Hindawi Publishing Corporation
Journal of Drug Delivery
Volume 2012, Article ID 167896, 14 pages
doi:10.1155/2012/167896
Review Article
Nanomaterials Toxicity and Cell Death Modalities
Daniela De Stefano,1 Rosa Carnuccio,1 and Maria Chiara Maiuri1, 2
1 Dipartimento
di Farmacologia Sperimentale, Facoltà di Scienze Biotecnologiche, Università degli Studi di Napoli Federico II,
Via D. Montesano 49, 80139 Napoli, Italy
2 INSERM U848, IGR, 39 Rue C. Desmoulins, 94805 Villejuif, France
Correspondence should be addressed to Maria Chiara Maiuri, mcmaiuri@unina.it
Received 10 September 2012; Accepted 7 November 2012
Academic Editor: Giuseppe De Rosa
Copyright © 2012 Daniela De Stefano et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
In the last decade, the nanotechnology advancement has developed a plethora of novel and intriguing nanomaterial application
in many sectors, including research and medicine. However, many risks have been highlighted in their use, particularly related
to their unexpected toxicity in vitro and in vivo experimental models. This paper proposes an overview concerning the cell death
modalities induced by the major nanomaterials.
1. Introduction
Nanotechnologies are emerging for important new applications of nanomaterials in various fields. Nanomaterials
are defined as substances which have one or more external
dimension in the nanoscale (1–100 nm). Nanomaterials,
especially nanoparticles and nanofibres, show higher physical
and chemical activities per unit weight. These properties
explain their large application not only in industry but also
in the scientific and medical researches. In fact, in these
areas, the use of many kinds of manufactured nanoparticles
products is in development, such as metal oxide nanoparticles (cerium dioxide, cupric oxide, titanium dioxide, zinc
oxide, etc.), metal nanoparticles (gold, silver, platinum, palladium, etc.), C60 fullerenes nanocrystals, carbon nanotubes
(CNTs), and quantum dots. Initially, the nanomaterials were
believed to be biologically inert, but a growing literature
has highlighted the toxicity and potential risks of their use.
Extrapolations from the field of toxicology of particulate
matter (less than 10 nm) confirm that nanoparticles present a
range of harmful effects [1, 2]. In most cases, enhanced generation of reactive oxygen species (ROS), leading to oxidative
stress which in turn may trigger proinflammatory responses,
is assumed to be responsible for nanomaterials toxicity,
although nonoxidative stress-related mechanisms have also
been recently reported (see the extensive and interesting
reviews [3–10]). However, despite intensive investigations,
the understanding of nanomaterials-induced cellular damage
remains to be clarified. The literature in the field suggests
correlations between different physicochemical properties
and the biological and toxicological effects of cells and tissues
exposure to nanomaterials. First of all, nanomaterials are
characterized by high specific surface area that correlates
with high interfacial chemical and physical reactivity that,
in turn, translates to biological reactivity [11]. The addition
of different types of nanoparticles to various primary cell
cultures or transformed cell lines may result in cell death
or other toxicological outcomes, depending on the size of
the nanomaterial. Quantum dots were reported to localize to
different cellular compartment in a size-dependent manner
[12]. Silica nanoparticles (40–80 nm) can enter into the
nucleus and localize to distinct subnuclear domains in
the nucleoplasm, whereas thin and coarse ones located
exclusively in the cytoplasm [13]. Gold nanocluster (1.4 nm)
intercalates within the major groove of DNA and is a potent
inducer of cell death in human cancer cells [14]. Growing
evidence suggests that the state of nanoparticles aggregation
cannot be ignored; in fact, the toxicity may depend on the
size of the agglomerate and not on the original nanoparticle
size itself [15, 16]. For example, in rats exposed by inhalation
to 20 nm or 250 nm titanium dioxide (TiO2 ) particles, the
half-times for alveolar clearance of polystyrene test particles
2
Journal of Drug Delivery
Cell-site specific
accumulation
(nucleus/cytoplasm)
Size/shape
Aggregation
Biodegradability
Nanomaterials features leading
to toxicity
Surface
changes
Chemical nature
Adsorption of
proteins, ions, etc.
Figure 1
were proportional to the TiO2 particle surface area per
million of macrophages [17, 18]. Clearly, a surface impurity,
resulting from air or water contaminants such as bacterial
endotoxin, could contribute to the cellular responses induced
by nanomaterials, in particular immunological responses
[16]. The same consideration is true for residual materials
(surfactants or transition metals) arising from the synthetic
process [6, 19, 20]. Nevertheless, the adsorption ability and
surface activity are also involved in cellular influences of
nanomaterials. When dispersed in culture medium, some
metal oxide nanoparticles and CNTs could adsorb proteins,
often called “protein corona” such as serum albumin, or
calcium, which could change the biological activity of nanomaterials. This adsorption could be particle size and time
dependent. In these conditions, many nanoparticles form
secondary particles, which are a complex of nanoparticles
and medium components [21–26]. For example, adsorbed
albumin on the CNT was involved in phagocytosis of
the macrophage via scavenger receptor [27]. A surfaceengineered functionalization also may be linked with the
biological nanomaterials activity, although in this item that is
a wanted effect. Moreover, examples of dose-dependent toxicity also are evaluated [6, 28, 29]. As pointed out in a recent
review [6], the degree of recognition and internalization of
nanomaterials likely influences their distribution and may
determine also their toxic potential. It has been reported that
the number of internalized quantum dots (the intracellular
dose) correlates with the toxicity in human breast cancer
cell line [30]. Furthermore, the toxicity and cell death fate
appear to correlate with the type of crystal structures [16,
31]. Finally, the nanomaterials degradability should also be
taken into account (Figure 1). Nondegradable nanomaterials
can accumulate into the cells and/or organs and exert
damage effect as well as their degradation products [32–34].
