Sustainable Nanostructured Materials for Culturing of Various Biological Cells

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Sustainable Nanostructured Materials for Culturing of Various Biological Cells MANASH D. DEY, PHD, NPDF • RUPAK MUKHOPADHYAY, PHD • SUJOY K. DAS, PHD, MRSC

INTRODUCTION Nanostructured materials have always ignited the innovative cores of scientific intelligentsia as has already been discussed in the preceding chapters. Broadly, nanoparticles are classified into carbon based, metal based, dendrimers, and composites. The role and impact of nanostructured materials is varied and promises to deliver all human demands of the present and the future generations such as medicine, protein detection, magnetic resonance imaging (MRI) contrast enhancement, phagokinetic study (Salata, 2004), and water purification (Parandhaman et al., 2017). The traditional method of the synthesis of nanomaterials has a profound impact on the biological systems and the environment. Green chemical synthesis with its tenets attempts to circumvent the toxicity aspect of nanomaterial synthesis and deliver biocompatible nanomaterials for various applications. Biocompatibility in its core implies compatibility with the biological cells. Hence, the study of interaction of biological cells with engineered nanoparticles gains importance in light of its possible impact. Biological cells represent a live system of multiple components acting together to keep up with the life processes. Introduction of nanostructured materials into the lumen of live cells is a unique situation open for detailed investigation of its impact and consequences. Development of newer nanomaterials has added to the nanomaterial research on its consequences on the cellular behavior and its response evaluation. In this chapter, we will discuss such aspects of nanostructured materials on environment and biological cells. Scheme 5.1 represents the various aspects of nanostructured materials by the cell, and for the cell.

TOXICITY CONCERN AND SAFETY ISSUES OF NANOSTRUCTURED MATERIALS: INTERACTION WITH BIOLOGICAL CELLS AND CELLULAR BEHAVIOR Logarithmic increase in the use of engineered nanomaterials with unique properties has led to increased availability of nanomaterials in the environment, which was otherwise minimal. The high surface area to volume ratio of nanoparticles results in physiochemically dynamic and reactive materials in the environment. The consequences are generally multifaceted such as reaction with biomacromolecules, aggregation, redox reaction, and dissolution that might occur in both biotic and abiotic systems (Lowry et al., 2012). Conventional chemical or physical processes of nanomaterial formulation yield desirable products serving various purposes. However, the exposure of nanomaterial and its constituents to the environment at the stages of formulation, application, and disposal, results in a serious challenge to the biological system. Additionally, a perceivable unavailability of a globally accepted standard analytical protocol for scoring pollution caused by individual contaminants makes it extremely difficult to assess the individual nanomaterial contaminants in real time (Goodwin et al., 2018). There is a lack of global consensus on tests for determining nanomaterial pollution and assigning pollution types to certain categories of nanoparticles. Although individual nanoparticles behave independently and may cause a different type of pollution, a general lack of procedures for determining categories of nanomaterial-mediated pollution aggravates the pollution problems and health issues. Nanocomposite materials and its contents get exposed to the

Dynamics of Advanced Sustainable Nanomaterials and Their Related Nanocomposites at the Bio-Nano Interface. https://doi.org/10.1016/B978-0-12-819142-2.00005-7 101 Copyright © 2019 Elsevier Inc. All rights reserved.

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SCHEME 5.1 Schematic representation of various aspects of nanostructured materials in biological cells.

environment at various stages, viz. during manufacturing, at application point and final disposal of used nanocomposite materials. Primarily, the exposure of constituent entities poses a threat to the environment at the point of contact; secondarily, persistence of a robust chemical entity in the environment further inflates its impact on the environment (Bennett et al., 1999). With an aim to elucidate the impact of nanomaterials on the biological system, this section elaborates on some of the widely reported nanostructured materials and their possible impact on the biological cells and cellular behaviors. Among various nanomaterials, graphene-based nanomaterials are the talk of the hour and have attracted much of scientific attention owing to its unique attributes. Graphene (reduced graphene oxide) is produced from graphene oxide, which is obtained from exfoliated graphite oxide, a product of oxidized graphite. All these operations required many toxic chemicals! For example, exfoliation of natural graphite requires application of organic solvents such as N,Ndimethylformamide (DMF) and N-methylpyrrolidone (NMP) along with strong oxidizing agents to oxidized graphite, which are known to have negative impact on environment and human health (Chen et al., 2012). Acute exposure to DMF has been observed to damage

