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1 Critical Evaluation of Toxicity Tests in CopyrightedContext to Engineered Nanomaterials: An Introductory Overview

Madan Lal Verma

CONTENTS Materials 1.1 Introduction...... 1 1.2 Methods for Visualizing Cellular Uptake and Biodistribution of Engineered Nanomaterials...... 3 1.3 Evaluation of Transport and Uptake of Engineered Nanomaterials In Vitro...... 4 1.4 In Vivo Uptake, Transport, and Detection...... 4 1.5 In Vitro Cytotoxicity Tests: Cytotoxicity Tests, Genotoxicity Tests...... 6 1.6 In Vivo Toxicity Testing: Toxicokinetics,- Immunological Response, Chronic Toxicity, and CarcinogenicityTaylor...... 8 1.7 Considerations for Selection of Toxicity Tests for Engineered Nanomaterials—In Vitro versus In Vivo...... 10 1.8 Conclusions...... 11 References...... and 12

1.1 Introduction Francis Nanomaterials exist naturally in the environment such as dust storms, volcanic ash, and soot from forest fires or are the incidental byproducts of combustion processes (e.g., diesel engines, welding, etc.). They are usually physicochemical heterogeneous and are often termed ultrafine particles (Donaldson et al. 2005; Ning et al. 2006; Buzea et al. 2007). Thus, human beings are exposed to naturally occurring nanomaterials. However, with the start of the tailor made synthesis of nanomaterials at research and development centers, there are pros and cons associated with these nanomaterials. Top down and bottom up approaches are employed for the production of nano- materials. The number of products keeps increasing day by day and is becoming a part of our life. The efficiency of many processes

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including bioprocessing, , and so on is improving multi-fold with the inclusion of nanomaterials. However, excessive exposure of nanomaterials to different consumers, ranging from research personnel to the common man, has become a of great concern due to the poten- tial nanotoxic effects (Kermanizadeh et al. 2013; Singh and Ramarao 2013; CopyrightedGuadagnini et al. 2015). Thus, it is pertinent to identify the potential risk fac- tors that are harmful to human health and the environment (Kermanizadeh et al. 2013). Current literature analysis sheds on the toxicity of engi- neered nanomaterials revealing that some nanomaterials are relatively safe as compared to other nanomaterials, which are harmful (Magdolenova et al. 2012; Kumar et al. 2014). The toxicity properties of such nanomaterials are directly associated with the physical and chemical properties of the con- cerned nanomaterials (Figure 1.1). The present chapter provides a concise and critical review of the various parameters of engineered nanomaterials (ENMs) employed for the detection and evaluation of toxicity via in vitro and in vivo assays. RecentMaterials considerations for toxicity tests for ­engineered nano- materials are also discussed.

- Size Taylor

Chemical Shape composition and

ENMs toxicity factors Francis

Surface Dose of ENMs covering administration

Route of ENMs administration

FIGURE 1.1 Various factors responsible for the toxicity of engineered nanomaterials (ENMs). Critical Evaluation of Toxicity Tests in Context to Engineered Nanomaterials 3