However, it is not yet clear which of these parameters mainly
influences the nanomaterials toxicity or if all of these features
act together [35]. It is important to note that in the literature
conflicting results are present. These are likely caused by
variations in type, composition, size, shape, surface charge,
and modifications of nanoparticles employed; use of various
in vivo and in vitro models (the cell death mode may be
also cell type dependent); experimental procedures (different
methods to evaluate cell death; nanomaterials dose, concentrations and efficiency of cellular uptake, and time of exposure). This paper aims to give a critical overview concerning
the different cell death modalities induced by nanomaterials.
Deregulated cell death is a common element of several
human diseases, including cancer, stroke, and neurodegeneration, and the modulation of this cellular response can be
an optimal target for an effective therapeutic strategy. Many
cytotoxic agents are potent anticancer therapeutics, whereas
cytoprotective compounds may be used to elude unwanted
cell death in the context of stroke, myocardial infarction or
neurodegenerative disorders [36, 37]. The complex molecular mechanisms and signalling pathways that control cell
death are increasingly becoming understood, and it is now
clear that different cell death subroutines play a critical role
in multiple diseases. In many instances, the modality by
which cells die is crucial to the cell death achievement at
the organism level. The Nomenclature Committee on Cell
Death (NCCD) has recently formulated a novel systematic
classification of cell death based on morphological characteristics, measurable biochemical features and functional
considerations [38]. We will consider these definitions of cell
death in order to summarize and organize the molecular
mechanisms underlying the nanomaterials toxicity. We could
not report all the studies, and we apologize for this; we will
describe the most recently, accurate, and representative ones
in term of the described molecular mechanisms.
2. Nanomaterials and Apoptosis
Apoptosis is a form of cellular suicide that can be classified
into extrinsic and intrinsic apoptosis. Extrinsic apoptosis
indicates the cell death, caspase dependent, stimulated by
extracellular stress signals that are sensed and propagated
by specific transmembrane receptors. Three major lethal
signalling cascades have been reported: (i) death receptor
signalling and activation of the caspase-8 (or -10) and
then caspase-3 cascade; (ii) death receptor signalling and
activation of the caspase-8 then BH3-interacting domain
Journal of Drug Delivery
death agonist (BID), mitochondrial outer membrane permeabilization (MOMP), caspase-9 and caspase-3 pathways; and
(iii) ligand deprivation-induced dependence receptor signalling followed by (direct or MOMP-dependent) activation
of the caspase-9 and after caspase-3 cascade [38]. Intrinsic
apoptosis can be triggered by a plethora of intracellular stress
conditions, such as DNA damage, oxidative stress, and many
others. It results from a bioenergetic and metabolic catastrophe coupled to multiple active executioner mechanisms. This
process could be caspase-dependent or- independent and
is mediated by MOMP associated with the generalized and
irreversible dissipation of the mitochondrial transmembrane
potential, release of mitochondrial intermembrane space
proteins into the cytosol (and their possible relocalization
to other subcellular compartments), and the respiratory
chain inhibition [38]. Apoptosis plays a fundamental role
in development and for maintenance of tissue homeostasis
in the adult organism. In addition, impairment of apoptosis
may contribute to tumour progression.
Nanomaterials are described as triggers of extrinsic and
intrinsic apoptotic pathways; however, the oxidative stress
paradigm of nanomaterials-induced cell death linked to
intrinsic apoptotic network is by far the most accepted, in
fact many in vitro studies have identified increased ROS
generation as an initiating factor of toxicity in nanomaterials
exposed cells [3, 6, 7, 10, 39]. Although it is well established
that the mode of cell death depends on the severity of the cellular insult (which may, in turn, be linked to mitochondrial
function and intracellular energy), it has been difficult to set
up a comprehensive mechanism of nanomaterials cell death
based on conflicting observations present in the literature.
Furthermore, in most of the studies, the molecular mechanisms underlying cell death are not investigated. Finally,
another problem is the nonhomogeneity of the studies, in
terms of materials and experimental methods used, which
makes it difficult to compare.
Sarkar and colleagues showed that the nano-copper
induces intrinsic apoptotic cell death in mice kidney tissue
(via the increase of ROS and reactive nitrogen species
production, regulation of Bcl-2 family protein expression,
release of cytochrome c from mitochondria to cytosol, and
activation of caspase-3), but, in addition, they observed the
activation of FAS, caspase-8, and tBID, suggesting also the
involvement of extrinsic pathways [40]. The exposure to
nano-copper dose-dependently caused oxidative stress and
led to hepatic dysfunction in vivo. Nano-copper caused the
reciprocal regulation of Bcl-2 family proteins, disruption of
mitochondrial membrane potential, release of cytochrome c,
formation of apoptosome, and activation of caspase-3.