the liver cells in animals and humans alike. Other symptoms of human exposure to DMF include nausea, abdominal pain, jaundice, alcohol intolerance, and rashes. Occupational exposure to DMF has been reported to cause digestive disturbances and liver damage among workers. Occupational exposure to NMP has been reported to cause stillbirth (Solomon et al., 1996). These reports univocally suggests that chemical exfoliation of natural graphite (for the manufacture of nanocomposites) is a procedure involving considerable risk of exposure to the biological system. Furthermore, oxidized graphite is formed by oxidation of the available natural graphite in the initial stages. Oxidation reaction of natural graphite is a harsh treatment involving reaction of natural graphite with concentrated nitric acid and sulfuric acid in a ratio of 1:2 (V/V) (Wang et al., 2008). When graphene production is scaled up, the amount of acids required will scale up proportionally. The unused corrosive acids thus generated after the reaction are released into the environment causing harm to all the biological cells and systems coming in its way. Nitric and sulfuric acids are known corrosive, irritant, and permeators in nature. Hence, these acids do permeate the biological cells and leads to the exudation of the cellular content into the external milieu, causing immediate cell death. Thereafter, the oxidized graphite

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is treated with reducing agents such as hydroquinone for production of graphene (reduced graphene oxide) (Wang et al., 2008; Zhang et al., 2009). There have been multiple approaches by researchers to find a suitable reducing agent with minimal effect on the environment. Production of high quality graphene sheets could be achieved by sonicating tetrabutylammonium hydroxide (TBA) and oleum-intercalated graphite in DMF (Chen et al., 2012). TBA, however, is known for having adverse effects on human health, involving high risk in skin contact, eye contact, and ingestion. Stankovich et al. (2007) reported formulation of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide by the use of hydrazine hydrate. However, hydrazine exposure is known to be toxic to blood, nervous system, kidneys, lungs, and mucous membranes (Gilbert, 2014). Similarly, Eigler et al. (2013) reported various wet chemical methods of graphene synthesis. However, the chemical precursors used for reduction of graphite oxide to graphene are corrosive chemicals such as potassium permanganate, sodium nitrate, and sulfuric acid. Such chemicals when released into the environment post graphene production lead to drastic impact on the environment owing to their corrosive nature. Post disposal, such graphene-containing nanocomposite materials meet the large water bodies and the organisms living in there. Long-term exposure of planktonic microorganism to the graphene nanocomposite was reported to have permanent inactivation effects on the planktons and biofilms (Mejias et al., 2012). Phytoplanktons are known for production of more than half of the world oxygen (Falkowski, 2012). Wastes harming planktonic population are known to affect the global oxygen content. Again silver nanoparticles, owing to its potent antimicrobial and other beneficial properties have multiple uses and hence have gained inroads into almost all of the cosmetic products of daily use as an essential ingredient (Gajbhiye and Sakharwade, 2016). As such, regular release of silver nanoparticles into the environment is expected. Zebrafish is a widely accepted model for the assessment of environmental toxicity. Osborne et al. (2015) examined the impact of 20 and 110 nm citrate-coated silver nanoparticles on the gills and intestine of Zebrafish. The research group reported prominent silver deposition in the basolateral membranes for the 20 nm only. Furthermore, they linked the site of tissue deposition to the disruption of sodiume potassium ion channel, which is also located on the basolateral membrane, thereby concluding that silver toxicity in zebrafish is directly proportional to the size of the nanomaterial (Osborne et al., 2015). Silver

