Trends in Analytical Chemistry 84 (2016) 160171

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Trends in Analytical Chemistry

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a c

Analytical methodologies for nanotoxicity assessment

Encarnacin Caballero-Daz *, Miguel Valcrcel Cases
Department of Analytical Chemistry, University of Crdoba, Annex Marie Curie Building, C.U. Rabanales, E-14071, Crdoba, Spain

A R T I C L E I N F O A B S T R A C T

Keywords:
Comprehensive research on toxicological effects of nanoparticles is of vital importance owing to their
Nanotoxicity
increasing manufacturing and use. This review outlines critical aspects in nanotoxicity assessment, from
Nanoparticles
In vitro
the primordial characterization of physicochemical properties of nanoparticles, to the most important
In vivo analytical strategies/techniques for elucidating their biological behaviour at cellular and organismal levels.
Assays 2016 Elsevier B.V. All rights reserved.
Inuential factors
Nanoparticle characterization
Analytical methods

Contents

1. Introduction ........................................................................................................................................................................................................................................................ 161

2. Cellular pathways for nanoparticle internalization .............................................................................................................................................................................. 161
3. Determining factors in nanoparticle-induced toxicity ......................................................................................................................................................................... 162
3.1. Nanoparticle intrinsic properties ................................................................................................................................................................................................... 162
3.1.1. Chemical composition ....................................................................................................................................................................................................... 162
3.1.2. Size and shape ..................................................................................................................................................................................................................... 162
3.1.3. Surface features ................................................................................................................................................................................................................... 163
3.1.4. Crystalline structure .......................................................................................................................................................................................................... 163
3.1.5. Agglomeration propensity ............................................................................................................................................................................................... 163
3.2. Other external factors ........................................................................................................................................................................................................................ 164
4. Nanotoxicity assessment ................................................................................................................................................................................................................................ 164
4.1. Conventional in vitro assessment protocols ............................................................................................................................................................................... 164
4.1.1. Genotoxicity .......................................................................................................................................................................................................................... 164
4.1.2. Oxidative stress ................................................................................................................................................................................................................... 164
4.1.3. Cell death ............................................................................................................................................................................................................................... 164
4.1.4. Reduced metabolic activity ............................................................................................................................................................................................. 165
4.1.5. Proinammatory cytokine release ................................................................................................................................................................................ 165
4.2. High-throughput screening tests ................................................................................................................................................................................................... 165
4.3. Advanced omics techniques ......................................................................................................................................................................................................... 166
4.3.1. Genomics ............................................................................................................................................................................................................................... 166
4.3.2. Proteomics ............................................................................................................................................................................................................................ 166
4.3.3. Metabolomics ...................................................................................................................................................................................................................... 166

Abbreviations: AFM, atomic force microscopy; BSA, bovine serum albumin; CFME, carbon ber microelectrode; CME, clathrin-mediated endocytosis; CvME, caveolin-
mediated endocytosis; EC50, median effective concentration; EDX, energy dispersive X-ray; EIS, electrochemical impedance sensing; DCFH-DA, dichlorodihydrouorescein
diacetate; DLS, dynamic light scattering; FBS, fetal bovine serum; FCS, fetal calf serum; FTIR, Fourier transform infrared spectroscopy; GSH, glutathione; HCS, high content
screening; HTS, high-throughput screening; ICP, inductively coupled plasma; LDH, lactate dehydrogenase; MDA, malondialdehyde; MTT, 3-(4,5-dimethylthiazolyl-2-yl)-
2,5-diphenyltetrazolium bromide; NBT, nitroblue tetrazolium; NMR, nuclear magnetic resonance; NPs, nanoparticles; NTA, nanoparticle-tracking analysis; PI, propidium
iodide; ROS, reactive oxygen species; RTCA, real time cell analyzer; SEM, scanning electron microscopy; SOD, superoxide dismutase; SRCD, synchrotron radiation circular
dichroism; SRXAS, synchrotron radiation X-ray absorption spectroscopy; SRXRF, synchrotron radiation X-ray uorescence; TEM, transmission electron microscopy; TUNEL,
terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling; XANES, X-ray absorption near-edge structure; XPS, X-ray photoelectron spectroscopy;
XRD, X-ray diffraction; XRF, X-ray uorescence.
* Corresponding author. Tel.: +34 957218616; Fax: +34 957218616.
E-mail address: nayara104@hotmail.com (E.C. Daz).

http://dx.doi.org/10.1016/j.trac.2016.03.007
0165-9936/ 2016 Elsevier B.V. All rights reserved.
 E. Caballero-Daz, M. Valcrcel Cases / Trends in Analytical Chemistry 84 (2016) 160171 161

4.4. Electrochemical techniques ............................................................................................................................................................................................................. 167

4.4.1. Carbon ber microelectrodes (CFMEs) ....................................................................................................................................................................... 167
4.4.2. Electrochemical impedance sensing (EIS) ................................................................................................................................................................. 167
4.5. In vivo nanotoxicity evaluation ...................................................................................................................................................................................................... 167
4.5.1. Nanoparticle uptake and biodistribution ................................................................................................................................................................... 167
4.5.2. Histological analysis .......................................................................................................................................................................................................... 168
4.5.3. Blood chemistry analysis ................................................................................................................................................................................................. 168
4.5.4. Clearance pathways ........................................................................................................................................................................................................... 168
5. Conclusions ......................................................................................................................................................................................................................................................... 169
Conict of interest ............................................................................................................................................................................................................................................ 169
Acknowledgements .......................................................................................................................................................................................................................................... 169
References ............................................................................................................................................................................................................................................................ 169

1. Introduction Finally, there are two stages that comprise in vivo evaluation. Usually,
testing of organ toxicity and other biological responses induced by
Dramatic advances in Nanoscience and Nanotechnology (N&N) NP exposure are performed in model animals.
have provoked a growing manufacturing and use of engineered NPs This review summarizes those more relevant aspects to be con-
(NPs) inevitably leading to direct and indirect emissions into the sidered in nanotoxicity studies, making special reference to the main
environment. As a consequence, evaluation of potential risks for cellular pathways for NP uptake, inuential factors, and different
human health and environment derived from exposure to NPs is a analytical approaches available for NP-induced toxicity assess-
matter of social concern these days. Donaldson et al. [1] coined for ment, from classical evaluation protocols to novel omics techniques.
the rst time in 2004 the term nanotoxicology to establish a new
subcategory of toxicology focuses on lling gaps in knowledge of
NP-induced toxicity, emphasising research efforts on those NPs more 2. Cellular pathways for nanoparticle internalization
likely to come into contact with human being. Fig. 1 illustrates the
principal work stages involve in any nanotoxicity assessment study Different cellular internalization mechanisms have been pro-
[2]. As can be appreciated, NP characterization must be taken into posed to explain the entrance of NPs through plasma membrane.
account in preliminar studies with the purpose of correlating the Fig. 2 shows a general scheme including the main mechanisms re-
biological response obtained with well-characterized and dened sponsible for cellular uptake of NPs.
NPs. Subsequently, cell-based systems are used as in vitro assess- Most research in this eld concludes that endocytic pathways
ment plataforms to elucidate the toxic impact of NPs at cellular level. constitute the main processes of transport of NPs into cells.

Fig. 1. Illustration of work stages involved in research of nanotoxicity. Reproduced with permission of [2].

Fig. 2. Schematic representation of the principal cellular uptake mechanisms of NPs.

