazzo et al., 2017). Their features also allow the use of CNTs in theranostic nanomedicine, integrating the therapy with the diagnosis by the co-delivery of diagnostic agents in addition to therapeutic agents. This allows us to monitor the effectiveness of pharmacological treatment at the cellular and molecular levels. Diagnostic agents commonly used in theranostic nanomedicine and suitable to be combined with CNTs in in vivo and ex vivo assessments include fluorescent probes for optical imaging, paramagnetic metals, such as iron oxides for magnetic resonance imaging, radionuclides for nuclear imaging, iodine and other elements (Ye and Chen, 2011).
could negatively affect the cognitive abilities of rats, particularly their learning ability and memory. Concerning the latter, the field excitatory postsynaptic potential (fEPSP) slopes were significantly reduced in the treated group, suggesting an impairment of the hippocampal long-term potential (LTP), an essential functional indicator of synaptic plasticity. This effect can be explained by a significant reduction of the N-methyl D-aspartate receptor subtype 2B (NMDAR2B) that regulates the activity of the N-methyl D-aspartate (NMDA) receptor and plays a vital role in LTP induction of learning and memory function. Also, the protein synaptophysin (SYP), an important membrane protein of synaptic vesicles, which is closely connected with synaptic plasticity and cognitive processes, was decreased. Moreover, the exposure to MWCNTs damaged pyramidal neurons in the CA1 hippocampal region, an area linked to conditioned reflex and memory (Gao et al., 2015). The biopersistence of SWCNT-PEG in CNS was studied in vivo by Dal Bosco et al. that, after infusion in the hippocampus, did not observe changes in the spatial recognition memory and locomotor activity in rats treated seven days before. At the same time by Raman analysis of the hippocampal homogenates they established the presence of SWCNT-PEG, despite histological analysis did not show remarkable morphological alterations (2015a). In a short time study, the same reaserch group observed that SWCNT-PEG caused lipid peroxidation in the hippocampus. However this effect was transient and overcome by the mobilization of antioxidant defenses already after 24 h (Dal Bosco et al., 2015b). Overall, the analysis of the studies so far performed shows a potential health risk as well as the possible onset of neurological disorders. Considering the widespread presence of CNTs in new materials produced by using nanotechnologies, the risk should not be underestimated. 1.6. Potential use of CNTs in neurology Despite their intrinsic cytotoxicity as reported above, several features of CNTs make their use in the medical field extremely attractive. Due to the high hydrophobicity, in addition to high length to diameter ratio, CNTs efficiently cross the membranes without damaging them and accumulate in intracellular compartments (Mu et al., 2009). This makes them innovative nanoplatforms for drug delivery, allowing unprecedented opportunities in the therapeutic treatment of neurological diseases, including infections, brain and spinal trauma, stroke and brain cancer as shown in several in vitro and in vivo models of neurological disorders (Liopo et al., 2006; Santos et al., 2014; Costa et al., 2016; Mehra and Palakurthi, 2016; Wang et al., 2016). Other than CNTs, carbon-based nano platforms experimented in nanomedicine include, fullerene and, more recently, graphene quantum dot and graphene oxide (GO) (Iannazzo et al., 2017). To today, however, few experimental models have been used these latter in neurological field, despite their capability to delivery biologically active cargoes into CNS. The pharmacological treatment of brain-related disorders by the systemic route is notoriously difficult, and several drugs, proteins, peptides and genetic materials cannot be administered parenterally. As previously reported, the needle-like shape of CNTs makes them suitable to cross the BBB, overcoming the issue due to drug insolubility, poor biodistribution and inability of several bioactive molecules to pass through BBB. In particular, the low permeability of anti-tumour drugs across the BBB when administered systemically has opened up new possibilities for CNT-based modalities in neuroncology. This feature of CNTs avoids problems related to the invasiveness of several therapeutic treatments and patient compliance, which complicate clinical applications in neurology. Compared to free drug and to many other nanoplatforms recently developed such as polymeric NPs, liposomes, dendrimers, nanoshells, micelles, nanogold particles and superparamagnetic particles, both SWCNTs and MWCNTs show a greater drug loading capacity, targeting and efficacy in neurological disorders (Costa et al., 2016). Their large
1.6.1. CNTs-based NIR therapy in neuroncology An inherent optical feature of the CNTs that has remarkable perspectives in neuroncology is the absorption of near-infrared radiation (NIR) not adsorbed by biological tissues. Time and dose dependent adsorbed radiation is converted to heat, allowing a local increase of temperature, i.e. hyperthermia (Santos et al., 2014), exclusively in the cancer cells where CNTs have been engulfed thanks to presence of targeting ligands. Due to preferential internalisation of CNTs, the cytotoxicity is extremely selective and in the malignant cells hyperthermia triggers apoptosis and other death pathway. Instead, the surrounding healthy cells do not suffer any cytotoxic effect (Santos et al., 2014). Compared to conventional cancer therapies, experimental data based on the CNT use as heating devices for hyperthermal therapy provides interesting perspective in neuro-oncology due to the lack of systemic side effects and the ability to preserve the functionality of neuronal tissue (Santos et al., 2014). As targeting ligand adsorbed to CNTs to recognise and eradicate by hyperthermia specific cells can be used monoclonal antibody. However, some aspects, still unresolved, must be underlined before the method can be applied in clinical trials. As above reported CNTs can be administrated by systemic route but is still necessary to optimize the NIR radiation step considering the poor tissue penetration of NIR radiation by external heat sources. 1.6.2. CNTs in neuroprosthetic devices and in neuroregenerative applications Stroke, heat stress, head and spinal cord trauma and bleeding that occurs in the brain, considerably worse the quality of life of the survivors, causing neuronal deterioration. One promising strategy for treatment of these conditions is to support and promote neurite and 28
Environmental Toxicology and Pharmacology 65 (2019) 23–30
A. Facciolà et al.
cells. In fact, Mehra and Palakurthi. used the tripeptide arginine-glycine-aspartic acid (RGD) to decorate SWCNTs considering that RGV has a high affinity for the αvβ3 integrins that are overexpressed on cancer cells. Similarly, Asn-Gly-Arg (NGR), by binding to CD13, have been exploited as ligand to target CNTs cargos to cancer cells, overexpressing the receptor (2016). The covalent insertion of COOH groups, other than to make dispersible and hydrophilic CNT suspensions, allows to add further functional groups to CNTs as amine and thiols, necessary to anchor bioactive molecules (Antonelli et al., 2010). In comparison to covalent functionalization, CNTs coated by peptides, nucleic acids and polymers enhance considerably the cargo biocompatibility, maintaining cellular viability almost unchanged. Moreover, it is useful to underline that in non covalent functionalization the weak interactions between the complex components do not ensure a sufficient biostability which is essential for their medical use. All that highlights as a complete and detailed physicochemical and biological characterization of the CNTs is needful for their use in medical field (Antonelli et al., 2010; Costa et al., 2016).
axonal growth by implanting nanometer-scale scaffolds using tissueengineering approaches (Liopo et al., 2006). Owing to their electrical conductivity and to the morphological features, that mimic tubular structures of CNS, such as axons and dendrites, CNTs are a potential regenerative matrices of neuronal tissue, allowing neuronal growth, promoting neuronal functions and stimulating neuronal interconnections. By using suitably coated CNTs as substrates, these very promising features were highlighted in in vitro model of nerve cells (Mehra and Palakurthi, 2016). CNT-based nanomaterials seem to form an optimal scaffold to increase number and length of neurite outgrowth and branching. The property can be further enhanced by adsorbing the CNTs with bioactive molecules such as nerve growth factor (NGF), or brain-derived neurotrophic factor (BDNF) that allow cell differentiation and neuron survival (Matsumoto et al., 2007). Particularly interesting and attributable to the high electrical conductivity of these engineered nanomaterials are the observed increases of neuronal circuits. When nerve cells are cultured into CNT substrates a neuronal network for the transmission of electrical signals within neurons is formed. As observed by Malarkey et al. (2009) the conductivity of CNTs-play a key role in the based neuronal growth and elongation of dendrites. However, as shown by the same group, only CNTs with a narrow range of conductivity (< 0.3 S cm−1) induce neuronal growth.
2. Conclusion Due to their morphological and physicochemical properties, CNTs are highly versatile, enhancing the structural properties of several materials and, therefore, they are produced in great volumes. For these reasons, they can represent a serious concern for human health due to professional, consumer and environmental exposures. Despite their reported intrinsic cytotoxicity, several features of CNTs make their use in the medical field extremely attractive, such as drug delivery, allowing unprecedented opportunities in the therapeutic treatment of neurological diseases, including infections, brain and spinal trauma, stroke and brain cancer. However, some still unresolved aspects must be addressed and clarified before these NPs can be applied in clinical trials. In particular, it is crucial to determine the long-term toxicological outcomes of CNTs and their fate within living systems.