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emission from different sources has already been reported for causing pollution at various levels. Eckelman and Graedel (2007) compared the silver emission profiles of 64 countries (Fig. 5.1A) and recorded the highest level of silver in the landfills arising by waste disposal. The pronounced incorporation of silver nanoparticles into consumer products poses a definite risk for the consumers. The effects of silver nanoparticles and ions on caco-2/TC7:HT2-MTX intestinal coculture model was studied by Georgantzopoulou et al. (2016). Ag nanoparticles of 20 and 200 nm size were taken up by the cells. AgNPs of 20 nm were mainly localized in organelles with high sulfur content. Fig. 5.1BeE depicts the elemental distribution in Caco-2/TC7:HT29-MTX cells in 90:10 cocultures. Thermal decomposition or incineration of nanocomposites post use has been reported to contain nanofillers (such as Fe2O3) in the aerosols and residual ash. The aerosols are released into the atmosphere and residual ashes end up in landfills, thereby raising concerns about the applicability of incineration as a waste disposal method for nanocomposites (Singh et al., 2016a,b). Such nanoparticles containing aerosols also meet various plant and animal cells owing to the process of respiration or topological exposure. Owing to their nanoscale structures, these nanomaterials have the capacity to exert untoward effects such as allergy response in biological cells and distort the normal cellular behavior. Realizing such extensive impact of nanomaterials and its constituents on the environment, investigators started looking for safer methods of nanostructured material formulation. This led to green chemical synthesis procedures for the formulation of nanocomposite hydrogel.

ASSESSMENT OF NANOTOXICITY THROUGH CELL CULTURE Assessment of nanotoxicity through cell culture method is now a universally accepted, established in vitro procedure of assessing and predicting the possible consequences of nanomaterial application on the proposed subjects and its toxicity potential when released into the environment. However, selection of cells for scoring the toxicity is a major concern. Therefore, it is suggested to collect standard growth curve data to determine baseline growth properties of selected cells. For comparative studies between different cell lines for nanotoxicity assessment, individual growth rates can be used for classification and appropriation, which in turn may help to explain results of cytotoxicity experiments. Generally, nonmalignant cell lines undergo all the three phases

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FIG. 5.1 Comparisons of silver emissions (A) to the environment for 64 countries. The units are logarithms of the silver flows in Mg (metric tons). Elemental distribution of 31P, 34S, and 107Ag (green) in 300 nm cuts. Caco-2/ TC7:HT29-MTX cells in 90:10 coculture were exposed to Ag 20 nm (C), Ag 200 nm (D), and AgNO3 (E) for 24 h, while (B) represents the untreated control cells. Scale bar is 5 mm. The color scales indicated are for P and S images. For Ag images, the secondary ion intensity is expressed in green scale. ((A) Reproduced with permission from Eckelman, M. J., Graedel, T. E. 2007. Silver emissions and their environmental impacts: a multilevel assessment. Environ. Sci. Technol. 41, 6283e6289, copyright American Chemical Society 2007. (BeE) sourced from open access of Georgantzopoulou, A., Serch, T., Camber, S., Leclerq, C. C., Renaut, J., Shao, J., Kruszewski, M., Lentzen, E., Grysan, P., Eswara, S., Audinot, J. N., Contal, S., Zebel, J., Guignard, C., Hoffmann, L., Murk, A. T. J., Gutleb, A. C., 2016. Effects of silver nanoparticles and ions on a co-culture model for the gastrointestinal epithelium. Part. Fibre Toxicol. 13, 9e26, copyright Georgantzopoulou et al. 2016.)

of growth curve (viz. lag, exponential, and stationary), each of which can reflect different responses to the same amount of nanomaterial insult (Hillegass et al., 2010). The procedures used for nanotoxicity assessment through cell culture may be classified based on the agent used for the assessment. Table 5.1 summarizes all the available viability assays in place and their working principle.

