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Development of nanotoxicology: Implications for drug delivery and medical
devices
Sourav Bhattacharjee* and David J. Brayden
UCD School of Veterinary Medicine and UCD Conway Institute, University College
Dublin, Dublin, Ireland
*Correspondence: Tel: +353 1 716 6233; Email: [email protected]
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Abstract
Toxicology assessments of nanomaterials are currently challenged by sub-
optimal in vitro models, lack of in vitro-in vivo correlations, deficits in both material
purity and physicochemical characterization, and variability within in vitro
nanotoxicological protocols. As a result of these issues, reliable nanomaterial toxicity
and mechanistic understanding of nanomaterials interactions in physiological systems
are required for health and toxicity risk assessments. Much in vitro toxicological data
is inconclusive in designating whether nanomaterials for drug delivery and medical
device implants are truly safe. A critique is presented to analyse the interface between
toxicology and nanopharmaceuticals. Deficiencies of existing practices in toxicology
are reviewed and useful emerging techniques (e.g. lab-on-a-chip technique, tissue
engineering, atomic force microscopy, high-content analysis, and multivariate
analysis), as well as better defined in vitro models are highlighted. Cross-fertilisation
between disciplines will aid development of biocompatible delivery and implant
platforms. Finally, improvements are suggested for development of advanced
protocols in nanotoxicology, with implications for translation.
Key words
Nanotoxicology, biomaterials, high content analysis, lab-on-a-chip, predictive
toxicology
Glossary
Nanotoxicology: in vitro and in vivo study of toxicity of nanomaterials, typically
<100 nm in at least one dimension.
Nanomedicine: the medicinal applications of nanomaterials obtained with
nanotechnology in diagnostics, medical devices, and delivery of therapeutics.
QSAR: (Quantitative Structure-Activity Relationship) is a statistical modelling tool
used in chemical and biological sciences to develop in silico models.
ROS: Reactive oxygen species: superoxides, hydroxyls, and peroxides, produced in
cells as a consequence of biochemical reactions
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Microfluidics: the design of devices, which contain processes with fluid flow
ranging from µl to nl volumes with application in chemistry, biology and
‘nanobiotechnological’ research.
Tissue spheroids: 3D conglomerates of living cells that mimic the evolution,
viability and biochemical processes comparable to original source tissues.
HCA: High Content Analysis is an automated technique used in discovery and in
delivery research, which produces quantifiable data from live cells on a range of
cellular and biochemical indicators of toxicity.
Abbreviations
AAS, atomic absorption spectroscopy; AFM, atomic force microscopy; AUC,
analytical ultracentrifugation; BET, Brunauer-Emmett-Teller; CdTe, cadmium
tellurite; CNT, carbon nanotube; DIC, disseminated intravascular coagulation; DLS,
dynamic light scattering; DMA, differential mobility analyzer; DSC, dynamic
scanning calorimetry; ECM, extra-cellular matrix; FFF, field flow fractionation;
FRET, Förster resonance energy transfer; FTIR, fourier transformation infrared
spectroscopy; GelMA, gelatin methacrylate; GFP, green fluorescent protein; GPC,
gas phase chromatography; HCA, high-content analysis; HNDF, human neonatal
dermal fibroblast cells; HPLC, high performance liquid chromatography; HTS, high-
throughput screening; HUVEC, human umbilical vein endothelial cells; ICP-MS,
inductively couples plasma mass spectrometry; iPSC, induced pluripotent stem cells;
I.V., intravenous; LDH, lactate dehydrogenase; LOC, lab-on-a-chip; MANOVA,
multivariate analysis of variance, MS, mass spectroscopy; MWCNT, multi-walled
carbon nanotube; MVA, multivariate analysis; NMR, nuclear magnetic resonance;
NP, nanoparticle; PAMAM, poly(amidoamine); PCA, principal component analysis,
PDMS, polydimethylsiloxane; QD, quantum dot; QSAR, quantitative structure-
activity relationship; RES, reticulo-edothelial system; RFP, red fluorescent protein;
ROS, reactive oxygen species; SEM, scanning electron microscopy, SOP, standard
operating protocol; SPM, scanning probe microscopy; TEM, transmission electron
microscopy; TEER, trans-epithelial electrical resistance; TGA, thermogravimetric
analysis; TIPS, thermally induced phase separation; TIRF, total internal reflection
fluorescence; TMRM, tetramethyl rhodamine methyl ester; MFA, multifactor
analysis; UV-Vis, ultraviolet-visible spectroscopy; XPS, X-ray photoelectron
spectroscopy; XRD, X-ray diffraction; ZO, zonula occludens.
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Executive Summary
Nanopharmaceuticals are being introduced mainly for cancer therapy, with other
prototypes being tested in preclinical study of delivery systems for a variety of
conditions.
Uncertainty regarding the safety and compatibility of nanopharmaceuticals hinder
translation.
The search for criteria that define the toxicity of nanomaterials remains elusive.
Nanoparticle features (e.g. surface charge, particle size) and non-nanoparticle-
related factors (e.g. protein corona) influence biological behavior of nanoparticles.
The protein corona adsorbed to nanoparticles is a highly dynamic and
heterogeneous layer and its composition is yet to be fully realized.
The role of oxidative stress as a mechanism of toxicity for nanoparticles needs to
be better understood.
Lab-on-a-chip techniques, tissue engineering, computational toxicology, high-
content analysis (HCA), atomic force microscopy, and multivariate analysis assist
nanotoxicological assessments, and these methods are being established in drug
delivery research.