1.6.3. Requirements for CNT use in neurology Only highly purified, well-tailored and suitably coated CNTs show improved safety. A post synthetic step, needful for the CNTs use in medical application, is their purification from both metals and carbonaceous particles. These contaminants are byproduct of the synthesis process, often responsible for the CNT toxicity (Bussy et al., 2015). In particular, as widely demonstrated, the pro-oxidant effect of metals (Fe, Ni, Co etc.), used as catalyst in the CNTs synthesis, decreases cell viability, causing lipid peroxidation and oxygen radical formation (Visalli et al., 2015b). The purification treatments remarkably cut down CNT toxicity in the CNS, as reported in several in vitro study (Pacurari et al., 2016). To reduce the length to diameter ratio of the CNT, that is responsible for cytotoxicity, shortening procedures include ultrasonication, steam-purification and mechanical methods, in addition to strong acidic oxidation (Bussy et al., 2015). Moreover, a prerequisite for their application in the biological field is provided by the surface engineering of CNTs (i.e., functionalisation). In the physiological environment pristine CNTs tend to agglomerate making their uptake in the cells of interest uneven. As well as increasing the dispersibility, the functionalisation allows the bonding of bioactive molecules to the groups added on the graphene surface. Moreover, functionalization improves interfacial adhesion, promoting CNTs-cell membranes interactions and it is essential to regulate biological halflife, biodistribution and pharmacokinetics of CNTs (Antonelli et al., 2010). As above reported, to make these nanoplatforms more biocompatible, several biocompatible hydrophilic compounds have been tested as coatings (Karimi et al., 2015; Mehra and Palakurthi, 2016), including biological macromolecules. While the CNT-DNA complexes are based on the π–π interactions between the aromatic bases of DNA and the CNTs surface, the supramolecular biocompatible complexes formed by CNT and peptides and/or proteins are based on hydrophobic interaction. In addition to natural protein such as human serum albumin (HAS), many synthetic peptides are used to decorate CNTs, representing an efficient dispersion agent for CNTs. In particular, specific amino acids, such as tryptophan, phenylalanine and tyrosine, play a pivotal role in peptides/proteins- CNTs interactions while the presence of multiple amino groups, enhances the water solubility of complexes and they work as a linker for drug compounds. In addition, specific peptides could be used to target these nanoplatforms for drug delivery to specific
Funding This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. Conflict of interest The authors declare that they have no conflict of interest. Transparency document The Transparency document associated with this article can be found in the online version. References Antonelli, A., Serafini, S., Menotta, M., Sfara, C., Pierigé, F., Giorgi, L., Ambrosi, G., Rossi, L., Magnani, M., 2010. Improved cellular uptake of functionalized single-walled carbon nanotubes. Nanotechnology 21 (42), 425101. https://doi.org/10.1088/09574484/21/42/425101. Arnoldussen, Y.J., Skogstad, A., Skaug, V., Kasem, M., Haugen, A., Benker, N., Weinbruch, S., Apte, R.N., Zienolddiny, S., 2015. Involvement of IL-1 genes in the cellular responses to carbon nanotube exposure. Cytokine 73 (1), 128–137. Bardi, G., Nunes, A., Gherardini, L., Bates, K., Al-Jamal, K.T., Gaillard, C., Prato, M., Bianco, A., Pizzorusso, T., Kostarelos, K., 2013. Functionalized carbon nanotubes in the brain: cellular internalization and neuroinflammatory responses. PLoS One 8 (11), e80964. https://doi.org/10.1371/journal.pone.0080964. Bussy, C., Al-Jamal, K.T., Boczkowski, J., Lanone, S., Prato, M., Bianco, A., Kostarelos, K., 2015. Microglia determine brain region-specific neurotoxic responses to chemically functionalized carbon nanotubes. ACS Nano 25 (8), 7815–7830 9. Chen, T., Yang, J.J., Zhang, H., Ren, G.G., Yang, Z., Zhang, T., 2014. Multi-walled carbon nanotube inhibits CA1 glutamatergic synaptic transmission in rat’s hippocampal slices. Toxicol. Lett. 229, 423–429. Costa, P.M., Bourgognon, M., Wang, J.T., Al-Jamal, K.T., 2016. Functionalised carbon
29
Environmental Toxicology and Pharmacology 65 (2019) 23–30
A. Facciolà et al.
a drug delivery perspective. Drug Discov. Today 21 (4), 585–597. https://doi.org/10. 1016/j.drudis.2015.11.011. Migliore, L., Uboldi, C., Di Bucchianico, S., Coppedè, F., 2015. Nanomaterials and neurodegeneration. Environ. Mol. Mutagen. 56 (2), 149–170. Mu, Q., Broughton, D.L., Yan, B., 2009. Endosomal leakage and nuclear translocation of multiwalled carbon nanotubes: developing a model for cell uptake. Nano Lett. 12, 4370–4375. Nowack, B., David, R.M., Fissan, H., Morris, H., Shatkin, J.A., Stintz, M., Zepp, R., Brouwer, D., 2013. Potential release scenarios for carbon nanotubes used in composites. Environ. Int. 59, 1–11. Oberdörster, G., Sharp, Z., Atudorei, V., Elder, A., Gelein, R., Lunts, A., Kreyling, W., Cox, C., 2002. Extrapulmonary translocation of ultrafine carbon particles following wholebody inhalation exposure of rats. J. Toxicol. Environ. Health A 65 (20), 1531–1543. Pacurari, M., Lowe, K., Tchounwou, P.B., Kafoury, R., 2016. A review on the respiratory system toxicity of carbon nanoparticles. Int. J. Environ. Res. Public Health 13 (3). https://doi.org/10.3390/ijerph13030325. pii: E325. Palomäki, J., Välimäki, E., Sund, J., Vippola, M., Clausen, P.A., Jensen, K.A., Savolainen, K., Matikainen, S., Alenius, H., 2011. Long, needle-like carbon nanotubes and asbestos activate the NLRP3 inflammasome through a similar mechanism. ACS Nano 5 (9), 6861–6870. Pantarotto, D., Singh, R., McCarthy, D., Erhardt, M., Briand, J.P., Prato, M., Kostarelos, K., Bianco, A., 2004. Functionalized carbon nanotubes for plasmid DNA gene delivery. Angew. Chem. Int. Ed. Engl. 43 (39), 5242–5246. Santos, T., Fang, X., Chen, M.T., Wang, W., Ferreira, R., Jhaveri, N., Gundersen, M., Zhou, C., Pagnini, P., Hofman, F.M., Chen, T.C., 2014. Sequential administration of carbon nanotubes and near-infrared radiation for the treatment of gliomas. Front. Oncol. 4, 180. Selvaraj, K., Gowthamarajan, K., Karri, V.V.S.R., 2017. Nose to brain transport pathways an overview: potential of nanostructured lipid carriers in nose to brain targeting. Artif. Cells Nanomed. Biotechnol. 28, 1–8. https://doi.org/10.1080/21691401.2017. 1420073. Thorne, R.G., Pronk, G.J., Padmanabhan, V., Frey 2nd, W.H., 2004. Delivery of insulinlike growth factor-I to the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration. Neuroscience 127 (2), 481–496. Trovato, M.C., Andronico, D., Sciacchitano, S., Ruggeri, R.M., Picerno, I., Di Pietro, A., Visalli, G., 2018. Nanostructures: between natural environment and medical practice. Rev. Environ. Health. https://doi.org/10.1515/reveh-2017-0036. Visalli, G., Baluce, B., Bertuccio, M., Picerno, I., Di Pietro, A., 2015a. Mitochondrialmediated apoptosis pathway in alveolar epithelial cells exposed to the metals in combustion-generated particulate matter. J. Toxicol. Environ. Health A 78 (11), 697–709. Visalli, G., Currò, M., Iannazzo, D., Pistone, A., Pruiti Ciarello, M., Acri, G., Testagrossa, B., Bertuccio, M.P., Squeri, R., Di Pietro, A., 2017a. In vitro assessment of neurotoxicity and neuroinflammation of homemade MWCNTs. Environ. Toxicol. Pharmacol. 56, 121–128. https://doi.org/10.1016/j.etap.2017.09.005. Visalli, G., Bertuccio, M.P., Iannazzo, D., Piperno, A., Pistone, A., Di Pietro, A., 2015b. Toxicological assessment of multi-walled carbon nanotubes on A549 human lung epithelial cells. Toxicol. In Vitro 29, 352–362. Visalli, G., Facciolà, A., Iannazzo, D., Piperno, A., Pistone, A., Di Pietro, A., 2017b. The role of the iron catalyst in the toxicity of multi-walled carbon nanotubes (MWCNTs). J. Trace Elem. Med. Biol. 43, 153–160. Wang, J.T., Rubio, N., Kafa, H., Venturelli, E., Fabbro, C., Ménard-Moyon, C., Da Ros, T., Sosabowski, J.K., Lawson, A.D., Robinson, M.K., Prato, M., Bianco, A., Festy, F., Preston, J.E., Kostarelos, K., Al-Jamal, K.T., 2016. Kinetics of functionalised carbon nanotube distribution in mouse brain after systemic injection: Spatial to ultrastructural analyses. J. Control. Release 224, 22–32. https://doi.org/10.1016/j. jconrel.2015.12.039. Ye, Y., Chen, X., 2011. Integrin targeting for tumor optical imaging. Theranostics 1, 102–126. Zhang, H., Liu, H., Davies, K.J., Sioutas, C., Finch, C.E., Morgan, T.E., Forman, H.J., 2012. Nrf2-regulated phase II enzymes are induced by chronic ambient nanoparticle exposure in young mice with age-related impairments. Free Radic. Biol. Med. 52 (9), 2038–2046. https://doi.org/10.1016/j.freeradbiomed.2012.02.042.
