Available online at www.sciencedirect.com
Nanomedicine: Nanotechnology, Biology, and Medicine xx (2010) xxx – xxx
www.nanomedjournal.com
Review Article
Pulmonary toxicity of carbon nanotubes: a systematic report
Jitendra Kayat, MPharm, Virendra Gajbhiye, MPharm,
Rakesh Kumar Tekade, MPharm, Narendra Kumar Jain, PhD⁎
Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar, India
Received 7 October 2009; accepted 8 June 2010
Abstract
Carbon nanotubes (CNTs) are nanosized cylindrical hollow tubes consisting entirely of the element carbon. Currently, CNTs are playing
an important role in drug delivery as a carrier system because of their several unique physical and chemical properties. Studies show that
CNTs are toxic and that the extent of that toxicity depends on properties of the CNTs, such as their structure (single wall or multiple wall),
length and aspects ratios, surface area, degree of aggregation, extent of oxidation, bound functional group(s), method of manufacturing,
concentration, and dose. People could be exposed to CNTs either accidentally by coming in contact with the aerosol form of CNTs during
production or by exposure as a result of biomedical use. Numerous in vitro and in vivo studies have shown that CNTs and/or associated
contaminants or catalytic materials that arise during the production process may induce oxidative stress, prominent pulmonary inflammation,
apoptosis in different cell types, and induction of cytotoxic effects on lungs. Studies on the toxicity of CNTs have mainly focused on the
pulmonary effects of intratracheal or pharyngeally administered CNTs. This review examines the potential pulmonary toxicity of CNTs.
© 2010 Published by Elsevier Inc.
Key words: Carbon nanotubes; Lung toxicity; SWCNTs; MWCNTs; Granulomas
Carbon nanotubes (CNTs), a distinct molecular form of
carbon atoms that was discovered in the late 1980s,1 were
exhaustively discovered by Sumio Iijima in 1991.2 Essentially,
CNTs are cylindrical molecules composed solely of carbon
atoms and basically exist in two classes (Figure 1). Single-walled
carbon nanotubes (SWCNTs) are long wrapped graphene sheets
that have a length-to-diameter ratio of about 1000; thus, their
structure can be considered as nearly one-dimensional.3 Multiwalled carbon nanotubes (MWCNTs) are larger and consist of
many single-walled tubes stacked one inside the other. CNTs are
distinct from carbon fibers, which are not single molecules but
strands of layered graphite sheets. The unique and diverse
properties of CNTs, in addition to the wide range of functionality
afforded by chemical modification, allow for many exciting
applications. Their nanometer dimensions give CNTs the
potential to interact with macromolecules such as proteins and
DNA.4 They can be thought of as a seamless cylinder formed
from a graphite sheet with a hexagonal lattice structure.5
No conflict of interest was reported by the authors of this article.
⁎Corresponding author: Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar 470
003, India.
E-mail address: [email protected] (N.K. Jain).
CNTs exhibit several unique physical and chemical properties.
These materials have been considered for use in numerous
technological applications,6-10 including electronic devices, field
emission devices,11 and composite materials12; in addition, they
have numerous biological and medical applications. The CNTs have
a unique absorption in the near-infrared region, which could be used
for biological sensing.13,14 A brief account of the available reports
on the toxicity of CNTs on the respiratory tract is presented below.
Respiratory tract
The respiratory system is one of the most critical organ
systems of the body that supply the body with oxygen and rid it
of carbon dioxide. This process also removes metabolic wastes
and maintains the pH balances of the body. The organs involved
are the airways, lungs, and muscles that mediate the movement
of air into and out of the body. The respiratory tract system is the
main route for dust entering the human body, followed by
ingestion. Nanoparticulate entities can enter living organisms
through inhalation (respiratory tract), ingestion (gastrointestinal
tract), dermal absorption (skin), and injection (blood circulation).