Trypan Blue Exclusion Assay Trypan blue is a diazo dye. Live viable cells with intact cell membrane do not take up the dye and hence is not colored. However, dead cells, with perforated cell membrane, takes up the dye and are hence distinguished from the live. Therefore, the unstained cells reflect the total number of viable cells in a target culture flask. The advantage of this method lies in the fact that this indicates the actual number of viable cells in the medium, thereby giving a direct interpretation for increase or decrease in the number of viable cells as a response to the nanomaterial insult. Typically in this

assay, cells are first subjected to nanomaterial exposure, trypsinized, and subsequently stained with trypan blue to score the live-to-dead cell ratio (Strober, 2015). Mittal et al. (2015) reported biosynthesis of silver nanoparticles using extracts of medicinal plant, Potentilla fulgens and evaluated the apoptotic effect of synthesized nanoparticles on normal and cancer cells using trypan blue exclusion assay. The results indicated that the as-prepared nanoparticles exhibited selective toxicity toward the malignant cell lines (MCF-7 and U87 cells) in comparison with primary normal cell lines (lymphocytes).

MTT Assay MTT or the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide-based cell viability assay is a qualitative method of scoring cell viability. This test does not provide direct information of the total cell numbers, but measure the viability of a cell population relative to the untreated control. The principle of this assay relies on the ability of NAD(P)H-

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TABLE 5.1

Cell Viability Assays and Their Working Principles. Name of the Assay

Principle Parameter

Working Principle

Trypan blue

Trypan blue

Quantitative assay. Trypan blue is a dye molecule that can penetrate the dead cell membrane only, thereby selectively distinguishing the live from the dead.

MTT assay

3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT)

Qualitative assay. NAD(P)H-dependent cellular oxidoreductase enzymes are capable of reducing the tetrazolium dye MTT to its insoluble purple-colored formazan, thereby giving a proportional color intensity to the amount of live cells present in the solution.

Colony forming efficiency

Number of colonies formed

Quantitative assay. After incubating with test molecule, the cells are allowed to grow in plate. Only reproductively viable cells are able to make colonies.

TUNEL and apostain assay

TdT (terminal deoxynucleotidyl transferase) dUTP (deoxy uridine triphosphate), apostain

Qualitative assay. TdT dUTP labels ends of fragmented DNA, which can be detected by streptavidin-horseradish peroxidise and a diaminobenzedine chromogen by light microscopy. Alternatively fluorescent labeling can also be done. Apostain on the other hand labels condensed chromatin. A combination of both TUNEL and apostain can provide assured assessment of apoptosis.

Lactate dehydrogenase assay

Lactate dehydrogenase enzyme

Qualitative assay. Lactate dehydrogenase is a cytosolic enzyme which is released when the cell is ruptured. Scoring the amount of lactate dehydrogenase indicates the extent of cell death and hence is used as a marker of cell death.

Resazurin assay

Resazurin

Qualitative assay. Resazurin is a blue-colored dye, reduced to resorufin, a pink-colored substance upon reduction wth NADH/Hþ. Hence, it is used as a marker for live cells.

dependent cellular oxidoreductase enzymes, which are capable of reducing the tetrazolium dye MTT to its insoluble purple-colored formazan, thereby providing a colorimetric assay for determining cell viability. Skladanowski et al. (2016) reported formulation of silver nanoparticles from Streptomyces sp. NH28 strain. The cytotoxicity of the as-prepared nanoparticles was accessed using L929 mouse fibroblast via MTT assay. These silver nanoparticles at a concentration of 1 and 5 mg mL
1 did not show any cytotoxic effect, and the viability of the test samples was comparable to that of the control. Raising the concentrations of asprepared silver nanoparticles (>50 mg mL
1) registered marked cytotoxic effect with IC50 established at 64.5 mg mL
1.

Clonogenic Assay or Colony Forming Efficiency This test allows assessment of decreased or increased survival and proliferation over a period of time (weeks). After implanting at a very minimal density, cells are

allowed to grow until colonies are observed (ranging from 10 days to 3 weeks). They are either pretreated with the agent under investigation or are treated following planting. An assumption is made that each colony originates from a single-plated cell and hence the name of the assay. Colonies can be stained with nuclear stains and quantified according to numbers and/or size. In short, this method is a protocol of choice to determine cell reproductive death after treatment with the test material. Bendale et al. (2017) reported the evaluation of cytotoxic activity of green-synthesized platinum nanoparticles using clonogenic assay. The authors reported that green-synthesized platinum nanoparticles treatment caused cell death and inhibited colony formation capability in the PA-1 cell population in a concentration-dependent manner. Following treatment with various concentrations (50, 100, and 200 mg mL
1), the plating efficiency of PA-1 cells declines, as showed by the reduction in the number of colonies formed.