162 E. Caballero-Daz, M. Valcrcel Cases / Trends in Analytical Chemistry 84 (2016) 160171
Endocytosis is a form of active transport by which a cell internal-
izes objects by enclosing them in vesicles or vacuoles that are
generated from its own cytoplasmic membrane [3]. There are two
general modalities of endocytosis: phagocytosis and pinocytosis.
Phagocytosis (cell eating) is the predominant mechanism for
uptake of NPs larger than 500 nm by immune response cells (i.e.
monocytes, macrophages, neutrophils and dendritic cells) [4]. This
entry pathway is rstly activated by opsonization processes in the
bloodstream, in which foreign substances are recognized by small
proteins (opsonins) that make them visible to immune response
cells. Protein-coated NPs are then bound to receptors on the cell
plasma membrane, triggering a signal cascade in the target cell.
As a result, a cell surface extension is formed engulng NP in an
intracellular vesicle (0.5 to 1 m in size) called phagosome. These
vesicles will be later fusioned with endosomes and lysosomes for
digestion. Once NPs are degraded, receptors are cycled back to the
cell membrane [4,5]. Pinocytosis (cell drinking) is an entrance
route that occurs in most cellular types constantly and indepen-
dently from the needs of cell [4]. Through this mechanism, plasma
membrane invaginates and engulfs materials by forming vesicles
0.55 m in size [6]. There are four modes of pinocytosis, namely:
macropinocytosis, receptor-mediated endocytosis, adsorptive pi-
nocytosis, and other alternative routes [5]. In general terms,
macropinocytosis allows uids and other essential generic mate-
rials (e.g. salts, glucose and amino acids) to be taken up into cells
in a nonspecic manner [6]. During process, those NPs with a size
about 1 m and located near the plasma membrane may be also
internalized coincidentally [5]. Macropinocytosis has been de-
scribed as a signicant mechanism for the cellular uptake of
positively charged NPs [3]. Among highly selective receptor-
mediated mechanisms, clathrin-mediated endocytosis (CME) is
generally accepted as the primary internalization process for NPs
in the range of 100120 nm through specic regions of the plasma
membrane enriched in clathrin proteins [3]. During CME, clathrin
proteins bind to proteins known as adaptor proteins 2 (AP-2) forming
vesicles that swallow NPs and transport them into cytoplasm.
After endocytosis, clathrin proteins dissociate from vesicles and
return to the cell membrane [6]. Other typical receptor-mediated
pathway in endothelial cells is the caveolin-mediated endocytosis
(CvME). In CvME, the protein caveolin integrates itself within the
cholesterol rafts present in hydrophobic domains of the plasma
membrane, whereby encouraging curvature and formation of ask-
shaped invaginations of 5080 nm in size [7]. These vesicles nally
detach from the membrane bringing their content to compart-
ments called caveosomes [4].
Unlike these selective and specic mechanisms, NPs may be also
internalized by non-specic interactions with complementary
binding sites on the cell surface. This process is known as adsorp-
tive pinocytosis. Cationic NPs show a high binding anity to the
negatively charged cell surface and thus are good candidates to be
taken up through this mechanism [5].
Several endocytic routes seem to be clathrin- and caveolin-
independent and involve regulation by other proteins such as Ras
homolog family member A, ADP-ribosylation factor 6, or the cell
division control protein 42 homolog, Cdc42 [5].
Finally, NPs may also penetrate into cells by non-endocytic
mechanisms of passive diffusion involving a chemical concentra-
tion gradient between intra and extracellular medium. Jiang et al.
[8] recently evaluated the cellular internalization of 2, 4 and 6 nm
core gold NPs featuring neutral (zwitterionic), anionic and cat-
ionic headgroups. Variable results from these studies were explained
by a modulation of uptake pathway depending on surface proper-
ties of NPs. According to authorsconclusions, zwitterionic gold NPs
were primarily internalized through passive diffusion, while the in-
ternalization of cationic and anionic NPs was dominated by multiple
endocytic routes.
3. Determining factors in nanoparticle-induced toxicity
The potential harmful effects associated with NPs have been dem-
onstrated to be highly varying depending on multiple inuential
factors such as physicochemical characteristics of the tested NPs and
exposure conditions. This might lead to misleading and little con-
clusive results that prevent from extrapolating ndings beyond each
case of study. In order to overcome this bottleneck, adequate con-
trols and complementary assays involving multiple cell types and
readouts are strongly required.
3.1. Nanoparticle intrinsic properties
It should be highlighted that one of the most important chal-
lenges of nanotoxicology research is to consider the highly dynamic
chemical composition and physical properties of NPs. And it is that,
once synthesized, NPs experience an evolution process as a result
of chemical and physical transformations, what determines their
interaction mechanisms with biological systems and resulting
nanotoxicity. In addition, new generations of NPs are appearing with
a great chemical and structural diversity what leads to highly vari-
able modes of interaction with living systems and to a growing
diculty for establishing standard protocols of evaluation [9]. There
is a minimum set of NP properties that are commonly agreed in char-
acterization studies for nanotoxicity assessment purposes. These
include chemical composition, size, shape, surface, crystallinity, and
agglomeration/aggregation state.
3.1.1. Chemical composition
One of critical physicochemical characteristics inuencing the
NP toxicity is their chemical composition, since elements them-
selves show different chemical reactivity and toxicity in biological
media. Analytical techniques suitable for analysis of chemical com-
position of NPs include X-ray photoelectron spectroscopy (XPS),
raman spectroscopy, X-ray uorescence spectroscopy (XRF), energy
dispersive X-ray analysis (EDX), inductively coupled plasma (ICP)
analyses, fourier transform infrared spectroscopy (FTIR) and nuclear
magnetic resonance (NMR) [10].
It is logical to think that NPs composed of elements with a low
associated toxicity will induce less harmful effects than those made
up of highly toxic elements. In this context, Lanone et al. [11] tested
the toxicity of 24 engineered NPs of similar equivalent spherical di-
ameter but different elemental composition in two human
pulmonary cell lines. Authors found that among all the tested NPs,
copper and zinc-based NPs presented the highest toxicity whatev-
er their oxidation status. In contrast, titania, alumina, ceria and
zirconia NPs showed moderate toxicity, while no toxicity was ob-
served for those tungsten carbide-based NPs. Other research works
also conrmed a higher cytotoxicity for zinc oxide NPs in compar-
ison with other NPs [12].
3.1.2. Size and shape
NP size and shape result to be crucial factors as they dene both
preferential internalization mechanisms and uptake eciency, con-
ditioning thus the toxicological behaviour of NPs in biological
environments. A variety of analytical techniques are appropiate for
NP size and shape determination including transmission electron
microscopy (TEM), scanning electron microscopy (SEM), atomic force
microscopy (AFM) and X-ray diffraction (XRD) [10,1316].
Size-dependent nanotoxicity of silver NPs has been recently ex-
amined by Chen et al. [17] in studies with sh red blood cells in
order to elucidate their suitability for biomedical applications. While
silver NPs-50 showed the highest level of adsorption and uptake,
suggesting an optimal nanoparticle size for passive uptake by red
blood cells, smallest NPs (silver NPs-15) were found to be more cy-
totoxic as a result of oxidative stress phenomena. Research with
 E. Caballero-Daz, M. Valcrcel Cases / Trends in Analytical Chemistry 84 (2016) 160171 163