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Contents
1. Introduction
2. Issues with current nanotoxicological in vitro methods
2.1 Discrepancies in operating protocols and in NP-characterization
2.2 Inadequate surface analysis of NPs and cells
2.3 Interactions between proteins and NPs
2.3.1 Role of protein corona around NPs in nanotoxicology
2.3.2 In vivo effects of protein-NP interactions: physiological relevance
2.4 Understanding oxidative stress in cytotoxic mechanisms
2.5 Unfulfilled translation of nanotechnologies to date
3. Upcoming technologies and testing platforms
3.1 Computational nanotoxicology
3.2 Transition towards 3D testing platforms
3.2.1. Nanotox-on-a-chip
3.2.2 Scope for tissue engineering
3.2.3 3D printing
3.3 High-content analysis (HCA)
3.4 Multivariate analysis (MVA)
3.5 Atomic force microscopy (AFM)
4. Future Perspectives
5. References
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1. Introduction
Nanotoxicology is a topic of importance in the current era of nanomedicine and
drug delivery. Interest in nanotechnology has grown rapidly in academia and
industry, as well as with policy makers, funding agencies and the media; the
worldwide market is expected to surpass the $ trillion landmark by 2015 [1]. There is
much effort is in development of “nano-bio” materials that have useful properties and
are safe and effective in drug delivery systems and medical devices. Nanotoxicology
is a subset area of nanomedicine and is used to assess acute and chronic
biocompatibility of nanomaterials; it is in a growth phase, as reflected by large
funding for consortia dispersed from the EU FP7- and other programmes. Due to the
size factor, nanoparticles (NPs) have unique features that influence toxicity, and this
differentiates nanotoxicology from conventional particle toxicology [2].
Nanotoxicology has however, failed to yield significant breakthroughs in
extrapolation of in vitro data to in vivo, despite the volume of literature on
nanomaterial safety published in the last decade [3]. The surge of articles in
nanotoxicology does not necessarily reflect in depth assessment of nanomaterials.
While the number of novel nanomaterials with potential use in diagnostics, tissue- and
cell-based scaffold implants, and delivery of therapeutics is rapidly expanding [4],
most research on their cytotoxicity has applied traditional cytotoxic cell death assays
in cell lines to predict outcomes in vivo, with decidedly mixed results. In addition, a
major challenge for in vitro assay development is to model is sub-lethal toxicity that
may be caused by nanomaterials at low concentrations during chronic exposure in
vivo.
2. Issues with current nanotoxicological in vitro methods
The factors that influence nanomaterial cytotoxicity can be classified as follows: (1)
Factors related to the nanomaterials: e.g. surface charge, particle size, porosity,
surface functionalization, crystallinity, shape and composition; (2) Factors related to
experiment: e.g. presence of opsonins, interactions with culture media components,
surface adsorption to cells or to system supports, cell selection, the role of cellular
receptors, and the bioassays used. While these factors are multifactorial, there is a
tendency to express cytotoxicity primarily in respective of single parameters. For
example, a positive surface charge on NPs is recognized to promote cellular
interaction and can promote increased cytotoxicity compared to neutral or negatively-
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charged NPs [5-7]. However, surface charge does not represent the entire cytotoxic
potential of NPs and further depends on surface functionalization [8] and the spatial
configuration with reference to the surrounding steric bulk [9]. In addition, the charge
density on the surface needs to be taken into account [10, 11]. Undue focus on surface
charge per se lead to errors in conclusions, and the broader physico-chemical
intricacies of surface charge need to be better investigated [12].
Apart from surface charge, particle size is also a critical factor in nanotoxicology
[13, 14]. An aqueous dispersion of NPs is typically a colloid, not accurately reflected
by electron micrograph images. Interpretation is especially complex when NPs are
incubated in culture medium, comprising a gamut of multiple salts, ions,
immunoglobulins, lipids and proteins. They compete for adsorption sites on the
particle, which causes size to fluctuate over time. Adsorption impacts surface charge
and can change overall surface chemistry [15, 16]. Singling out individual factors
therefore will provide limited data, in the event that multiple parameters likely
combine to contribute to overall nanotoxicity.
2.1 Discrepancies in operating protocols for NP characterization
The main obstacle towards gaining insights regarding in vitro nanotoxicology is
questionable methodology [17]. The field lacks many validated SOPs (standard
operating procedures) and of those that are, they are not widely implemented. While
there is debate over which discipline nanotoxicology is closest to – pharmacology and
toxicology, chemistry, or the physical sciences, tackling its challenges requires multi-
disciplinary collaborations. Current nanotoxicology research suffers from lack of
adequate physicochemical characterization of NPs [18, 19], and there are no
universally-agreed assays with designated acceptance criteria [20]. Furthermore,
although there are attempts to provide guidelines on characterization of nanomaterials
in biological media [21, 22], there is no consensus. Scattered knowledge dissemination
in the literature may contribute in part to the lack of standardization.
Nanotoxicological research is published in a wide variety of journals including those
in pure toxicology, pharmaceutics, and polymer and lipid chemistry. There is a
tendency to focus on journals within one’s own expertise rather than on less familiar
journals.
In order to assess cytotoxicity of NPs in cellular environments, useful data can be
achieved from techniques in physical chemistry and colloid sciences. Such
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laboratories are well equipped with instruments essential to characterize
nanomaterials. These include dynamic light scattering (DLS) to measure
hydrodynamic size of NPs, zeta potential measurement to assess the stabilities of
suspensions, and the Brunauer-Emmett-Teller (BET) technique to measure surface
area and porosity. They also contribute X-ray diffraction (XRD) to investigate the
crystal structure, transmission/scanning electron microscopy (TEM/SEM), and atomic
force microscopy (AFM) to image the NPs, as well as spectroscopic nuclear magnetic
resonance (NMR), infrared (IR), ultraviolet (UV-VIS) and chromatographic liquid
chromatography/mass spectroscopy (LC/MS) to understand compositions. Common
techniques employed to characterize NPs are listed (Table 1).