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TUNEL and Apostain Assay

Resazurin Assay

TdT (Terminal deoxynucleotidyl transferase) dUTP (deoxy uridine triphosphate) Nick End labeling (TUNEL) and Apostain assay is widely used to detect apoptosis. Apoptosis is a form of programmed cell death characterized by mitochondrial DNA damage, cell membrane bleeding, nuclear and cytoplasmic shrinkage, chromatin condensation, fragmentation into apoptotic bodies, and DNA fragmentation. The TUNEL assay labels the ends of fragmented DNA, resulting in biotinylated dUTP at the 30 -OH end that can be detected by the application of streptavidin-horseradish peroxidase and a diaminobenzedine chromogen by light microscopy. Alternatively, the incorporated dUTP nucleotides can be labeled with a fluorescent dye and evaluated using fluorescent microscope. Apostain, on the other hand, labels condensed chromatin. Therefore, TUNEL assay in combination with Apostain assay provides an assured assessment and scoring of apoptotic cell death. Sun et al. (2018) assessed the toxicological profile of gold nanoparticles using TUNEL assay. Poly(ethylene glycol)-coated gold nanoparticles (13 nm) were reported to accumulate in the liver and were also found to elicit apoptosis 7 days after injection as showed by TUNEL assay analysis.

Resazurin (7-hydroxy-3H-phenoxazin-3-one 10 oxide) is a blue-colored dye (weakly fluorescent) until it is reduced (by NADH/Hþ) irreversibly to pink color and highly red fluorescent dye resofurin. This mechanism is used as an oxidation-reduction indicator in cell viability assays for anaerobic/aerobic respiration. Yang et al. (2017) evaluated cell viability of HeLa and MCF7 cells exposed to gold nanoparticles. The results showed concentration-dependent cell death in vitro. As the concentration of the target nanoparticles were increased, the resazurin assay scored higher cell death.

Lactate Dehydrogenase Assay Lactate dehydrogenase (LDH) is a soluble cytosolic enzyme that acts as an indicator of cell lysis-mediated cell death, as it is released into the extracellular medium following cellular membrane damage resulting from apoptosis or necrosis. Popularly accepted as cell death marker, it should be understood that this test is simply a test of cell membrane integrity, and in certain tests, can be positive even when the total cell count is not significantly altered. Hussain et al. (2005) evaluated in vitro cytotoxicity of different sizes of nanoparticles such as silver (Ag; 15, 100 nm), molybdenum (MoO3; 30, 150 nm), aluminum (Al; 30, 103 nm), iron oxide (Fe3O4; 30, 47 nm), and titanium dioxide (TiO2; 40 nm) via LDH assay using rat liver-derived cell line (BRL 3A). LDH leakage was reported to significantly rise in cells exposed to Ag nanoparticles (10e50 mg mL
1), while the other nanoparticles displayed LDH leakage only at higher doses (100e250 mg mL
1). Based on such results, Hussain et al. (2005) concluded that silver nanoparticles were much toxic in comparison to other nanoparticles used in the test.

SUSTAINABLE SYNTHESIS OF NANOSTRUCTURED MATERIALS USING VARIOUS BIOLOGICAL CELLS Conventional syntheses of nanoparticles using various chemical and physical methods have been found detrimental to the environment due to leaching and exposure of the nanoparticles themselves, or the ingredients required to synthesize such nanoparticles, as described in the earlier section of this chapter. To combat such exposure of disastrous chemicals (corrosive acids or harsh alkalis) to the environment during production of nanoparticles, researchers have brought into use the living cells of various origins (eukaryotic and prokaryotic) and employed them as nanofactories for the production of nanostructured materials (Singh et al., 2016). Interestingly, in case of metal nanoparticles synthesis, the reduction ability of the living cells most often help in the reduction of metal ion to metal nanoparticles. This eliminates the use of corrosive and harsh chemicals, thereby being categorized as green synthesis method of nanostructured material production. Fig. 5.2 schematically represents biosynthesis of nanostructured materials and their subsequent applications. Investigators have also reported to use viruses for the production of nanostructured materials.