different sized gold NPs revealed that 1.4 nm NPs were the most toxic serum compounds resulting in a reduced endocytosis rate and
for four cell lines compared with 15 nm NPs which were shown to cytotoxicity.
be non-toxic at up to 60-fold higher concentrations [18].
Nanotoxic effects have been also shown to vary depending on
NP shape. Spherocylindrical NPs have demonstrated to exhibit a 3.1.4. Crystalline structure
greater ability of translocation inside cells in comparison with spher- The crystallinity of NPs might also play a relevant role in their
ical, pyramidal, conic, cubic or rod-like NPs [6]. Other demonstration physicochemical behaviour and toxicological impact. Differences in
of the inuence of nanoparticle shape on nanotoxicity was re- the orientation of the atoms result in variations in chemical sta-
ported by Zheng et al. [19] when compared the nanotoxicity induced bility of NPs and in their resulting biological response. XRD is one
by several optical NPs (gold nanospheres and nanorods, silver of the classical and most used techniques to provide information
nanospheres and triangular nanoplates, and quantum dots) to human about the crystallographic structure of NPs, which is dened by the
carcinoma cells. Authors found that silver and gold nanospheres were pattern, position, intensity and shape of the diffraction peaks [21].
more toxic than silver triangular nanoplates and gold nanorods, A vast majority of studies about the inuence of NP crystallin-
respectively. ity on nanotoxicity have been conducted in model aquatic organisms
[30]. In this context, Iswarya et al. [31] investigated the possible toxic
3.1.3. Surface features impact of titania NPs in its two crystalline phases (i.e. anatase and
Surface properties of NPs (i.e. surface area, chemistry and charge) rutile) to freshwater microalgae Chlorella sp. EC50 values (i.e. con-
strongly determine their interaction with cellular membranes as rst centration of a substance that causes a certain effect in 50% of the
step for induced cytotoxicity mechanisms. Analytical techniques for tested organisms) were found to be strongly dependent on the NP
determining surface chemistry of NPs are the same that those pre- crystallinity, resulting to be 3.36 and 6.25 mg L1 for anatase and
viously commented in section 3.1.1 for chemical composition analysis. rutile NPs, respectively.
Among them, ICP analysis constitutes the most appropiate choice
for metal and metal oxide NPs when the quantication of metal ions
3.1.5. Agglomeration propensity
released into cellular environments is imperative [20]. Regarding
The NP aggregation is a quite common phenomenon in biolog-
surface charge, zeta potential of NPs is usually determined by light-
ical media containing salts and proteins that might lead to the
scattering electrophoresis or electro-acoustophoresis methods.
formation of aggregates/agglomerates with much larger hydrody-
Finally, surface area and porosity might be investigated by the
namic diameter, and shape, surface, colloidal stability and
Brunauer-Emmett-Teller analysis method which is based on gas
homogeneity very different to those of the parental material. In an
adsorption/desorption isotherms [21].
extreme case, NPs may precipitate at the bottom of the plate re-
Cytotoxicity values highly dependent on surface area of NPs were
sulting in a local concentration dose distinct from the originally
reported in cellular studies with silicon-based NPs [22]. In terms
intended one [21]. NP-tracking analysis (NTA) and dynamic light
of surface chemistry, NP functionalization might have serious effects
scattering (DLS) are the most utilized techniques for NP sizing in
on the resulting toxicity given that certain functional groups confer
liquids yielding information about changes in NP hydrodynamic
a greater biocompatibility to NPs or, on the contrary, a greater toxic
radius arising from bioconjugation events [13,32]. Whereas DLS pro-
potential. Hauck et al. [23] modulated the mammalian cellular uptake
vides a NP size distribution resulting from combination of signals
of gold nanorods by manipulating the surface charge and function-
of all NPs present in the liquid matrix, NTA allows for visualiza-
al groups located on NP surface. Authors found that the absolute
tion and monitoring of single NPs or aggregates.
quantity of uptake and consequent cytotoxicity, was dependent on
Most of research studies dene the toxicity of aggregated NPs
the chemistry of the molecules adsorbed onto the nanorod surface.
as a function of the tested NP concentration. For instance, the ag-
Other ndings reported for studies with silver NPs demonstrated
gregation behaviour of zinc oxide NPs was correlated with their toxic
that the viability of NIH/3T3 broblasts and cellular uptake were
potential to RAW 264.7 cells in a NP concentration-dependent
both dependent on NP surface chemistry. In addition, further surface
manner. According to authors, the aggregation process reduced the
modication of NPs with PEG molecules was found to decrease the
ion release from NPs and partially covered up their cytotoxic po-
NP internalization and also the cytotoxicity values [24]. As re-
tential to cells. Fig. 3 illustrates the agglomeration effect on the
ported by these authors, other publications have also demonstrated
cytotoxicity induced by zinc oxide NPs as a function of NP concen-
that the functionalization of NPs with PEG corona provides multi-
tration [33].
ple benets such as reducing nonspecic protein adsorption, cellular
uptake and cytotoxicity [25,26].
Concerning nanoparticle surface charge, it is crucial to always
investigate whether the zeta potential of NPs is positively or neg-
atively charged since this fact marks the interactions with biological
systems. Numerous reports have concluded that positively charged
NPs induced more toxic effects on cells than their negatively charged
counterparts [27]. To mention a more detailed example, Baek et al.
[28] determined the correlation between physicochemical proper-
ties of zinc oxide NPs and their cytotoxicity to human lung A549
cells by testing NPs featuring two different sizes (20 nm and 70 nm)
and charges (positive and negative). In vitro assays showed that pos-
itively charged NPs of small size exhibited the highest toxicity
probably due to their greater interaction with the negatively charged
plasma membrane. It is worthy to note in this point that the for-
mation of protein corona surrounding the NP might alter its surface
charge leading to variable biological responses. For instance, Merhi
et al. [29] found that both fetal calf serum (FCS) and bovine serum
albumin (BSA) sharply reduced the positive surface charge of NPs Fig. 3. Implications of zinc oxide NP aggregation on their cytotoxicity to murine mac-
by associations between the cationic surface of NPs and the anionic rophages RAW 264.7 cells. Reproduced with permission of [33].
164 E. Caballero-Daz, M. Valcrcel Cases / Trends in Analytical Chemistry 84 (2016) 160171
3.2. Other external factors
Some discrepancies have been found in results from in vitro pro-
tocols when NPs with similar physicochemical properties are
evaluated. This fact might be explained by the undeniable impact
of cell type/line and conditions and constituents of the culture
medium on colloidal stability of NPs in physiological matrices, what
generates important consequences on the toxicological prole of
identical NPs. Nafee et al. [34] checked the colloidal stability and
cytotoxicity of chitosan-modied PLGA NPs in culture medium under
variable conditions of study. Cell type was found to be a determin-
ing factor in all assays, dening a different biological response for
same incubation conditions and tested NPs. Equally, different pH
values of medium exerted variable cytotoxicity in terms of lactate
dehydrogenase (LDH) content. Specically, NPs acquired positive
surface charge in acidic conditions what led to a greater harmful
effect on cells membranes. Authors also observed that the pres-
ence of FBS (fetal bovine serum) in culture medium increased the
cell viability in treated cells given that FBS formed a protective shell
around the NPs shielding their original harmful effects. As demon-
strated in these studies, cellular type and line, even its differentiation
stage, might make cells to show a different sensitivity to the cyto-
toxic effects of NPs. It has been extensively reported in literature
that the induction and type of NP-induced toxicity mechanisms
depend on the tested cells [3537].
4. Nanotoxicity assessment
Taking into account those challenging factors associated with re-
search in nanotoxicity, that were previously commented in section
3.1, nanotoxicologists have opted for two different strategies for
nanotoxicity assessment purposes. In general terms, the rst meth-
odology consists in using conventional in vitro protocols through
acquisition of commercially available kits that measure a specic
cellular endpoint likely to be altered by NPs. And the second, and
most interesting approach, has as objective to develop and use ad-
vanced analytical methodologies with a greater degree of adaptation
and specicity to the assessment of nanotoxic effects. Recently,
Gunsolus and colleagues [9] have extensively reviewed the analyt-
ical techniques most used in NP toxicity research, making special
reference to NP physicochemical properties and transformations,
and their biological impact.
4.1. Conventional in vitro assessment protocols
4.1.1. Genotoxicity
The potential of some NPs to damage the cellular DNA is com-
monly evaluated by alkaline comet assay or single-cell gel
electrophoresis. The basis of this rapid and sensitive assay lies in
the separation of DNA fragments from lysed cells in agarose gel due
to their different migration distances. Once separated, DNA frag-
ments are visually analyzed by uorescence microscopy following
staining with ethidium bromide dye, mainly. The extent of migra-
tion is nally interpreted as a reection of the DNA damage [38,39].
By using this in vitro test, recent studies have demonstrated the
genotoxic potential of some NPs, such as silver and metal oxide NPs
[4042]. TUNEL (terminal deoxynucleotidyl transferase deoxyuridine
triphosphate nick end labeling) assay is also useful for detecting DNA
damage-induced apoptosis mechanisms. TUNEL assays have been
reported for apoptosis assessment and DNA damage in cells exposed
to carbon nanotubes, and silver and zinc oxide NPs [4345]. Other
approaches also available to assess the mutagenic activity of NPs
comprise micronucleus assay, which examines the ability of NPs to
induce the formation of micronuclei, and the evaluation of expres-
sion of proteins related to DNA repair [46,47].
4.1.2. Oxidative stress
One of the most widely reported nanotoxicity mechanisms is the
generation of intracellular reactive oxygen species (ROS). The over-
production of ROS overwhelms the antioxidant defenses of cells
leading to oxidative stress and other side damages such as alter-
ation of proteins, lipid peroxidation, DNA breakage and gene
expression modulation [48]. There are two ways to analyze oxida-
tive stress in cells: direct methods that quantify the amount of
intracellular ROS, and indirect methods that monitor other
side effects as a result of a prolonged oxidative stress. Both
dichlorodihydrouorescein diacetate (DCFH-DA) and nitroblue tet-
razolium (NBT) assays belong to the rst category. DCFH-DA is
oxidized into a green uorescent product in presence of intracel-
lular ROS and the obtained uorescence signal might be directly
correlated to the ROS level [39]. Copper oxide NPs have been re-
cently tested in hepatocellular carcinoma cells by DCFH-DA test and
results revealed signicant intracellular ROS generation of up to 242%
in poorly differentiated cells [49]. Less used, the colorimetric NBT
assay is based on the reduction of a tetrazolium salt to formazan
crystals in presence of superoxide anions. To give an example, Dubey
et al. [50] recently studied the impact of titanium dioxide and zinc
oxide NPs in cells from gill tissue of Wallago attu using this in vitro
test.
Concerning indirect methods, these basically contemplate the
evaluation of glutathione (GSH) content, malondialdehyde (MDA)
formation and superoxide dismutase (SOD) activity. While GSH is
an antioxidant and its depletion is indicative of ROS overproduc-
tion in cells, the increase of MDA, which is a byproduct of lipid
peroxidation, is clear evidence of cellular oxidative stress [39]. These
two biomarkers demonstrated the potential of silica NPs to induce
oxidative effects in human liver cells in terms of ROS generation,
lipid peroxidation and depletion of GSH following exposure [51].
Oxidative stress after exposure of aquatic organisms to silver NPs
has been also reported in studies with drosophila melanogaster and
zebrash [52,53]. SOD catalyzes the dismutation of superoxide anion
and its activity is a reection of superoxide production in cells. An
interesting study of Gao et al. [54] related the depletion of GSH in
HL7702 cells, following the treatment with gold NPs, with strong
Au-S bonding interactions between NP surface and antioxidant.
4.1.3. Cell death
Apoptosis and necrosis (i.e. programmed and accidental cell
death) reect the ability of NPs to trigger intracellular suicide mecha-
nisms or destroy cells. NP-induced cell death assessment focuses
on examining the mitochondrial membrane integrity or potential,
measuring the apoptotic protein levels/activation or evaluating DNA
fragmentation [55]. In this section, it will be discussed the princi-
pal screening techniques to evaluate the membrane integrity, given
that these constitute the most common ways to measure cell death-
related processes.
4.1.3.1. Trypan blue and propidium iodide staining tests. These vital
dyes can freely cross the cell membrane when it is compromised
and stain the intracellular components. Therefore, only damaged
cells will appear stained [38]. Fullerol NPs were tested in human
retinal epithelial cells by trypan blue (TB) assay. Experimentally,
treated cells were rstly stained by TB reagent and then loaded onto
hematocytometer chamber and analyzed by microscopy, yielding
information on the number of dead and viable cells. Results con-
rmed that NP concentrations above 10 M after 17 h treatment
resulted to be very cytotoxic to cells [56]. Similar scheme was fol-
lowed by Acar et al. [57] in order to investigate the toxicity of
titanium dioxide NPs in human amniotic uid cells.
Propidium iodide (PI) staining followed by ow cytometry anal-
ysis have also applied to examine NP-induced apoptosis in terms
of alteration in cell cycle progression [5860]. Particularly, the
 E. Caballero-Daz, M. Valcrcel Cases / Trends in Analytical Chemistry 84 (2016) 160171
execution of PI test in MCF-7 cancer cells treated with zinc oxide
NPs showed a signicant induction of apoptosis, as determined by
differences in the percentage of cells in each phase of the cellular
cycle compared with control cells [61].
4.1.3.2. Neutral red staining test. Neutral red dye is only incorpo-
rated into viable cells, consequently, unstained cells will correspond
to those with compromised membranes [39]. A variation of the con-
ventional neutral red test is the so-called neutral red retention time
(NRRT) assay that measures the retention of the dye over time. NRRT
assay followed by absorbance measurements has been applied to
evaluate the toxicity of copper, chromium, cobalt, gold and tita-
nium NPs [62].
4.1.3.3. Lactate dehydrogenase content test. In damaged cells, lactate
dehydrogenase (LDH) is excreted out of cellular membrane and can
be quantied through a coupled enzymatic assay where a tetrazo-
lium salt is converted into a red formazan product that is detected
spectrophotometrically. The absorbance of the reaction product is
thus related directly to the membrane integrity disruption [38]. Cell
death caused by copper oxide, gold and silver NPs have been
recently examined by using commercially available LDH tests
[6365].
4.1.3.4. Phosphatidylserine translocation monitored by Annexin
V. Phosphatidylserine is translocated to the outer leaet of the cel-
lular membrane in apoptotic cells, and can be monitored by using
uorescently-labelled annexin V that binds very specically to the
exposed phosphatidylserine allowing for its detection by ow
cytometry and uorescence microscopy [38]. Several examples in
literature describe the use of annexin-V/PI double-staining assays
in order to distinguish apoptotic cells from necrotic cells in re-
sponse to NPs [66,67]. Annexin-V FITC is commonly used as a marker
of apoptosis, while PI only recognizes necrotic cells. Data from double
staining assays are shown as percentages of unstained cells (viable
cells), those stained with PI (necrotic cells), those stained with
annexin-V FITC (apoptotic cells), and dual stained cells (late apop-
tosis) [68].
4.1.3.5. Mitochondrial membrane potential and apoptotic protein
level/activity. Mitochondrial membrane depolarization after NP ex-
posure is a clear indicative of iniciation of apoptosis processes. The
state of potential is normally examined by the use of membrane-
permeant uorescent dyes that are able to accumulate in the
mitochondria and modify its uorescence depending on the po-
tential state. For example, the typical uorescent probe JC-1 exhibits
red uorescence in healthy cells, whereas it shows green uores-
cence in cells with impaired mitochondria [69]. Consideration of
changes in activity/level of apoptosis-related proteins might help
nanotoxicologists to elucidate NP-induced cell death mechanisms.
In this sense, caspase-3 and caspase-7 activation are common in-
dicators evaluated in nanotoxicity assays since these enzymes play
a crucial role in apoptotic cascade. Increased activity/level of caspases