Table 1: Usual characterization techniques for NPs
Parameter Properties Techniques
Morphology
Size TEM, SEM, AFM, XRDShape/Structure TEM, SEM, AFM, DLS, FFF, AUC, HPLCSize distribution EM, SEM, AFM, DLS, AUC, FFF, HPLCMolecular weight AUC, GPC
Stability DLS, AUC, FFF, SEM, TEM
Surface features
Surface area BETSurface charge SPM, TitrationsSurface coating SPM, XPS, MS, FTIR, NMR
Surface coverage AFM, AUC, TGATopology SEM, SPM, MS
Chemistry
Composition XPS, MS, AAS, ICP-MS, FTIR, NMRPurity ICP-MS, AAS, AUC, HPLC, DSC
Crystallinity XRD, DSCStability MS, HPLC, FTIR
Drug deliveryDrug loading MS, HPLC, UV-VIS, Fluorescence
In vitro release UV-VIS, MS, HPLC, FluorescenceDeformability AFM, DMA
Abbreviations: AAS, atomic absorption spectroscopy; AFM, atomic force microscopy; AUC, analytical ultracentrifugation; BET, Brunauer-Emmett-Teller; DLS, dynamic light scattering; DMA, differential mobility analyzer; DSC, dynamic scanning calorimetry; EM, electron microscopy; FFF, field flow fractionation; FTIR, fourier transformation infrared spectroscopy; GPC, gas phase chromatography; HPLC, high performance liquid chromatography; ICP-MS, inductively coupled plasma mass spectrometry; MS, mass spectroscopy; NMR, nuclear magnetic resonance; SEM, scanning electron microscopy, SPM, scanning probe microscopy; TEM, transmission electron microscopy; TGA, thermogravimetric analysis; TIRF, total internal reflection fluorescence; UV-Vis, ultraviolet-visible spectroscopy; XPS, X-ray photoelectron spectroscopy; XRD, X-ray diffraction
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There are three major areas to further address in characterization of NPs:
(1) NP-interference – NPs are reactive and interact with components of in vitro
systems (e.g. salts, proteins, immunoglobulins). They have high surface area/mass
ratios, which facilitate molecule adsorption. For example, carbon nanotubes
(CNTs) can adsorb to the formazan in the [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] (MTT) assay and produce false positive cytotoxicity
[25]. Other examples of interference during assays occur with fluorescent NPs (e.g.
quantum dots, fluorescent latex beads), which can interfere with
spectrofluorometric assays [26]. Furthermore, NPs can adsorb salts and proteins in
culture medium [27], creating medium component concentrations that are too low
to support cell viability, thus indirectly causing cytotoxicity.
(2) Concentration considerations – compared to realistic exposure concentrations,
there is a tendency to use very high levels of NPs both in vitro and in vivo. Very
high concentrations of NP are of little toxicological relevance in making a risk
assessment. Furthermore, they can paralyze cellular physiology by establishing a
~500 nm layer of NPs on the plasma membrane, which deprives cells of nutrients
and oxygen [28, 29].
(3) Effect of solvents – many laboratories uses commercially-available NPs “as
received” from suppliers. Quantum dots (QDs) and latex beads arrive suspended in
a “vehicle mixture” containing surfactants, stabilizers and organic solvents (e.g.
tetrahydrofuran, THF), which may have inherent toxicity. For example, the
oxidative stress in cells observed from C60 fullerenes originated from the peroxides
produced from ageing THF, which were present to provide fullerene stability [30].
Effects of solvents therefore need to be taken into consideration in order to avoid
erroneous conclusions on cytotoxicity.
2.2 Inadequate surface analysis of NPs and cells
Surface chemistry is important in nanotoxicology not only because most of the
nanobio-interactions are surface-dependent, but also due to the growing use of
functionalized NPs. The surfaces of NPs can be coupled with functional groups via
conjugation or through attachment of surface coatings to facilitate passage through
biological barriers (e.g. human intestinal mucus, sub-cutaneous tissue). Therefore,
characterization of NPs is incomplete without detailed knowledge about surface
properties [31]. Surface charge has a significant effect on cellular internalization as
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well as on toxicity of NPs. Importantly, the entire surface chemistry (i.e. the combined
effects of curvature, surface area/volume ratio, the percentage of molecules expressed
on the surface, functionalization, and hydrophilicity) contributes to how NPs interact
with biological systems.
The consequences for the plasma membrane after exposure to NPs are also of
interest. With the help of AFM, the surfaces of rat macrophage NR8383 cells exposed
to 100 nm positively- and negatively-charged latex beads were visualized [32]. The
positively-charged NPs created holes in the cell membrane and increased the overall
roughness of the cell surface, which were associates with cytotoxicity. In contrast,
non-toxic negatively charged NPs caused few changes in cell surface topography.
These results showed how NPs affect the surface properties of the cells and also
suggested an additional mechanism of toxicity for the cationic NP: disruption of the
surface congruity of cell membranes.
Unfortunately, it is a challenge to fully characterize the surfaces of either NP or
cells. One major reason is the typically harsh conditions (e.g. ultra-high vacuum, high
pressure, dessication) that are employed in the current surface analysis tools (e.g. XPS
(X-ray photoelectron spectroscopy), LEED (low energy electron diffraction),
TEM/SEM, SIMS (secondary ion mass spectrometry), and Auger electron
spectroscopy. Another contributing factor arises from sub nm structures, (e.g. ion
channels), which participate in such cell-NP interactions, but current surface analytical
tools do not have the resolution to map them, although this is being addressed. The
presence of water in biological as well as NP environments is also a restricting factor
towards the applicability of such tools.
2.3 Interactions between proteins and NPs
2.3.1 Role of protein corona around NPs in nanotoxicology
The concept of a protein corona forming on NPs in serum-rich culture medium has
gained general acceptance [33-37]. The protein corona is a highly dynamic and
heterogeneous adsorbed layer; its composition further depends on the chemistry of the
NP and determines the mechanism of interaction [38], agglomeration states, and as to
whether NPs can stimulate specific receptor-mediated internalization [39].
The role of protein corona in cellular interactions of NPs however, needs to be
analyzed further [40]. DMEM and RPMI-1640 are mixtures of different amino acids,
lipids, salts (calcium chloride, potassium chloride, phosphates), carbohydrates, and
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vitamins (folic acid, nicotinamide, riboflavin). In medium, such molecules also adsorb
onto NP surfaces. Molecules with smaller masses (e.g. lipids) move faster and thereby
are adsorbed on the NP-surface quicker than the large proteins in fetal bovine serum
(FBS). However, with the arrival of proteins at surfaces, the lower mass molecules are
gradually displaced in a dynamic process [41]. Lower molecular weight molecules
however, contribute more than proteins in building up the corona at early time points,
during which cell interaction and uptake of NP may be at their highest level. The role
of non-protein molecules in cell culture medium may therefore contribute significantly
to initial interactions between cells and NPs, as well as to cytotoxicity. To our
knowledge, there is very little published on their potentially important role. The
occurrence of protein adsorption on biomaterials in biologically relevant media
including like blood has been reported before, but there is a tendency for some
researchers to ignore literature [42-45]. The topics of particle curvature, defects and
energy isotherms on NP-surfaces, as well as analytical issues remain challenging for
the corona, although the basic principles for adsorption are well established. The
composition of protein corona on NP-surfaces is dynamic and depends on a multitude
of factors including protein size and concentration. Unfortunately, the composition of
protein corona on NP-surfaces is yet not well studied due to lack of adequate tools.