These results indicate that nano-copper induces hepatic dysfunction and cell death via the oxidative stress-dependent
signalling cascades and mitochondrial event [41].
Metallic nickel nanoparticles induced apoptotic cell
death through an FAS/caspase-8/BID mediated, cytochrome
c-independent pathway in mouse epidermal cells [42]. Nickel
oxide nanoparticles excited in dose-dependent manner
the increase of ROS production, lipid peroxidation, and
caspase-3 activation in human airway epithelial and breast
cancer cells [43]. Moreover, nickel ferrite nanoparticles
3
provoked apoptosis in human lung epithelial cells through
ROS generation via upregulation of p53 and Bax as well as
the activation of caspases cascade [44].
In vitro, silicon dioxide (SiO2 ) nanoparticles increased
ROS and RNS (reactive nitrogen species) production that,
in turn, can induce the intrinsic apoptotic machinery [45].
Furthermore, Wang and collaborators showed that p53
plays a key role in silica-induced apoptosis in vitro (mouse
preneoplastic epidermal cells and fibroblasts) and in vivo
(p53 wild-type and deficient mice) [46].
TiO2 nanoparticles, sized less than 100 nm, triggered
apoptotic cell death through ROS-dependent upregulation of
FAS and activation of Bax in normal human lung fibroblast
and breast epithelial cell lines [47]. Moreover, it was also
demonstrated that TiO2 nanoparticles induced apoptosis
through the caspase-8/BID pathway in human bronchial
epithelial cells and lymphocytes as well as in mouse preneoplastic epidermal cells [48, 49]. Some reports indicated that
TiO2 induced also lipid peroxidation, p53-mediated damage
response, and caspase activation [50, 51]. In contrast, there
are also reports demonstrating that TiO2 nanoparticles did
not induce oxidative stress on mouse macrophages [52] as
well as did not shown cytotoxicity in human dermal fibroblasts and lung epithelial cells [31].
A number of studies have been published concerning the
effects of CNTs on apoptosis. Multiwall carbon nanotubes
(MWCNTs) induced an increase of ROS, cell cycle arrest,
decrease in mitochondrial membrane potential, determining
apoptosis in different in vitro models [53–56]. In contrast,
another study reported that these nanotubes were nontoxic
[57]. Accordingly, it has been observed that MWCNTs did
not stimulate cell death in vitro after acute exposure and
neither after the continuous presence of their low amounts
for 6 months [58]. Instead, apoptotic macrophages have been
observed in the airways of mice after inhalation of SWCNTs
(single-walled carbon nanotubes) [6]. Accordingly, several
studies in vivo suggest that the exposure to SWCNTs leads
to the activation of specific apoptosis signalling pathways
[59, 60]. For more details, recent interesting reviews focus
on the nanomaterials toxicity in vivo studies [6, 34].
Nanoparticles are frequently detected in lysosomes upon
internalization, and a variety of nanomaterials have been
associated with lysosomal dysfunction [61]. It has been
established that lysosomal destabilization triggers the mitochondrial pathway of apoptosis [62, 63]. Carbon nanotubes
were shown to induce lysosomal membrane permeabilization and apoptotic cell death in murine macrophages and
human fibroblasts [64, 65]. Carbon black nanoparticles
elicited intrinsic apoptosis in human bronchial epithelial
cells with activation of Bax and release of cytochrome c from
mitochondria, whereas TiO2 nanoparticles induced apoptosis through lysosomal membrane destabilization and cathepsin B release, suggesting that the pathway of apoptosis
differs depending on the nanomaterials chemical nature [66].
The lysosomal destabilization induced by TiO2 is also confirmed in mouse fibroblasts [67]. SiO2 and several cationic
nanoparticles, such as cationic polystyrene nanospheres
and cationic polyamidoamine (PAMAM) dendrimers, have
also shown the same mode of action [68–70]. However,
4
also the micromaterials are able to destabilize lysosomes,
in fact silica microparticles have been demonstrated to
induce apoptosis in mouse alveolar macrophages by this
molecular mechanism [70]. A comparative study of nanoversus microscale gold particles demonstrated that nanoparticles present a higher potency in the induction of lysosomal
membrane destabilization [71].
Chronic or unresolved endoplasmic reticulum (ER)
stress can also cause apoptosis [72, 73]. Zhang and colleagues
reported that the ER stress signalling is involved in silver
nanoparticles-induced apoptosis in human Chang liver cells
and Chinese hamster lung fibroblasts [74]. Using omic
techniques and systems biology analysis, Tsai and collaborators demonstrated that upon ER stress, cellular responses,
including ROS increase, mitochondrial cytochrome c release,
and mitochondria damage, chronologically occurred in
the gold nanoparticles-treated human leukemia cells. This
treatment did not induce apoptosis in the normal human
peripheral blood mononuclear cells [75]. It has been shown
that poly(ethylene glycol)-phosphoethanolamine (PEG-PE),
an FDA-approved nonionic diblock copolymer widely used
in drug delivery systems, accumulated in the ER and
induced ER stress and apoptosis only in cancer cells (human
adenocarcinomia alveolar basal epithelial), whereas it did not
have effect in normal cells (secondary human lung fibroblasts
and embryonic kidney cells) [76].