37 has been reported in cell-based assays following incubation with
nickel oxide and gold NPs [70,71].
4.1.4. Reduced metabolic activity
Cellular damage also might lead to a reduction in metabolic ac-
tivity of cells. One of the most common analytical methods is MTT
[3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide]
assay where a tetrazolium salt is reduced to formazan product as
a result of the mitochondrial activity in alive cells. Formazan is a
colored compound that can be spectrophotometrically quantied
at 500600 nm, resulting its absorbance value proportial to per-
centage of viable cells in samples. The cytotoxicity of several NPs
has been assayed by comparing the absorbance values of samples
165
with controls [72,73]. However, some interferences between this
assay and various NPs have been reported in literature. In this line,
the Lupus group [74] conrmed that titanium dioxide NPs induced
the transformation of MTT to formazan in experiments without cells
due to the photocatalytic properties of these NPs. Equally, Kroll et al.
[75] demonstrated that both carbon black and titanium dioxide NPs
interfered with the light absorption of formazan product in MTT
assays.
Cell viability for nanotoxicity assessment purposes might be also
evaluated by using alamar blue or resazurin assay in which a
nonuorescent compound (alamar blue) is converted into a pink
uorescent product in metabolically active cells. Using this method,
Gliga et al. [76] found cytotoxicity for 10 nm citrate- and PVP-
coated silver NPs in BEAS-2B cells after 24 h exposure.
Adenosine triphosphate (ATP) luminiscence assays are also other
available alternatives to measure cell viability by quantifying the
luminiscent signal derived from a conversion reaction of luciferin
to oxyluciferin in presence of ATP. Huang et al. [77] reported a sig-
nicant decrease in ATP content of human dermal broblasts in
response to gold NPs.
4.1.5. Proinammatory cytokine release
NPs may also trigger immune response in cells by the release
of cytokines that are important biomarkers, mainly expressed by
macrophages, that play a key role in the regulation of the immune
response, inammatory reaction and phagocytosis [78]. Levels of
IL-1, IL-6 and TNF- cytokines in culture medium were evalu-
ated following the exposure of human monocytic cells to iron oxide
NPs. Results showed no increases in cytokine secretion what was
attributed to the protective protein corona shield of NPs [79].
4.2. High-throughput screening tests
Taking into consideration the increasing production of NPs with
tuneable physicochemical features, and multiple factors that de-
termine their toxicity in real environments, there is an urgent need
for methods that allow for testing multiple samples simultane-
ously. Current research in nanotoxicology proposes high-throughput
screening (HTS) platforms as an ecient alternative to overcome
this bottleneck and speed up NP risk assessment. Fig. 4 shows a ow
chart illustrating the work stages required for performing the high
throughput nanotoxicity screening. Firstly, NPs are acquired from
a supplier that usually distributes them as dry powders. NPs are then
characterized as-produced, in terms of chemical composition (purity),
size, shape, crystallinity and surface features. Subsequently, NP dis-
persions in liquid media (e.g. water or culture medium) are prepared
and further characterization is required in this stage in order to elu-
cidate the colloidal stability of NPs and any change in their surface
as a result of interactions with other entities present in solution.
Once characterized, NPs are tested in HTS platforms and nally, the
set of obtained data is mined and processed by advanced statisti-
cal tools [21].
Numerous reports in literature have conrmed the usefulness
of HTS assays to simultaneously evaluate multiple cellular injury
pathways across an array of different NPs and incubation condi-
tions (e.g. doses and exposure times) [80,81].
Available HTS approaches can be classied into two modali-
ties: plate reader-based assays that use luminiscence, uorescence
or absorption readouts, and high content screening (HCS) methods.
For the rst category, NP-induced cytotoxicity might be assessed
by monitoring the biological response after treatment using a cock-
tail of compatible dyes each one of the which is specic for
examinating a cellular endpoint [21]. Following the scheme of the
plate reader-based HTS assays, the toxicity of different metal and
metal oxide NPs was assayed in two cell types and Zebrash embryos
through several mechanisms related to oxidative stress, cationic
166 E. Caballero-Daz, M. Valcrcel Cases / Trends in Analytical Chemistry 84 (2016) 160171
Fig. 4. Flow chart with the different stages in HTS applied to nanotoxicity. Reproduced with permission of [21].
injury and metal ion shedding. Fluorescence readout-based HTS anal-
ysis was performed by using a cocktail of three compatible dyes,
each one of which was used to examine different cellular end-
points such as mitochondrial depolarization and membrane
permeability. The analysis of uorescence at varying wavelengths
allowed for obtaining a heat map of responses, where colder and
warmer colours indicated, respectively, lower and higher percent-
ages of cells positive to the response under different incubation
conditions. Final HTS data demonstrated the highest lethality for
quantum dots and zinc oxide NPs in cell-based assays [82].
HCS assays are proposed as advanced tools in the integration and
automation of quantitative uorescence microscopy and image anal-
ysis. Jan and colleagues [83] conducted a comprehensive investigation
of cytotoxicity of CdTe quantum dots to HepG2 human hepatocel-
lular carcinoma cells using various HCS assays. Fluorescence images
from multiplexed cytotoxicity assays (i.e. cell count, nuclear area,
mitochondrial membrane potential and intracellular free calcium
concentration) were acquired, processed and analyzed by HCS plat-
form and image analysis software, providing a more sensitive and
informative assessment of nanotoxic mechanisms.
As well as the screening at cellular level, whole-organism screen-
ing has been succesfully applied to nanotoxicity eld. Recently, it
has been described a HTS system to measure population- and
organism-level toxicity of 20 types of NPs using Caenorhabditis
elegans as model organism. Results showed that most of NPs caused
toxicity with the exception of fullerene, fullerol, titanium dioxide
and cerium oxide NPs [84].
4.3. Advanced omics techniques
Some biomarkers, such as genes, mRNA transcripts, proteins and
metabolites, might be differentially expressed in an organism when
it has been exposed to a xenobiotic, yielding information about the
specic pathways that have been altered because of exposure. Mo-
lecular indicators not only provide insight into triggered toxicity
mechanisms, but also inform about the modulation of the general
molecular response of a cell, tissue or organism in response to NPs.
4.3.1. Genomics
The use of global gene expression patterns in microarrays and
next-generation sequencing technologies allows for analysis of a mul-
titude of genes and pathways all at once and with no preconceived
idea about those more signicant genes [85]. Nanotoxicity has been
widely investigated by using gene expression proling as expo-
sure biomarkers [8688]. To cite an example, Coccini et al. [89]
examined the renal effect of cadmium-containing silica NPs in rats
exposed intratracheally using DNA microarray technology. Renal gene
expression showed that 139 and 153 genes were differentially ex-
pressed at 7 and 30 day, respectively, compared with control
organisms. For the shorter exposure time, genes related to acute in-
ammation, immunity and defence processes were down-regulated,
while 30 days after dosing, the altered gene expression was asso-
ciated to cell regulation and apoptosis mechanisms.
4.3.2. Proteomics
Research efforts in proteomics focus on delving into those spe-
cic cellular pathways related to protein expression that explain the
biological response provoked by NPs. Western blot analysis is the
most used approach to investigate the expresion of proteins of in-
terest, which in most of cases are related to apoptosis processes.
With this purpose, Qin et al. [90] investigated the cytotoxicity of
graphene quantum dots (GQDs) in THP-1 macrophages. GQDs re-
sulted to decrease the expression level of anti-apoptosis protein Bcl-
2, and to increase the expression of apoptotic proteins Bax and Bad.
Western blot analysis of Bcl-2 and Bax proteins was also per-
formed for evaluating the harmful effects caused by copper
nanoclusters in C2C12 myoblasts. Findings reported a marked de-
crease in Bcl-2 anti-apoptotic protein expression level, while Bax
apoptotic protein expression level was increased after treatment with
NPs [91].
4.3.3. Metabolomics
Metabolomics constitutes an ideal tool for investigating NP-
organism interaction at different molecular levels by tracking the
altered expression pattern of small molecules (e.g. amino acids or
sugars) involved in metabolic and biological processes in re-
sponse to external stimuli. To date, the main analytical techniques
used for metabolomic studies are NMR, gas chromatography/mass
spectrometry (GC/MS), liquid chromatography/mass spectrom-
etry (LC/MS), and capillary electrophoresis/mass spectrometry (CE/
MS) [92].
Some advantages of metabolomics over other omics tech-
niques (i.e. genomics, transcriptomics and proteomics) are: the
reduced number of molecules of interest, the possibility of quan-
titative and qualitative interpretation of the results obtained from
the other techniques, and the more generic nature of the provided
information [93]. Briey, for metabolomic analysis, biological samples
are collected (i.e. biouids, tissues or cells cultures) and metabo-
lites are extracted and analyzed by spectroscopic detection, NMR
 E. Caballero-Daz, M. Valcrcel Cases / Trends in Analytical Chemistry 84 (2016) 160171
mainly [94]. Raw data are then processed by advanced chemometric
analysis and differential small molecules are identied and dened
as biomarkers. Changes in biomarker concentrations before and after
treatment enable researchers to map the main metabolic path-
ways involved in the perturbations caused by exposure to stimuli
[93].
As of now, applications of metabolomics to nanotoxicity eld have
been barely described in literature in spite of the fact that this an-
alytical technique yields crucial information concerning specic NP-
induced toxicity mechanisms at molecular level. In this line, Lei et al.
[94] investigated the biochemical effects of copper NPs by analyz-
ing urine, serum, and liver and kidney tissues from exposed rats by
a NMR-based metabolomic approach. Biomarkers were correlated
with mitochondrial failure, enhanced ketogenesis, fatty acid
-oxidation, and glycolysis, what was nally associated with hepa-
totoxicity and nephrotoxicity mechanisms. Toxicity of several metal
oxide NPs have been also assayed by comparison of metabolic pro-
les of controls and samples [92,95]. More specically, metabolomic
abnormalities in the concentration of various cellular amino acids
and gluthatione antioxidant revealed oxidative stress mecha-
nisms in MRC-5 human fetal lung broblasts after treatment with
silica NPs [96].
4.4. Electrochemical techniques
Analytical electrochemistry has arisen as a simple and useful
methodology for shedding light on those biochemical and cellular
events involved in nanotoxicological response. Additionally, elec-
trochemical methods also provide insight into evaluation of
physicochemical properties of NPs that inuence in their biologi-
cal reactivity, such as their surface, size, oxidation state and
dissolution kinetics. However, up to date, electrochemical ap-
proaches have been sparingly dedicated to the nanotoxicity
framework despite of multiple advantages of these methodolo-
gies offer over conventional evaluation protocols. In addition, most
of related publications have been devoted to in vitro nanotoxicity,
while applications to in vivo models are still lacking [97].
4.4.1. Carbon ber microelectrodes (CFMEs)
The principal application of carbon ber microelectrodes (CFMEs)
is monitored electrochemical species related to physiological and
neurological processes which have been altered by NPs. CFMEs allow
for observation of biochemical changes and exocytosis processes in
single cells or model animals with high temporal and spatial res-
olution [97]. zel and colleagues [98] quantied electrochemically
the intestinal nitric oxide (NO) concentration in live zebrash using
a CFME implanted in the intestine of embryos. Changes in NO con-
centration following exposure to copper oxide and cerium oxide NPs
were monitored and correlated with results from conventional assays
based on uorescent dyes specic for NO. Copper oxide NPs were
found to increase NO concentration to almost 4 times higher com-
pared with unexposed embryos, suggesting thus an intestinal
oxidative damage in organisms, while low concentrations of cerium
oxide NPs showed scavenging effect against NO.
Concerning the application of CFME amperometry to cell-
based analyses, most of publications focus on examining effects of
NPs on exocytosis function [99]. In this context, the Maurer-Jones
group [100] evaluated the exocytosis perturbation in mast cells as
a result of exposure to titanium dioxide NPs by using CFMEs. Real-
time electrochemical measurements revealed that NP-induced
oxidative stress under UV light exposure was correlated to pertur-
bations in exocytotic cellular function so more ROS present caused
a greater decrease in the number of serotonin molecules being
released.
167
4.4.2. Electrochemical impedance sensing (EIS)
In view of the interferences reported between some types of NPs
and classical endpoint methods with colorimetric and uorimet-
ric readouts, impedance-based systems have appeared as label-
free and high throughput alternatives providing dynamic responses
in real time for nanotoxicity screening purposes. Real time cell ana-
lyzer (RTCA) systems measure the electrical impedance through
microelectrodes integrated on the bottom of culture plates. The
sensing mechanism is based on the measurement of electrode
surface impedance which is sensitive to cellular changes that affect
to cell adhesion degree to the plate, such as cell number, viability
and morphological alterations. The applicability of RTCAs have been
successfully demonstrated to evaluate the cytotoxicity of multiple
NPs [101103]. Hondroulis et al. [104] fabricated a EIS chip to assess
the toxicity of graphene to blood brain barrier (BBB) cells. EIS chip
was designed with an array of eight gold electrodes inserted in the
bottom of well plate where cells were later seeded and incubated
with NPs.
4.5. In vivo nanotoxicity evaluation
Although most of research has been devoted to in vitro
nanotoxicity evaluation, biological effects observed in cells neces-
sarily require further corroboration in in vivo models where new
factors and mechanisms come into play and contribute to dene
the toxicological prole of NPs. The concept of pharmacokinetics
emcompasses those biological processes which NPs are subjected
to, such as absorption, distribution, metabolism and excretion.
4.5.1. Nanoparticle uptake and biodistribution
Analysis of uptake and biodistribution of NPs in organisms enables
researchers to elucidate the target tissues and organs. For this
purpose, conventional microscopy techniques are considered as
useful analysis tools to quantify and map NPs in treated animals.
Particularly, electron microscopy techniques (i.e. SEM and TEM) are
good candidates to examine the uptake and presence of NPs in
tissues, however, their low sensitivity and potential interferents of
biological samples make uorescence microscopy a more appropiate
approach for analysis. However, uorescence techniques also exhibit
some handicaps for in vivo analysis including reduced light
penetration, interfering compounds, and photobleaching and de-
stabilization of the dyes [105]. Microscopy techniques have been
extensively used for investigating the tracking and uptake of mul-
tiple NPs [106108]. ICP analytical techniques have been also proven
for monitoring metal and metal oxide NPs in in vivo models, yield-
ing information on the elemental content in tissues and organs
[109,110]. Whereas these techniques provide low detection limits,
multielemental analysis, high precision and possibility of monitor-
ing changes over time, they are also limited when there are native
elements in biological systems that coincide with those from the
tested NPs [105].
New analytical approaches, such as radiotracer and synchro-
tron techniques have arisen as potential tools for monitoring the
biodistribution of NPs in animals. In radiotracer techniques, NPs are
rstly labelled with radiactive isotopes (e.g. 125I) and then moni-
tored in vivo with a gamma-ray detector. By using radiotracer
technique, distribution of silver NPs, nanodiamonds and graphene
oxide has been shown in model organisms [111113]. Concerning
synchrotron radiation techniques, there are three different modali-
ties: synchrotron radiation X-ray uorescence (SRXRF), synchrotron
radiation X-ray absorption spectroscopy (SRXAS) or also known as
X-ray absorption near-edge structure (XANES), and synchrotron ra-
diation circular dichroism (SRCD). The principal attribute of these
techniques is the use of a tuneable, very bright and focused light
source that improves the signal to noise ratio, reduces analysis time
and provides single cell resolution [114]. Plainly, the former
168 E. Caballero-Daz, M. Valcrcel Cases / Trends in Analytical Chemistry 84 (2016) 160171

