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Development of nanotoxicology: implications for drug delivery and medical devices
Current nanotoxicology research suffers from suboptimal in vitro models, lack of in vitro–in vivo correlations, variability within in vitro protocols, deficits in both material purity and physicochemical characterization. Reliable nanomaterial toxicity and mechanistic insights 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 analyze the interface between toxicology and nanopharmaceuticals. Deficiencies of existing practices in toxicology are reviewed and useful emerging techniques (e.g., lab-on-a-chip, tissue engineering, atomic force microscopy, high-content analysis) are highlighted. Cross-fertilization between disciplines will aid development of biocompatible delivery and implant platforms while improvements are being suggested for better translation of nanotoxicology.
Sourav Bhattacharjee*,1 & David J Brayden1,2 Conway Institute, University College Dublin (UCD), Dublin, Ireland 2 School of Veterinary Medicine, University College Dublin (UCD), Dublin, Ireland * Author for correspondence: Tel.: +353 1 716 6233 sourav.bhattacharjee@ ucd.ie 1
Keywords: biomaterials • high-content analysis • lab-on-a-chip • nanotoxicology • predictive toxicology
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 US$ 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 programs. 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
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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 sublethal toxicity that may be caused by nanomaterials at low concentrations during chronic exposure in vivo. Issues with current nanotoxicological in vitro methods The factors that influence nanomaterial cytotoxicity can be classified as follows: first, factors related to the nanomaterials: for example, surface charge, particle size, porosity, surface functionalization, crystallinity, shape
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Review Bhattacharjee & Brayden and composition; and second, factors related to experiment: for example, 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 increase cytotoxicity compared with neutral or negatively 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 leads 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 microscopy 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 toxicity. Discrepancies in operating protocols for NP characterization
The main obstacle toward 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 multidisciplinary 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
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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 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 [23,24] . Common techniques employed to characterize NPs are listed in Table 1. There are three major areas to further address in characterization of NPs: NP interference: NPs are reactive and interact with components of in vitro systems (e.g., salts, proteins and 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 and 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; Concentration considerations: compared with realistic exposure concentrations, there is a tendency to use very high levels of NPs both in vitro and in vivo. Very high concentrations of NPs are of little toxicological relevance in making a risk assessment. Furthermore, they can paralyze cellular physiology by establishing an approximately 500-nm layer of NPs on the plasma membrane, which deprives cells of nutrients and oxygen [28,29] ; Effect of solvents: many laboratories use 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
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Development of nanotoxicology: implications for drug delivery and medical devices
Review
Table 1. Usual characterization techniques for nanoparticles. Parameter
Properties
Techniques
Morphology
Size
TEM, SEM, AFM, XRD
Shape/structure
TEM, SEM, AFM, DLS, FFF, AUC, HPLC
Size distribution
EM, SEM, AFM, DLS, AUC, FFF, HPLC
Molecular weight
AUC, GPC
Stability
DLS, AUC, FFF, SEM, TEM
Surface features
Surface area
BET
Surface charge
SPM, titrations
Surface coating
SPM, XPS, MS, FTIR, NMR
Surface coverage
AFM, AUC, TGA
Topology
SEM, SPM, MS
Chemistry
Composition
XPS, MS, AAS, ICP-MS, FTIR, NMR
Purity
ICP-MS, AAS, AUC, HPLC, DSC
Crystallinity
XRD, DSC
Stability
MS, HPLC, FTIR
Drug delivery
Drug loading
MS, HPLC, UV-Vis, fluorescence
In vitro release
UV-Vis, MS, HPLC, fluorescence
Deformability
AFM, DMA
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; 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; UVVis: Ultraviolet-visible spectroscopy; XPS: X-ray photoelectron spectroscopy; XRD: X-ray diffraction.
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. Inadequate surface analysis of NPs & 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 and subcutaneous tissue). Therefore, characterization of NPs is incomplete without detailed knowledge about surface properties [31] . Surface charge has a significant effect on cellular internalization as 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
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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 surfaces, which were associated with cytotoxicity. In contrast, nontoxic 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 NPs: disruption of the surface congruity of cell membranes. Unfortunately, it is a challenge to fully characterize the surfaces of either NPs or cells. One major reason is the typically harsh conditions (e.g., ultrahigh vacuum, high pressure and desiccation) 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 participates 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 toward the applicability of such tools.