The respiratory tract acts as the main pathway for
nanoparticle entry. The chief mechanism for deposition of
inhaled nanosized particles in the respiratory tract is diffusion
1549-9634/$ – see front matter © 2010 Published by Elsevier Inc.
doi:10.1016/j.nano.2010.06.008
Please cite this article as: J. Kayat, V. Gajbhiye, R.K. Tekade, N.K. Jain, Pulmonary toxicity of carbon nanotubes: a systematic report. Nanomedicine:
NBM 2010;xx:1-10, doi:10.1016/j.nano.2010.06.008
2
J. Kayat et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2010) xxx–xxx
Figure 1. Basic types of CNTs SWCNTs (top left) and MWCNTs (top right) with typical transmission electron micrographs below. (Adapted from Donaldson
et al28 with permission from publisher.)
due to displacement when they collide with air.8 Several defense
mechanisms exist throughout the respiratory tract aimed at
keeping the mucosal surfaces free from cell debris and particles
deposited by inhalation. Once deposited, nanosized particles in
contrast to larger sized particles appear to translocate readily to
extrapulmonary sites and reach other target organs by different
transfer routes and mechanisms. Once the particles have reached
pulmonary interstitial sites, uptake into the blood circulation, in
addition to lymphatic pathways, can occur. In addition to particle
size, the extent of extrapulmonary translocation is highly
dependent on particle surface characteristics and chemistry.15
Body distribution of CNTs from respiratory tracts
CNTs are cylinders of one or several coaxial graphite layer
(s) with a diameter in the order of nanometers.16 CNTs are in
the nanometer size range and hence easily enter into the lungs
via the respiratory tract with air inhalation. After entering the
lungs they distribute rapidly in the central nervous system,
peripheral nervous system, lymph, and blood (Figure 2). They
show rapid distribution in heart, spleen, kidney, bone marrow,
and liver.15 The ability of nanomaterials to move in the body
may depend on their chemical reactivity, surface characteristics,
and ability to bind the body proteins. Depending upon size and
physical structure of nanosized particles, they are deposited in
the different regions of the respiratory tract.17 After deposition,
the nanosized particles are translocated to the extrapulmonary
site and reach the target organ site by various transfer routes and
mechanisms. The nanomaterials access the blood circulation
probably by transcytosis across the respiratory tract into the
interstitium. Clearance of the deposited particles in the
respiratory tract takes place for the most part by two
mechanisms: (1) physical translocation of particles by different
mechanisms and (2) chemical clearance processes. Chemical
dissolution is directed at the components of the particles, which
are either the lipid-soluble or soluble in the intracellular or
extracellular fluids. The soluble components of the particles
then undergo absorption and diffusion into the proteins and
other subcellular components.18 Oberdorster et al19 found
significant amounts (80 or 180 μg/m3) of 13C-labeled carbon
particles (22–30 nm in diameter) in the livers of rats after 6
hours of inhalation exposure. Colvin20 found that inhaled 13Clabeled carbon particles reached the olfactory bulb and also the
cerebrum and cerebellum. The results suggested that translocation to the brain occurred through the nasal mucosa along with
the olfactory nerve.20 In another study, the intraperitoneal
administration of 50-nm fluorescent magnetic nanoparticles was
found to penetrate the blood-brain barrier without causing
significant toxicity.21
CNT-mediated lung toxicity
People could be exposed to CNTs through accidental
exposure by coming in contact with the aerosol form of CNTs
during production or exposure as a result of biomedical use.