To date, few animal studies investigating the role of surface charge in
biodistribution and bioavailability of charged NPs have noted any significant effect of
their surface charge, either following oral [46, 47] or parenteral administration [48, 49]
There is a lack of any consensus on the effect of surface charge on biodistribution of
NPs after oral delivery. This is in contrast to in vitro data [50]. One explanation for the
apparent discrepancy between in vitro and ex-/in vivo intestinal epithelial data may be
the role of mucous overlying the intestinal epithelium, which impedes NP-uptake by
enterocytes, depending on the model [51]. The role of the surface charge in surface
adsorption of proteins can also be an explanation for this lack of in vitro-ex-/in vivo
correlation [52]. The protein molecules harbor both cationic and an anionic binding
sites, which adhere to NPs thereby altering the surface properties and masking effects
observed in vitro [53]. Therefore, the in vivo understanding of the protein corona
around NPs has not yet been worked out adequately.
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2.3.2 In vivo effects of protein-NP interactions: physiological relevance
Following I.V. injection, NPs appear to interact with proteins in plasma in both
surface charge- and size-dependent ways. For example, Greish et al. [54] tested non-
porous silica NPs (50, 200 nm; amine- and acid-terminated), PAMAM
[poly(amidoamine)] dendrimer generations (G3.5-G7; sizes (3.2-8 nm), as well as
surface functionalized analogues (amine, hydroxyl and acid-terminated) in CD-1 mice
following I.V. injection. Of the silica NPs, only the largest 200 nm silica NPs caused
in vivo cytotoxicity, irrespective of their surface charge. In contrast, amine-terminated
dendrimers induced lethality at a dose of >10 mg/kg, confirming the cytotoxicity of
cationic but not the anionic dendrimers. The cationic dendrimers caused disruption of
the clotting cascade, intestinal hemorrhage, as well as disseminated intravascular
coagulation. In a similar study, cationic PAMAM dendrimers induced platelet
dysfunction while inhibiting thrombin formation [55]; a mechanism mediated by
interaction with P-selectin, RANTES and PF4 (platelet factor) recognition sites on
platelets. Insights into the role of NP-protein interactions in in vivo toxicology in such
examples will provide better understanding of nanotoxicity in the circulation and will
assist the translational value of NPs after parenteral exposure.
2.4 Understanding oxidative stress in cytotoxic mechanisms
Oxidative stress is a common mechanism of cytotoxicity of NPs [56-59], since NP
are highly reactive and interact with a variety of proteins and lipids. If internalized,
NPs may trigger enzymatic pathways capable of producing reactive oxygen species
(ROS) including superoxides, hydroxyls and peroxides. There are doubts however,
over whether oxidative stress is a primary or secondary toxicological mechanism.
Charged NPs disrupt the mitochondrial membrane permeability [60, 61], making them
leaky. The mitochondrial membrane is the site for the electronic transport chain
(ETC), which quenches oxygen free radicals [62]. Therefore, a disturbance to the
mitochondrial membrane by charged NPs will hamper the ETC, causing leach of
oxygen radicals into cytoplasm. Hence, oxidative stress can manifest as a secondary
feature of disrupted mitochondrial physiology caused by charged NPs. In addition,
mitochondria is a source of intracellular calcium [63], and disruption will also cause
calcium release into the cytoplasm leading to overload [64, 65]. Calcium overload can
in turn trigger apoptotic pathways via production of secondary cellular messengers
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including NF-κB [66]. Other cellular cytotoxic pathways exist within cells in response
to NPs, and perhaps systems biology mapping can add value [67, 68].
2.5 Unfulfilled translation of nanotechnologies to date
In spite of the large amount of nanotoxicology data available, building safe and
sustainable drug delivery platforms based on NP constructs remains largely unfulfilled
[69-76]. 10,000 papers on nanotoxicology were published from 2011-2014 [26], but
few constructs translated into clinical approvals. Three major reasons are highlighted:
(1) The deficiency in correlation between in vitro and in vivo – current in vitro models
in nanotoxicology continue to be inadequate for in vivo correlation, yet in vitro
studies have dominated the nanotoxicology literature, due largely to the difficulties
associated with in vivo experimentation, including inadequate animal models,
stringent licensing procedures, and significant Facility investment.
(2) Sequestration of NPs into organs – more than 90% of the bioavailable dose of NPs
fails to reach target organs due to sequestration by the liver or spleen [77]. Coating
NPs with PEG (polyethylene glycol) of different chain lengths is a valid way to
alter surface hydrophilicity in order to change distribution by avoiding uptake by
the RES (reticulo-endothelial system) [78].
(3) Inability of engineered NPs to cross biological barriers – many NPs fail to cross
biological barriers (e.g. blood-brain-barrier, small intestine). NPs with high surface
charge density show better flux through mucus [79], but such muco-penetrating
NPs then need further design to promote epithelial cell internalization, unless
release of payload close to the epithelium is enough for efficacy. In many studies
Caco-2 cell monolayers grown on inserts are used to measure passage of NPs [80],
but there are discrepancies between protocols, with no account taken for lack of
mucous. Most of the mucous-covered epithelial constructs have reproducibility
issues and do not elaborate the same mucous composition as in vivo.