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Review Bhattacharjee & Brayden Interactions between proteins & NPs 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 NPs 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 and phosphates), carbohydrates and vitamins (folic acid, nicotinamide and 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 NPs may be at their highest level. The role of nonprotein 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
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between in vitro and ex/in vivo intestinal epithelial data may be the role of mucus 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. In vivo effects of protein-NP interactions: physiological relevance
Following intravenous injection, NPs appear to interact with proteins in plasma in both surface charge- and size-dependent ways. For example, Greish et al. [54] tested nonporous 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 analogs (amine, hydroxyl and acid-terminated) in CD-1 mice following intravenous 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 more than 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. Understanding oxidative stress in cytotoxic mechanisms
Oxidative stress is a common mechanism of cytotoxicity of NPs [56–59] , since NPs 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
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Development of nanotoxicology: implications for drug delivery and medical devices
the site for the electronic transport chain (ETC), which quenches free oxygen 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 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] . 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] . Approximately 10,000 papers on nanotoxicology were published from 2011 to 2014 [27] , but only few constructs translated into clinical approvals. Three major reasons are highlighted: 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; 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 of different chain lengths is a valid way to alter surface hydrophilicity in order to change distribution by avoiding uptake by the reticulo-endothelial system (RES) [78] ; Inability of engineered NPs to cross biological barriers: many NPs fail to cross biological barriers (e.g., blood–brain barrier and intestinal mucus). 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 mucus. Most of the mucus-covered epithelial constructs have reproducibility issues and do not elaborate the same mucus composition as in vivo.
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Upcoming technologies & testing platforms 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 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 multilayered 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 toward 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) can be developed, which can assist development of NP-based delivery technologies [92] . Transition toward 3D testing platforms 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
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Review Bhattacharjee & Brayden 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 mixing between fluids. As a result, the chances of bacterial infection are reduced compared with 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 with 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 highthroughput 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 (iPSCs) [109] to develop new techniques in cell therapy and the study of cell-cell, or cell–extracellular matrix (ECM) interactions. Some organ-on-a-chip devices (e.g., lung-on-a-chip [110] , liver-on-a-chip [111] and guton-a-chip [112]) are already being used in nanotoxicology and this is relevant for NP-based delivery systems. There are efforts to develop a ‘human-on-a-chip’ microdevice 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) (Figure 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
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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. 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 5 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 with traditional in vitro set-ups [115] . 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 microfluidic 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] . 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 bioartificial 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
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Development of nanotoxicology: implications for drug delivery and medical devices
Epithelium
Review
Air Upper layer
Endothelium Membrane
Stretch Porous membrane
PDMS etchant
Lower layer Vacuum Side chambers
Vacuum
Side chambers
Capillaries
Air Alveolus
Diaphragm
Pip
Pip
Figure 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 and flexible PDMS membrane coated with extracellular matrix. 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. PDMS: Polydimethylsiloxane. Reproduced with permission from [110] © American Association for the Advancement of Science (AAAS; 2010). For color figures, please see online at www.futuremedicine.com/doi/full/10.2217/NNM.15.69
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 and polycaprolactone) [128] , or from natural materials (e.g., collagen, chitosan, polysaccharides and 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 distributions [131] . Other progress is in the use of CNTs [132] as scaffold materials. They can provide a
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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 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 microtissue spheroids can be achieved [137] . Additionally, with the use of different growth factors, microtissue spheroids with altered vascularity can be generated [138] . As a
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Review Bhattacharjee & Brayden result, the nanotoxicity data derived from such microtissue spheroids may turn out to be more consistent and extrapolative to in vivo conditions compared with 2D systems. Application of approximately 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 (Figure 2) . Significant differences were found between the 2D and 3D systems, whereby NP toxicities were overestimated in the former; toxicity data obtained from NPs in 3D systems are of higher quality compared with the 2D [140] . In a second example, toxicity of polypyrrolidonecoated 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 with 2D, and it decreased faster over time. Computer-based design has also been used to predict the diffusion of NPs through microtissue 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.