Toxicity of CNTs is related to properties of the CNT material,
J. Kayat et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2010) xxx–xxx
3
19
liver, and spleen (Figure 3). Toxicity of CNTs has been
attributed to the following reasons:
• They are nanoparticulate and so could have more toxicity
than larger sized particles,27
• They are fiber shaped and so might behave like asbestos
and other pathogenic fibers, which have toxicity associated with their needle-like shape,28 and
• They are essentially graphitic and so are expected to be
biopersistent.28
Figure 2. Distribution of CNTs in the body. Iκβ, inhibitor of κβ; IL-10,
interleukin 10; NF-κβ, nuclear factor κβ; TGF-β, transforming growth
factor β.
such as their structure (SWCNTs or MWCNTs), length and
aspects ratio, surface area, degree of aggregation, extent of
oxidation, bound functional group(s), method of manufacturing
(which can leave the catalytic residues and produced impurities),
as well as to their concentration and dose. Numerous in vitro and
in vivo studies have shown that CNTs and/or associated
contaminants or catalytic materials that arise during the
production process may induce oxidative stress and prominent
pulmonary inflammation. Recent studies also suggest some
similarities between the pathogenic properties of MWCNTs and
those of asbestos fibers.16 Studies on the toxicity of CNTs have
mainly focused on the pulmonary effects of CNTs administered
through the intratracheal or pharyngeal routes.22 Several studies
demonstrated that both SWCNTs and MWCNTs might induce
cytotoxic effects and apoptosis in different cell types.23,24
Muller et al also suggested that CNTs show cytotoxic effects.25
The lung deposition of a nanosized material depends upon its
surface area-to-mass ratio. A study on the toxicity of the
nanosized particles reports the effect of high-purity carbon black
(CB; 14 nm) and CB (16 nm) on pulmonary tissue, in which the
high-purity CB caused increased oxidative stress26 in human
type II alveolar epithelial cells in vitro and increased murine
alveolar macrophage migration in fetal calf serum nearly
twofold, as compared with high-purity CB (260 nm). On the
other hand, CB (16 nm) causes the development of pulmonary
tumor in rats through subchronic inhalations. Although C60
fullerenes do not yet show significant toxicity, they show rapid
distribution in rats and deposition in many tissues like brain,
Muller et al25 found that agglomerates of intact CNTs
remained entrapped in the largest airway, whereas ground
nanotubes were much better dispersed in the lung tissue. Upon
reaching the respiratory tract, CNTs cause pulmonary inflammation, pulmonary fibrosis, induced accumulation of neutrophils
and eosinophils, mechanical blockage, and increase in various
cytotoxicity/inflammatory markers in the lungs25 (Figure 4).
These include a significant increase in total bronchoalveolar
lavage cells and polymorphonuclear leukocytes and also protein,
lactate dehydrogenase (LDH), tumor necrosis factor-α (TNF-α),
interleukin-1β (IL-1β), and mucin levels.29 Jacobsen et al30
compared the effects of instillation of three carbonaceous
particles; CB, fullerenes C60 (C60), and SWCNTs, as well as
gold particles and quantum dots in ApoE–/– mice. Characterization of the instillation media revealed that all particles were
delivered as agglomerates and aggregates. Significant increases
in Il-6, Mip-2, and Mcp-1 messenger RNA were detected in lung
tissue, 3 hours and 24 hours following instillation of SWCNTs,
CB, and quantum dots, whereas gold and C60 particles caused
much weaker inflammatory responses.30 It has been found that
nanotubes induced an inflammatory response that to a certain
extent was greater than with ground CNTs. Administration of
CNTs as well as ground CNTs induced a dose-dependent
increase in LDH release, which is a marker of cell toxicity during
the inflammation of lungs. Pulmonary fibrosis can be measured
by the lung hydroxyproline (OH-proline) and soluble collagen
type I contents. After administration of CNTs, the OH-proline
levels were increased significantly and dose-dependently. CNTs
as well as ground CNTs induced a significant increase of the
type I collagen lung levels in comparison with the control rats.
The measurements indicated that the fibrotic response to
nanotubes was dose-dependent, and the intensity of the fibrotic
response induced by 5 mg of ground CNTs was equivalent to
that induced by 2 mg of CNTs, suggesting that ground CNTs
may be safer. The results indicated that ground CNTs were
cleared more rapidly, but a significant fraction of the
administered dose (36%) still remained in the lung after 60
days. Ground CNTs reached the alveolar spaces and induced the
formation of parenchymal granulomas.25
Pulmonary toxicity of SWCNTs
SWCNTs have a diameter ranging from 0.7 to 1.5 nm.