modality focuses on investigating the microdistribution of trace el-
ements in biological matrices. SRXAS or XANES yields insight into
the chemical speciation of NPs in biological environments for a better
understanding of their interactions with other biological entities.
And nally, SRCD is useful to examine protein-NP interactions [115].
These techniques have been already applied to explore the physi-
cochemical changes experimented by quantum dots in vivo, the
biodistribution of titanium dioxide NPs in mice, and the interac-
tions of protein corona with gold nanorods [116118].
4.5.2. Histological analysis
Histological examination of tissues allows for investigation of
damages, inammation or lesions in organs as a consequence of NP
treatment. As a general rule, organ sections are rstly xed with a
chemical xative (e.g. formalin), embedded in paran, cut in thin
sections with a microtome, and then mounted on glass slides. Finally,
sections are stained with a dye, commonly hematoxylin and eosin
(H&E), and examined under microscopy [119]. The most used organs
for histological analysis comprise brain, kidney, stomach, liver, lung,
heart and spleen. Song et al. [120] reported histological abnormali-
ties in gills of three species of freshwater sh following incubation
with copper NPs. Fig. 5 shows images from biodistribution study
and histological analysis conducted by Zhang and colleagues [117]
in mice after administration of titanium dioxide NPs. Fluores-
cence images obtained by SRXRF clearly indicate how NPs
preferentially accumulate in lungs.
4.5.3. Blood chemistry analysis
Any positive or negative deviation in blood constituents of NP-
treated animals compared with control groups is a clear nanotoxicity
indicator [121]. Within this context, Jenkins et al. [122] analyzed
blood samples from mice injected with gold-iron oxide NPs 14 days
after administration. As blood chemistry markers, researchers se-
lected Na+, K+, glucose, creatinine and urea nitrogen. In addition, other
parameters were also contemplated for analysis including, total bili-
rubin, white blood cell count, mean corpuscular count and mean
corpuscular hemoglobin. Findings conrmed no toxicity of these NPs
towards animals under the treatment conditions.
4.5.4. Clearance pathways
Of special importance for nanomaterial biosafety is to take into
account the excretion routes from the body, since this might de-
termine the unexpected appearance of long-term toxic effects or,
on the contrary, the reversibility of the observed acute toxicity. Moni-
toring of NP excretion can be performed by ICP analyses of
urine and feces samples collected at different time points after