1.2 Methods for Visualizing Cellular Uptake and Biodistribution of Engineered Nanomaterials During intended and unintended exposure to nanomaterials, engineered Copyrightednanomaterials (ENMs) may come in contact with the biological fluids of the human body system through different routes such as inhalation, ingestion, and the . Once inside the human body, ENMs will get the opportunity to interact with various biomacromolecules such as proteins, sugars, lipids, and nucleic acids. Immediate crowding of the macromolecules, primarily proteins on the surface of the ENMs occurs; this phenomenon is known as protein corona (Shang et al. 2014). The nature of protein corona is dynamic and depends on the individual components and their affinities toward the macromolecules in the biological fluids. Cellular uptake (internalization) may involve transport across the membrane which can be of two types: receptor mediated activeMaterials transport and passive transport. The cellular uptake of ENMs was determined fluorimetrically using Coumarin 6 as a fluorescent model drug (Panyam et al. 2002). The unique physical and chemical properties of ENMs render the benefits of increased absorption that lead to the enhanced cellular uptake of ENMs (Mundargi et al. 2008; Adair et al. 2010). However, this enhanced cellular uptake can also lead to the increased interaction of ENMs with subcellular organelles which results in the provocation of various- signaling pathways. This also evokes a stress response in the cell that includesTaylor free radical formation, cel- lular-organelle damage, and even cell death (Bayles et al. 2010, Wang et al. 2011a,b). The degree of cytotoxicity of the nanomaterials, either low or high, depends on their cellular uptake (Ryman-Rasmussen et al. 2007; Geys et al. 2008). The low toxicity of is demonstrated due to inefficient cellular uptake (Singh and Ramarao 2013). Researchersand studied the cellular uptake of a fluorescent drug (Coumarin 6) using polymeric nanoparticles. In vitro release of Coumarin 6 from nanoparticles showed that less than 1% dye leached from nanoparticles in 24 h. The confocal microscopyFrancis study revealed that nanoparticles are effectively and quickly internalized in the cells of macrophage cell lines (RAW 264.7). ENMs entered into the cytoplas- mic compartment rather than the cell nucleus. Since 2-dimensional imag- ing cannot exactly trace the location of intracellular versus surface-bound ENMs, the cellular uptake of ENMs was confirmed by using 3-dimensional imaging. However, the punctuate shows that the ENMs are localized in cellular organelles such as lysosomes; the diffused cytoplasmic fluorescence confirms the presence of ENMs in the cytoplasm. The ENMs are trafficked to the lysosomal compartment where they may undergo charge reversal resulting in lysosomal escape (Panyam et al. 2002; Cartiera et al. 2009). Thus, in addition to cytoplasm, the ENMs may be present in cell organelles. 4 Nanotoxicology

1.3 Evaluation of Transport and Uptake of Engineered Nanomaterials In Vitro Transport and uptake of the ENMs becomes crucial for evaluation when they Copyrightedenter into specialized tissues, for example, the brain and fetuses. Such investi- gation required in vitro studies using techniques with specialized transwell apparatus to provide access to apical as well as basal compartments (Kettiger et al. 2013). The evaluation of transport and uptake of ENMs of dif- ferent chemical compositions can be systematically done by using cell lines of different origins. Such cell lines represent various compartments of target tissue such as macrophage, hepatocyte, renal epithelial, pulmonary epithelial, and neuronal cells (Singh and Ramarao 2013). A systematic study to deter- mine the high or low concentrations’ effects of ENMs was needed to evaluate the cell viability effects. For example, Singh and Ramarao, (2013) studied the concentration effect Materialsof ENMs on cell viability by using a series of different cell lines originated from RAW 264.7 (macrophage), Hep G2 (hepatocyte), A549 (lung epithelial), A498 (kidney epithelial), and Neuro 2A (neuronal). One researcher reported a novel fluorescence recovery after quenching (FRAQ) assay to determine intracellular degradation of ENMs (Singh and Ramarao 2013). ENMs showed toxicity at the highest doses in all cell lines. Moreover, ENMs were efficiently internalized by RAW 264.7 cells and stim- ulated reactive oxygen species and tumor- necrosis factor-alpha production (Figure 1.2). However, the stability of intracellularTaylor organelles such as lyso- somal and mitochondrial organelles remained unaffected. The intracellular degradation of ENMs was determined by monitoring changes in osmolality of the culture medium and a novel fluorescence recovery after quenching assay (Ryman-Rasmussen et al. 2007; Geys et al. 2008; Singh and Ramarao 2013). Cell death showed a good correlation with osmolalityand of the culture medium suggesting the role of increased osmolality in cell death. Energy inhibition assay is commonly employed for understanding the transport and uptake of cellular mechanisms. Energy inhibition studies workFrancis on diverse metabolic conditions and inhibitors in coupling with imaging tools via ­confocal or electron microscopy (Kaweeteerawat et al. 2015).