Intratracheal instillation of SWCNTs in the lungs of rats resulted
in the formation of lung granulomas and produced mortality in
∼15% of instilled rats within 24 hours postinstillation due to the
enhanced blockage of the large airways.31 Alveolar macrophages
4
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Figure 3. Potential pathways after the inhalation of CNTs. (Adapted from Jain et al18 with permission from publisher.)
constitute the first line of immunological defense against
invading particles in the lung; researchers have conducted a
cytotoxicity study of CNTs with macrophages as well. SWCNTs
can induce pulmonary injury in mice, as recently confirmed by
Chou et al.32 The intratracheal instillation of 0.5 mg of SWCNTs
into male imprinting control region (ICR) mice induced alveolar
macrophage activation, various chronic inflammatory responses,
and severe pulmonary granuloma formation.32 The SWCNTs
have a strong tendency to agglomerate following intratracheal
exposures.33 SWCNTs were found to induce interstitial
granulomas and pulmonary injuries in a dose-dependent
manner.34 Jia35 observed profound dose-dependent cytotoxicity
of SWCNTs in alveolar macrophages isolated from guinea pigs
in vitro for 6 hours. The macrophages exposed to SWCNTs or
MWCNTs showed characteristic features of apoptosis at
different dosages, toxic response being more with SWCNTs
than with the MWCNTs, quartz, or fullerene used in this study.
There are reports showing contradictory results of CNT
cytotoxicity to macrophages. Kalbacova et al36 found SWCNTs
toxicity to monocytes/macrophage (THP-1) cells, while Fiorito
et al37 reported insignificant toxicity of SWCNTs to human
macrophages. The intratracheal or pharyngeal instillation of
SWCNT suspension in mice caused a persistent accumulation of
CNTs aggregates in the lung, followed by the rapid formation
of pulmonary granulomatous and fibrous tissues at the site; it
also produced cardiovascular toxicity.38 Macrophage uptake of
SWCNTs was observed after intratracheal instillation of 0.5 mg
of SWCNTs, which activated various transcription factors such
as nuclear factor κB (NF-κB) and activator protein 1 (AP-1).
This leads to oxidative stress, release of proinflammatory
J. Kayat et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2010) xxx–xxx
5
Figure 4. Effect of grinding on the dispersion of CNTs in the lungs after intratracheal administration. The panels present macroscopic views and H&E-stained
lung sections from saline (A, D), CNTs (B, E; 2 mg per rat), or ground CNTs (C, F; 2 mg per rat) rats after intratracheal instillation. Arrows indicate the
dispersion of grinding CNTs. (Adapted from Muller et al25 with permission from publisher.)