Synthesis of Nanostructured Materials by Bacterial Cells Bacteria represent the most versatile category of microorganisms that are found in every habitable zones of the planet. As such, they need to protect themselves from various reactive metal ions that are present in the environment. Reduction of such reactive metal

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FIG. 5.2 Schematic representation of biosynthesis of nanoparticles and their subsequent applications. (Reproduced with permission from Singh, P., Kim, Y., Zhang, D., Yang, D., 2016. Biological sunthesis of nanoparticles from plants and microorganisms. Trends Biotechnol. 34 (7), 588e599, copyright Elsevier 2016.)

ions to zero valence metal nanoparticles renders these assaulting ions to benign nanostructured materials. This propensity of metal ion reduction by the bacteria cells have been brought to task for the synthesis of nanostructured materials. However, bacterial cells may produce nanoparticles in two major fashions: extracellular and intracellular. In case of extracellular nanoparticles production, isolation of as-prepared nanostructured particles becomes easy and hence proves to be a user-friendly process. However, in certain cases, the nanoparticles so produced are done in the intracellular milieu of the bacteria concerned. In such cases, removal of as-prepared nanostructured materials becomes a bit tedious. Otari et al. (2015) reported intracellular synthesis of silver nanoparticles using Rhodococcus spp. Transmission electron microscopy study of the Rhodococcus spp. revealed that the synthesis of silver nanoparticles (ranging from 5 to 50 nm) was occurring inside the cytoplasm. The nanoparticles were found to be coated

with peptides that rendered them stable in colloidal solutions. Interestingly, these silver nanoparticles showed excellent bactericidal and bacteriostatic activity against pathogenic Gram-positive (Staphylococcus aureus) and Gram-negative (Klebsiella pneumonia, Proteus vulgaris, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli) microorganisms. Ozden et al. (2017) reported the use of bacteria as a bio-template for the synthesis of 3D carbon nanotube architectures. The authors demonstrated the use of Magnetospirillum magneticum (AMB-1) bacteria as a bio-template to fabricate 3D lightweight solid structure of carbon nanotubes. The resulting porous scaffold showed good mechanical stability and extensively large surface area caused by the pore interconnection and high porosity. Singh et al. (2018) reported production of extracellular silver nanoparticles using Pseudomonas sp. THG-LS1.4, a strain isolated from soil. These silver nanoparticles exhibited antibacterial activity against Bacillus cereus, Staphylococcus aureus, Candida tropicalis, Vibrio parahaemolyticus,

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Escherichia coli, and Pseudomonas aeruginosa. In addition, these nanostructured materials were shown to exhibit biofilm inhibition activity.