The predisposition of some nanoparticles to target mitochondria, ER, or lysosomes and initiate cell death could be
used as a new cancer chemotherapy principle.
Interestingly, nanoparticles (polystyrene nanoparticles of
20–40 nm with two different surface chemistries, carboxylic
acid, and amines) may also induce apoptosis in individual
cells (differentiated human colorectal adenocarcinoma) that
then propagates to other neighbouring cells through a
“bystander killing effect.” The authors of this study suggest
that ingested nanoparticles represent a potential health risk
due to their detrimental impact on the intestinal membrane
by destroying their barrier protection capability over time
[77].
Surely in this context, a common incentive to synchronize the studies and research efforts is needed. The understand
why cancer cells and distinctive normal cells have different
cell fates as a result of nanomaterials exposure, focusing on
the underlying mechanisms, will allow a better prediction of
the consequences of exposure to nanomaterials and a safer
assessment of the risks (Figure 2).
3. Nanomaterials and Mitotic Catastrophe
Recently, Vitale and colleagues suggested a novel definition of
mitotic catastrophe based on functional consideration [78].
They proposed to consider mitotic catastrophe not a “pure”
cell death executioner pathway but as an oncosuppressive
mechanism that is triggered by perturbations of the mitotic
apparatus, is initiated during the M phase of the cell cycle, is
paralleled by some degree of mitotic arrest, and induces cell
death (apoptosis or necrosis) and senescence [78].
It has been reported that several nanomaterials, such
as SiO2 , TiO2 , cobalt-chrome (CoCr) metal particles, and
Journal of Drug Delivery
carbon nanotubes, interact with structural elements of the
cell, with an apparent binding to the cytoskeleton and
in particular the tubulins [79, 80]. In this setting, some
evidence in vitro demonstrated that carbon nanotubes mimic
or interfere with the cellular microtubule system, thereby
disrupting the mitotic spindle apparatus and leading to
aberrant cell division [81–83]. In particular, the perturbation
of centrosomes and mitotic spindles dynamics caused by
these nanoparticles results in monopolar, tripolar, and
quadripolar divisions, that, in turn, could determinate
aneuploidy [78], an event closely linked to the carcinogenesis. Tsaousi and collaborators found that alumina ceramic
particles increase significantly in micronucleated binucleate
cells [84], which is considered a morphological marker
of mitotic catastrophe [78]. Interestingly, this increase was
much greater after exposure of primary human fibroblasts
to CoCr metal particles, suggesting that these nanoparticles
are particularly efficient in affecting the mitotic machinery
[84]. Apparently, the genotoxic effect of CoCr nanoparticles
is size dependent. Indeed, CoCr nanoparticles induced more
DNA damage than microsized ones in human fibroblasts
(Figure 3). In fact, the mechanism of cell damage appears
to be different after nano- or microparticles exposure. The
enhanced oxidative DNA damage by the microparticles may
result from a stronger ability of large particles to activate
endogenous pathways of reactive oxygen species formation,
for example, involving NADPH oxidases or mitochondrial
activation. It also suggests that the observed genotoxic effect
of the nanoparticles in the comet assay and the micronucleus
assay (i.e., stronger aneugenic effect) is due to mechanisms
other than oxidative DNA attack. A different mechanism of
DNA damage by nanoparticles and microparticles is further
suggested by measures of DNA damage from the comet
and micronucleus assays. The comet assay revealed more
damage in nanoparticle-exposed than in microparticle cells.
In contrast, the micronucleus assay revealed slightly less
centromere-negative micronuclei in nanoparticle exposed
than in microparticle-exposed cells. This assay measures
clastogenic, that is, double strand breakage events. Although
some micronuclei in nanoparticle-exposed cells might not
have been seen as a result of inhibition of cell division from
greater cytotoxicity, these results point to a greater complexity of DNA damage caused by exposure to nanoparticles
compared to microparticles [85]. A genotoxic effect has also
described for silver nanoparticles that induced chromosomal
aberrations, damage of metaphases, and aneuploidy in medaka (Oryzias latipes) cell line [86].
Further studies are needed to validate this dangerous
potential effect of the nanomaterials. Obviously, close attention to safety issues will be required, also in the light of
the potential interference between engineered nanomaterials
and the environment.
4. Nanomaterials and Autophagy or
“Autophagic Cell Death”
Autophagy is a highly conserved homeostatic process,
involved in the recognition and turnover of damaged/aged
Journal of Drug Delivery
5
Nanoparticles
CNTs/TiO2 /SiO2
Lysosomal
dysfunction
Lysosomal
membrane
permeabilization
Carbon black
nanoparticles
PEG-PE/Au
nanoparticles
ROS
Cathepsin B
Mithocondrial
pathway of
apoptosis
ER stress
Cancer cells
Apoptosis
Figure 2
CoCr/TiO2 /SiO2 /CNTs
Binding to
cytoskeletal
tubulin
Disruption
of mitotic
apparatus/
perturbation
of centromers
Alumina
ceramic
Aberrant cell
division
Aneuplody/
carcinogenesis
CoCr
Mitotic
catastrophe
Figure 3
proteins and organelles. During autophagy, parts of the
cytoplasm are sequestered within characteristic double- or
multi-membraned autophagic vacuoles (named autophagosomes) and are finally delivered to lysosomes for bulk
degradation. This process is dynamically regulated by ATG
(Autophagy-related gene) gene family and is finely controlled
by several signalling pathways [87]. Autophagy constitutes a
cytoprotective response activated by cells in the challenge to
cope with stress. In this setting, pharmacological or genetic
inhibition of autophagy accelerates cell death. On the basis of
morphological features, the term “autophagic cell death” has
widely been used to indicate instances of cell death that are
accompanied by a massive cytoplasmic vacuolization [38].