cytokines, recruitment of leukocytes, induction of protective and
antiapoptotic gene expression, and the activation of T cells. The
SWCNTs induced alveolar macrophage activation, various
chronic inflammatory responses, and severe pulmonary granuloma formation.32
Stoker et al39 assessed the health risk of CNTs on the human
respiratory system by using co-culture of normal bronchial
epithelial cells and normal human fibroblasts. They incubated an
aqueous solution of SWCNTs having an average length and
diameter of about 500 nm and less than 10 nm, respectively, with
the above co-culture. The result indicated increased production of
nitrous oxide and decreased cell viability after exposure to
different concentrations of SWCNTs. The above observations
were associated with inflammatory and cytotoxic effect of
SWCNTs, respectively.39 The dose administered by inhalation
produced greater respiratory toxicity than the same dose
administered by aspiration. The SWCNTs (about 1 nm in
diameter and between 100 and 1000 nm in length), in both a
6
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Table 1
Studies of pulmonary toxicity of single-walled carbon nanotubes (SWCNTs)
Animal species
Objective
Outcomes or results
Reference
B6C3F1 mice
To investigate pulmonary toxicity of SWCNTs containing
different types and amounts of residual catalytic metals
To explore unusual pulmonary effects elicited by
pharyngeal aspiration of SWCNTs
Production of dose-dependent interstitial granulomas and
pulmonary injuries
Caused granulomatous inflammation, interstitial fibrosis
with alveolar wall thickening, damage to pulmonary cells,
increased numbers of alveolar type II (AT-II) cells
Caused formation of lung granuloma and produced
mortality in ∼15% of instilled rats within
24 hours postinstillation
Caused formation of pulmonary granulomatous and
fibrous tissues and production of cardiovascular toxicity
Caused acute inflammation (enhanced total number of
inflammatory cells, number of polymorphonuclear
leukocytes, released LDH, total protein content, and levels
of proinflammatory cytokines, TNF-α and IL-6) and
enhanced profibrotic responses (elevation of TGF-β
and collagen deposition)
Induced alveolar macrophage activation, various chronic
inflammatory responses, and severe pulmonary
granuloma formation
34
Caused cytotoxic/inflammatory responses and barrier
function of the human lung layers
39
Production of respiratory toxicity, increased mucus
secretion, enlarged mucocytes on the gills, and elevated
ventilation rates
44
C57BL/5 mice
Wistar rats
C57BL/6 mice
C57BL/6 mice
CR mice
Co-culture of normal
human bronchial
epithelial cells
and normal
human fibroblasts
Fish (rainbow trout)
To compare pulmonary toxicity of intratracheally instilled
SWCNTs with positive and negative control particles like
quartz and carbonyl iron particles, respectively
To analyze the cardiovascular adverse effects of SWCNTs
on respiratory exposure
To explore the pulmonary inflammatory reactions to
aspired SWCNTs
To demonstrate SWCNT-induced alveolar macrophage
activation, chronic inflammatory responses, and severe
pulmonary granuloma formation by
intratracheal instillation
To analyze changes in airway physiological function
following exposure to different
concentrations of SWCNTs
To observe toxicity of SWCNTs on aquatic animals
43
33
38
41
32
Abbreviations: IL-6, interleukin-6; LDH, lactate dehydrogenase; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α.
single-dose aspiration study and a 4-day inhalation study, caused
an initial inflammatory response followed by granulomas,
fibrosis, and decreased rates of respiration, as well as activation
of a gene that produces lung cancer.40 Exposure of mice to
SWCNTs induces an unusually robust pulmonary inflammatory
response with an early onset of fibrosis, which is accompanied by
oxidative stress and antioxidant depletion. Marked decrease in the
level of pulmonary antioxidants was found in SWCNT-treated
vitamin E–deficient mice as compared with controls. This result
was associated with a higher sensitivity to SWCNT-induced
acute inflammation (total number of inflammatory cells, number
of polymorphonuclear leukocytes, released LDH, total protein
content, and levels of proinflammatory cytokines, TNF-α and IL6), and enhanced profibrotic responses [elevation of transforming
growth factor-β (TGF-β) and collagen deposition].41
Shvedova et al42 demonstrated that SWCNTs caused
inflammatory responses and fibrosis. Timely elimination of
polymorphonuclear neutrophils (PMNs) through apoptosis and
their subsequent clearance by macrophages is a necessary stage
in the resolution of pulmonary inflammation whereby NADPH
oxidase contributes to control of apoptotic cell death and
clearance of PMNs. It is found that NADPH oxidase–null
mice responded to SWCNT exposure with a marked