3. Upcoming technologies and testing platforms
3.1 Computational nanotoxicology
Quantitative structure-activity relationship (QSAR) models have been proposed as a
promising tool to model and predict NP cytotoxicity [81, 82]. In silico models may
predict cytotoxicities of NPs to a satisfactory degree [83]. One of the key features is
interaction between NPs and cell membrane-bound receptor pathways. These
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receptors, including clathrin and caveolin pathways [84, 85], are the basis of
internalization of a wide range of NPs.
With knowledge on the energetics and mechanisms of endocytic pathways
emerging, reliable computational models can be developed [86]. However, we must
first acquire improved characterization of NPs as the quality of the data input will
determine the quality of results that can be extracted from these models. QSAR
models may provide insight into receptor-NP interactions and, for example, can
predict behavior of multi-layered phospholipid layers after being exposed to charged
NPs [87]. With increasing numbers of NPs being incorporated into consumer products,
it is extremely difficult to test them all in vivo [88]. Taking such points into
consideration, QSAR can be an effective model towards predictive nanotoxicology in
future, although it will still need in vivo datasets to compare with.
Another aspect of computer design-aided systems is the potential for protocols in artificial
intelligence (AI) domains. Some of this research is related to designing computer-human
brain interfaces [89, 90]. Similarly, other human model systems (e.g. skin) [91] are being
developed digitally, and such models may predict nanomaterial toxicity in a more defined
way. Through these informatics-based designs, interesting models of human physiological
barriers [e.g. blood-brain-barrier (BBB)] can be developed, which can assist development of
NP-based delivery technologies [92].
3.2 Transition towards 3D testing platforms
3.2.1 Nanotox-on-a-chip
Microfluidics-based approaches are finding new applications in biology. Improvements in
surface fabrication and lithography techniques (e.g. wet and dry etching, sputtering, anodic
and fusion bonding) [93] can produce surfaces (e.g. silicon, glass and polymers) with
embedded channels and reservoirs of µm dimensions. These surfaces can be used to produce
chips synthesized from materials including polydimethylsiloxane (PDMS) [94]; µm
dimensional structures are then transferred onto chip materials. With advanced
instrumentation and introduction of modern automation including robotics, sophisticated
microfluidics-based devices are being developed with precise control over process- and fluid
flow. Lab-on-a-chip (LOC) technology has potential in drug delivery [95, 96], and especially
within the fields of bio-MEMS (biomedical microelectromechanical systems) [97, 98] and
µTAS (micro total analysis systems) [99, 100]. One of the advantages of fluid flow in
microchannels is the laminar flow and absence of turbulence [101], thereby preventing
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mixing between fluids. As a result, the chances of bacterial infection are reduced compared to
conventional in vitro techniques [102]. Additionally, bio-MEMS platforms require few
reagents, produce low level waste, and have a high degree of integration, sensitivity, and
portability. The surface fabrication costs are becoming manageable and this will contribute to
growth [103].
In last few years, bio-MEMS have been used extensively in biomedical engineering
and drug discovery. One of the important developments in this field is the designing of
‘organ-on-a-chip’ devices [104]. These 3D microfluidics-based devices are tissue
culture systems, which simulate the functions and physiologies of different organs.
They offer more flexibility compared to 2D conventional counterparts, in addition to a
low contamination risk. Organ-on-a-chip devices are being used extensively for in
vitro studies investigating chemotaxis, stem cell differentiation, axon guidance and
embryonic development [105-108]. They offer the advantages of multiplexing with
capability of high-throughput screening (HTS) and hence, are being used in the
development of biosensors, point-of-care diagnostics, and “omics” studies. Apart from
that, they are being used with induced pluripotent stem cells (iPSC) [109] to develop
new techniques in cell therapy and the study of cell-cell, or cell-ECM (extra-cellular
matrix) interactions. Some organ-on-a-chip devices (e.g. lung-on-a-chip [110], liver-
on-a-chip [111], gut-on-a-chip [112]) are already being used in nanotoxicology and
this is relevant for NP delivery systems. There are efforts to develop a “human-on-a-
chip” µdevice which will integrate all the major systems in human body [113].
A human lung-on-a-chip model was reported in 2010 [110]. Although some sporadic
attempts of designing human lung-on-a-chip have been done before [114], this model may
potentially be used for testing pulmonary toxicity of NPs. The chip was produced in PDMS
containing µm-dimensional structures and contained a three layer arrangement (PDMS-
membrane-PDMS) (Fig. 1). The membrane sandwiched between the two PDMS layers was
porous and, after the chip assembly, epithelial and endothelial cellular monolayers were
grown on opposite sides of the membrane. Intact structural aspects of the monolayers were
confirmed by TEER (trans-epithelial electrical resistance) and staining by a ZO-1 (zonula
occludens) antibody to the tight junction protein. Two parallel hollow channels were at the
sides of the cell-containing chambers in order to exert cyclic stretching by application of
periodic vacuum to mimic the effect of diaphragmatic movement on the alveoli during
respiration in man.
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In related work, a novel human gut-on-a-chip was reported in 2012 [112]. Caco-2
cells were grown on a porous PDMS membrane (coated with rat collagen type I and
Matrigel as ECM) sandwiched between two PDMS layers from top and bottom. Two
hollow side chambers were also kept in order to apply periodic longitudinal stretching
in order to mimic the peristaltic movements. The viability of the Caco-2 monolayers
was confirmed by measuring the activity of aminopeptidase enzyme which is
expressed in the brush borders of differentiated cells. A strain of Lactobacillus
rhamnosus GG (LGG) was then added as a co-culture to model gut microflora and
monolayers were maintained for five days at a flow rate of 30 µl/h. Undoubtedly,
these devices also have scope for use with cells/tissues from real patients, and they
have high levels of integration with tight control over experimental conditions, and a
lower waste compared to traditional in vitro set ups [115].
Fig 1. “Biologically inspired design of a human breathing lung-on-a-chip microdevice. (A) The microfabricated lung mimic device uses compartmentalized PDMS microchannels to form an alveolar-capillary barrier on a thin, porous, flexible PDMS membrane coated with ECM.