1.4 In Vivo Uptake, Transport, and Detection The primary target of ENMs is the respiratory organs followed by other organs such as the gastrointestinal tract. ENMs enter the gastrointestinal tract through different routes: (i) an indirect route via mucociliary move- ment and (ii) a direct route via the oral intake of water, food, cosmetics, drugs, and systems (Meng et al. 2007). The interactions of the Critical Evaluation of Toxicity Tests in Context to Engineered Nanomaterials 5

NP

Copyrighted Internalization

Degradation

ROS cytokines

Materials

FIGURE 1.2 Mechanism of cytotoxicity of ENMs. Abbreviations: NP, ; ROS, reactive oxygen species. (Adapted from Singh, R. P. and P. Ramarao. 2013. Toxicol Lett 136(1):131–143.) - physico-chemical properties (e.g., size, shape,Taylor surface chemistry, composi- tion, and aggregation) of ENMs with biological systems inside the body in order to elucidate the relationship for induction of toxic biological responses can be summarized as follows: (a) The main entry for ENMs to the body occurs primarily by six routes: intravenous, dermal, subcutaneous, inhala- tion, intraperitoneal, and oral; (b) absorption takes placeand where the ENMs first interact with complex biological components (proteins, cells) to form the biological corona of the nanoparticles; (c) afterward, ENMs spread to various organs of the body and may retain the pristine structure of the nanoparticlesFrancis or even be modified/metabolized; (d) ENMs enter the cells and reside in the initial organ before moving to other body organs or finally being excreted (Fischer and Chan 2007; Maynard 2006). ENMs’ interaction with biological systems may cause toxic effects such as , cytotoxicity, fibrosis, allergy, tissue damage, and organ failure (Maynard 2006; Nel et al. 2006; Singh et al. 2009). In vivo uptake and detection of the ENMs becomes complex due to biological interactions, for example, opsonization (Iversen et al. 2011). The biodistribution of ENMs within the tissue relies on various factors asso- ciated with nanomaterials such as size and surface chemistry and requires long durations to monitor the full profile of in vivo uptake and transport of cellular mechanisms. Sophisticated instruments such as inductively coupled mass spectrometry (ICP-MS) are used to evaluate tissue levels for a range of targeted nanomaterials (Laborda et al. 2016). 6 Nanotoxicology

1.5 In Vitro Cytotoxicity Tests: Cytotoxicity Tests, Genotoxicity Tests In vitro tests, either cytotoxicity or genotoxicity are commonly employed to Copyrightedobtain initial information on engineered nanomaterials’ toxicity (Table 1.1). In vitro cytotoxicity tests are performed using cell lines or isolated primary human cells, for example, macrophages. In vitro cytotoxicity studies for evaluation of ENMs have been carried out systematically by using standard methods such as MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Coomassie blue (CB) assays (Singh et al. 2012; Singh and Ramarao 2012). Cells were incubated with various concentrations of ENMs for 72 h. After incubation, cells were washed extensively with phosphate buf- fer saline (PBS) solutions to remove ENMs, then cell viability was determined by standard assay methods. In brief, culture supernatants from control or ENMs-containing samplesMaterials were collected and cells were incubated with MTT. The formazan was dissolved in organic such as dimethyl sulf- oxide (DMSO) and absorbance was measured at 550 nm. While in CB assay, the reaction mixture containing culture supernatants and Bradford reagent was incubated and absorbance was determined at 595 nm. The absorbance of control samples was assumed to be 100% and cell viability of treated samples was determined with respect to control samples (Singh and Ramarao 2013). DCFDA (2′,7′-dichlorofluorescin diacetate)- assay was done to evaluate the free radicals. Free radical production was determinedTaylor by monitoring the pro- duction of reactive oxygen species and reactive nitrogen species in the mac- rophage cell lines (Singh and Ramarao 2012). RNS (Reactive Nitrogen Species) production was determined by nitrite assay in culture supernatants using Griess reagent (Tsikas 2005). A reaction mixture containing equal volumes of culture supernatant and the Griess reagent was incubatedand at room temper- ature for 30 min and resulted in diazo salt formation. The absorbance was measured at 540 nm. The nitrite concentration was calculated from a standard graph constructed using sodium nitrite (Singh et al. 2012; Singh andFrancis Ramarao 2012). Cytokine productions (TNF-α and IL-6 levels) were measured in culture supernatants by colorimetric enzyme linked immunosorbent assay (ELISA). Mitochondrial stability assay was performed to observe changes in mitochon- drial membrane potential. This stability assay is done using standard methods such as Rh123 and Safranin O. The Rh123 fluorescence intensity was deter- mined at 530 nm excitation and 590 nm emission. The absorbance of Safranin O was determined at 523 and 555 nm and the ratio of intensities was calculated (Deryabina et al. 2001; Severin et al. 2010). Lysosomal stability was determined by leakage of acridine orange from acridine orange-loaded lysosomes and the accumulation of neutral red in intact lysosomes. Acridine orange, a lyso- motropic agent, preferentially accumulates in intracellular organelles such as lysosomes that show a red fluorescence. This dye leaks out into the cytoplasm Critical Evaluation of Toxicity Tests in Context to Engineered Nanomaterials 7