accumulation of PMNs and elevated levels of apoptotic cells
in the lungs, production of proinflammatory cytokines,
decreased production of the anti-inflammatory and profibrotic
cytokine TGF-β, and significantly lower levels of collagen
deposition, as compared with C57BL/6 control mice, and the
results indicated the role for NADPH oxidase–derived reactive
oxygen species in determining the course of pulmonary
response to SWCNTs.42
By using specific pathogen–free adult female C57BL/6 mice,
Shvedova et al43 detected two morphologically distinct responses
in the lung as early as 7 days postexposure of SWCNTs, delivered
by pharyngeal aspiration.43 CNT material was clearly visualized
with granulomatous inflammation, and interfacing bundles of
fibrous connective tissue were observed within discrete granulomas. In lung regions alterations were predominantly diffuse
interstitial fibrosis with alveolar wall thickening. They further
investigated whether the exposure caused significant damage to
pulmonary cells or not. Increased numbers of alveolar type II
(AT-II) cells, the progenitor cells that replicate following alveolar
type I (AT-I) cell death, were noted in response to SWCNTs. This
proliferation of AT-II cells indicates either AT-I cell injury,
proliferation of AT-II cells to cover new basement membrane
associated with the nodules, or both.43 After the exposure of fish
to SWCNTs, signs of gill irritation and mucus secretion were
detected. Visual inspection of the gills and mucus smears on day 4
showed a thin layer of secreted mucus on the surface of gills from
fish treated with SWCNTs, but not the controls. It was found that
exposure to SWCNTs caused respiratory toxicity in trout,
increased mucus secretion, and enlarged mucocytes on the
gills; elevated ventilation rates compared with the controls were
observed, but no mortalities and gill injuries (swollen tips of
lamellae, enlarged mucocytes, edema) had occurred at the fourth
day Table 1.44
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7
Table 2
Pulmonary toxicity of multiwalled carbon nanotubes (MWCNTs)
Species
Objective
Outcomes or results
Reference
Sprague-Dawley rats
To investigate potential toxicity of MWCNTs
to humans
25
Wistar rats
To explore the toxic responses with
progressively and selectively modified MWCNTs
To examine effect of structural properties
on the toxicity of MWCNTs
Caused significant protein exudation and granulomas on
the peritoneal side of the diaphragm, induced inflammatory
and fibrotic reactions, stimulated the production of TNF-α
Induced alveolitis, fibrosis, and genotoxicity in epithelial cells
46
Acute pulmonary toxicity
47
Wistar rats
Abbreviation: TNF-α, tumor necrosis factor-α.
Pulmonary toxicity of MWCNTs
MWCNTs samples with high aspect ratio caused significant
PMN or protein exudates (fluid rich in protein and cellular
elements that leach out of blood vessels due to inflammation) and
granulomas on the peritoneal side of the diaphragm. The
MWCNTs and ground MWCNTs were found to be present in
the lung after 60 days, and both induced inflammatory and
fibrotic reactions. After 2 months collagen-rich granulomas were
observed protruding in the bronchial lumen, which was also
associated with alveolitis in surrounding tissues of the
pulmonary tract. Both CNTs and ground CNTs stimulated the
production of TNF-α in the lung of treated animals. In vitro,
ground CNTs induced the overproduction of TNF-α by
macrophages, and results suggested that CNTs may be
potentially toxic to humans.25 Experimental studies indicate
that CNTs have the potential to induce adverse pulmonary
effects, including alveolitis, fibrosis, and genotoxicity in
epithelial cells. In vitro experiments on rat lung epithelial cells
showed that the acute pulmonary toxicity and the genotoxicity of
CNTs were reduced upon heating but restored upon grinding,
suggesting that the intrinsic toxicity of CNTs is mainly mediated
by the presence of defective sites in their carbon framework.45-47
After 30- and 60-day inhalation exposure, the pulmonary toxicity
of MWCNTs was assessed using biochemical indices in
bronchoalveolar lavage fluid and pathological examination. It
was found that the aerosolized MWCNTs did not induce obvious
pulmonary toxicity in the 30-day exposure group but induced
severe pulmonary toxicity in the 60-day exposure group.48
Poland49 studied two types of MWCNTs (one with straight fibers
longer than 20 μm and the other consisting of low aspect ratio—
i.e., shorter length—tangled aggregates) by administering them
into the peritoneal (abdominal) cavity of mice. The results
indicated increased levels of protein exudation and the formation
of scarlike structures (lesions) called granulomas. MWCNTs
samples with high aspect ratio caused significant PMN or protein
exudation and granulomas on the peritoneal side of the