nanotubes: From intracellular uptake and cell-related toxicity to systemic brain delivery. J. Control. Release 241, 200–219. https://doi.org/10.1016/j.jconrel.2016.09. 033. Crowe, T.P., Greenlee, M.H.W., Kanthasamy, A.G., Hsu, W.H., 2018. Mechanism of intranasal drug delivery directly to the brain. Life Sci. 195, 44–52. https://doi.org/10. 1016/j.lfs.2017.12.025. Dal Bosco, L., Weber, G.E., Parfitt, G.M., Paese, K., Gonçalves, C.O., Serodre, T.M., Furtado, C.A., Santos, A.P., Monserrat, J.M., Barros, D.M., 2015a. PEGylated carbon nanotubes impair retrieval of contextual fear memory and alter oxidative stress parameters in the rat hippocampus. Biomed Res. Int., 104135. https://doi.org/10. 1155/2015/104135. Dal Bosco, L., Weber, G.E., Parfitt, G.M., Cordeiro, A.P., Sahoo, S.K., Fantini, C., Klosterhoff, M.C., Romano, L.A., Furtado, C.A., Santos, A.P., Monserrat, J.M., Barros, D.M., 2015b. Biopersistence of PEGylated carbon nanotubes promotes a delayed antioxidant response after infusion into the rat hippocampus. PLoS One 15 (6), e0129156. https://doi.org/10.1371/journal.pone.0129156. 10. De Volder, M.F., Tawfick, S.H., Baughman, R.H., Hart, A.J., 2013. Carbon nanotubes: present and future commercial applications. Science 339 (6119), 535–539. Dhuria, S.V., Hanson, L.R., Frey, W.H., 2009. Novel vasoconstrictor formulation to enhance intranasal targeting of neuropeptide therapeutics to the central nervous system. J. Pharmacol. Exp. Ther. 328, 312–320. Di Pietro, A., Visalli, G., Baluce, B., Micale, R.T., La Maestra, S., Spataro, P., De Flora, S., 2011. Oxidative damage in human epithelial alveolar cells exposed in vitro to oil fly ash transition metals. Int. J. Hyg. Environ. Health 214 (2), 138–144. Donaldson, K., Poland, C.A., Murphy, F.A., MacFarlane, M., Chernova, T., Schinwald, A., 2013. Pulmonary toxicity of carbon nanotubes and asbestos-similarities and differences. Adv. Drug Deliv. Rev. 65, 2078–2086. Gao, J., Zhang, X., Yu, M., Ren, G., Yang, Z., 2015. Cognitive deficits induced by multiwalled carbon nanotubes via the autophagic pathway. Toxicology 337, 21–29. https://doi.org/10.1016/j.tox.2015.08.011. Gottschalk, F., Nowack, B., 2011. The release of engineered nanomaterials to the environment. J. Environ. Monit. 13 (5), 1145–1155. Hartz, A.M., Bauer, B., Block, M.L., Hong, J.S., Miller, D.S., 2008. Diesel exhaust particles induce oxidative stress, proinflammatory signaling, and P-glycoprotein up-regulation at the blood-brain barrier. FASEB J. 22, 2723–2733. Iannazzo, D., Pistone, A., Salamò, M., Galvagno, S., Romeo, R., Giofré, S.V., Branca, C., Visalli, G., Di Pietro, A., 2017. Graphene quantum dots for cancer targeted drug delivery. Int. J. Pharm. 518 (1–2), 185–192. https://doi.org/10.1016/j.ijpharm. 2016.12.060. Kafa, H., Wang, J.T., Rubio, N., Klippstein, R., Costa, P.M., Hassan, H.A., Sosabowski, J.K., Bansal, S.S., Preston, J.E., Abbott, N.J., Al-Jamal, K.T., 2016. Translocation of LRP1 targeted carbon nanotubes of different diameters across the blood-brain barrier in vitro and in vivo. J. Control. Release 225, 217–229. https://doi.org/10.1016/j. jconrel.2016.01.031. Karimi, M., Solati, N., Amiri, M., Mirshekari, H., Mohamed, E., Taheri, M., Hashemkhani, M., Saeidi, A., Estiar, M.A., Kiani, P., Ghasemi, A., Basri, S.M., Aref, A.R., Hamblin, M.R., 2015. Carbon nanotubes part I: preparation of a novel and versatile drug-delivery vehicle. Expert Opin. Drug Deliv. 12 (7), 1071–1087. https://doi.org/10.1517/ 17425247.2015.1003806. Liopo, A.V., Stewart, M.P., Hudson, J., Tour, J.M., Pappas, T.C., 2006. Biocompatibility of native and functionalized single-walled carbon nanotubes for neuronal interface. J. Nanosci. Nanotechnol. 6 (5), 1365–1374. Malarkey, E.B., Fisher, K.A., Bekyarova, E., Liu, W., Haddon, R.C., Parpura, V., 2009. Conductive single-walled carbon nanotube substrates modulate neuronal growth. Nano Lett. 9 (1), 264–268. Matsumoto, K., Sato, C., Naka, Y., Kitazawa, A., Whitby, R.L., Shimizu, N., 2007. Neurite outgrowths of neurons with neurotrophin-coated carbon nanotubes. J. Biosci. Bioeng. 103 (3), 216–220. McAlpine, F.E., Lee, J.K., Harms, A.S., Ruhn, K.A., Blurton-Jones, M., Hong, J., Das, P., Golde, T.E., LaFerla, F.M., Oddo, S., Blesch, A., Tansey, M.G., 2009. Inhibition of soluble TNF signaling in a mouse model of Alzheimer’s disease prevents pre-plaque amyloid-associated neuropathology. Neurobiol. Dis. 34 (1), 163–177. Mehra, N.K., Palakurthi, S., 2016. Interactions between carbon nanotubes and bioactives:
30
Contents lists available at ScienceDirect
Environmental Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/etap
Carbon nanotubes and central nervous system: Environmental risks, toxicological aspects and future perspectives
T
Alessio Facciolàa, Giuseppa Visallib, Sebastiano La Maestrac, Manuela Ceccarellia, ⁎ Francesco D’Aleoa, Giuseppe Nunnaria, Giovanni Francesco Pellicanòa, Angela Di Pietrob, a
Department of Clinical and Experimental Medicine, Unit of Infectious Diseases, University of Messina, Italy Department of Biomedical and Dental Sciences and Morphofunctional Imaging, University of Messina, Italy c Department of Health Sciences, University of Genoa, 16132, Genoa, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: Carbon nanotubes Environmental exposure Neurotoxicity Drug delivery
Due to their morphological and physicochemical properties, carbon nanotubes (CNTs) enhance the structural properties of several materials and are produced in great volumes. The production and the manufacturing of CNTs-incorporated products can lead to the potential environmental release of CNTs. For these reasons, CNTs can represent a serious concern for human health. Humans are exposed to nanoparticles through inhalation, ingestion and skin uptake. After their entrance, the particles can reach the Central Nervous System (CNS) through three different pathways: the systemic, olfactory and trigeminal pathways. In the first, through systemic blood circulation, nanoparticles cross both the blood-brain and blood-spinal cord barriers, which are highly selective semipermeable barriers that protect the CNS compartments. The second is the step from the nose to brain route and occurs along axons and via nerve bundles that cross the cribriform plate to the olfactory bulb. In the third, the compounds diffuse through the nasal cavity mucosa to reach the branches of the trigeminal nerve in the olfactory and respiratory regions, and they reach brain stem via axonal transport. After their entrance, CNTs reach the CNS where they may cause cytotoxicity of selected neurons in several CNS regions, impairing molecular pathways and contributing to the onset and progression of chronic brain inflammation, microglia activation and white matter abnormalities with an increased risk for autism spectrum disorders, lower IQ in children, neurodegenerative diseases and stroke. The large surface area to mass ratio of CNTs greatly increases surface reactivity. Despite this property considerable contributes to their toxicological profile in biological systems, also makes CNTs very attractive in the medical field, where they can be used as carriers of bioactive molecules, contrast agents, biological platforms and for many other applications in medicine.
1. Introduction Carbon nanotubes (CNTs) are engineered nanoparticles (NPs) formed by graphene (one atom-thick carbon sheet) that can be single rolled sheet, named single-walled (SWCNTs), or concentric multiple sheets (from 2 to 50, holden together by van der Waals interactions) named multi-walled (MWCNTs). The morphological and physicochemical properties of CNTs allow to enhance the structural properties of plastics, rubbers and composite materials (De Volder et al., 2013). Similar to all NPs, the large surface area to mass ratio of CNTs greatly increases surface reactivity. Despite this property considerable contributes to their toxicological profile in biological systems, also makes CNTs very attractive in the medical field, where they can be used as
⁎
carriers of bioactive molecules, contrast agents, biological platforms and for many other applications in medicine (Liopo et al., 2006; Ye & Chen, 2011; Mehra and Palakurthi, 2016). Worldwide CNT production ranges from 350 tons/year to 500 tons/ year and it is assumed that the global CNT market will increase from $2.26 billion in 2015 to $5.64 billion in 2020 (Nowack et al., 2013). The substantial increase in production and the widespread usage of CNTs in industrial applications and consumer products creates the potential for their release, causing an increase of human and environmental exposures to CNTs (Gottschalk and Nowack, 2011) Even if little is known about the fate of CNTs during the full life cycle of polymer/ CNTs nanocomposites, the release has definitely increased (Nowack et al., 2013). During use and after disposal, these materials are exposed
Corresponding author. E-mail address: [email protected] (A. Di Pietro).
https://doi.org/10.1016/j.etap.2018.11.006 Received 27 July 2018; Received in revised form 5 November 2018; Accepted 22 November 2018 Available online 22 November 2018 1382-6689/ © 2018 Published by Elsevier B.V.