Copyrighted References Carlsson et al. (1993) Oostingh et al. (2011), Sadik et al. (2009), Oostingh et al. (2011), Casey et al. (2007), Laaksonen et al. (2006), (2007), Worle-Knirsch and Inman (2006) Monteiro-Riviere (2010), Kocbach et al. (2008), Veranth (2010), Kocbach et al. (2008), Veranth and et al. (2007), Monteiro-Riviere Inman (2006) Kain et al. (2012) Hillegass et al. (2010) Hillegass et al. (2010), Altman et al. (1993) Hillegass et al. (2010), Oh et al. (2014), Kroll et al. (2012), Oh et al. (2014), Kroll Ong et al. (2014), Repetto (2008) Kroll et al. (2012), Wang et al. (2011a,b), et al. (2011a,b), et al. (2012), Wang Kroll Guadagnini et al. (2015), Brown et al. Guadagnini et al. (2015), Brown Ferraro et al. (2016), Karlsson (2015), Ferraro Magdolenova et al. (2012),

Materials Disadvantages - n Vitro Assays LDH activity inhibition intereference possibility of reduction, for possibility of reduction, optical example, superoxide, interference nanomaterials detection; with interference formamidopyrimidine DNA glycosylase, photocatalytic breakage DNA ENMs increase after uv light exposure nanomaterial endocytosis Time consuming Time Time consuming, LDH adsorption, Time Dye adsorption and optical Formazan adsorption by ENMs; Adsorption of cytokines DNA and engineered and engineered DNA Taylor Cytochalasin B decreases

and anomaterials via I Francis Advantages ngineered N cells/dead cells lysosomal uptake of dyes assay involves conversion to formazan that enables measurement of functional cells response detection and estimate response cytokines concentration single- and double-strand breaks frequency of micronucleus formation of micronucleus frequency Dyes selectively stains non-living Colorimetrical measurement of LDH Colorimetrical measurement Measurement of viable cells after Measurement Tetrazolium based colorimetrical Tetrazolium Colorimetrical assay for immune Single cell gel electrophoresis damage either Detects DNA Measurement of changes in the Measurement ytotoxicity E of C ssay A itro immunosorbent assays TABLE 1.1 TABLE Determining the In V blue assay Trypan LDH assay Neutral red assay Neutral red MTS and MTT assays Enzyme-linked Comet assay Micronuclei assay Micronuclei 8 Nanotoxicology