of printing organs with defined vascularity [145] . However, it is worth pointing out a few current drawbacks. The structures produced by 3D printing require postprocessing, 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 (Figure 3) [149] . 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]-labeled human neonatal dermal fibroblast cells (HNDFs) and red fluorescent protein [RFP]-labeled human umbilical vein endothelial cells [HUVECs]) 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, which in turn will lead to better validation, improved optimization and better extrapolation to in vivo. High-content analysis
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: bonding-based inkjet printing, whereby a particle-based material is deposited in layers along with binder molecules. After processing, this technique produces biomaterials with designs that can be used as scaffolds in tissue engineering; and bioinkjet printing where instead of particles, biologically relevant materials can be used. This is an exciting way
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The concept of high-content analysis (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 with conventional cytotoxicity assays. Another important characteristic of HCA analysis is that in contrast to cell death assays, it successfully detects sublethal 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;
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Development of nanotoxicology: implications for drug delivery and medical devices
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 (Figure 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 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] . Multivariate analysis
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 parameters (e.g., production of intracellular ROS, reduction of
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Figure 2. Comparison of 2D and 3D culture of HepG2 cells after 12 h of CdTe nanoparticle 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). (E– H) Confocal images of live/dead-stained normal (E) 2D and (G) 3D spheroid cultures; live cells are green and dead cells are red. Most cells in both cultures showed excellent viability. (F) Again after CdTe nanoparticle exposure, 2D culture revealed that a significant number of cells were dead. (H) Although a few cells located on the surface of spheroids were dead, overall the number is much smaller than the 2D culture. Reproduced with permission from [139] © Wiley-VCH (2009).
cellular glutathione and 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 attention. Two recent studies used MVA to investigate cytotoxicities of carbon NPs (multiwalled CNTs [MWCNTs]) and C60
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Figure 3. 3D-printed tissue construct with vascularization. (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) and HUVECs (red). (G) Cell-viability assay results of printed 10T ½ fibroblast-laden and HNDF-laden GelMA features compared with a control sample (200–300 μm thick) of identical composition. *p < 0.05 obtained from Student’s t-test. HNDF: Human neonatal dermal fibroblast cell; HUVEC: Human umbilical vein endothelial cell; MF: Mouse fibroblast. Reproduced with permission from [149] © Wiley-VCH (2014).
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 datasets were generated and analyzed. Through scanning of specific regions of the spectra and further comparison with control data, a larger picture of intracellular
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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. 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 prepara-
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Development of nanotoxicology: implications for drug delivery and medical devices
tion and absence of any requirements of harsh conditions as high vacuum or pressure, integration possibilities with fluorescence and microscopy techniques like confocal, TIRF and Förster resonance energy transfer (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 (
Development of nanotoxicology: implications for drug delivery and medical devices
Current nanotoxicology research suffers from suboptimal in vitro models, lack of in vitro–in vivo correlations, variability within in vitro protocols, deficits in both material purity and physicochemical characterization. Reliable nanomaterial toxicity and mechanistic insights 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 analyze the interface between toxicology and nanopharmaceuticals. Deficiencies of existing practices in toxicology are reviewed and useful emerging techniques (e.g., lab-on-a-chip, tissue engineering, atomic force microscopy, high-content analysis) are highlighted. Cross-fertilization between disciplines will aid development of biocompatible delivery and implant platforms while improvements are being suggested for better translation of nanotoxicology.
Sourav Bhattacharjee*,1 & David J Brayden1,2 Conway Institute, University College Dublin (UCD), Dublin, Ireland 2 School of Veterinary Medicine, University College Dublin (UCD), Dublin, Ireland * Author for correspondence: Tel.: +353 1 716 6233 sourav.bhattacharjee@ ucd.ie 1
Keywords: biomaterials • high-content analysis • lab-on-a-chip • nanotoxicology • predictive toxicology
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 US$ 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 programs. 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
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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 sublethal toxicity that may be caused by nanomaterials at low concentrations during chronic exposure in vivo. Issues with current nanotoxicological in vitro methods The factors that influence nanomaterial cytotoxicity can be classified as follows: first, factors related to the nanomaterials: for example, surface charge, particle size, porosity, surface functionalization, crystallinity, shape