Environmental Toxicology and Pharmacology 65 (2019) 23–30
A. Facciolà et al.
The sidewall covalent functionalization is obtained by some chemical reactions and allows the attachment of organic group to the tips and wall surface of CNT such as the amino groups, added by 1,3-dipolar cycloaddition that keeps unchanged the length and the surface of the CNTs, thus not altering their electronic properties More simply, the addition of carboxylic group is obtained by strong acidic oxidation which also causes a drastic reduction in the length to diameter ratio of the CNT, increasing their biocompatibility (Visalli et al., 2015b). However, carboxylation, requiring strong reaction condition, disrupts the sp2 hybridisation of the carbon atoms and erodes the graphene external layer. In addition to unavoidable surface defects in hexagon ring produced during CNT synthesis, this simple and quick post synthetic process increases surface reactivity, which may affect safety of CNTs (Visalli et al., 2017a, 2017b). Non-covalent functionalization involves van der Waals forces, π-π interactions, electrostatic force and hydrogen bonds between the nanotube surface and hydrophobic/aromatic regions of the amphiphilic organic molecules, forming supramolecular complexes. Non-covalent methods for surface functionalization of CNTs preserve the aromatic structure of CNT and is performed by coating CNTs with several hydrophilic materials such as surfactants, polymers, or, limited to the medical field, biological macromolecules such as protein/peptides and nucleic acids, especially single-stranded (ss) DNA. Anionic, cationic and nonionic surfactants (sodium dodecyl sulphate: SDS, Sodium dodecylbenzene sulphonate: SDBS, sodium cholate: SC, Triton X-100, formed by the hydrophilic polyethylene oxide chains and by an aromatic hydrophobic group) have been applied in the postsynthetic treatment of CNTs and, as shown by electronic transmission microscopy (TEM), surfactants cover CNTs that are maintained uniformly dispersed. However, the surfactants, notoriously able to permeabilize plasma membranes, are cytotoxic not allowing their use in medical fields. More recently, biocompatible surfactants are available. These include the nonionic triblock copolymers named Pluronics that, as a function of the length of their chain, effectively allow CNT dispersion, preserving cell integrity. Several stable nontoxic polymers have been used for the noncovalent functionalization of CNTs. Among others, they include polyethyleneimine: PEI poly (diallyldimethylammonium chloride: PDDC, poly (acrylic acid): PAA, polyoxyethylene sorbitan monooleate: PS-80, poly-m-aminobenzene sulphonic acid: PABS, polyethylene glycol: PEG and, polyvinylpyrrolidone: PPY (Antonelli et al., 2010). Other than to increase water solubility, these nanocomposite materials show a high stability in biological media. Due to the limited cytotoxicity, the water soluble highly hydrated PEG, is the hydrophilic polymer more commonly used. (i.e. PEGylation) to coating CNTs for medical use. Furthermore, depending on the length of the PEG chain, PEGylation increases half-life of CNT in blood vessels, preventing the immunological clearance, due to opsonisation.
to a wide range of environmental agents, such as ultraviolet radiation, ozone and microbial agents that could cause the release of CNTs from the polymer matrices. The potential mechanisms of release during the polymer/CNTs nanocomposite lifecycle include biodegradation, mechanical damage (abrasion, scratching and sanding), washing, diffusion, matrix degradation (photo-, thermo- and hydrolytic) and incineration. For these reasons, CNTs can represent a serious concern for environmental and human health (Pacurari et al., 2016). Novack et al. recognises different settings of CNT release and subsequent human exposure distinguished in professional, consumer and environmental exposures. For example, the professional exposure is unlikely for injection moulding but very likely for manufacturing. The consumer exposure is unlikely for sports equipment and electronics but very likely for tires and textiles. The environmental exposure is unlikely or very unlikely for injection moulding, manufacturing, sports equipment and electronics but very likely for tires and textiles. In general, it can be concluded that the expected release of CNTs from products and articles is unlikely, except for in the manufacturing and subsequent processing of tires and textiles and in recycling operations (2013). However, except for high energy machining processes, which affect production workers only that can also be accidentally exposed to high CNTs concentrations, most likely the resulting exposure for the general population will be low. 1.1. Synthesis and postsynthetic treatment of CNTs SWCNTs and MWCNTs have similar lengths that range from a few hundred nanometres to several tens of micrometres, while the diameter is considerably different and equal to 1–3 nm and 10–200 nm for SWCNTs and MWCNTs, respectively. The extremely high length to diameter ratio (i.e., aspect ratio) awards to these engineered NPs distinctive characteristics. Referring to more specific reviews (Karimi et al., 2015), we briefly report that CNTs are synthesized by several methods developed over time and with variable yield and degree of purity. These include: i) electric arc discharge, in an inert gas atmosphere formed by Ar or He, ii) laser ablation and, iii) chemical vapour deposition (CVD) using hydrocarbons as carbon source (acetylene, propylene, ethylene or methane) and metal catalysts such as iron, nickel and cobalt or rare-earth metals. In turn, CVD methods include several synthesis processes such as hot filament CVD (HFCVD), plasma enhanced CVD (PECVD), radio frequency plasma-enhanced CVD (RF-PECVD), microwave plasma-enhanced CVD (MPECVD), water-assisted CVD (WA-CVD). Whatever is the process of synthesis, one must consider that major batch-to-batch variations in length, width and number of walls exist between different CNT-preparations since it is difficult to control all the parameters (De Volder et al., 2013). A prerequisite for their application in almost all fields and, above all, in medicine, is given by surface engineering of CNTs, i.e. functionalization, that, modifying the morphological and physicochemical characteristics of CNTs, also changes their impact on health and has a dual purpose. Due to the van der Waals forces occurring at the surface, pristine CNTs tend to agglomerate in aqueous environment. On the one hand, the functionalization mitigates the strong hydrophobicity, enhancing their dispersibility in water solutions and, on the other, it allows the bond of the graphene surface to form composite materials. Functional groups are attached to the CNT surface by covalent bonds or non-covalent interactions. The strong chemical bonds between carbon atoms in the hexagonal ring, conferring an outstanding chemical stability, allow several functionalization treatments that, generally, maintain unabridged the honeycomb structure of the CNT walls. The covalent insertion of surface charged organic functional groups, attached onto the CNT backbone, create electrostatic repulsive forces between the single tubes, promoting dispersibility. As assessed by zeta potential measurement, the electrostatic repulsion between particles stabilize aqueous colloidal systems, hindering the particle aggregation.
1.2. Cellular uptake of CNTs Due to their shape, CNTs can enter through the skin or lungs and reach other sites. In general, these NPs can penetrate into cells through two different ways: the endocytic pathway and/or passive diffusion (Fig. 1) and then translocate into different subcellular compartments. In comparison to pristine, functionalized CNTs, obtained by adding hydrophilic groups are preferentially singly dispersed and cross cell membranes by a passive diffusion pathway. Unlike the endocytosismediated internalisation of CNT tangles, this mechanism reduces cytotoxicity not causing lysosomial content leakage by overload endosomes (Visalli et al., 2017a, 2017b). Moreover, functionalization improves interfacial adhesion, promoting CNTs-cell membranes interactions and it is essential to regulate biological half-life, biodistribution and pharmacokinetics of CNTs (Mu et al., 2009) In the first mechanism, CNTs are internalised in endosomes that 24
Environmental Toxicology and Pharmacology 65 (2019) 23–30
A. Facciolà et al.
perivascular microglia survey the systemic blood environment for adverse stimuli, such as inflammatory cytokines and soluble/particulate pollutants that reach the systemic circulation via respiratory tract penetration. Small lipophilic molecules easily enter into the blood and cross the BBB compared to high molecular weight and hydrophilic molecules (Kafa et al., 2016). Several “in vitro” studies highlighted a similar capability of CNTs to cross the BBB. Kafa et al., using a BBB co-culture model comprised of primary porcine brain endothelial cells and primary rat astrocytes, revealed that functionalised MWCNTs (f-MWCNTs) were quickly internalised via endocytosis and then released from endocytic vesicles near the abluminal side of the cells within 24–48 h. This process did not damage the cell membranes and did not involve the tight junctions during translocation, suggesting a transcellular route to cross the BBB. The same group investigated the role played by the diameter of CNTs on BBB translocation. Particularly, they showed that “wide” MWCNTs (∼35.9 nm diameter) translocated in a higher percentage than the “thin” MWCNTs (∼9.2 nm diameter) (Kafa et al., 2016). Moreover, other studies demonstrated the capacity of CNTs to penetrate biological membranes without perturbing the membrane integrity (Wang et al., 2016). Similarly, “in vivo” studies provided the evidence of the capacity of CNTs to reach the brain tissues beyond the BBB. In particular, although in vitro data suggested that wide f-MWCNTs are more efficient in crossing the BBB, the available in vivo data suggests that uptake in healthy brain tissues after systemic injection is favoured by f-MWNTs with smaller diameters. In any case, wider MWCNTs exhibit better brain retention, and it was demonstrated that the conjugation of CNTs with angiopeptin-1 (ANG) enhances brain uptake of wider MWCNTs but not of thinner ones. ANG modified f-MWCNTs of wider diameter seem to be the most suitable candidates among the ones studied for BBB and brain tumour double targeting (Costa et al., 2016). Wang et al. have studied the brain accumulation of f-MWCNTs using single photon emission computed tomography/computed tomography (SPECT/CT) imaging and autoradiography after systemic intravenous administration of radiolabelled f-MWCNTs. The accumulation was evident, especially in the mid-brain region, at the early time points between 0.5 h and 4 h (2016).
Fig. 1. Different mechanisms of CNTs cellular uptake.
then merge with lysosomes to form endolysosomes. For all types of CNTs (SWCNTS and MWCNTs), the process is energy-dependent and clathrin-dependent (Mu et al., 2009). Instead, passive diffusion, also called “needle-like penetration”, is an energy-independent process that occurs after the interaction between CNTs and the plasma membrane surface, which is promoted by the high hydrophobicity of the CNTs. By crossing the phospholipid bilayer, CNTs penetrate into the cytoplasm where, similarly to all foreign particles and damaged cellular components, they can be subjected to autophagocytic processes over time. Particularly, clusters of CNTs are internalised through endocytic processes, whereas single CNTs mainly use passive membrane diffusion (Costa et al., 2016). The internalisation mechanism of CNTs is dependent not only on their physicochemical properties but also on the phagocytic nature of cells. In professional phagocytic cells, CNTs penetrate mainly through phagocytosis, and they accumulate in the lysosomal compartment, even if the stoppage of this pathway still allows the uptake of CNTs by passive diffusion. Conversely, it was established that SWCNTs penetrate the cell membrane of non-phagocytic cells by passive diffusion and accumulate in the mitochondria (Costa et al., 2016). Antonelli et al. investigated the differential uptake of CNTs into human monocyte-derived macrophages of fluorescently labelled nanotubes as a function of their length (2010). The results showed that CNTs > 400 nm in length were mainly localised in endosomes, while the fluorescent signal from shorter CNTs was diffuse throughout the cytosol, supporting their extravesicular localisation. This suggests that the shortening of CNTs allows for their uptake by passive diffusion, even in phagocytic cells (Antonelli et al., 2010).