in damaged lysosomes and renders a green fluorescence. The intensity of the green fluorescence is directly proportional to the degree of lysosomal damage. Further, the increment in the intensity of green fluorescence by cytoplasmic acridine orange appears early compared with the decrement in the intensity of red fluorescence by lysosomal acridine orange (Antunes et al. 2001; Castino Copyrightedet al. 2007). The intensity of the degree of fluorescence was measured at 488 nm excitation and 540 nm emission. Neutral red assay is based on the accumulation of the dye in intact lysosomes. A reduction in viable lysosomes leads to a reduc- tion in neutral red uptake by cells and is done by taking absorbance at 540 nm. Genotoxicity of nanoparticles refers to the toxicity against the genetic mate- rial of the cell. It affects DNA integrity that ultimately leads to DNA damage. This may cause mutagenicity and carcinogenicity in some cases (Nesslany and Benameur 2015). The genotoxic properties of ENMs render DNA damage due to oxidative stress resulting from the hyper-production of reactive oxy- gen species and reactive nitrogen species (Kisin et al. 2007; Barnes et al. 2008). Induction of oxidativeMaterials stress by ENMs is the mechanism most responsible for the cause of potential toxicity (Li et al. 2010; Manke et al. 2013). ENMs-mediated reactive oxygen species and reactive nitrogen species production mechanisms can be divided into three groups: intrinsic production, production by interac- tion with cell targets, and production mediated by the inflammatory reaction. These three groups share responsibility for most of the primary or secondary genotoxic effects observed so far with ENMs (Nesslany and Benameur 2016). Currently, evaluation of engineered- nanomaterials is done by in vitro genotoxicity tests including the Ames, Taylormicronucleus, and HPRT (hypo- xanthine phosphorybosyl transferase) mutation assays. These tests can do a safe assessment of nanomaterials-induced DNA damage. However, the Organisation for Economic Co-operation and Development (OECD)-based genotoxicity assays for engineered nanomaterials are not universal, for example, the Ames test is not applicable for the assessmentand of engineered nanomaterials, while in vitro HPRT and micronucleus assays for nanomate- rial assessment require specific protocols. Thus, there is a requirement for strategic planning to deal with in vitro genotoxicity testing (DoakFrancis et al. 2012).

1.6 In Vivo Toxicity Testing: Toxicokinetics, Immunological Response, Chronic Toxicity, and Carcinogenicity ENMs behave quite differently in the complex environment of living sys- tems. Thus, ENMs work differently in vitro versus in vivo studies. Evaluation of ENMs using in vivo study provides a more realistic outcome of the poten- tial toxicity (Kumar et al. 2012). Compared to in vitro tests, in vivo analyses are laborious and expensive. Various factors such as route of administration, biodistribution, biodegradability, short- or long-term disposition, induction Critical Evaluation of Toxicity Tests in Context to Engineered Nanomaterials 9

of developmental defects, and activation of the compliment and/or immune system are all major issues in determining in vivo nanotoxicity, and cannot possibly be done through in vitro assays (Table 1.2; Rizzo et al. 2013; Kettiger et al. 2013). Recently, zebrafish embryo assay has been employed to assess the acute toxic effects as well as the “long-term” developmental defects resulting Copyrightedfrom exposure to engineered nanomaterials (George et al. 2011). Exposure of metal and metal oxide ENMs such as silver (Ag), gold (Au), silicon