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Review Bhattacharjee & Brayden and composition; and second, factors related to experiment: for example, 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 increase cytotoxicity compared with neutral or negatively 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 leads 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 microscopy 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 toxicity. Discrepancies in operating protocols for NP characterization
The main obstacle toward 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 multidisciplinary 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
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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 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 [23,24] . Common techniques employed to characterize NPs are listed in Table 1. There are three major areas to further address in characterization of NPs: NP interference: NPs are reactive and interact with components of in vitro systems (e.g., salts, proteins and 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 and 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; Concentration considerations: compared with realistic exposure concentrations, there is a tendency to use very high levels of NPs both in vitro and in vivo. Very high concentrations of NPs are of little toxicological relevance in making a risk assessment. Furthermore, they can paralyze cellular physiology by establishing an approximately 500-nm layer of NPs on the plasma membrane, which deprives cells of nutrients and oxygen [28,29] ; Effect of solvents: many laboratories use 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
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Development of nanotoxicology: implications for drug delivery and medical devices
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Table 1. Usual characterization techniques for nanoparticles. Parameter
Properties
Techniques
Morphology
Size
TEM, SEM, AFM, XRD
Shape/structure
TEM, SEM, AFM, DLS, FFF, AUC, HPLC
Size distribution
EM, SEM, AFM, DLS, AUC, FFF, HPLC
Molecular weight
AUC, GPC
Stability
DLS, AUC, FFF, SEM, TEM
Surface features
Surface area
BET
Surface charge
SPM, titrations
Surface coating
SPM, XPS, MS, FTIR, NMR
Surface coverage
AFM, AUC, TGA
Topology
SEM, SPM, MS
Chemistry
Composition
XPS, MS, AAS, ICP-MS, FTIR, NMR
Purity
ICP-MS, AAS, AUC, HPLC, DSC
Crystallinity
XRD, DSC
Stability
MS, HPLC, FTIR
Drug delivery
Drug loading
MS, HPLC, UV-Vis, fluorescence
In vitro release
UV-Vis, MS, HPLC, fluorescence
Deformability
AFM, DMA
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; 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; UVVis: Ultraviolet-visible spectroscopy; XPS: X-ray photoelectron spectroscopy; XRD: X-ray diffraction.
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. Inadequate surface analysis of NPs & 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 and subcutaneous tissue). Therefore, characterization of NPs is incomplete without detailed knowledge about surface properties [31] . Surface charge has a significant effect on cellular internalization as 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
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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 surfaces, which were associated with cytotoxicity. In contrast, nontoxic 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 NPs: disruption of the surface congruity of cell membranes. Unfortunately, it is a challenge to fully characterize the surfaces of either NPs or cells. One major reason is the typically harsh conditions (e.g., ultrahigh vacuum, high pressure and desiccation) 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 participates 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 toward the applicability of such tools.
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Review Bhattacharjee & Brayden Interactions between proteins & NPs 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 NPs 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 and phosphates), carbohydrates and vitamins (folic acid, nicotinamide and 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 NPs may be at their highest level. The role of nonprotein 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
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between in vitro and ex/in vivo intestinal epithelial data may be the role of mucus 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. In vivo effects of protein-NP interactions: physiological relevance
Following intravenous injection, NPs appear to interact with proteins in plasma in both surface charge- and size-dependent ways. For example, Greish et al. [54] tested nonporous 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 analogs (amine, hydroxyl and acid-terminated) in CD-1 mice following intravenous 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 more than 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. Understanding oxidative stress in cytotoxic mechanisms
Oxidative stress is a common mechanism of cytotoxicity of NPs [56–59] , since NPs 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
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Development of nanotoxicology: implications for drug delivery and medical devices
the site for the electronic transport chain (ETC), which quenches free oxygen 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 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] . 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] . Approximately 10,000 papers on nanotoxicology were published from 2011 to 2014 [27] , but only few constructs translated into clinical approvals. Three major reasons are highlighted: 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; 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 of different chain lengths is a valid way to alter surface hydrophilicity in order to change distribution by avoiding uptake by the reticulo-endothelial system (RES) [78] ; Inability of engineered NPs to cross biological barriers: many NPs fail to cross biological barriers (e.g., blood–brain barrier and intestinal mucus). 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 mucus. Most of the mucus-covered epithelial constructs have reproducibility issues and do not elaborate the same mucus composition as in vivo.