Fig. 5. Distribution of titanium element in lungs (A and B), liver (C and D) and spleen (E and F) after 1 day of administration in mice at a dose of 1 mg Kg1. X-ray uores-
cence image (right) is paired with the corresponding histological image (left) obtained with H&E staining. Reproduced with permission of [117].
 E. Caballero-Daz, M. Valcrcel Cases / Trends in Analytical Chemistry 84 (2016) 160171
administration. Inductively coupled plasma optical emission spec-
trometry (ICP-OES) is normally used to measure the elemental
content from NPs in biological samples. For example, Fu et al. [123]
analyzed by ICP-OES the silicon content in liver, spleen, kidney, lung,
muscle, intestine, feces and urine samples 24 h and 7 days after ad-
ministration of silica NPs to mice. Data demonstrated that the vast
majority of NPs were excreted through feces. In other study, ICP-
MS data of samples from mice treated with different-sized gold NPs
revealed that the primary clearance via for all NPs was the renal
route given that, urine gold concentration was signicantly high at
2 and 5 h after administration, while the gold concentration in feces
remained low throughout the study [124].
5. Conclusions
Properly evaluating of risks associated with NP exposure is a
matter of social concern due to the growing use and applications
of nanomaterials in the current society, what becomes likely the
fact that human being can come into contact with NPs somehow.
Much research efforts have been dedicated to design and synthe-
size safer NPs for which, it is strongly required to evaluate the effects
of the manufactured NPs on human health and environment and
to propose alternative synthesis methods that allow for obtaining
more biocompatible NPs. As a general rule, before performing any
nanotoxicity analysis, it is mandatory to execute a previous and ex-
haustive characterization study of the NPs of interest, where a
minimum set of physicochemical features should be tested, includ-
ing chemical composition, size, shape, surface, crystallinity and
colloidal stability. Equally, multiple inuential factors such as con-
ditions of culture medium, or the cell type should be considered
for their impact on NP-induced biological response. Numerous an-
alytical methodologies are currently used for nanotoxicity assessment
purposes comprising from the more conventional testing proto-
cols, most of which are commercially available kits for determining
a specic cellular endpoint, to advanced analytical techniques, such
as omics techniques (i.e. genomics, transcriptomics, proteomics
and metabolomics), or high throughput electrochemical screen-
ing approaches. While most of publications in this eld focus on
investigating cellular responses based on in vitro studies, further cor-
roboration of the observed effects with in vivo models would provide
further understanding on the toxicological pattern of NPs in living
systems. Overall, there is an urgent need for: (i) new advances in
analytical techniques for testing nanotoxicity, (ii) consideration of
inuential factors, (iii) special attention to the limitations of the
current assessment protocols, and (iv) appropiate controls and com-
plementary studies. All these factors will contribute to establish a
better evaluation scenario yielding reliable information on poten-
tial risks of NPs.
Conict of interest
The authors have declared no conict of interest.
Acknowledgements
Authors would like to express their gratitude to the Spains Min-
istry of Innovation and Science for Proyect CTQ2014-52939R.
References
[1] K. Donaldson, V. Stone, C.L. Tran, W. Kreyling, P.J.A. Borm, Nanotoxicology,
Occup. Environ. Med. 61 (2004) 727728.
[2] S.W. Shin, I.H. Song, S.H. Um, Role of physicochemical properties in
nanoparticle toxicity, Nanomaterials 5 (2015) 13511365.
[3] F. Zhao, Y. Zhao, Y. Liu, X. Chang, C. Chen, Y. Zhao, Cellular uptake, intracellular
tracking, and cytotoxicity of nanomaterials, Small 7 (2011) 13221337.
169
[4] A. Panariti, G. Miserocchi, I. Rivolta, The effect of nanoparticle uptake on cellular
behavior: disrupting or enabling functions?, Nanotechnol. Sci. Appl. 5 (2012)
87100.
[5] H. Kettiger, A. Schipanski, P. Wick, J. Huwyler, Engineered nanomaterial uptake
and tissue distribution: from cell to organism, Int. J. Nanomed. 8 (2013)
32553269.
[6] C.M. Beddoes, C.P. Case, W.H. Briscoe, Understanding nanoparticle cellular
entry: a physicochemical perspective, Adv. Colloid Interface Sci. 218 (2015)
4868.
[7] K.G. Rothberg, J.E. Heuser, W.C. Donzell, Y.-S. Ying, J.R. Glenney, R.G.W.
Anderson, Caveolin, a protein component of caveolae membrane coats, Cell
68 (1992) 673682.
[8] Y. Jiang, S. Huo, T. Mizuhara, R. Das, Y.-W. Lee, S. Hou, et al., The interplay of
size and surface functionality on the cellular uptake of sub-10 nm gold NPs,
ACS Nano 9 (2015) 99869993.
[9] I.L. Gunsolus, C.L. Haynes, Analytical aspects of nanotoxicology, Anal. Chem.
(2015) doi:10.1021/acs.analchem.5b04221 Just accepted manuscript.
[10] K.-M. Kim, J.H. Song, M.-K. Kim, S.-T. Chung, J. Jeong, J.-Y. Yang, et al.,
Physicochemical analysis methods for nanomaterials considering their
toxicological evaluations, Mol. Cell. Toxicol. 10 (2014) 347360.
[11] S. Lanone, F. Rogerieux, J. Geys, A. Dupont, E. Maillot-Marechal, J. Boczkowski,
et al., Comparative toxicity of 24 manufactured NPs in human alveolar
epithelial and macrophage cell lines, Part. Fibre Toxicol. 6 (2009) 14.
[12] H. Liu, D. Yang, H. Yang, H. Zhang, W. Zhang, Y. Fang, et al., Comparative study
of respiratory tract immune toxicity induced by three sterilisation
nanoparticles: silver zinc oxide and titanium dioxide, J. Hazard. Mater. 248249
(2013) 478486.
[13] M.V.D.Z. Park, W. Annema, A. Salvati, A. Lesniak, A. Elsaesser, C. Barnes, et al.,
In vitro developmental toxicity test detects inhibition of stem cell
differentiation by silica nanoparticles, Toxicol. Appl. Pharm. 240 (2009)
108116.
[14] R.G. Mendes, B. Koch, A. Bachmatiuk, A.A. El-Gendy, Y. Krupskaya, A. Springe,
et al., Synthesis and toxicity characterization of carbon coated iron oxide
nanoparticles with highly dened size distributions, Biochim. Biophys. Acta
2014 (1840) 160169.
[15] D. Sekar, M.L. Falcioni, G. Barucca, G. Falcioni, DNA damage and repair following
in vitro exposure to two different forms of titanium dioxide nanoparticles on
trout erythrocyte, Environ. Toxicol. 29 (2014) 117127.
[16] M. Baalousha, A. Prasad, J.R. Lead, Quantitative measurement of the
nanoparticle size and number concentration from liquid suspensions by atomic
force microscopy, Environ. Sci. Process. Impacts 16 (2014) 13381347.
[17] L.Q. Chen, L. Fang, J. Ling, C.Z. Ding, B. Kang, C.Z. Huang, Nanotoxicity of silver
nanoparticles to red blood cells: size-dependent adsorption, uptake and
hemolytic activity, Chem. Res. Toxicol. 28 (2015) 501509.
[18] Y. Pan, S. Neuss, A. Leifert, M. Fischler, F. Wen, U. Simon, et al., Size-dependent
cytotoxicity of gold nanoparticles, Small 3 (2007) 1941.
[19] Q. Zheng, D. Shao, W. Ji, J. Li, L. Chen, J. Song, The nanotoxicity investigation
of optical nanoparticles to cultured cells in vitro, Toxicol. Rep. 1 (2014)
137144.
[20] Y. Araj, T. Miyayama, S. Hirano, Difference in the toxicity mechanism between
ion and nanoparticle forms of silver in the mouse lung and in macrophages,
Toxicology 328 (2015) 8492.
[21] R. Damoiseaux, S. George, M. Li, S. Pokhrel, Z. Ji, B. France, et al., No time to
lose-high throughput screening to assess nanomaterial safety, Nanoscale 3
(2011) 13451360.
[22] V. Rabolli, L.C. Thomassen, F. Uwambayinema, J.A. Martens, D. Lison, The
cytotoxic activity of amorphous silica nanoparticles is mainly inuenced by
surface area and not by aggregation, Toxicol. Lett. 206 (2011) 197203.
[23] T.S. Hauck, A.A. Ghazani, W.C.W. Chan, Assessing the effect of surface chemistry
on gold nanorod uptake, toxicity, and gene expression in mammalian cells,
Small 4 (2008) 153159.
[24] E. Caballero-Daz, C. Pfeiffer, L. Kastl, P. Rivera-Gil, B. Simonet, M. Valcrcel,
et al., The toxicity of silver nanoparticles depends on their uptake by cells
and thus on their surface chemistry, Part. Part. Syst. Char. 30 (2013) 1079
1085.
[25] M. Piazza, M. Colombo, I. Zanoni, F. Granucci, P. Tortora, J. Weiss, et al., Uniform
lipopolysaccharide (LPS)-loaded magnetic nanoparticles for the investigation
of LPS/TLR4 signaling, Angew. Chem. Int. Ed. Engl. 50 (2011) 622626.
[26] A. Orlando, M. Gregori, E. Cazzaniga, M. Colombo, A. Panariti, D. Prosperi, et al.,
Iron oxide nanoparticles surface coating and cell uptake affect biocompatibility
and inammatory responses of endothelial cells and macrophages, J. Nanopart.
Res. 17 (2015) 351.
[27] S. Bhattacharjee, I.M.C.M. Rietjens, M.P. Singh, T.M. Atkins, T.K. Purkait, Z. Xu,
et al., Cytotoxicity of surface-functionalized silicon and germanium
nanoparticles: the dominant role of the surface charges, Nanoscale 5 (2013)
48704883.
[28] M. Baek, M.K. Kim, H.J. Cho, J.A. Lee, J. Yu, H.E. Chung, et al., Factors inuencing
the cytotoxicity of zinc oxide nanoparticles: particle size and surface charge,
J. Phys. Conf. Ser. 304 (2011) 012044.
[29] M. Merhi, C.Y. Dombu, A. Brient, J. Chang, F. Le Curieux, D. Marzin, et al., Study
of serum interaction with cationic nanoparticle: implication for in vitro
endocytosis, cytotoxicity and genotoxicity, Int. J. Pharm. 423 (2012) 3744.
[30] F. Seitz, R.R. Rosenfeldt, S. Schneider, R. Schulz, M. Bundschuh, Size-, surface-
and crystalline structure composition-related effects of titanium dioxide
nanoparticles during their aquatic life cycle, Sci. Total Environ. 493 (2014)
891897.
170 E. Caballero-Daz, M. Valcrcel Cases / Trends in Analytical Chemistry 84 (2016) 160171
[31] V. Iswarya, M. Bhuvaneshwari, S.A. Alex, S. Iyer, G. Chaudhuri, P.T.
Chandrasekaran, et al., Combined toxicity of two crystalline phases (anatase