The device recreates physiological breathing movements by applying vacuum to the side chambers and causing mechanical stretching of the PDMS membrane forming the alveolar-
capillary barrier. (B) During inhalation in the living lung, contraction of the diaphragm causes a reduction in intrapleural pressure (Pip), leading to distension of the alveoli and
physical stretching of the alveolar-capillary interface. (C) Three PDMS layers are aligned and irreversibly bonded to form two sets of three parallel microchannels separated by a 10-μm-
thick PDMS membrane containing an array of through-holes with an effective diameter of 10 μm. Scale bar, 200 μm. (D) After permanent bonding, PDMS etchant is flowed through the
side channels. Selective etching of the membrane layers in these channels produces two large side chambers to which vacuum is applied to cause mechanical stretching. Scale bar, 200 μm. (E) Images of an actual lung-on-a-chip microfluidic device viewed from above.” (from [110])
Reproduced from Ref. 110 with permission from the American Association for the Advancement of Science (AAAS).
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In spite of the potential of organ-on-a-chip models, significant strides still need to
be made before they are suitable for nanotoxicity testing [116-118]. Commonly used
chip materials such as PDMS can cause cytotoxicity and interfere with biological
assays. Several models are based on oversimplified principles, which do not reproduce
the structural complexities of tissue. However, improving current in vitro conditions
does not automatically mean higher extrapolative values to in vivo. Most of these
organ-on-a-chip models are built on selected cell lines and none have been validated
for their tissue phenotypes, genetic expressions, or metabolism. It is therefore a
challenge to expect that cells grown on a chip in a closed µfluidic environment will
mimic in vivo or even conventional in vitro systems. To address this, there is renewed
interest in use of “tissue-on-a-chip” models built on perfused ex vivo tissue slices from
toxicologically relevant organs (e.g. liver) [119, 120].
3.2.2 Scope for tissue engineering
From being a simple branch of cell biology and testing biomaterials, tissue engineering
has experienced unprecedented growth in the last two decades. Innovations including “in
vitro meat” [121], and generation of bio-artificial organs [122-124]) are paving the way.
Usually, the rationale for developing tissues in vitro is to harvest cells onto natural or
synthetic scaffolds [125, 126]. These scaffolds provide mechanical support for cells to
develop 3D aspects within the tissue systems and also for multiple cell types to differentiate.
Therefore, such cells have higher differentiation and increased robustness compared to
traditional 2D systems [127]. To embed cells within scaffolds and to ensure supply of
nutrients and oxygen, it is essential that the scaffolds are porous and exhibit surface
roughness. The scaffolds are usually made from synthetic biodegradable materials (e.g.
polylactic acid, polyglycolic acid, polycaprolactone) [128], or from natural materials (e.g.
collagen, chitosan, polysaccharides, glycosaminoglycans) [129]. They can be prepared by
electrospinning, thermally-induced phase separation (TIPS), emulsification/freeze drying, or
gas foaming [130]. Recently computer aided design was used to produce scaffolds with
uniform pores and controllable pore distribution [131]. Other progress is in the use of carbon
CNTs [132] as scaffold materials. They can provide a mesh with nm-range roughness, which
can be optimal for seeded cells to grip and coalesce upon, before growing into a tissue mass.
Additionally, the conductivity of CNTs can be used further to stimulate cell growth and
vasculature development [133]. The hanging drop technique is now being used to produce
microtissues which can then be used for toxicity testing [134]. The significant growth in
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surface lithography techniques and materials science is enabling development of novel
biomaterials, which can be used to harvest cells in order to promote differentiation of
spheroids [135, 136]. Control of oxygen tension, supply of nutrients, pH, humidity,
temperature, along with succinct and defined specificities and negligible variations between
such µtissue spheroids can be achieved [137]. Additionally, with the use of different growth
factors, microtissue spheroids with altered vascularity can be generated [138]. As a result, the
nanotoxicity data derived from such microtissue spheroids may turn out to be more consistent
and extrapolative to in vivo conditions compared to 2D systems. Application of ~100 µm
spheroids produced from human hepatocarcinoma-derived HepG2 cells in
polyacrylamide hydrogel inverted colloidal crystal (ICC) scaffolds in the estimation of
NP toxicity was recently carried out [139]. Spheroids were exposed for 12 h to CdTe
(cadmium tellurite)-QDs, as well as gold NPs, and lactate dehydrogenase (LDH) and
MTT assays were used as read-outs. The authors compared the NPs in the scaffolds
with a 2D system (Fig. 2). Significant differences were found between the 2D and 3D
systems, whereby NP-toxicities were over-estimated in the former; toxicity data
obtained from NPs in 3D systems are of higher quality compared to the 2D [140].
Fig 2. “Comparison of 2D and 3D culture of HepG2 cells after 12 h of CdTe NP exposure. A–D) Optical images of normal A) 2D and C) 3D spheroid cultures. After CdTe NP introduction,
the 2D culture showed a dramatically different morphology (B), while it was hard to distinguish any change in the 3D culture under an optical microscope (D).” (from [139]).
Reproduced with permission, Copyright: Wiley-VCH.
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In a second example, toxicity of polypyrrolidone-coated silver (Ag) NPs was tested
in murine macrophage RAW 264.7 cells in 2D cell culture and in 3D spheroid culture
systems. Again, the toxicity of AgNPs was comparatively lower in spheroids
compared to 2D, and it decreased faster over time. Computer-based design has also
been used to predict the diffusion of NPs through micro-tissue spheroids [141]. Even
with the limited amount of literature available from nanotoxicological investigations
performed in 3D cell cultures to date, the differences between toxicity between 2D and
3D systems are clearly apparent [142]. A likely explanation is that differentiated cells
grown within 3D systems are different from those within 2D systems, both
morphologically and physiologically. This also raises concerns about the huge amount
of in vitro data in nanotoxicology that have been published in the last two decades, as
most of these data were based on 2D models.