diaphragm. However, the mesothelial lining on the pleural side
of the diaphragm was normal, and short CNTs did not mimic the
behavior of long CNTs Table 2.49
Toxicity due to metallic contamination of CNTs on lungs
CNTs are generally produced by three main techniques: (1)
arc discharge method, (2) laser ablation method, and (3)
chemical vapor deposition method. In the arc discharge method
a vapor is created by an arc discharge between two electrodes
with or without catalyst. In the laser ablation method, a highpower laser beam impinges on a volume of carbon-containing
feedstock gas (methane or carbon monoxide). It is impossible to
remove catalyst metal contaminants in CNTs entirely without
destroying the structural entity. The CNTs were found to contain
a large proportion of metal catalyst (iron and nickel), which
contribute significantly to the oxidative stress, indicated by the
formation of free radicals and accumulation of peroxidative
products, depletion of total antioxidant reserve, and a loss of cell
viability.50 Transition metals such as iron can be singled out as
those that are important in the toxicity of a range of pathogenic
dusts through their ability to redox-cycle and cause oxidative
stress.51-53 Later Pulskamp et al54 confirmed the role of metal
catalyst in reactive oxygen species formation using rat macrophages NR8383 and human A549 lung cell lines. SWCNTS rich
in 30 wt% iron were reported to cause oxidative stress and loss of
cell viability, including ultrastructural and morphological
changes in human epidermal keratinocytes (HaCaTs),50 and 26
wt% iron-rich SWCNTs resulted in a significant loss of
intracellular low- molecular-weight thiols (GSH) and accumulation of lipid hydroperoxides in murine macrophages.55 None of
a group of animals treated with low dose (0.1 mg) of CNTs
(containing nickel and yttrium) showed any overt clinical signs.
However, 5/9 mice treated with a high dose (0.5 mg) of this
product died. All deaths occurred 4 to 7 days after instillation of
CNTs. The deaths were preceded by lethargy, inactivity, and loss
of body weight. The iron-containing raw nanotubes and pristine
(purified) nanotubes did not cause death in mice but produced
mild signs of inactivity, hypothermia, piloerection, and occasionally shivering. These effects were most noticeable 8 to 12
hours post treatment with the high dose (0.5 mg) of raw
nanotubes as compared to pristine nanotubes.34 The above
animal studies suggested that the iron catalyst might contribute to
the toxicity of CNTs.
Toxicity of functionalized nanotubes on lungs
Functionalization renders CNTs more biocompatible with
physiological systems and hence reduces their toxicity compared
with pristine CNTs. It has been found that the functionalized
SWNT-phenyl-SO3H and SWNT-phenyl-(COOH)2, covalently
bound sidewall functional groups, are less cytotoxic than the
functionalized SWNTs in 1% Pluronic F108, which is stabilized
8
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in a micellar solution without covalent functionalization.56 The
comparison of the toxicity of hydrophobic unmodified
MWCNTs with that of nitric acid–functionalized MWCNTs
revealed that the oxidized nanotubes were significantly more
toxic than their unmodified counterparts. The oxidized
MWCNTs were more highly dispersed in aqueous solution,
which provided a higher probability for interaction with cells.23
Kam et al57 examined endocytosis (intracellular localization) of
SWCNTs and SWCNTs-biotin-streptavidin conjugates within
human promyelocytic leukemia (HL60) cells and human T
(Jurkat) cells. They found that unfunctionalized SWCNTs
exhibited little toxicity, but the SWCNTs-biotin-streptavidin
complexes caused extensive cell death.57 Magrez assessed the
cellular toxicity of MWCNTs and other carbon-based nanomaterials as a function of their aspect ratio and surface chemistry
using lung tumor cells in vitro and found that the carbon
nanomaterials were toxic and cytotoxicity was enhanced when
the surface of the particles was functionalized after an acid
treatment.58 It was shown that the lower concentration of 5 ng/
mL of 6-aminohexanoic acid–derivatized SWCNTs (AHASWCNTs) maintained cell viability and induced a mild
cytotoxicity, but 50,000 ng/mL of AHA-SWCNTs demonstrated
an irritation response by an increase in IL-8.59
Gao et al60 reported that toxicity of cationic functionalized
CNTs (f-CNTs) is a function of CNT surface cation density, and
their observations indicated that soluble f-CNTs are incompatible
and show a significantly improved toxicity profile compared
with pristine CNTs.60 Research shows that f-CNTs are
noncytotoxic and preserve the functionality of primary immune
cells. Two types of f-CNTs were prepared, following the 1,3dipolar cyclo addition reaction (f-CNTs 1 and 2) and the
oxidation/amidation treatment (f-CNTs 3 and 4), respectively.