1.3.2. Olfactory pathway The nasal cavity provides an alternative transport pathway for direct brain delivery of some inhaled particles and airborne pollutants that are strongly related to cognitive impairment and neurodegenerative diseases. The nose to brain pathway involves both the olfactory nerve and trigeminal nerve (Selvaraj et al., 2017). The nasal cavity consists of three regions: vestibule, respiratory and olfactory regions with a surface area of approximately 160 cm2 in humans. The first is the anterior external region that opens to the outside and is not involved in absorptive functions. The second is formed by different cellular types (ciliated and non-ciliated columnar cells, mucus secreting goblet cells and basal cells) and is mainly involved in the absorption of pollutants/ drugs The third region is the olfactory region and includes the olfactory receptor and basal and sustentacular cells with a surface area of 10 cm2. The olfactory system originates with specialised bipolar neurons, named olfactory neurons, found within the olfactory epithelium that lines this portion of the nasal cavity. Projections from the olfactory neurons form the olfactory nerve (cranial nerve I), which ultimately terminates in the olfactory bulb after the nerve fibres pass through the skull. Molecules reach the olfactory receptor neurons by a paracellular or transcellular mechanism (Selvaraj et al., 2017). The integrity of nasal epithelium along with the tight junctions, desmosomes, adherent junctions and spaces between the epithelial cells only partially prevent the entry of compounds by paracellular transport. The olfactory pathway is the determining step of the nose to brain route and occurs along axons and via nerve bundles that cross the cribriform plate to the olfactory bulb. From the olfactory nerve fibres, the substances can reach the cerebrospinal fluid (CSF) and olfactory bulb (Dhuria et al., 2009).
1.3. CNT uptake in central nervous system Humans are exposed to NPs through inhalation, ingestion and skin uptake. After their entrance, the particles can reach the central nervous system (CNS) through three different pathways: the systemic, olfactory and trigeminal pathways (Selvaraj et al., 2017) (Fig. 2). 1.3.1. Systemic pathway Though the systemic blood circulation, NPs cross the blood- brain barrier (BBB) and blood-spinal cord barrier (BSCB), which are the highly selective semipermeable barriers that protect the compartments of the CNS. These structures limit the entry to small lipophilic molecules or those for which an active and specific transporter is expressed (e.g., glucose, leptin, ghrelin or transferrin-bound iron). Different cell types compose these structures: endothelial cells, pericytes and astrocytes. Age, diseases, pollutants and toxins can compromise the protective role of the BBB, which normally hinders the entry of macromolecules, toxins and small organic drugs (Selvaraj et al., 2017). Some brain regions have a more fenestrated BBB; in these areas the 25
Environmental Toxicology and Pharmacology 65 (2019) 23–30
A. Facciolà et al.
Fig. 2. Different pathways of CNTs neurological uptake.
pollutants escape the metabolic barrier of the olfactory epithelium, reaching the brain with neurotoxic effects. Oberdörster et al. demonstrated that the olfactory bulb was a site of NP accumulation after its exposure via inhalation (2002).
The molecules are distributed from the CSF to the brain by mixing with interstitial fluid in the cerebral parenchyma (Selvaraj et al., 2017). Olfactory transport involves two different pathways: one intracellular and the other extracellular. The intracellular mechanism involves the internalisation of materials by an olfactory neuron in an endocytic vesicle that reaches the neuron's projection site, and, finally, it is released via exocytosis. In the extracellular pathway, the materials cross the nasal epithelium and reach the lamina propria, before being transported externally along the length of the neuronal axon. The intracellular transport requires hours to days to reach different regions of the brain. Instead, the extracellular, involving transport through perineural channels, is very fast and takes only a few minutes to directly reach the brain (Crowe et al., 2018). Transport of airborne xenobiotics along the olfactory nerve is a route to reach the CNS that bypasses the protective BBB. Various metals (e.g., manganese, iron, cadmium, thallium, mercury, cobalt and zinc) as well as carbon particles appear to move into the brain following inhalation or intranasal/tracheal instillation exposure. However, not all studies confirm these transport routes, and they highlighted how interspecies differences are important. In humans, the relative surface area of the nasal olfactory mucosa is much smaller than that of rodents (5% vs. 50%), making humans less susceptible to airborne pollutants. In nasal mucosa, there is a “metabolic” barrier present and phase II enzymes, such as UFP-glucuronosyltransferase, epoxide hydrolase, sulfotransferases and glutathione S-transferases, detoxify reactive metabolites originating following phase 1 reactions, or, in any case, they conjugate harmful pollutants after the contact between mucosal cells and xenobiotics (Zhang et al., 2012). This barrier provides a first line of defence for the brain, limiting axonal transport of exogenous material from the olfactory nerve cells to the olfactory bulb. However, several airborne
1.3.3. Trigeminal pathway The trigeminal nerve (i.e., cranial nerve V) is formed by three branches—ophthalmic, maxillary and mandibular, respectively. The ophthalmic and maxillary branches play an important role in nose to brain transport that can occur by intracellular transport (endocytosis) or by axonal transport. Neurons from these branches reach the nasal mucosa and some segments end in the olfactory bulb. Once the compounds diffuse through the nasal cavity mucosa, they reach the branches of the trigeminal nerve in the olfactory and respiratory regions; and, via axonal transport, they reach the brain stem. This pathway was demonstrated by Thorne et al. who reported that the intranasal administration of insulin-like growth factor-I (IGF-I) rapidly reached the brain via the trigeminal neuronal pathway (2004). After going through the mucus and bypassing mucociliary clearance, there are several mechanisms involved in transport through the mucosa: paracellular, transcellular, carrier-mediated transport, receptor-mediated transport and transcytosis. While the paracellular route occurs between adjacent cells through intercellular spaces, the transcellular route refers to transport across cells and may occur by carrier-mediated transport or by endocytosis. 1.4. Toxicological effects of CNTs in biological systems The high hydrophobicity combined with the high aspect ratio enables CNTs to efficiently cross biological membranes, and their uptake, 26
Environmental Toxicology and Pharmacology 65 (2019) 23–30
A. Facciolà et al.
by the activation of the NF-κB or AP-1 transcription factors (Palomäki et al., 2011). The microglia activation, due to prolonged release of proinflammatory cytokines, has a pathogenic role in neurodegenerative diseases leading to a loss of neuronal cells. A two-fold faster rate of decline in AD patients is associated with systemic inflammation as determined via increases in serum TNF-α. The mechanism is confirmed by McAlpine et al. who showed the inhibition of TNF-α signalling significantly reduced amyloid beta protein deposition in the brain in a mouse model of AD (2009). Bardi et al. investigated the neurotoxicity of different types of CNTs through cortical stereotactic injection in mouse brain. Particularly, oxMWCNTs-NH3+ caused cytokine and glial cell activation, suggesting that oxidation can contribute to a sustained inflammatory reaction in healthy brain, while MWCNTs-NH3+ was better tolerated, causing only a local and transient inflammatory response. This different effect was observed despite the uptake levels in different brain tissue cells (astrocytes, microglia and neurons) being similar for the two engineered NPs. Compared to MWCNTs-NH3+, the release of the proinflammatory cytokines TNF-α and IL-1β and microglia activation were significantly higher after injection of ox-MWCNTs-NH3+ (2013). A recent follow-up study revealed that microglia are involved in the cytotoxic response generated by f-MWCNTs in brain tissue. This study showed that exposure to various types of f-MWCNTs (ox-MWCNTs, oxMWCNTs-NH3+ and ox-MWCNTs-am-NH3+) resulted in different effects in base on cellular types. At doses up to 100 μg/mL, they did not induce any damage in neuronal cultures isolated from the two different brain regions studied (frontal cortex (FCO) and striatum (ST) within 24 h. In contrast, the assayed f-MWCNTs caused cytotoxicity in microglia-containing cultures. The authors concluded that the observed brain region-specific sensitivity to MWCNTs exposure most likely was related to the number of microglial cells in different brain regions (Bussy et al., 2015). CNT-induced cytotoxicity was observed even in neuronal cell cultures. The toxic effect, reported by Bussy et al. in human neuroblastoma cells (SH-SY5Y) incubated with co-polymer (Pluronic F127)-coated nanotubes, was dependent on the dispersibility, concentration, incubation time (up to 2 weeks) and contaminant content of CNTs. Particularly, concerning the impurities, CNTs with high iron catalyst content decreased cell viability of pheochromocytoma cells and disrupted the cytoskeleton (Bussy et al., 2015). Visalli et al., using differentiated SH-SY5Y, highlighted the neurotoxic and proinflammatory effects of pristine (p) and functionalised (f) MWCNTs (i.e., submitted to strong acid oxidation to allow the covalent insertion of terminal carboxyl groups into CNTs). The authors observed significant oxidative damage in the neuronal-like cells confirmed by the analysis of DNA damage using the comet assay. Moreover, they highlighted that MWCNT-induced ROS overproduction activated a proinflammatory response as shown by transcript levels of IL-1β, IL-6 and TNF-α, confirmed by the cytokine levels detected in the cell supernatants. The authors hypothesised that the superimposable pro-oxidant activity of both CNTs was imputable to excessive lengths with regard to the p-MWCNTs (10–20 μm vs. 200–1000 nm) and to higher surface reactivity with regard to the f-MWCNTs (2017). Even if the presence of carboxyl groups enhances water dispersibility and causes a reduction in the length to diameter ratio of CNTs, making them more biocompatible, these effects are nullified by the erosion acid-induced by the graphene external layers. This increases the surface reactivity and, consequently, the cellular toxicity (Visalli et al., 2015b). CNT-induced neurotoxic effects include a decrease in cell activity, as shown by Chen et al. that demonstrated the inhibition effect on CA1 glutamatergic synaptic transmission in rat hippocampal slices in vitro (Chen et al., 2014). Gao et al. explored the in vivo relationship between the autophagic flux and synaptic plasticity damage caused by MWCNTs, observing autophagy enhancement and synaptic plasticity damage in the CA1 area (2015). The same authors demonstrated that exposure to MWCNTs