3.3.3 3D printing
3D printing produces digital structures that were considered previously to be impossible to
create [143], and it has great potential in tissue engineering. Sophisticated structures of
varying geometries can be producing by the layer-by-layer deposition of materials, which
offer opportunities for nanotoxicology [144]. With the capability in developing
structures/scaffolds with high precision, 3D printing technology may help eliminate
deficiencies in methodologies in nanotoxicology. There are two main techniques to
manufacture biomaterials using such methods: (1) Bonding-based inkjet printing, whereby a
particle-based material is deposited in layers along with binder molecules. After post-
processing, this technique produces biomaterials with designs that can be used as scaffolds in
tissue engineering; (2) Bioink-jet printing where instead of particles, biologically relevant
materials can be used. This is an exciting way of printing organs with defined vascularity
[145]. However, it is worth pointing out a few current drawbacks. The structures produced by
3D printing require post-processing, which can compromise biocompatibilities and cell
viability. Most 3D printing products are also porous in structure, which can hamper their use
as sustainable biological models [146]. A lot of tissue engineering techniques, especially in
bone tissue engineering [147], are already being used with the help of 3D printing. A TED-
talk was used as a recent forum to demonstrate how to print a human kidney using cells as
bio-ink [148]. Others also showed how cell-laden tissue constructs with grown
vascularization were developed with the help of 3D printing (Fig. 3) [149].
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Fig 3. “a,b) Schematic views of the top-down and side views of a heterogeneous engineered tissue construct, in which blue, red, and green filaments correspond to printed 10T1/2 fibroblast-laden
GelMA, fugitive, and GFP HNDF DEFINE -laden GelMA, inks, respectively. The gray shaded region corresponds to pure GelMA matrix that encapsulates the 3D printed tissue construct. Note: The red filaments are evacuated to create open microchannels, which are endothelialized with RFP DEFINE
HUVECs. c) Bright field microscopy image of the 3D printed tissue construct, which is overlayed with the green fluorescent channel. d) Image showing the spanning and out-of-plane nature of the 3D
printed construct. e) Image acquired during fugitive ink evacuation. f) Composite image (top view) of the 3D printed tissue construct acquired using three fluorescent channels: 10T1/2 fibroblasts (blue), HNDFs (green), HUVECs (red). g) Cell-viability assay results of printed 10T ½ fibroblast-laden and
HNDF-laden GelMA features compared to a control sample (200–300 μm thick) of identical composition. The asterisks indicate differences with p < 0.05 obtained from student's t-test” (from
[149]). Reproduced with permission, Copyright: Wiley-VCH.
To print the vasculature, tri-block copolymer Pluronic F127 [poly(ethylene oxide)-
poly(propylene oxide)-poly(ethylene oxide)] was used. Gelatin methacrylate (GelMA) was
used as an ink for the ECM. Different cells [fibroblastic 10T1/2 cells, green fluorescent
protein (GFP)-labelled human neonatal dermal fibroblast cells/HNDF and red fluorescent
protein (RFP)-labelled human umbilical vein endothelial cells/HUVEC] were used. The
main motivation behind 3D platforms is associated with their capabilities in
reproducing the intrinsic physiological complexities at an in vitro tissue/organ level.
This addresses some current inadequacies of 2D in vitro platforms in nanotoxicology
[150]. A shift in focus to better 3D models represents the physiological complexities
and intricacies at an organ-level to a much greater degree and with more control. The
in vitro data derived from these 3D models will be of comparatively better quality,
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which in turn will lead to better validation, improved optimization and better
extrapolation to in vivo.
3.3 High-content analysis (HCA)
The concept of HCA is relatively new in nanotoxicology research for drug delivery
and stems from drug discovery [151-153]. HCA is an integrated automated platform
comprising fluorescence microscopy and advanced imaging software for live cells. It
confers improved sensitivities and specificities compared to conventional cytotoxicity
assays. Another important characteristic of HCA analysis is that in contrast to cell
death assays, it successfully detects sub-lethal cellular changes simultaneously in
relation to concentration and exposure time. In one of the first HCA studies in drug
delivery, the cytotoxicity of melittin (a peptide from bee venom under investigation as
an intestinal permeation enhancer) was tested in Caco-2 cells using a four-dye mixture
of Hoechst 33342 (to detect cell number, nuclear intensity and nuclear area), Fluo-4
AM (to detect intracellular calcium), tetramethyl rhodamine methyl ester (TMRM) (to
detect mitochondrial membrane potential) and TOTO®-3 iodide (to detect plasma
membrane permeability) [154]. The data revealed a structure-activity relationship for
single amino acid replacements in melittin and proved that permeation enhancement
and cytotoxicity mechanism were associated. HCA offers scope for multiplexing and
HTS, as it counts the changes in individual cells and then provides a mean value
whereas traditional toxicity assays provide a single mean point for the entire
population of cells, irrespective of varying degrees of developmental and
differentiation of all the cells present. This can be of importance in nanotoxicology as
it is known that toxic effects of NPs differ depending on the differentiation stages.
Therefore, it is possible that the toxicity of NPs occurs only in a subset of the cellular
population, which can be missed by traditional in vitro techniques. Additionally, HCA
offers the possibility for rapid screening of nanoparticulates, which can be very useful
in the current context of the high numbers of emerging novel biomaterials. A recent
study used HCA to compare toxic CdTe-QDs and innocuous gold (Au) NPs [155].
Murine neuroblastoma NG108-15 (exposed to QDs) and HepG2 (exposed to AuNPs)
were used as cell models. Cell viability, calcium leakage, neurite growth, and
apoptosis were measured by HCA (Fig. 4). The results showed escalation of apoptosis
in NG108-15 cells induced by QDs, with varying responses in differentiated and
undifferentiated cells, whereas the AuNPs induced intracellular calcium release in
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HepG2 cells and altered sub-lethal parameters to a lesser extent. A similar study where
HCA has been used to assess toxicities of NPs was also published recently with
investigation on toxicity of iron oxide NPs [156].
Fig 4. “Representative fluorescence and bright field images of a healthy (green outline), an apoptotic (red outline), and a necrotic (yellow outline) cell. Cells were stained simultaneously with Hoechst 33342 (blue channel, first column) and propidium iodide (red channel, middle column). Outline and classification of cells were generated by the IN Cell Investigator image
analysis software using the supervised classification capability.” NG108-15 murine neuroblastoma cells were exposed to thioglycolic acid/gelatin coated QDs (from [155]).
Reproduced with permission from Elsevier.