Both types of f-CNTs were taken up by B and T lymphocytes as
well as macrophages in vitro, without affecting cell viability. It
was discovered that f-CNTs 1, which are highly water-soluble,
did not influence the functional activity of immunoregulatory
cells. f-CNTs 3, which instead possess reduced solubility and
form mainly stable water suspensions, preserved lymphocytes'
functionality while provoking secretion of proinflammatory
cytokines by macrophages. One important conclusion from this
study is that certain types of CNTs functionalized with lipids are
highly water-soluble, which would facilitate their movement
through the human body and would also reduce the risk of
blockage of vital body organ pathways, thus making them more
attractive as drug delivery vehicles.61
MWCNTs. Similarly, ultrafine/nanoparticles were found to
produce enhanced toxicity responses compared with larger sized
particles of similar chemical composition. These investigators
have also indicated transmigration of ultrafine/nanoparticles to
the pulmonary interstitium escaping alveolar macrophages.62-64
The surface area was found to be an important variable that best
predicts the potential toxicity of these refined CNTs. SWCNTs
produced by high-pressure carbon monoxide chemical vapor
deposition process65 having greater surface area showed higher
toxicity compared with SWCNTs produced by arc discharge
method, in media depletion experiments.66,67 Not only size, but
also the surface characteristics play an important role in the
toxicity68 of CNTs. However, the degree of inflammatory
response in subcutaneous tissue in rats showed lengthdependent inflammation. Carbon nanofibers, being lengthier
than SWCNTs, were found to be comparatively more cytotoxic
in a study involving mesenchymal stem cells and monocyte/
macrophage cell line THP-1.69
CNTs have recently been explored as an important drug
delivery system and are considered for use in numerous
technological applications, including electronic devices, field
emission devices, and composite materials; in addition, they
have potential for numerous biological and medical applications.
The horizon of biomedical applications of CNTs is likely to
widen in the future to include multifunctional drug delivery
systems with intended diagnosis and targeting purposes.
However, the unambiguous safety of these CNTs is yet to be
proved, a prerequisite for categorizing them as “generally
regarded as safe.”. Available literature suggests that as compared
with nonfunctionalized CNTs, functionalization of CNTs by
nitric acid, 6-aminohexanoic acid, Pluronic F108, biotinstreptavidin complex, as well as cationic functionalization
increases the cytotoxicity of CNTs on different cell lines,
whereas functionalization of CNTs by phenyl-SO3H, phenyl(COOH)2, and lipids renders them biologically safer. A logical
conclusion from the available meager literature is that the
SWCNTs may exert greater toxic responses as compared with
MWCNTs. It is recommended that the issue regarding toxicity of
CNTs (both single- and multiwalled) be resolved on priority
before taking up any venture in the pharmaceutical and medical
fields. This would also warrant development of a protocol(s) for
the toxicity (more specifically, pulmonary toxicity) evaluation of
CNTs, for both SWCNTs and MWCNTs.
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