and rutile) of titania nanoparticles towards freshwater microalgae: chlorella
sp, Aquat. Toxicol. 161 (2015) 154169.
[32] D. Bartczak, P. Vincent, H. Goenaga-Infante, Determination of size- and
number-based concentration of silica nanoparticles in a complex biological
matrix by online techniques, Anal. Chem. 87 (2015) 54825485.
[33] N. Tripathy, T.-K. Hong, K.-T. Ha, H.-S. Jeong, Y.-B. Hahn, Effect of ZnO
nanoparticles aggregation on the toxicity in RAW 264.7 murine macrophage,
J. Hazard. Mater. 270 (2014) 110117.
[34] N. Nafee, M. Schneider, U.F. Schaefer, C.-M. Lehr, Relevance of the colloidal
stability of chitosan/PLGA nanoparticles on their cytotoxicity prole, Int. J.
Pharm. 381 (2009) 130139.
[35] P.J. Chueh, R.-Y. Liang, Y.-H. Lee, Z.-M. Zheng, S.-M. Chuang, Different cytotoxic
effects of gold nanoparticles in different mammalian cell lines, J. Hazard. Mater.
264 (2014) 303312.
[36] I.-Y. Kim, E. Joachim, H. Choi, K. Kim, Toxicity of silica nanoparticles depends
on size, dose, and cell type, J. Nanomed. Nanotechnol. 11 (2015) 14071416.
[37] B.B. Manshian, A. Al-Ali, J.S. Jenkins, S.H. Doak, Cell type-dependent changes
in CdSe/ZnS quantum dot uptake and toxic endpoints, Toxicol. Sci. 144 (2015)
246258.
[38] J.L. Luque-Garcia, R. Sanchez-Daz, I. Lopez-Heras, P. Martin, C. Camara,
Bioanalytical strategies for in-vitro and in-vivo evaluation of the toxicity
induced by metallic nanoparticles, Trends Analyt. Chem. 43 (2013) 254268.
[39] E. Caballero-Daz, M. Valcrcel, Toxicity of gold nanoparticles, in: M. Valcrcel, [66] L. Wei, J. Tang, Z. Zhang, Y. Chen, G. Zhou, T. Xi, Investigation of the cytotoxicity
A.I. Lpez-Lorente (Editors), Gold nanoparticles in analytical chemistry,
Elsevier, Netherlands, 2014, pp. 207254.
[40] K.K. Awasthi, R. Verma, A. Awasthi, K. Awasthi, I. Soni, P.J. John, In vivo
genotoxic assessment of silver nanoparticles in liver cells of Swiss albino mice
using comet assay, Adv. Mater. Lett. 6 (2015) 187193.
[41] M. Khan, A.H. Naqvi, M. Ahmad, Comparative study of the cytotoxic and
genotoxic potentials of zinc oxide and titanium dioxide nanoparticles, Toxicol.
Rep. 2 (2015) 765774.
[42] K.K. Awasthi, A. Awasthi, R. Verma, N. Kumar, P. Roy, K. Awasthi, et al.,
Cytotoxicity, genotoxicity and alteration of cellular antioxidant enzymes in
silver nanoparticles exposed CHO cells, RSC Adv. 5 (2015) 3492734935.
[43] S.L. Montes-Fonseca, E. Orrantia-Borunda, A. Aguilar-Elguezabal, C.
Gonzlez-Horta, P. Talams-Rohana, B. Snchez-Ramrez, Cytotoxicity of
functionalized carbon nanotubes in J774A macrophages, J. Nanomed.
Nanotechnol. 8 (2012) 853859.
[44] V. Sharma, P. Singh, A.K. Pandey, A. Dhawan, Induction of oxidative stress, DNA
damage and apoptosis in mouse liver after sub-acute oral exposure to zinc
oxide nanoparticles, Mutat. Res-Gen. Tox. En. 745 (2012) 8491.
[45] L. Huo, R. Chen, L. Zhao, X. Shi, R. Bai, D. Long, et al., Silver nanoparticles activate
endoplasmic reticulum stress signaling pathway in cell and mouse models:
the role in toxicity evaluation, Biomaterials 61 (2015) 307315.
[46] P.V. Asharani, G.L.K. Mun, M.P. Hande, S. Valiyaveettil, Cytotoxicity and
genotoxicity of silver nanoparticles in human cells, ACS Nano 3 (2009)
279290.
[47] C.-T. Ng, L.Y.L. Yung, H.L.-F. Swa, R.W.-Y. Poh, J. Gunaratne, B.H. Bay, Altered
protein expression prole associated with phenotypic changes in lung
broblasts co-cultured with gold nanoparticle treated small airway epithelial
cells, Biomaterials 39 (2015) 3138.
[48] P.P. Fu, Q. Xia, H.-M. Hwang, P.C. Ray, H. Yu, Mechanisms of nanotoxicity:
generation of reactive oxygen species, J. Food Drug Anal. 22 (2014) 6475.
[49] M.-L. Kung, S.-L. Hsieh, C.-C. Wu, T.-H. Chu, Y.-C. Lin, B.-W. Yeh, et al., Enhanced
reactive oxygen species overexpression by CuO nanoparticles in poorly
differentiated hepatocellular carcinoma cells, Nanoscale 7 (2015) 18201829.
[50] A. Dubey, M. Goswami, K. Yadav, D. Chaudhary, Oxidative stress and nano-
toxicity induced by TiO2 and ZnO on WAG cell line, PLoS ONE 10 (2015)
e0127493.
[51] J. Ahmad, M. Ahamed, M.J. Akhtar, S.A. Alkokayan, M.A. Siddiqui, J. Musarrat,
et al., Apoptosis induction by silica nanoparticles mediated through reactive
oxygen species in human liver cell line HepG2, Toxicol. Appl. Pharm. 259 (2012)
160168.
[52] M. Ahamed, R. Posgai, T.J. Gorey, M. Nielsen, S.M. Hussain, J.J. Rowe, Silver
nanoparticles induced heat shock protein 70, oxidative stress and apoptosis
in Drosophila melanogaster, Toxicol. Appl. Pharm. 242 (2010) 263269.
[53] J.E. Choi, S. Kim, J.H. Ahn, P. Youn, J.S. Kang, K. Park, et al., Induction of oxidative
stress and apoptosis by silver nanoparticles in the liver of adult zebrash,
Aquat. Toxicol. 100 (2010) 151159.
[54] W. Gao, K. Xu, L. Ji, B. Tang, Effect of gold nanoparticles on glutathione
depletion-induced hydrogen peroxide generation and apoptosis in HL7702
cells, Toxicol. Lett. 205 (2011) 8695.
[55] S.A. Love, M.A. Maurer-Jones, J.W. Thompson, Y.-S. Lin, C.L. Haynes, Assessing
nanoparticle toxicity, Annu. Rev. Anal. Chem. (Palo Alto Calif) 5 (2012) 181
205.
[56] A.R. Wielgus, B. Zhao, C.F. Chignell, D.-N. Hu, J.E. Roberts, Phototoxicity and
cytotoxicity of fullerol in human retinal pigment epithelial cells, Toxicol. Appl.
Pharm. 242 (2010) 7990.
[57] M.S. Acar, Z.B. Bulut, A. Ates, B. Nami, N. Koak, B. Yildiz, Titanium dioxide
nanoparticles induce cytotoxicity and reduce mitotic index in human amniotic
uid-derived cells, Hum. Exp. Toxicol. 34 (2015) 7482.
[58] J.A. Kim, C. Aberg, G. de Crcer, M. Malumbres, A. Salvati, K.A. Dawson, Low
dose of amino-modied nanoparticles induces cell cycle arrest, ACS Nano 7
(2013) 74837494.
[59] Y.-X. Yang, Z.-M. Song, B. Cheng, K. Xiang, X.-X. Chen, J.-H. Li, et al., Evaluation
of the toxicity of food additive silica nanoparticles on gastrointestinal cells,
J. Appl. Toxicol. 34 (2014) 424435.
[60] J.-H. Kim, J. Sanetuntikul, S. Shanmugam, E. Kim, Necrotic cell death caused
by exposure to graphitic carbon-coated magnetic nanoparticles, J. Biomed.
Mater. Res. A 103 (2015) 28752887.
[61] R. Wahab, M.A. Siddiqui, Q. Saquib, S. Dwivedi, J. Ahmad, J. Musarrat, et al.,
ZnO nanoparticles induced oxidative stress and apoptosis in HepG2 and MCF-7
cancer cells and their antibacterial activity, Colloid. Surface. B 117 (2014)
267276.
[62] W. Hu, S. Culloty, S. Lynch, J. Davenport, S. Ramirez-Garcia, K. Dawson, et al.,
Neutral red retention time assay in determination of toxicity of nanoparticles,
Mar. Environ. Res. 111 (2015) 158161.
[63] X. Jing, J.H. Park, T.M. Peters, P.S. Thorne, Toxicity of copper oxide nanoparticles
in lung epithelial cells exposed at the air-liquid interface compared with in
vivo assessment, Toxicol. In Vitro 29 (2015) 502511.
[64] M.S.P. Boyles, T. Kristl, A. Andosch, M. Zimmermann, N. Trac, E. Casals, et al.,
Chitosan functionalisation of gold nanoparticles encourages particle uptake
and induces cytotoxicity and pro-inammatory conditions in phagocytic cells,
as well as enhancing particle interactions with serum components, J.
Nanobiotechnol. 13 (2015) 84.
[65] F. Sambale, S. Wagner, F. Stahl, R.R. Khaydarov, T. Scheper, D. Bahnemann,
Investigations of the toxic effect of silver nanoparticles on mammalian cell
lines, J. Nanomater. 2015 (2015) 136765.
mechanism of silver nanoparticles in vitro, Biomed. Mater. 5 (2010) 044103.
[67] K. Solarska, A. Gajewska, G. Bartosz, K. Mitura, Induction of apoptosis in human
endothelial cells by nanodiamond particles, J. Nanosci. Nanotechnol. 12 (2012)
51175121.
[68] W. Chunyan, S. Valiyaveettil, Correlation of biocapping agents with cytotoxic
effects of silver nanoparticles on human tumor cells, RSC Adv. 3 (2013) 14329.
[69] L.A. Austin, S. Ahmad, B. Kang, K.R. Rommel, M. Mahmoud, M.E. Peek, et al.,
Cytotoxic effects of cytoplasmic-targeted and nuclear-taretes gold and silver
nanoparticles in HSC-3 cells-A mechanistic study, Toxicol. In Vitro 29 (2015)
694705.
[70] W.-X. Duan, M.-D. He, L. Mao, F.-H. Qian, Y.-M. Li, H.-F. Pi, et al., NiO
nanoparticles induce apoptosis through repressing SIRT1 in human bronchial
epithelial cells, Toxicol. Appl. Pharm. 286 (2015) 8091.
[71] M. Sathishkumar, S. Pavagadhi, A. Mahadevan, R. Balasubramanian,
Biosynthesis of gold nanoparticles and related cytotoxicity evaluation using
A549 cells, Ecotoxicol. Environ. Saf. 114 (2015) 232240.
[72] I.-L. Hsiao, Y.-J. Huang, Effects of various physicochemical characteristics on
the toxicities of ZnO and TiO2 nanoparticles toward human lung epithelial
cells, Sci. Total Environ. 409 (2011) 12191228.
[73] M.S. Stan, I. Memet, C. Sima, T. Popescu, V.S. Teodorescu, A. Hermenean, A.
Dinischiotu, Si/SiO2 quantum dots cause cytotoxicity in lung cells through redox
homeostasis imbalance, Chem. Biol. Interact. 220 (2014) 102115.
[74] A.R. Lupu, T. Popescu, The noncellular reduction of MTT tetrazolium salt by
TiO2 nanoparticles and its implications for cytotoxicity assays, Toxicol. In Vitro
27 (2013) 14451450.
[75] A. Kroll, D. Hahn, M.H. Pillukat, J. Schnekenburger, Interference of engineered
nanoparticles with in vitro toxicity assays, Arch. Toxicol. 86 (2012) 11231136.
[76] A.R. Gliga, S. Skoglund, I.O. Wallinder, B. Fadeel, H.L. Karlsson, Size-dependent
cytotoxicity of silver nanoparticles in human lung cells: the role of cellular
uptake, agglomeration and Ag release, Part. Fibre Toxicol. 11 (2014) 11.
[77] Y. Huang, X. L, Y. Qu, Y. Yang, S. Wu, MicroRNA sequencing and molecular
mechanisms analysis of the effects of gold nanoparticles on human dermal
broblasts, Biomaterials 37 (2015) 1324.
[78] B. Mostaghaci, J. Susewind, G. Kickelbick, C.-M. Lehr, B. Loretz, Transfection
system of amino-functionalized calcium phosphate nanoparticles: in vitro
ecacy, biodegradability, and immunogenicity study, ACS Appl. Mater.
Interfaces 7 (2015) 51245133.