as described above, has been observed in several mammalian cell lines (Pantarotto et al., 2004; Visalli et al., 2015a, 2015b). After their internalisation and according to their physicochemical properties and functionalisation, CNTs accumulate in various subcellular compartments, such as the cytosol, endosomes, perinuclear region, mitochondria and nucleus (Pantarotto et al., 2004; Mu et al., 2009). Whereas lightweight nanosized CNTs aerosolise their unintentional inhalation is highly probable and has raised considerable concern about the possible impact on human health (Pacurari et al., 2016). In the first years, toxicological studies focused almost exclusively on the effects of CNTs in the respiratory system. This is attributable to the morphological similarity of CNTs, especially MWCNTs, to asbestos amphiboles (Palomäki et al., 2011; Donaldson et al., 2013). Like asbestos fibres, CNTs active several pathways, causing apoptosis, fibrosis, genotoxicity, tumourigenesis and inflammation in the lungs as highlighted in a previous review (Trovato et al., 2018). Briefly, following inhalation, epithelial cells and macrophages of the respiratory system are exposed to CNTs which are phagocytized by macrophages, triggering the response of other immune cells. The macrophages also release the inflammasome NLRP3, a multiprotein complex whose activation is caused by many different signals, including pathogenassociated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) (Palomäki et al., 2011). Long, needle-like CNTs activate NLRP3 by reactive oxygen species (ROS) overproduction. The inflammatory cascade continues and NLRP3, in turn, induces the expression of IL-1 gene production of IL-1β and cytokines (Palomäki et al., 2011; Arnoldussen et al., 2015). Also lung epithelial cells actively contribute to the inflammatory response since CNT exposure causes the activation of the nuclear factor kappa β (NF-κβ) (Arnoldussen et al., 2015). In addition to proinflammatory effects, a common mechanism of CNT toxicity is the oxidative damage due to redox imbalance that causes in exposed cells, other than DNA oxidative damage, metabolic impairment and, due to mitochondrial djsfunction, apoptosis, as observed in alveolar cells exposed to asbestos (Hartz et al., 2008; Palomäki et al., 2011; Donaldson et al., 2013) and to oil fly ash (Di Pietro et al., 2011; Visalli et al., 2015a, 2015b). 1.5. Toxicological effects of CNTs on CNS Recent studies indicate that airborne pollutants alter BBB function. Following exposure, endothelial cell damage in the cerebral vasculature, is confirmed by the increases of both intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression. Moreover, in vitro studies using whole brain rat capillaries have shown that treatment with particulate matter (also formed by NPs) causes production of proinflammatory cytokines and ROS and decreases expression of various tight junction proteins (Hartz et al., 2008). Owing to their high hydrophobicity, CNTs may cross the BBB to reach several CNS regions where they may damage selected neurons. CNTs impair molecular pathways and contribute to the onset and progression of chronic brain inflammation, microglia activation and white matter abnormalities with an increased risk for autism spectrum disorders, lower IQ in children, neurodegenerative diseases (Parkinson’s disease (PD) Alzheimer’s disease (AD) and multiple sclerosis) and stroke (Bussy et al., 2015; Migliore et al., 2015; Costa et al., 2016; Kafa et al., 2016). The pathological effects CNT-induced of brain tissue are due to ROS overproduction, mitochondrial dysfunction, genotoxicity and neuroinflammation (Bussy et al., 2015). Considering the high lipid levels and the elevated aerobic metabolic activity of nervous tissue in addition to the proportionally lower antioxidant content, the brain is mostly susceptible to redox imbalance (Migliore et al., 2015). The byproducts of lipid peroxidation, such as reactive aldehydes (malondialdehyde (MDA) and 4-hydroxynonenal (HNE), contribute to neurodegeneration caused by the ROS-induced damage of all biological macromolecules. Moreover, also in the CNS, CNTs trigger the inflammatory response 27
Environmental Toxicology and Pharmacology 65 (2019) 23–30
A. Facciolà et al.
surface area, ranging from 50 to 1315 m2/g, coupled to very low weight makes CNTs efficient drug delivery platform considering that also the inner cavity of CNTs can be filled with drugs or probe molecules. CNTs may also provide protection from enzymatic degradation or clearance, thus increasing the half-life of the drug within the biological fluids. As reported above, by the endocytic and/or passive diffusion pathways CNTs are internalised in cell compartments. When the CNTs are finely dispersed, the uptake happens preferentially by passive penetration while the endocytosis-mediated internalisation happens for CNT tangles (Mu et al., 2009). Regardless of the uptake pathway inside the cells, the loaded bioactive molecules are released as a result of the low pH and the lysosomal enzymes present in endolysosomes and autophagosomes, respectively. Then, free drug will be distributed in the interested cell compartment as shown by Iannazzo et al. for the intercalating drug doxorubicin (2017), which causes cancer cell death and could allow the treatment of gliomas and other brain cancer types. The possibility of attacking many molecules of different types, including targeting ligands that are recognised only by the cells to be treated (as antibodies, peptides or proteins, vitamins, etc.) allow to reduce drug dosages and, consequently, any side effects, maximizing the therapeutic effect (Mehra and Palakurthi, 2016). Monoclonal antibodies, disialoganglioside GD2, vitamins and all other compounds recognised by receptors expressed on the phospholipid bilayer of nerve cells can be used as targeting ligands to recognise specific cells in the CNS (Iannazzo et al., 2017). Their features also allow the use of CNTs in theranostic nanomedicine, integrating the therapy with the diagnosis by the co-delivery of diagnostic agents in addition to therapeutic agents. This allows us to monitor the effectiveness of pharmacological treatment at the cellular and molecular levels. Diagnostic agents commonly used in theranostic nanomedicine and suitable to be combined with CNTs in in vivo and ex vivo assessments include fluorescent probes for optical imaging, paramagnetic metals, such as iron oxides for magnetic resonance imaging, radionuclides for nuclear imaging, iodine and other elements (Ye and Chen, 2011).
could negatively affect the cognitive abilities of rats, particularly their learning ability and memory. Concerning the latter, the field excitatory postsynaptic potential (fEPSP) slopes were significantly reduced in the treated group, suggesting an impairment of the hippocampal long-term potential (LTP), an essential functional indicator of synaptic plasticity. This effect can be explained by a significant reduction of the N-methyl D-aspartate receptor subtype 2B (NMDAR2B) that regulates the activity of the N-methyl D-aspartate (NMDA) receptor and plays a vital role in LTP induction of learning and memory function. Also, the protein synaptophysin (SYP), an important membrane protein of synaptic vesicles, which is closely connected with synaptic plasticity and cognitive processes, was decreased. Moreover, the exposure to MWCNTs damaged pyramidal neurons in the CA1 hippocampal region, an area linked to conditioned reflex and memory (Gao et al., 2015). The biopersistence of SWCNT-PEG in CNS was studied in vivo by Dal Bosco et al. that, after infusion in the hippocampus, did not observe changes in the spatial recognition memory and locomotor activity in rats treated seven days before. At the same time by Raman analysis of the hippocampal homogenates they established the presence of SWCNT-PEG, despite histological analysis did not show remarkable morphological alterations (2015a). In a short time study, the same reaserch group observed that SWCNT-PEG caused lipid peroxidation in the hippocampus. However this effect was transient and overcome by the mobilization of antioxidant defenses already after 24 h (Dal Bosco et al., 2015b). Overall, the analysis of the studies so far performed shows a potential health risk as well as the possible onset of neurological disorders. Considering the widespread presence of CNTs in new materials produced by using nanotechnologies, the risk should not be underestimated. 1.6. Potential use of CNTs in neurology Despite their intrinsic cytotoxicity as reported above, several features of CNTs make their use in the medical field extremely attractive. Due to the high hydrophobicity, in addition to high length to diameter ratio, CNTs efficiently cross the membranes without damaging them and accumulate in intracellular compartments (Mu et al., 2009). This makes them innovative nanoplatforms for drug delivery, allowing unprecedented opportunities in the therapeutic treatment of neurological diseases, including infections, brain and spinal trauma, stroke and brain cancer as shown in several in vitro and in vivo models of neurological disorders (Liopo et al., 2006; Santos et al., 2014; Costa et al., 2016; Mehra and Palakurthi, 2016; Wang et al., 2016). Other than CNTs, carbon-based nano platforms experimented in nanomedicine include, fullerene and, more recently, graphene quantum dot and graphene oxide (GO) (Iannazzo et al., 2017). To today, however, few experimental models have been used these latter in neurological field, despite their capability to delivery biologically active cargoes into CNS. The pharmacological treatment of brain-related disorders by the systemic route is notoriously difficult, and several drugs, proteins, peptides and genetic materials cannot be administered parenterally. As previously reported, the needle-like shape of CNTs makes them suitable to cross the BBB, overcoming the issue due to drug insolubility, poor biodistribution and inability of several bioactive molecules to pass through BBB. In particular, the low permeability of anti-tumour drugs across the BBB when administered systemically has opened up new possibilities for CNT-based modalities in neuroncology. This feature of CNTs avoids problems related to the invasiveness of several therapeutic treatments and patient compliance, which complicate clinical applications in neurology. Compared to free drug and to many other nanoplatforms recently developed such as polymeric NPs, liposomes, dendrimers, nanoshells, micelles, nanogold particles and superparamagnetic particles, both SWCNTs and MWCNTs show a greater drug loading capacity, targeting and efficacy in neurological disorders (Costa et al., 2016). Their large
1.6.1. CNTs-based NIR therapy in neuroncology An inherent optical feature of the CNTs that has remarkable perspectives in neuroncology is the absorption of near-infrared radiation (NIR) not adsorbed by biological tissues. Time and dose dependent adsorbed radiation is converted to heat, allowing a local increase of temperature, i.e. hyperthermia (Santos et al., 2014), exclusively in the cancer cells where CNTs have been engulfed thanks to presence of targeting ligands. Due to preferential internalisation of CNTs, the cytotoxicity is extremely selective and in the malignant cells hyperthermia triggers apoptosis and other death pathway. Instead, the surrounding healthy cells do not suffer any cytotoxic effect (Santos et al., 2014). Compared to conventional cancer therapies, experimental data based on the CNT use as heating devices for hyperthermal therapy provides interesting perspective in neuro-oncology due to the lack of systemic side effects and the ability to preserve the functionality of neuronal tissue (Santos et al., 2014). As targeting ligand adsorbed to CNTs to recognise and eradicate by hyperthermia specific cells can be used monoclonal antibody. However, some aspects, still unresolved, must be underlined before the method can be applied in clinical trials. As above reported CNTs can be administrated by systemic route but is still necessary to optimize the NIR radiation step considering the poor tissue penetration of NIR radiation by external heat sources. 1.6.2. CNTs in neuroprosthetic devices and in neuroregenerative applications Stroke, heat stress, head and spinal cord trauma and bleeding that occurs in the brain, considerably worse the quality of life of the survivors, causing neuronal deterioration. One promising strategy for treatment of these conditions is to support and promote neurite and 28