dioxide (SiO2), magnesium oxide (MgO), titanium oxide (TiO2), and zinc oxide (ZnO) to a living system induce a low in vivo toxicity (Auffan et al. 2009; Lu et al. 2009; Warheit et al. 2009; Zhu et al. 2009). However, factors such as the chemi- cal stability of the nanoparticles are mainly responsible for causing toxicity at the cellular level. The oxidized/reduced/solubilized properties of ENMs are potentially toxic and need special consideration before use (Auffan et al. 2009). Sung et al. (2009) reported the effects of Ag nanoparticles in Sprague-Dawley rats. A low dose wasMaterials non-toxic. However, higher doses produced severe effects TABLE 1.2 Showing Possible Engineered Nanomaterials (ENMs) Effects as the Basis for Pathophysiology and Toxicity Experimental NM Effects Possible Pathophysiological Outcomes ROS generation Protein, DNA and membrane injury, oxidative stress- Oxidative stress TaylorII enzyme induction, inflammation, mitochondrial perturbation Mitochondrial perturbation Inner membrane damage, permeability transition (PT), pore opening, energy failure, apoptosis, apo-necrosis, cytotoxicity Inflammation Tissue infiltration with inflammatory cells, fibrosis, , andatherogenesis, acute phase protein expression (e.g., C-reactive protein) Uptake by reticulo-endothelial system Asymptomatic sequestration and storage in liver, spleen, lymph nodes, possible organ enlargement and dysfunction Francis Protein denaturation, degradation Loss of enzyme activity, auto-antigenicity Nuclear uptake DNA damage, nucleoprotein clumping, autoantigens Uptake in neuronal tissue Brain and peripheral nervous system injury Perturbation of phagocytic function, Chronic inflammation, fibrosis, granulomas, “particle overload,” mediator release interference in clearance of infectious agent Endothelial dysfunction, effects on Atherogenesis, thrombosis, stroke, myocardial clotting infarction Generation of neoantigens, breakdown in Autoimmunity, adjuvant effects immune tolerance Altered cell cycle regulation Proliferation, cell cycle arrest, senescence DNA damage Mutagenesis, metaplasia, carcinogenesis Source: Adapted from Nel, A. et al. 2006. Science 311:622–627. 10 Nanotoxicology

on organs such as the lungs and liver. Ag nanoparticles also induced inflam- matory responses at higher concentrations. This in vivo study with ENMs has shown dose-dependent cytotoxic effects. Cho et al. (2009) investigated the tox- icity effects of Au nanoparticles in mice and found the induction of inflam- matory immune- and metabolic-process responses in the liver of mice. Huang Copyrightedet al. (2009) reported a minimal cytotoxic effect on the surface of some organs by modified nanoparticles such as carboxymethyl dextran-coated iron oxide in the brain of mice. Kobayashi et al. (2009) studied the effects of variable sizes of

TiO2 nanoparticles on rat lungs. Different degrees of agglomeration with vari- able sizes of ENMs developed different toxicity effects such as higher reversible inflammation. Simberg et al. (2009) investigated the effects of superparamag- netic iron oxide nanoparticles in the mouse model. In vivo studies have shown that high concentrations of superparamagnetic iron oxide nanoparticles in the lumen caused thrombosis in the blood vessels. Further, entrapment of ENMs in the growing intravascular thrombi led to cell death. Therefore, tumor-targeted ENMs inhibited tumorMaterials growth. Zhu et al. (2008) reported oxidative stress in the lungs of male Sprague-Dawley rats using intratracheal administration of mag- netic nanoparticles. Such ENMs-induced oxidative stress produced a series of lung problems that includes follicular hyperplasia, protein effusion, pulmo- nary capillary vessel hyperaemia, and alveolar lipoproteinosis. Sayes et al. (2007) demonstrated the effects of ZnO nanoparticles in rats. The in vivo study showed pulmonary toxicity effects in reversible inflam-

mation. Warheit et al. (2009) observed -toxicity effects of TiO2 such as acute dermal irritation in rabbits. Liu et al. (2008)Taylor investigated the effect of intra- venously administered modified single-walled nanotubes in mice. The ENMs were found to be nontoxic and excreted in feces via the biliary and renal pathways. The possible mechanism for non-toxicity was due to the biological inertness provided to the ENMs with the aid of surface modifica- tion with polyethylene glycol. Ma-Hock et al. (2009) studiedand the toxic effects of multi-walled carbon nanotubes in Wistar rats and found them to be safe even with exposure for more than three months. However, the immuno- logical response of neutrophil production was reported at higherFrancis levels of ENMs. Thus, the above discussed recent studies of in vivo toxicity effects in different animal models show the response of immunological systems and chronic toxicity of exposure to ENMs at high doses. However, ENMs are found to be quite safe at low concentrations.