[79] V. Escamilla-Rivera, M. Uribe-Ramrez, S. Gonzlez-Pozos, O. Lozano, S. Lucas,
A. De Vizcaya-Ruis, Protein corona acts as a protective shield against Fe3O4-PEG
inammation and ROS-induced toxicity in human macrophages, Toxicol. Lett.
240 (2016) 172184.
[80] T. Tong, C.T.T. Binh, J.J. Kelly, J.-F. Gaillard, K.A. Gray, Cytotoxicity of commercial
nano-TiO2 to Escherichia coli assessed by high-throughput screening: effects
of environmental factors, Water Res. 47 (2013) 23522362.
[81] S. Lin, Y. Zhao, Z. Ji, J. Ear, C.H. Chang, H. Zhang, et al., Zebrash high-throughput
screening to study the impact of dissolvable metal oxide NPs on the hatching
enzyme, ZHE1, Small 9 (2013) 17761785.
[82] S. George, T. Xia, R. Rallo, Y. Zhao, Z. Ji, S. Lin, et al., Use of a high-throughput
screening approach coupled with in vivo zebrash embryo screening to
develop hazard ranking for engineered nanomaterials, ACS Nano 5 (2011)
18051817.
[83] E. Jan, S.J. Byrne, M. Cuddihy, A.M. Davies, Y. Volkov, Y.K. Gunko, et al.,
High-content screening as a universal tool for ngerprinting of cytotoxicity
of nanoparticles, ACS Nano 2 (2008) 928938.
[84] S.-K. Jung, X. Qu, B. Aleman-Meza, T. Wang, C. Riepe, Z. Liu, et al., A multi-
endpoint, high-throughput study of nanomaterial toxicity in Caenorhabditis
elegans, Environ. Sci. Technol. 49 (2015) 24772485.
[85] R. Klaper, D. Arndt, J. Bozich, G. Dominguez, Molecular interactions of
nanomaterials and organisms: dening biomarkers for toxicity and high-
throughput screening using traditional and next-generation sequencing
approaches, Analyst 139 (2014) 882895.
 E. Caballero-Daz, M. Valcrcel Cases / Trends in Analytical Chemistry 84 (2016) 160171
[86] G. Gao, Y. Ze, B. Li, X. Zhao, T. Zhang, L. Sheng, et al., Ovarian dysfunction and
gene-expressed characteristics of female mice caused by long-term exposure
to titanium dioxide nanoparticles, J. Hazard. Mater. 243 (2012) 1927.
[87] H.C. Poynton, H.J. Allen, J.M. Lazorchak, A. Loguinov, C.A. Impelliteri, J.L.
Heckman, et al., Toxicogenomic responses of nanotoxicity in daphnia magna
exposed to silver nitrate and coated silver nanoparticles, Environ. Sci. Technol.
46 (2012) 62886296.
[88] S.I.L. Gomes, S.C. Novais, J.J. Scott-Fordsmand, W. De Coen, A.M.V.M. Soares,
M.J.B. Amorim, Effect of Cu-nanoparticles versus Cu-salt in Enchytraeus albidus
(Oligochaeta): differential gene expression through microarray analysis, Comp.
Biochem. Physiol. C Toxicol. Pharmacol. 155 (2012) 219227.
[89] T. Coccini, E. Roda, M. Fabbri, M.G. Sacco, L. Gribaldo, L. Manzo, Gene expression
proling in rat kidney after intratracheal exposure to cadmium-doped
nanoparticles, J. Nanopart. Res. 14 (2012) 110.
[90] Y. Qin, Z.-W. Zhou, S.-T. Pan, Z.-X. He, X. Zhang, J.-X. Qiu, et al., Graphene
quantum dots induce apoptosis, autophagy, and inammatory response via
p38 mitogen-activated protein kinase and nuclear factor-kB mediated signaling
pathways in activated THP-1 macrophages, Toxicology 327 (2015) 6276.
[91] Y. Liu, J. Liang, Q. Wang, Y. He, Y. Chen, Copper nanoclusters trigger muscle
cell apoptosis and atrophy in vitro and in vivo, J. Appl. Toxicol. (2015) Article
in press.
[92] Y. Liu, Y. Zhang, W. Jia, Y. Cheng, T. Chen, X. Wang, et al., GC/TOFMS analysis
of endogenous metabolites in mouse broblast cells and its application in TiO2
nanoparticle-induced cytotoxicity study, Chromatographia 75 (2012) 1301
1310.
[93] M. Lv, W. Huang, Z. Chen, H. Jiang, J. Chen, Y. Tian, et al., Metabolomics
techniques for nanotoxicity investigations, Bioanalysis 7 (2015) 15271544.
[94] R. Lei, C. Wu, B. Yang, H. Ma, C. Shi, Q. Wang, et al., Integrated metabolomic
analysis of the nano-sized copper particle-induced hepatotoxicity and
nephrotoxicity in rats: a rapid in vivo screening method for nanotoxicity,
Toxicol. Appl. Pharm. 232 (2008) 292301.
[95] M. Tang, T. Zhang, Y. Xue, S. Wang, M. Huang, Y. Yang, et al., Dose dependent
in vivo metabolic characteristics of titanium dioxide nanoparticles, J. Nanosci.
Nanotechnol. 10 (2010) 85758583.
[96] S.-M. Huang, X. Zuo, J.J. Li, S.F.Y. Li, B.H. Bay, C.N. Ong, Metabolomics studies
show dose-dependent toxicity induced by SiO2 nanoparticles in MRC-5 human
fetal lung broblasts, Adv. Healthc. Mater. 1 (2012) 779784.
[97] R.E. zel, X. Liu, R.S.J. Alkasir, S. Andreescu, Electrochemical methods for
nanotoxicity assessment, Trends Analyt. Chem. 59 (2014) 112120.
[98] R.E. zel, R.S.J. Alkasir, K. Ray, K.N. Wallace, S. Andreescu, Comparative
evaluation of intestinal nitric oxide in embryonic zebrash exposed to metal
oxide nanoparticles, Small 9 (2013) 42504261.
[99] S.A. Love, Z. Liu, C.L. Haynes, Examining changes in cellular communication
in neuroendocrine cells after noble metal nanoparticle exposure, Analyst 137
(2012) 3004.
[100] M.A. Maurer-Jones, J.R. Christenson, C.L. Haynes, TiO2 nanoparticle-induced
ROS correlates with modulated immune cell function, J. Nanopart. Res. 14
(2012) 1291.
[101] L. Otero-Gonzlez, R. Sierra-Alvarez, S. Boitano, J.A. Field, Application and
validation of an impedance-based real time cell analyzer to measure the
toxicity of nanoparticles impacting human bronchial epithelial cells, Environ.
Sci. Technol. 46 (2012) 1027110278.
[102] X. Zhu, E. Hondroulis, W. Liu, C.-Z. Li, Biosensing approaches for rapid
genotoxicity and cytotoxicity assays upon nanomaterial exposure, Small 9
(2013) 18211830.
[103] A. Pandey, R.S. Chouhan, Y. Gurbuz, J.H. Niazi, A. Qureshi, S. cerevisiae
whole-cell based capacitive biochip for the detection of toxicity of different
forms of carbon nanotubes, Sensor. Actuat. B-Chem. 218 (2015) 253260.
[104] E. Hondroulis, Z. Zhang, C. Chen, C.Z. Li, Impedance based nanotoxicity
assessment of graphene nanomaterials at the cellular and tissue level, Anal.
Lett. 45 (2012) 272282.
171
[105] X. He, Y. Ma, M. Li, P. Zhang, Y. Li, Z. Zhang, Quantifying and imaging engineered
nanomaterials in vivo: challenges and techniques, Small 9 (2013) 1482
1491.
[106] X. Huang, L. Li, T. Liu, N. Hao, H. Liu, D. Chen, et al., The shape effect of
mesoporous silica nanoparticles on biodistribution, clearance, and
biocompatibility in vivo, ACS Nano 5 (2011) 53905399.
[107] G. Xie, C. Wang, J. Sun, G. Zhong, Tissue distribution and excretion of
intravenously administered titanium dioxide nanoparticles, Toxicol. Lett. 205
(2011) 5561.
[108] C.P. Vignardi, F.M. Hasue, P.V. Sartorio, C.M. Cardoso, A.S.D. Machado, T.C.A.
Santos, et al., Genotoxicity, potential cytotoxicity and cell uptake of titanium
dioxide nanoparticles in the marine sh Trachinotus carolinus (Linnaeus, 1766),
Aquat. Toxicol. 158 (2015) 218229.
[109] D.S. Anderson, E.S. Patchin, R.M. Silva, D.L. Uyeminami, A. Sharmah, T. Guo,
et al., Inuence of particle size on persistence and clearance of aerosolized
silver nanopartices in the rat lung, Toxicol. Sci. 144 (2015) 366381.
[110] P. Prabu, W.S. Vedakumari, T.P. Sastry, Time-dependent biodistribution,
clearance and biocompatibility of magnetic brin nanoparticles: an in vivo
study, Nanoscale 7 (2015) 96769685.
[111] C.-M. Zhao, W.-X. Wang, Biokinetic uptake and eux of silver nanoparticles
in Daphnia magna, Environ. Sci. Technol. 44 (2010) 76997704.
[112] X. Zhang, J. Yin, C. Kang, J. Li, Y. Zhu, W. Li, et al., Biodistribution and toxicity
of nanodiamonds in mice after intratracheal instillation, Toxicol. Lett. 198
(2010) 237243.
[113] B. Li, X.Y. Zhang, J.Z. Yang, Y.J. Zhang, W.X. Li, C.H. Fan, et al., Inuence of
polyethylene glycol coating on biodistribution and toxicity of nanoscale
graphene oxide in mice after intravenous injection, Int. J. Nanomed. 9 (2014)
46974707.
[114] Y.-F. Li, J. Zhao, Y. Qu, Y. Gao, Z. Guo, Z. Liu, et al., Synchrotron radiation
techniques for nanotoxicology, J. Nanomed. Nanotechnol. 11 (2015) 1531
1549.
[115] B. Wang, Z. Wang, W. Feng, M. Wang, Z. Hu, Z. Chai, et al., New methods for
nanotoxicology: synchrotron radiation-based techniques, Anal. Bioanal. Chem.
398 (2010) 667676.
[116] Y. Qu, W. Li, Y. Zhou, X. Liu, L. Zhang, L. Wang, et al., Full assessment of fate
and physiological behaviour of quantum dots utilizing Caenorhabditis elegans
as a model organism, Nano Lett. 11 (2011) 31743183.
[117] J. Zhang, B. Li, Y. Zhang, A. Li, X. Yu, Q. Huang, et al., Synchrotron radiation
X-ray uorescence analysis of biodistribution and pulmonary toxicity of
nanoscale titanium dioxide in mice, Analyst 138 (2013) 65116516.
[118] L. Wang, J. Li, J. Pan, X. Jiang, Y. Ji, Y. Li, et al., Revealing the binding structure
of the protein corona on gold nanorods using synchrotron radiation-based
techniques: understanding the reduced damage in cell membranes, J. Am.
Chem. Soc. 135 (2013) 1735917368.
[119] Z. Khatun, M. Nurunnabi, D.Y. Lee, Y.-J. Kim, Y. Byun, K.J. Cho, et al., Optical
imaging, biodistribution and toxicity of orally administered quantum dots
loaded heparin-deoxycholic acid, Macromol. Res. 23 (2015) 686695.
[120] L. Song, M.G. Vijver, W.J.G.M. Peijnenburg, T.S. Galloway, C.R. Tyler, A
comparative analysis on the in vivo toxicity of copper nanoparticles in three
species of freshwater sh, Chemosphere 139 (2015) 181189.
[121] B.J. Marquis, S.A. Love, K.L. Braun, C.L. Haynes, Analytical methods to assess
nanoparticle toxicity, Analyst 134 (2009) 425439.
[122] J.M. Jenkins, D.L. Halaney, K.V. Sokoloc, L.L. Ma, H.J. Shipley, S. Mahajan, et al.,
Excretion and toxicity of gold-iron nanoparticles, J. Nanomed. Nanotechnol.
9 (2013) 356365.
[123] C. Fu, T. Liu, L. Li, H. Liu, D. Chen, F. Tang, The absorption, distribution, excretion
and toxicity of mesoporous silica nanoparticles in mice following different
exposure routes, Biomaterials 34 (2013) 25652575.
[124] H. Yang, L. Du, X. Tian, Z. Fan, C. Sun, Y. Liu, et al., Effects of nanoparticle size
and gestational age on maternal biodistribution and toxicity of gold
nanoparticles in pregnant mice, Toxicol. Lett. 230 (2014) 1018.