Environmental Toxicology and Pharmacology 65 (2019) 23–30
A. Facciolà et al.
cells. In fact, Mehra and Palakurthi. used the tripeptide arginine-glycine-aspartic acid (RGD) to decorate SWCNTs considering that RGV has a high affinity for the αvβ3 integrins that are overexpressed on cancer cells. Similarly, Asn-Gly-Arg (NGR), by binding to CD13, have been exploited as ligand to target CNTs cargos to cancer cells, overexpressing the receptor (2016). The covalent insertion of COOH groups, other than to make dispersible and hydrophilic CNT suspensions, allows to add further functional groups to CNTs as amine and thiols, necessary to anchor bioactive molecules (Antonelli et al., 2010). In comparison to covalent functionalization, CNTs coated by peptides, nucleic acids and polymers enhance considerably the cargo biocompatibility, maintaining cellular viability almost unchanged. Moreover, it is useful to underline that in non covalent functionalization the weak interactions between the complex components do not ensure a sufficient biostability which is essential for their medical use. All that highlights as a complete and detailed physicochemical and biological characterization of the CNTs is needful for their use in medical field (Antonelli et al., 2010; Costa et al., 2016).
axonal growth by implanting nanometer-scale scaffolds using tissueengineering approaches (Liopo et al., 2006). Owing to their electrical conductivity and to the morphological features, that mimic tubular structures of CNS, such as axons and dendrites, CNTs are a potential regenerative matrices of neuronal tissue, allowing neuronal growth, promoting neuronal functions and stimulating neuronal interconnections. By using suitably coated CNTs as substrates, these very promising features were highlighted in in vitro model of nerve cells (Mehra and Palakurthi, 2016). CNT-based nanomaterials seem to form an optimal scaffold to increase number and length of neurite outgrowth and branching. The property can be further enhanced by adsorbing the CNTs with bioactive molecules such as nerve growth factor (NGF), or brain-derived neurotrophic factor (BDNF) that allow cell differentiation and neuron survival (Matsumoto et al., 2007). Particularly interesting and attributable to the high electrical conductivity of these engineered nanomaterials are the observed increases of neuronal circuits. When nerve cells are cultured into CNT substrates a neuronal network for the transmission of electrical signals within neurons is formed. As observed by Malarkey et al. (2009) the conductivity of CNTs-play a key role in the based neuronal growth and elongation of dendrites. However, as shown by the same group, only CNTs with a narrow range of conductivity (< 0.3 S cm−1) induce neuronal growth.
2. Conclusion Due to their morphological and physicochemical properties, CNTs are highly versatile, enhancing the structural properties of several materials and, therefore, they are produced in great volumes. For these reasons, they can represent a serious concern for human health due to professional, consumer and environmental exposures. Despite their reported intrinsic cytotoxicity, several features of CNTs make their use in the medical field extremely attractive, such as drug delivery, allowing unprecedented opportunities in the therapeutic treatment of neurological diseases, including infections, brain and spinal trauma, stroke and brain cancer. However, some still unresolved aspects must be addressed and clarified before these NPs can be applied in clinical trials. In particular, it is crucial to determine the long-term toxicological outcomes of CNTs and their fate within living systems.
1.6.3. Requirements for CNT use in neurology Only highly purified, well-tailored and suitably coated CNTs show improved safety. A post synthetic step, needful for the CNTs use in medical application, is their purification from both metals and carbonaceous particles. These contaminants are byproduct of the synthesis process, often responsible for the CNT toxicity (Bussy et al., 2015). In particular, as widely demonstrated, the pro-oxidant effect of metals (Fe, Ni, Co etc.), used as catalyst in the CNTs synthesis, decreases cell viability, causing lipid peroxidation and oxygen radical formation (Visalli et al., 2015b). The purification treatments remarkably cut down CNT toxicity in the CNS, as reported in several in vitro study (Pacurari et al., 2016). To reduce the length to diameter ratio of the CNT, that is responsible for cytotoxicity, shortening procedures include ultrasonication, steam-purification and mechanical methods, in addition to strong acidic oxidation (Bussy et al., 2015). Moreover, a prerequisite for their application in the biological field is provided by the surface engineering of CNTs (i.e., functionalisation). In the physiological environment pristine CNTs tend to agglomerate making their uptake in the cells of interest uneven. As well as increasing the dispersibility, the functionalisation allows the bonding of bioactive molecules to the groups added on the graphene surface. Moreover, functionalization improves interfacial adhesion, promoting CNTs-cell membranes interactions and it is essential to regulate biological halflife, biodistribution and pharmacokinetics of CNTs (Antonelli et al., 2010). As above reported, to make these nanoplatforms more biocompatible, several biocompatible hydrophilic compounds have been tested as coatings (Karimi et al., 2015; Mehra and Palakurthi, 2016), including biological macromolecules. While the CNT-DNA complexes are based on the π–π interactions between the aromatic bases of DNA and the CNTs surface, the supramolecular biocompatible complexes formed by CNT and peptides and/or proteins are based on hydrophobic interaction. In addition to natural protein such as human serum albumin (HAS), many synthetic peptides are used to decorate CNTs, representing an efficient dispersion agent for CNTs. In particular, specific amino acids, such as tryptophan, phenylalanine and tyrosine, play a pivotal role in peptides/proteins- CNTs interactions while the presence of multiple amino groups, enhances the water solubility of complexes and they work as a linker for drug compounds. In addition, specific peptides could be used to target these nanoplatforms for drug delivery to specific
Funding This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. Conflict of interest The authors declare that they have no conflict of interest. Transparency document The Transparency document associated with this article can be found in the online version. References Antonelli, A., Serafini, S., Menotta, M., Sfara, C., Pierigé, F., Giorgi, L., Ambrosi, G., Rossi, L., Magnani, M., 2010. Improved cellular uptake of functionalized single-walled carbon nanotubes. Nanotechnology 21 (42), 425101. https://doi.org/10.1088/09574484/21/42/425101. Arnoldussen, Y.J., Skogstad, A., Skaug, V., Kasem, M., Haugen, A., Benker, N., Weinbruch, S., Apte, R.N., Zienolddiny, S., 2015. Involvement of IL-1 genes in the cellular responses to carbon nanotube exposure. Cytokine 73 (1), 128–137. Bardi, G., Nunes, A., Gherardini, L., Bates, K., Al-Jamal, K.T., Gaillard, C., Prato, M., Bianco, A., Pizzorusso, T., Kostarelos, K., 2013. Functionalized carbon nanotubes in the brain: cellular internalization and neuroinflammatory responses. PLoS One 8 (11), e80964. https://doi.org/10.1371/journal.pone.0080964. Bussy, C., Al-Jamal, K.T., Boczkowski, J., Lanone, S., Prato, M., Bianco, A., Kostarelos, K., 2015. Microglia determine brain region-specific neurotoxic responses to chemically functionalized carbon nanotubes. ACS Nano 25 (8), 7815–7830 9. Chen, T., Yang, J.J., Zhang, H., Ren, G.G., Yang, Z., Zhang, T., 2014. Multi-walled carbon nanotube inhibits CA1 glutamatergic synaptic transmission in rat’s hippocampal slices. Toxicol. Lett. 229, 423–429. Costa, P.M., Bourgognon, M., Wang, J.T., Al-Jamal, K.T., 2016. Functionalised carbon