1.7 Considerations for Selection of Toxicity Tests for Engineered Nanomaterials—In Vitro versus In Vivo Ideally, every new ENM should be evaluated for potential toxicity; this will require an insight for the particular factors/characteristics responsible for Critical Evaluation of Toxicity Tests in Context to Engineered Nanomaterials 11

that particular nanotoxicity (Soenen et al. 2011; Carreira et al. 2015). With this in mind, an important initiative was taken by EU Framework 7 Health program in the form of a NanoTEST project. The main motive behind this project was to develop rapid assessment of the toxicological profiles of nanomaterials using in vitro and in silico methods (Dusinska et al. 2009). CopyrightedNanoTEST was also designed to evaluate the uptake and transport of medical diagnostics-relevant engineered nanomaterials, in particular in reference to checking ENMs’ potential to cross specific cell barriers. The penetrating nature of ENMs will allow further dissemination throughout the body or specific penetration to sensitive areas, for example, the fetus during pregnancy. The probability of invasive ENMs reaching secondary targets is quite high; this will require careful consideration of potential tox- icity (Iversen et al. 2012). A recent literature survey reveals that more stringent controls should be undertaken for nanotoxicological studies and guidelines should be pro- vided to reduce the Materialspotential for ENM-assay interactions along with linked aberrant results (Ong et al. 2014). Higher doses of ENMs in a concentration of 10 mg/L have a greater probability of interfering with assay function, and the use of such a high dose is not uncommon in toxicological stud- ies. Therefore, ENM concentration should be restricted in the final sample, recognizing that even with multiple washes/centrifugations ENMs could remain within cells or bound to membranes (Davoren et al. 2007; Monteiro- Riviere et al. 2009). Furthermore, the -use of centrifugation is counterpro- ductive in case the ENMs have tightly Taylorbound to the assay components, inadvertently removing dyes and/or proteins essential for accurate read- ings (Holder et al. 2012). Thus, it is strongly recommended that researchers should carefully consider the final dose of ENM concentration (Ong et al. 2014). Recently, nanomaterial synthesis via biogeneic routes has been based on principles that are the most sought-afterand alternative to chemical and physical methods (Dahl et al. 2007; Iravani 2011; Verma et al. 2013a,b). More efficient biogenic routes for microorganisms ( and fungi) and plants avoid harmful reagents and are even safer Francisfor human beings (Ravindran et al. 2003; Albrecht et al. 2006).

1.8 Conclusions Nanotechnology has an impact on the advance of the sciences including major benefits for society. The continuous production of a copious number of engineered nanomaterials has provided promising technical benefits to consumers and medical appliances. Despite the advantageous properties of nanomaterials such as being (antifungal, antiviral, and anti- biotic), drug carriers, contrasting agents, and so on, the susceptibility of 12 Nanotoxicology

nanoscale materials to be toxic to human health and the environment is quite high and exposes a major gap in knowledge (Matysiak et al. 2016). ENMs exposure generates harmful effects through interaction with biological sys- tems as revealed in several in vivo studies. The literature survey of the toxicity of engineered nanomaterials con- Copyrightedcludes that nanoparticles have the potential to be toxic. However, the degree of toxicity can be modulated by various factors of the engineered nanomate- rials such as size, shape, surface charge, modifications, and so on. The critical parameters of ENMs play a pivotal role in the degree of toxicity such as dose, route of administration, and exposure. Thus, every nanomaterial needs to pass rigorous testing before being considered safe. Hence, multidisciplinary collaborative research is required to fill the knowledge gaps in the research and development activities under the umbrella of nanotoxicity research. Employing holistic approaches such as biogenic synthesis and omics tech- niques will show solutionsMaterials for these curr­ ent concerns.

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