The expression “autophagic cell death” is highly prone to
misinterpretation and hence must be used with caution, but,
discussion this problem is beyond the scope of this paper,
and an excellent paper concerning this subject has been
published [88]. In any case, “autophagic cell death” is used
to imply that autophagy would execute the cell demise. In
the literature, it has been reported that several classes of
nanomaterials induce elevated levels of autophagic vacuoles
in different animals and human cell culture as well as in
vivo models (masterfully summarized in two recent reviews
[10, 61]). Such nanomaterials include alumina, europium
oxide, gadolinium oxide, gold, iron oxide, manganese,
neodymium oxide, palladium, samarium oxide, silica, terbium oxide, titanium dioxide, ytterbium oxide, and yttrium
oxide nanoparticles; nanoscale carbon black; fullerene and
fullerene derivate; and protein-coated quantum dots. The
induction of autophagy was evaluated using panoply of
established methods, including the electron microscopy
detection of autophagic vacuoles, the immunoblot detection
of ATG expression level and/or LC3-I to LC3-II conversion
(an established marker of autophagy activity) and/or cellular
immunolabeling of punctate LC3-II in cytoplasmic vacuoles.
These studies were performed in vivo but mainly in primary
cells and/or cell lines from rat (alveolar macrophages, kidney,
dopaminergic neuron, and glioma), mouse (macrophages
and neuroblasts), porcine (kidney), and human (lung, oral,
colon, breast, cervical and epithelial cancer cells as well as
fibroblasts, peripheral blood mononuclear, and endothelial
and mesenchymal stem cells). Nanomaterials may induce
6
autophagy via an oxidative stress mechanism, such as accumulation of damaged proteins and subsequent endoplasmic
reticulum or mitochondrial stress [39, 89–92] and altering
gene/protein expression and/or regulation, and interfering
with the kinase-mediated regulatory cascades [93–103]. The
increase in autophagic vacuoles in response to nanomaterials
may be an adaptive cellular response. There is evidence that
autophagy can selectively compartmentalize nanomaterials.
In fact, nanoparticles are commonly observed within the
autophagosome compartment, suggesting that activation of
autophagy is a targeted exertion to sequester and degrade
these materials following entrance into the cytoplasm [104].
It is possible that the cells might perceive nanomaterials as an
endosomal pathogen or an aggregation-prone protein (both
commonly degraded by the autophagy machinery). Recent
evidence supports ubiquitination of nanomaterials directly
or indirectly via colocalization with ubiquitinated protein
aggregates, suggesting that cells may indeed select nanomaterials for autophagy through a pathway similar to invading
pathogens [13, 98, 105]. Additionally, ubiquitinated proteins accumulate concomitantly with nanomaterial-induced
autophagic vacuoles [106].
It is important to underlie that nanoscale was a significant factor in eliciting the autophagic response. Autophagy
was not induced by quantum dots that had a tendency
to aggregate to microscale particles into the cells [107].
Nanoscale size dependence was also reported for neodymium
oxide nanoparticle, with larger particles inducing less autophagy [108]. Apparently, modifications of the surface properties might be able to alter the autophagy-inducing activity
of the nanomaterials. Cationic PAMAM dendrimers elicited
autophagy more than anionic ones in vitro [94]. Carbon nanotubes with carboxylic acid group could induce autophagy,
while those functionalized with poly-aminobenzene sulfonic
acid and polyethylene glycol groups were not [100]. Recently,
it has been published that a short synthetic peptide, RE1, binds to lanthanide-based nanocrystals, forms a stable
coating layer on the nanoparticles surface, and significantly
abolishes their autophagy-inducing activity. Furthermore,
the addition of an arginine-glycine-aspartic acid motif to
RE-1 enhances autophagy induced by lanthanide-based
nanocrystals [109].
It is also possible that nanomaterials cause a state of
autophagic dysfunction, correlated with a blockade of autophagy flux, and this may be involved in their mechanism
of toxicity [110, 111]. Nanoparticles could give rise to
autophagy dysfunction by overloading or directly inhibiting
lysosomal enzymes or disrupting cytoskeleton-mediated
vesicle trafficking, resulting in diminished autophagosomelysosome fusion [112]. Nanoparticles could also directly
affect lysosomal stability by inducing lysosomal oxidative
stress, alkalization, osmotic swelling, or causing detergentlike disruption of the lysosomal membrane (see the complete
review of Stern and colleagues [61] about this subject).
Disruption in autophagosome trafficking to the lysosome
has been implicated in several human pathologies, including
cancer development and progression as well as neurodegenerative diseases. As exposure to airborne pollution has been
associated with Alzheimer and Parkinson-like pathologies,
Journal of Drug Delivery
and nanoparticles are the primary particle number and
surface area component of pollution-derived particulates,
Stern and Johnson have recently postulated a relationship
between nanoparticle-induced autophagy dysfunction and
pollution-associated neurodegeneration [113].