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Upcoming technologies & testing platforms 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 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 multilayered 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 toward 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) can be developed, which can assist development of NP-based delivery technologies [92] . Transition toward 3D testing platforms 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
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Review Bhattacharjee & Brayden 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 mixing between fluids. As a result, the chances of bacterial infection are reduced compared with 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 with 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 highthroughput 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 (iPSCs) [109] to develop new techniques in cell therapy and the study of cell-cell, or cell–extracellular matrix (ECM) interactions. Some organ-on-a-chip devices (e.g., lung-on-a-chip [110] , liver-on-a-chip [111] and guton-a-chip [112]) are already being used in nanotoxicology and this is relevant for NP-based delivery systems. There are efforts to develop a ‘human-on-a-chip’ microdevice 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) (Figure 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
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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. 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 5 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 with traditional in vitro set-ups [115] . 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 microfluidic 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] . 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 bioartificial 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
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Development of nanotoxicology: implications for drug delivery and medical devices
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Figure 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 and flexible PDMS membrane coated with extracellular matrix. 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. PDMS: Polydimethylsiloxane. Reproduced with permission from [110] © American Association for the Advancement of Science (AAAS; 2010). For color figures, please see online at www.futuremedicine.com/doi/full/10.2217/NNM.15.69
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 and polycaprolactone) [128] , or from natural materials (e.g., collagen, chitosan, polysaccharides and 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 distributions [131] . Other progress is in the use of CNTs [132] as scaffold materials. They can provide a
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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 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 microtissue spheroids can be achieved [137] . Additionally, with the use of different growth factors, microtissue spheroids with altered vascularity can be generated [138] . As a
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Review Bhattacharjee & Brayden result, the nanotoxicity data derived from such microtissue spheroids may turn out to be more consistent and extrapolative to in vivo conditions compared with 2D systems. Application of approximately 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 (Figure 2) . Significant differences were found between the 2D and 3D systems, whereby NP toxicities were overestimated in the former; toxicity data obtained from NPs in 3D systems are of higher quality compared with the 2D [140] . In a second example, toxicity of polypyrrolidonecoated 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 with 2D, and it decreased faster over time. Computer-based design has also been used to predict the diffusion of NPs through microtissue 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.
of printing organs with defined vascularity [145] . However, it is worth pointing out a few current drawbacks. The structures produced by 3D printing require postprocessing, 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 (Figure 3) [149] . 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]-labeled human neonatal dermal fibroblast cells (HNDFs) and red fluorescent protein [RFP]-labeled human umbilical vein endothelial cells [HUVECs]) 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, which in turn will lead to better validation, improved optimization and better extrapolation to in vivo. High-content analysis
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: bonding-based inkjet printing, whereby a particle-based material is deposited in layers along with binder molecules. After processing, this technique produces biomaterials with designs that can be used as scaffolds in tissue engineering; and bioinkjet printing where instead of particles, biologically relevant materials can be used. This is an exciting way
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Nanomedicine (Lond.) (Epub ahead of print)
The concept of high-content analysis (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 with conventional cytotoxicity assays. Another important characteristic of HCA analysis is that in contrast to cell death assays, it successfully detects sublethal 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;
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Development of nanotoxicology: implications for drug delivery and medical devices
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 (Figure 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 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] . Multivariate analysis
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 parameters (e.g., production of intracellular ROS, reduction of
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Figure 2. Comparison of 2D and 3D culture of HepG2 cells after 12 h of CdTe nanoparticle 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). (E– H) Confocal images of live/dead-stained normal (E) 2D and (G) 3D spheroid cultures; live cells are green and dead cells are red. Most cells in both cultures showed excellent viability. (F) Again after CdTe nanoparticle exposure, 2D culture revealed that a significant number of cells were dead. (H) Although a few cells located on the surface of spheroids were dead, overall the number is much smaller than the 2D culture. Reproduced with permission from [139] © Wiley-VCH (2009).
cellular glutathione and 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 attention. Two recent studies used MVA to investigate cytotoxicities of carbon NPs (multiwalled CNTs [MWCNTs]) and C60
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Figure 3. 3D-printed tissue construct with vascularization. (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) and HUVECs (red). (G) Cell-viability assay results of printed 10T ½ fibroblast-laden and HNDF-laden GelMA features compared with a control sample (200–300 μm thick) of identical composition. *p < 0.05 obtained from Student’s t-test. HNDF: Human neonatal dermal fibroblast cell; HUVEC: Human umbilical vein endothelial cell; MF: Mouse fibroblast. Reproduced with permission from [149] © Wiley-VCH (2014).
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 datasets were generated and analyzed. Through scanning of specific regions of the spectra and further comparison with control data, a larger picture of intracellular
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Nanomedicine (Lond.) (Epub ahead of print)
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. 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 prepara-
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Development of nanotoxicology: implications for drug delivery and medical devices
tion and absence of any requirements of harsh conditions as high vacuum or pressure, integration possibilities with fluorescence and microscopy techniques like confocal, TIRF and Förster resonance energy transfer (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 (