29
Environmental Toxicology and Pharmacology 65 (2019) 23–30
A. Facciolà et al.
a drug delivery perspective. Drug Discov. Today 21 (4), 585–597. https://doi.org/10. 1016/j.drudis.2015.11.011. Migliore, L., Uboldi, C., Di Bucchianico, S., Coppedè, F., 2015. Nanomaterials and neurodegeneration. Environ. Mol. Mutagen. 56 (2), 149–170. Mu, Q., Broughton, D.L., Yan, B., 2009. Endosomal leakage and nuclear translocation of multiwalled carbon nanotubes: developing a model for cell uptake. Nano Lett. 12, 4370–4375. Nowack, B., David, R.M., Fissan, H., Morris, H., Shatkin, J.A., Stintz, M., Zepp, R., Brouwer, D., 2013. Potential release scenarios for carbon nanotubes used in composites. Environ. Int. 59, 1–11. Oberdörster, G., Sharp, Z., Atudorei, V., Elder, A., Gelein, R., Lunts, A., Kreyling, W., Cox, C., 2002. Extrapulmonary translocation of ultrafine carbon particles following wholebody inhalation exposure of rats. J. Toxicol. Environ. Health A 65 (20), 1531–1543. Pacurari, M., Lowe, K., Tchounwou, P.B., Kafoury, R., 2016. A review on the respiratory system toxicity of carbon nanoparticles. Int. J. Environ. Res. Public Health 13 (3). https://doi.org/10.3390/ijerph13030325. pii: E325. Palomäki, J., Välimäki, E., Sund, J., Vippola, M., Clausen, P.A., Jensen, K.A., Savolainen, K., Matikainen, S., Alenius, H., 2011. Long, needle-like carbon nanotubes and asbestos activate the NLRP3 inflammasome through a similar mechanism. ACS Nano 5 (9), 6861–6870. Pantarotto, D., Singh, R., McCarthy, D., Erhardt, M., Briand, J.P., Prato, M., Kostarelos, K., Bianco, A., 2004. Functionalized carbon nanotubes for plasmid DNA gene delivery. Angew. Chem. Int. Ed. Engl. 43 (39), 5242–5246. Santos, T., Fang, X., Chen, M.T., Wang, W., Ferreira, R., Jhaveri, N., Gundersen, M., Zhou, C., Pagnini, P., Hofman, F.M., Chen, T.C., 2014. Sequential administration of carbon nanotubes and near-infrared radiation for the treatment of gliomas. Front. Oncol. 4, 180. Selvaraj, K., Gowthamarajan, K., Karri, V.V.S.R., 2017. Nose to brain transport pathways an overview: potential of nanostructured lipid carriers in nose to brain targeting. Artif. Cells Nanomed. Biotechnol. 28, 1–8. https://doi.org/10.1080/21691401.2017. 1420073. Thorne, R.G., Pronk, G.J., Padmanabhan, V., Frey 2nd, W.H., 2004. Delivery of insulinlike growth factor-I to the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration. Neuroscience 127 (2), 481–496. Trovato, M.C., Andronico, D., Sciacchitano, S., Ruggeri, R.M., Picerno, I., Di Pietro, A., Visalli, G., 2018. Nanostructures: between natural environment and medical practice. Rev. Environ. Health. https://doi.org/10.1515/reveh-2017-0036. Visalli, G., Baluce, B., Bertuccio, M., Picerno, I., Di Pietro, A., 2015a. Mitochondrialmediated apoptosis pathway in alveolar epithelial cells exposed to the metals in combustion-generated particulate matter. J. Toxicol. Environ. Health A 78 (11), 697–709. Visalli, G., Currò, M., Iannazzo, D., Pistone, A., Pruiti Ciarello, M., Acri, G., Testagrossa, B., Bertuccio, M.P., Squeri, R., Di Pietro, A., 2017a. In vitro assessment of neurotoxicity and neuroinflammation of homemade MWCNTs. Environ. Toxicol. Pharmacol. 56, 121–128. https://doi.org/10.1016/j.etap.2017.09.005. Visalli, G., Bertuccio, M.P., Iannazzo, D., Piperno, A., Pistone, A., Di Pietro, A., 2015b. Toxicological assessment of multi-walled carbon nanotubes on A549 human lung epithelial cells. Toxicol. In Vitro 29, 352–362. Visalli, G., Facciolà, A., Iannazzo, D., Piperno, A., Pistone, A., Di Pietro, A., 2017b. The role of the iron catalyst in the toxicity of multi-walled carbon nanotubes (MWCNTs). J. Trace Elem. Med. Biol. 43, 153–160. Wang, J.T., Rubio, N., Kafa, H., Venturelli, E., Fabbro, C., Ménard-Moyon, C., Da Ros, T., Sosabowski, J.K., Lawson, A.D., Robinson, M.K., Prato, M., Bianco, A., Festy, F., Preston, J.E., Kostarelos, K., Al-Jamal, K.T., 2016. Kinetics of functionalised carbon nanotube distribution in mouse brain after systemic injection: Spatial to ultrastructural analyses. J. Control. Release 224, 22–32. https://doi.org/10.1016/j. jconrel.2015.12.039. Ye, Y., Chen, X., 2011. Integrin targeting for tumor optical imaging. Theranostics 1, 102–126. Zhang, H., Liu, H., Davies, K.J., Sioutas, C., Finch, C.E., Morgan, T.E., Forman, H.J., 2012. Nrf2-regulated phase II enzymes are induced by chronic ambient nanoparticle exposure in young mice with age-related impairments. Free Radic. Biol. Med. 52 (9), 2038–2046. https://doi.org/10.1016/j.freeradbiomed.2012.02.042.
nanotubes: From intracellular uptake and cell-related toxicity to systemic brain delivery. J. Control. Release 241, 200–219. https://doi.org/10.1016/j.jconrel.2016.09. 033. Crowe, T.P., Greenlee, M.H.W., Kanthasamy, A.G., Hsu, W.H., 2018. Mechanism of intranasal drug delivery directly to the brain. Life Sci. 195, 44–52. https://doi.org/10. 1016/j.lfs.2017.12.025. Dal Bosco, L., Weber, G.E., Parfitt, G.M., Paese, K., Gonçalves, C.O., Serodre, T.M., Furtado, C.A., Santos, A.P., Monserrat, J.M., Barros, D.M., 2015a. PEGylated carbon nanotubes impair retrieval of contextual fear memory and alter oxidative stress parameters in the rat hippocampus. Biomed Res. Int., 104135. https://doi.org/10. 1155/2015/104135. Dal Bosco, L., Weber, G.E., Parfitt, G.M., Cordeiro, A.P., Sahoo, S.K., Fantini, C., Klosterhoff, M.C., Romano, L.A., Furtado, C.A., Santos, A.P., Monserrat, J.M., Barros, D.M., 2015b. Biopersistence of PEGylated carbon nanotubes promotes a delayed antioxidant response after infusion into the rat hippocampus. PLoS One 15 (6), e0129156. https://doi.org/10.1371/journal.pone.0129156. 10. De Volder, M.F., Tawfick, S.H., Baughman, R.H., Hart, A.J., 2013. Carbon nanotubes: present and future commercial applications. Science 339 (6119), 535–539. Dhuria, S.V., Hanson, L.R., Frey, W.H., 2009. Novel vasoconstrictor formulation to enhance intranasal targeting of neuropeptide therapeutics to the central nervous system. J. Pharmacol. Exp. Ther. 328, 312–320. Di Pietro, A., Visalli, G., Baluce, B., Micale, R.T., La Maestra, S., Spataro, P., De Flora, S., 2011. Oxidative damage in human epithelial alveolar cells exposed in vitro to oil fly ash transition metals. Int. J. Hyg. Environ. Health 214 (2), 138–144. Donaldson, K., Poland, C.A., Murphy, F.A., MacFarlane, M., Chernova, T., Schinwald, A., 2013. Pulmonary toxicity of carbon nanotubes and asbestos-similarities and differences. Adv. Drug Deliv. Rev. 65, 2078–2086. Gao, J., Zhang, X., Yu, M., Ren, G., Yang, Z., 2015. Cognitive deficits induced by multiwalled carbon nanotubes via the autophagic pathway. Toxicology 337, 21–29. https://doi.org/10.1016/j.tox.2015.08.011. Gottschalk, F., Nowack, B., 2011. The release of engineered nanomaterials to the environment. J. Environ. Monit. 13 (5), 1145–1155. Hartz, A.M., Bauer, B., Block, M.L., Hong, J.S., Miller, D.S., 2008. Diesel exhaust particles induce oxidative stress, proinflammatory signaling, and P-glycoprotein up-regulation at the blood-brain barrier. FASEB J. 22, 2723–2733. Iannazzo, D., Pistone, A., Salamò, M., Galvagno, S., Romeo, R., Giofré, S.V., Branca, C., Visalli, G., Di Pietro, A., 2017. Graphene quantum dots for cancer targeted drug delivery. Int. J. Pharm. 518 (1–2), 185–192. https://doi.org/10.1016/j.ijpharm. 2016.12.060. Kafa, H., Wang, J.T., Rubio, N., Klippstein, R., Costa, P.M., Hassan, H.A., Sosabowski, J.K., Bansal, S.S., Preston, J.E., Abbott, N.J., Al-Jamal, K.T., 2016. Translocation of LRP1 targeted carbon nanotubes of different diameters across the blood-brain barrier in vitro and in vivo. J. Control. Release 225, 217–229. https://doi.org/10.1016/j. jconrel.2016.01.031. Karimi, M., Solati, N., Amiri, M., Mirshekari, H., Mohamed, E., Taheri, M., Hashemkhani, M., Saeidi, A., Estiar, M.A., Kiani, P., Ghasemi, A., Basri, S.M., Aref, A.R., Hamblin, M.R., 2015. Carbon nanotubes part I: preparation of a novel and versatile drug-delivery vehicle. Expert Opin. Drug Deliv. 12 (7), 1071–1087. https://doi.org/10.1517/ 17425247.2015.1003806. Liopo, A.V., Stewart, M.P., Hudson, J., Tour, J.M., Pappas, T.C., 2006. Biocompatibility of native and functionalized single-walled carbon nanotubes for neuronal interface. J. Nanosci. Nanotechnol. 6 (5), 1365–1374. Malarkey, E.B., Fisher, K.A., Bekyarova, E., Liu, W., Haddon, R.C., Parpura, V., 2009. Conductive single-walled carbon nanotube substrates modulate neuronal growth. Nano Lett. 9 (1), 264–268. Matsumoto, K., Sato, C., Naka, Y., Kitazawa, A., Whitby, R.L., Shimizu, N., 2007. Neurite outgrowths of neurons with neurotrophin-coated carbon nanotubes. J. Biosci. Bioeng. 103 (3), 216–220. McAlpine, F.E., Lee, J.K., Harms, A.S., Ruhn, K.A., Blurton-Jones, M., Hong, J., Das, P., Golde, T.E., LaFerla, F.M., Oddo, S., Blesch, A., Tansey, M.G., 2009. Inhibition of soluble TNF signaling in a mouse model of Alzheimer’s disease prevents pre-plaque amyloid-associated neuropathology. Neurobiol. Dis. 34 (1), 163–177. Mehra, N.K., Palakurthi, S., 2016. Interactions between carbon nanotubes and bioactives:
30