Several studies have been suggested also that the
nanomaterial-induced autophagy dysfunction is correlated
with mitochondrial damage [102, 114–118].
In the majority of the studies, autophagosome accumulation induced by nanomaterials treatment was associated with cell death, unfortunately the possibility of
autophagy inhibition was not often investigated (the block
of autophagy flux and autophagy induction both can determinate autophagosome accumulation) [119], and the mechanism of nanomaterial-induced autophagy accumulation in
many cases is unclear.
Interestingly, nanomaterials have been proposed also as
tools to monitor autophagy [120, 121]. In conclusion, a
growing body of the literature indicates that nanomaterials
impact the autophagy pathways, then the possible autophagic
response should be always taken into consideration in the
development of novel nanomaterials systems (Figure 4).
Moreover, further studies should be performed to clarify the
molecular mechanisms underlying the interaction between
nanomaterials and the autophagy machinery as well as to
expand the knowledge of the implications and biological
significance of this modulation.
5. Nanomaterials and Necrosis
Necrosis was, for a long time, considered as an accidental
form of cell death, but in recent years several studies clarified
that this process is regulated and may play a role in multiple
physiological and pathological settings [122]. Several triggers
can induce regulated necrosis, including alkylating DNA
damage, excitotoxins, and the ligation of death receptors [38,
122]. Indeed, when caspases are genetically or pharmacologically inhibited, RIP1 (receptor-interacting protein kinase 1)
and its homolog RIP3 are not degraded and engage in
physical and functional interactions that ultimately activate
the execution of necrotic cell death [38, 122]. It should
be noted that RIP3-dependent and RIP1-independent cases
of necrosis have been described, suggesting that there are
several subprograms of regulated necrosis [38, 122–124].
In a genome-wide siRNA screen, Hitomi and colleagues
elucidated the relationship between appotosis and necrosis
pointing out that some components of the apoptotic pathway
(e.g., the BH3-only protein Bmf) are also crucial in the
necrotic machinery [125]. Moreover, recent studies provide
evidence that apoptosis and necrosis are closely linked [126–
128]. The term “necroptosis” has been used as a synonym
of regulated necrosis, but it was originally introduced to
indicate a specific case of necrosis, which is induced by death
receptor ligation and can be inhibited by the RIP-1 targeting
chemical necrostatin-1 [38, 122, 129].
In the literature, there are confused and inconsistent
examples of necrosis induced by nanomaterials, because on
one hand only the loss of cell viability is often evaluated
without focalising into the cell death modalities and on
Journal of Drug Delivery
7
Alumina/metal oxides/CNTs/fullerene
Damaged
proteins
Inhibition/
overloading of
lysosomal
enzymes
ER/mithocondria
stress
Alteration of
gene/protein
expression/regulation
Interfering
with kinase
cascades
Disruption of
cytoskeletal-mediated
vesicle trafficking
Oxidative
stress
Reduced
autophagosome-lysosome
function
Autophagy
dysfunction
Figure 4
the other hand, there are no single discriminative biochemical markers available yet. Moreover, it should not be
underestimated that the induction of apoptosis in cell culture
is inevitably followed by secondary necrosis, and this could
lead to a misinterpretation of results. However, a recent study
demonstrated that water-soluble germanium nanoparticles
with allylamine-conjugated surfaces (4 nm) induce necrotic
cell death that is not inhibited by necrostatin-1 in Chinese
hamster ovary cells [130]. Although the mechanisms of ligand and surface chemistry, surface charge, and crystallinitybased toxicity are complex, studies are beginning to elucidate
certain surface functional groups and properties that can
effectively alter biological responses. In fact, the crystal structure, with the different forms, of nanomaterials can dictate its
cytotoxic potential. Braydich-Stolle and coworkers identify
that both size and crystal structure (rutile, anatase, and
amorphous) of TiO2 nanoparticles affect the mechanism of
cell death in mouse keratinocyte cell line [131]. They found
that 100% anatase TiO2 nanoparticles induced necrosis in
size-independent manner, whereas the rutile TiO2 nanoparticles elicited apoptosis. Pan and collaborators investigated
the size-dependent cytotoxicity exhibited by gold nanoparticles (stabilized with triphenylphosphine derivatives) in
several human cell lines. All cell types internalised gold
nanoparticles and showed signs of stress. Smaller particles
(<1.4 nm) were more toxic than their larger equivalents.
However, 1.4 nm nanoparticles cause predominantly rapid
cell death by necrosis, while closely related particles 1.2 nm in
diameter affect predominantly apoptosis [132, 133]. Besides,
it has been reported that small (10 nm) silver nanoparticles
had a greater ability to induce apoptosis than other-sized
ones (50 and 100 nm) in mouse osteoblastic cell line and
induce necrosis in rat phaeochromocytoma cells [134]. The
shape-dependent toxicity of polyaniline (PANI) nanomaterials with four different aspect ratios on human lung fibroblast
cells was evaluated. The toxicity increased with decreasing
aspect ratio of PANI nanomaterials; low aspect ratio PANI
nanomaterials induced more necrosis than others [135].