3.4 Multivariate analysis (MVA)
One of the new methodologies to analyze nanotoxicology data is multivariate analysis
(MVA), which is based on the principles of multivariate statistics and analyzes outcomes
taking multiple variables into account [157]. Owing to its capacity to deal with multiple
factors at a time, MVA offers improvements in understanding cell-NP interactions. It has
several variations: principal component analysis (PCA), multifactor analysis (MFA), and
multivariate analysis of variance (MANOVA). The choice of model will depend on
dependent or independent factors. The target of MVA is to build up a model statistical tool
through determining the regression trends that can be used to analyze a broad range of
datasets [158]. It can be used in specific nanomaterial toxicity assays, where individual
parameters including surface charge, composition, and particle size are correlated to toxicity
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parameters (e.g. production of intracellular ROS, reduction of cellular glutathione, disruption
of mitochondrial membrane integrity). It is still unknown to what extent these factors
contribute individually to the amalgamated toxic effects and therefore MVA approaches can
shed new light on this. Use of MVA to represent the NP-toxicity data is gaining traction. Two
recent studies used MVA to investigate cytotoxicities of carbon NPs (multi-walled
CNTs (MWCNTs) and C60 fullerenes on gram negative organisms (P. fluorescens and
M. vanbaalenii) [159, 160]. The authors used synchrotron radiation-based Fourier-
transform infrared (IR) spectroscopic techniques in order to assess cellular metabolic
activities, in association with cellular imaging. Control cells were scanned using
advanced IR spectroscopy to determine the fingerprint range for the biomolecules
within the cells. Next, with systematic scanning of cellular samples exposed to
different MWCNTs and C60 fullerenes, 64 different data sets were generated and
analyzed. Through scanning of specific regions of the spectra and further comparison
with control data, a larger picture of intracellular events including production of ROS
were identified. Furthermore, these data were depicted in 3D hyperspace after
uploading the computational model with the data and determining the vector. The 3D
depiction showed outcomes of clustered datasets, and advanced a novel way of
representing and analyzing data.
3.5 AFM
In the last two decades, AFM has evolved to be an extremely powerful and sensitive tool
to image surface topologies at even sub-nm scales. AFM offers advantages (e.g. real-time
mapping, capability to measure in aqueous environments, minimal sample preparation, and
absence of any requirements of harsh conditions as high vacuum or pressure, integration
possibilities with fluorescence and microscopy techniques like confocal, TIRF, FRET), which
makes it a suitable tool for biological specimens [161]. AFM sensitivity has seen growth in
mapping cell surface topography, an important aspect of nanotoxicology. The simplest
application of AFM is in NP-visualization. With advanced techniques and tiny diameter of
the tips, very high resolution is now possible, which enables visualization of NPs (<50 nm),
and examination of surface complexities and functionalization [162]. Adequate surface
analysis is essential before starting toxicity assays, so AFM can provide a broad range of
comprehensive surface analysis of both NPs and plasma membranes. It can also image
complex biomolecules (proteins, DNA) [163, 164], and can investigate their interactions with
NPs. It is also used to image membrane proteins, including elucidation of their repetitive sub-
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units and other structural properties [165], as well as mapping ion channels and receptor
patterns [166]. Furthermore, AFM was used to investigate cell mechanics by imaging
changes in cytoskeleton following exposure to toxicants including NPs [167]. With
functionalized tips, AFM is now able to provide hyper-resolution of sub-nm scale “bionano”-
interactions and also provides information on intermolecular interactions and adhesion forces.
This can be helpful especially in developing NPs for peptide delivery across the intestine,
where the most promising prototype should be able to penetrate mucus and reach the
epithelium. AFM can measure the adhesion forces between NPs with mucin and can rank
NPs on their propensity to permeate mucus. It has potential in nanotoxicology through its
interrogation of the complex interactions between NPs and cell membranes, provides
information about how NPs interact with receptors, and elucidates the processes of cellular
internalization.
4. Future Perspectives
Nanotoxicology has tremendous potential to facilitate NP-drug delivery research.
However, poorly disseminated SOPs, lack of rationale for selection of concentrations in vitro
and dose levels in vivo, poorly predictive in vitro models, insufficient NP characterization, as
well as system-based interferences hamper progress. These are all areas that need to be
addressed. Furthermore, NP designs for drug delivery are being continuously introduced. For
example, nanocrystals of the original drug can form NPs with very high drug loadings [168],
and is especially suitable for oral drugs with low solubility. Oral nanocrystals of many
important small molecules (e.g. clarithromycin [169], amphotericin [170], danazol [171] and
naproxen [172]) have been reported. As a second example, structural knowledge of viral
capsids is enabling use of viral NPs in delivery of DNA and RNA [173], peptides [174]) and
for imaging and diagnostics [175, 176]. Due to their extremely high surface charge densities,
viral particles may also diffuse through mucus, which is relevant for non-parenteral delivery
platforms. It is clear that multi-disciplinary NP-drug delivery collaborations between
nanotoxicologists, physical chemists, pharmaceutical formulators and pharmacologists will
lead to higher chances of translating such concepts.
5. Conclusion
Emerging concepts, including 3D printing, bio-MEMS, HCA, AFM, tissue engineering
and MVA can contribute significantly to nanotoxicology assay development and can assist to
derive better in vitro-in vivo correlation. In spite of investing so much effort in
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nanotoxicology, progress in understanding the intricacies of cytotoxicity of NPs still remains
insufficient for accurate predictions for man and this is especially relevant for drug delivery
application. There is an urgent need to establish standardized protocols in order to bring
comparability within nanotoxicology research. An inter-disciplinary research environment
needs to be established to produce more reliable safety profiles of engineered nanomedicine.
In doing so, nanotoxicology will facilitate the safety aspects to enable effective robust NP
platforms.
Acknowledgements
Research for this review has received funding from the European Union Seventh
Framework Programme (FP7 / 2007-2013) under grant agreement n° 281035 (TRANS-INT)
and also from the Science Foundation Ireland Centre for Medical Devices (CURAM) (13-
RC-2073).
Declaration
The authors declare no competing interests.
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