Furthermore, the surface charge seems to be a major factor
of how nanoparticles impact cellular processes. It has been
demonstrated that charged gold nanoparticles induced cell
death via apoptosis, whereas neutral nanoparticles caused
necrosis [136]. Clearly, other parameters may influence the
cell death modalities induced by nanomaterials, such as the
dose or the time of exposure. Depending on the concentration, nano-C60 fullerene caused ROS-mediated necrosis
(high dose), or ROS-independent autophagy (low dose) in
rat and human glioma cell cultures [137]. The type of cell
death induced by silver ions (Ag+ ) and silver nanoparticle
coated with polyvinylpyrrolidone were also dependent on the
dose and the exposure time, with Ag+ being the most toxic in
a human monocytic cell line [138]. The silver nanoparticles
concentrations required to elicit apoptosis were found to be
much lower than the concentrations required for necrosis in
human fibrosarcoma, skin, and testicular embryonal carcinoma cells [139, 140]. In conclusion, although the reports
are often contradictory, the cell death appears roughly cell
type, material composition, and concentration dependent.
For instance, it has been reported that TiO2 (5–10 nm),
SiO2 (30 nm), and MWCNTs (with different size: <8 nm, 20–
30 nm, and >50 nm, but same length 0.5–2 µm) induce cellspecific responses resulting in variable toxicity and subsequent cell fate in mouse fibroblasts and macrophages as well
as telomerase-immortalized human bronchiolar epithelial
cells. Precisely, the macrophages were very susceptible to
nanomaterial toxicity, while fibroblasts are more resistant at
all the treatments, whereas only the exposure of SiO2 and
MWCNT (<8 nm) induce apoptosis in human bronchiolar
epithelial cells. In the experimental conditions of this study,
the investigated nanomaterials did not trigger necrosis [65].
In the same mouse macrophage cell line, it has been
demonstrated that MWCNT (10–25 nm) and SWCNTs (1.2–
1.5 nm) induced necrosis in a concentration-dependent
manner [141]. CNTs have been demonstrated to induce
8
both necrosis and apoptosis in human fibroblasts [142]. In
contrast, Cui and co-workers found that SWNTs upregulate
apoptosis-associated genes in human embryo kidney cells
[143], and Zhu and colleagues showed that MWCNTs
induce apoptosis in mouse embryonic stem cells [144], while
Pulskamp and collaborators assert that commercial CNTs
do not induce necrosis or apoptosis in rat macrophages
[145]. Recently, a multilevel approach, including different
toxicity tests and gene-expression determinations, was used
to evaluate the toxicity of two lanthanide-based luminescent
nanoparticles, complexes with the chelating agent EDTA.
The study revealed that these nanomaterials induced necrosis
in human lymphoblasts and erythromyeloblastoid leukemia
cell lines, while no toxicity was observed in human breast
cancer cell line. Moreover, no in vivo effects have been
observed. The comparative analysis of the nanomaterials and
their separated components showed that the toxicity was
mainly due to the presence of EDTA [146].
The knowledge advances concerning the molecular characterization of necrosis will make necessary more precise and
accurate studies to confirm the ways in which nanomaterials
might cause necrotic death.
6. Nanomaterials and Pyroptosis
Pyroptosis described the peculiar death of macrophages infected by Salmonella typhimurium [147]. Several other bacteria triggering this atypical cell death modality have been
identified. Pyroptosis neither constitutes a macrophage-specific process nor a cell death subroutine that only results from
bacterial infection. Pyroptotic cells can exhibit apoptotic
and/or necrotic morphological features. The most distinctive
biochemical feature of pyroptosis is the early caspase1 activation associated with the generation of pyrogenic
mediators, such as Interleukin-1β (IL-1β) [38].
Recently, it has been shown that the exposure of macrophages (both a mouse macrophage cell line and primary
human alveolar macrophages) to carbon black nanoparticles
resulted in inflammasome activation as defined by cleavage
of caspase-1 to its active form and downstream IL-1β release.
The carbon black nanoparticles-induced cell death was
identified as pyroptosis through the inhibition of caspase1 and pyroptosis by specific pharmacological inhibitors.
The authors showed that, in this setting, TiO2 particles did
not induce pyroptosis or significantly activate the inflammasome [148]. In contrast, it has been shown that nanoTiO2 and nano-SiO2 , but not nano-ZnO (zinc oxide) and
carbon nanotubes, induced inflammasome activation but
not cell death in murine bone marrow-derived macrophages
and human macrophages cell line. Although the caspase-1
cleavage and IL-1β release was induced, the inflammation
caused by nanoparticles was largely caused by the biological
effect of IL-1α [149]. This apparent discrepancy could be
explained considering the different concentration and kind
of nanomaterials used in these studies; moreover, it is
possible that different macrophages perform differently in
response to nanomaterials. Future studies should address this
issue. However, the identification of pyroptosis as a cellular
Journal of Drug Delivery
response to carbon nanoparticles exposure is novel and
relates to health impacts of carbon-based particulates.
7. Conclusions and Perspectives
The continued expansion of the nanotechnology field requires a thorough understanding of the potential mechanisms of nanomaterial toxicity for proper safety assessment
and identification of exposure biomarkers. With increasing
research into nanomaterial safety, details on the biological effects of nanomaterials have begun to emerge. The
nanomaterials intrinsic toxicity has been attributed to their
physicochemical characteristics, that is, their smallness and
the remarkably large surface area per unit mass and high
surface reactivity. In fact, their type, composition and
modifications, size, shape, and surface charge should be
considered. However, the complex death paradigms may also
be explained by activation of different death pathways in a
context-dependent manner. In vitro experiments could be
influenced by a cell type-specific response, and ones in vivo
could be affected by the animal species and the model used
or by pharmacokinetic parameters (administration, distribution, metabolism, etc.). Moreover, the dose, concentrations,
and the time of exposure of a nanomaterial employed are
essential. In effect, the efficiency of cellular uptake of nanomaterials and the resultant intracellular concentration may
determine the cytotoxic potential. Elucidating the molecular
mechanisms by which nanosized particles induce activation
of cell death signalling pathways will be critical for the
development of prevention strategies to minimize the cytotoxicity of nanomaterials. Unfortunately, in the literature,
there are many conflicting data; the most plausible reason is
certainly the discrepancy of nanomaterials and experimental
models engaged. Although some authors have recently
alerted colleagues on these issues [3, 5, 8, 9, 150–152], it
has not yet been put in place a guideline, generally accepted
by the scientific community in the field, to address these
matters. In fact, harmonization of protocols for material
characterization and for cytotoxicity testing of nanomaterials
is needed. In addition, parallel profiling of several classes
of nanomaterials, combined with detailed characterization
of their physicochemical properties, could provide a model
for safety assessment of novel nanomaterials [153]. During
the past decade, owing to major technological advances
in the field of combinatorial chemistry in addition to the
sequencing of an ever increasing number of genomes, highcontent chemical and genetic libraries have become available,
raising the need for high-throughput screening (HTS) and
high-content screening (HCS) approaches. In response to
this demand, multiple conventional cell death detection
methods have been adapted to HTS/HCS, and many novel
HTS/HCS-amenable techniques have been developed [37,
154]. In the last years, several authors started to study the
nanotoxicity with this tools and highlighted the potential
of these approaches [9, 60, 75, 155–161]. An overall aim
should identify HTS/HCS assays that can be used routinely
to screen nanomaterials for interaction with the cell death
modalities system. HTS/HCS may accelerated the analysis
on a scale that commensurates with the rate of expansion
Journal of Drug Delivery
of new nanomaterials but in any case is a first validation
step, then it remains to confirm whether the same identified
mechanisms in vitro are responsible for their in vivo toxicity.
In conclusion, a multilevel-integrated uniform and consistent approach should contemplate for nanomaterial toxicity
characterization.
In spite of the recent advances in our understanding of
cell death mechanisms and associated signalling networks,
much work remains to be done before we can fully elucidate
the toxicological behaviour of the nanomaterials as well as
understand their participation in the determination of cell
fate. More and accurate results are needed for apoptosis,
autophagy, and necrosis induction by nanomaterials; further
studies are necessary to test if the novel strategic targets
identified could be affected either directly or indirectly by
nanomaterials. Moreover, no data are present in the literature
concerning the nanomaterials exposure and other forms of
cell death including anoikis, entosis, parthanatos, netosis,
and cornification. For example, although numerous studies
have been performed on keratinocytes, none of these has
rated cornification, a cell death subroutine restricted to
keratinocytes and functionally linked to the generation of
the stratum corneum of the epidermis [38]. It will be of
considerable interest to establish whether these various cell
death modalities are associated with the intent of identifying a structure-activity relationship and delineating the
mechanisms by which these interactions occur. In addition
to the established paradigms of nanomaterials toxicity, the
study of their interactions with the death signalling pathways
could potentially have many important human pathological
outcomes, including cancer, metabolic disorders, and neurodegenerative disorders.
Abbreviations
Ag+ :
ATG:
Bcl-2:
BH3:
BID:
Bmf:
CNTs:
CoCr:
DNA:
EDTA:
ER:
FDA:
HCS:
HTS:
IL:
MOMP:
Silver ions
Autophagy-related gene
B-cell lymphoma 2
Bcl-2 homology domain 3
BH3-interacting domain death agonist
Bcl-2-modifying factor
Carbon nanotubes
Cobalt-chrome
Deoxyribonucleic acid
Ethylenediaminetetraacetic acid
Endoplasmic reticulum
Food and Drug Administration
High-content screening
High-throughput screening
Interleukin
Mitochondrial outer membrane
permeabilization
MWCNTs: Multiwall carbon nanotubes
NADPH: Nicotinamide adenine dinucleotide phosphate
NCCD:
Nomenclature Committee on Cell Death
PAMAM: Cationic polyamidoamine
PANI:
Polyaniline
PEG-PE: Poly(ethylene glycol)-phosphoethanolamine
RIP:
Receptor-interacting protein kinase
9
RNA:
RNS:
ROS:
SiO2 :
siRNA:
SWCNTs:
tBID:
TiO2 :
ZnO:
Ribonucleic acid
Reactive nitrogen species
Reactive oxygen species
Silicon dioxide
Small interfering RNA
Single-walled carbon nanotubes
Truncated BID
Titanium dioxide
Zinc oxide.
Conflict of Interests
The authors declare that they have no conflict of interests.
Acknowledgment
This work is supported by the Italian Ministry of the University and Scientific Research.
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