Synthesis of Nanostructured Materials by Fungal Cells Fungal production of nanostructured materials is seen as yet another potential biological pathway for the synthesis of nanostructured materials. Fungus is found in humid and moist areas. Moist milieus provides reactive environment due to easy dissolution of metal ions and other chemical entities. Fungus secretes enzymes and proteins as reducing agents, which can be employed for the production of nanostructured materials. The extracellular and intracellular modes of nanomaterial synthesis (as discussed in the previous section) also hold good for the fungi-based synthesis of nanostructured materials. However, owing to the risk involved with fungal infection, this area has not been employed extensively in real time. Das et al. (2010) reported using cell free extract of Rhizopus oryzae for the synthesis of different shapes of gold nanostructures, for example triangular, hexagonal, pentagonal, spherical, spheroidal, urchin-like, 2D nanowires, and nanorods, by altering gold ion concentration, protein concentration, solution pH, and reaction time (Fig. 5.3AeF). Even the protein extract of R. oryzae was demonstrated for the reduction of chloroauric acid to gold nanoparticles (Das et al., 2012a,b). Das et al. (2012a,b) exhibited biomineralization of gold nanoparticles in R. oryzae. Fig. 5.3F shows graphically the pathway for the synthesis of gold nanoparticles using fungal mycelia. Reduction of gold occurred both on the cell wall of the fungal cell as well as in the cytoplasmic region of the fungus (Fig. 5.3G,H). The average size was determined to be 15 ̴ nm. The mechanism of gold nanoparticles synthesis was also probed, and it was found that it occurred through adsorption, initial reduction to Au(I), followed by complexation [AU(I) complexes], and final reduction to Au0. Protein profiling of the whole protein after growing the fungal mycelia in concomitantly increasing the dose of Au(III) was done. Two of the proteins bands with molecular weight MWw45 and w42 kDa exhibited upregulation in the cells grown in the presence of 70e130 mM Au(III) (Fig. 5.3I, lanes 5 and 6); whereas, two other protein bands with MW of w68 and w50 kDa were suppressed (Fig. 5.3I, lanes 2 and 3). Expression of protein bands w45 and w42 kDa proteins increased, while that of w68 and w50 kDa proteins decreased with increasing Au(III) concentrations, showing concentration-dependent

protein expression. Xue et al. (2016) reported biosynthesis of silver nanoparticles by the fungus Arthroderma fulvum. Biosynthesized silver nanoparticles showed significant activity against some fungal strains of Candida spp., Aspergillus spp., Fusarium spp., and Candida spp. Das et al. (2014) developed a facile approach for the biosynthesis of Pd, Pt, and Ag nanostructured materials through in situ reduction process on the biomass surface without using any harmful reagents. Pd and Pt biomineralization produced “flower”-like branched nanoparticles; whereas, Ag produced spherical nanoparticles.

Synthesis of Nanostructured Materials by Algal Cells Algae constitute a diverse group of photosynthetic eukaryotic polyphyletic organisms. It includes singlecelled microalgae (chlorella and the diatoms) to multicellular forms (giant kelp for example). Being the occupants of the first trophic level in the food chain, these microorganisms entrap solar energy through photosynthesis and have a very high energy quotient. Acutodesmus dimorphus has widely been reported for biofuel production. Chokshi et al. (2016) reported the use of residual biomass of A. dimorphus after lipid extraction, for the synthesis of silver nanoparticles. The biosynthesized silver nanoparticles demonstrated antioxidant potential evaluated using 2,2’azino-bis (3ethylbenzothiazoline6-sulphonic acid), that is, ABTS and 1,1-diphenyl-2-picrylhydrazyl, that is, DPPH, free radical scavenging assays. Such amalgamation of phycology and nanotechnology leads to the development of a new interdisciplinary advancement termed as “phyconanotechnology.” Li and Zhang, 2016 reported the biosynthesis of gold nanoparticles using green alga Pithophora oedogonia. Algal extract was used for reducing Au salt. The average size of gold nanoparticles was determined to be 32.06 nm using scanning electron microscopy and dynamic light scattering machine.

Synthesis of Nanostructured Materials by Viral Particles Viruses offer a confined milieu and unique protein surface topology (i.e., surface relief, residue charge, and polarity) for the synthesis of nanostructured materials. Viruses are also amenable to molecular biology manipulations. Thus, viruses provide a versatile platform for the synthesis of nanostructured materials tunable to the requirements of the end user. Plant viral capsids provide bio-templates for the production of novel nanostructured materials with inorganic/organic

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FIG. 5.3 TEM images of different shapes of gold nanoparticles synthesized under different conditions.

Formation of triangle (A), hexagon (B), pentagon (C), star (D), and wire-like (E) gold nanoparticles achieved upon fine-tuning the reaction condition. The schematic representation (F) of cell-free protein-mediated reduction of gold ions to AuNPs. The synthesis of gold nanoparticles was observed at the cell wall (G) and cytoplasm (H) of the R. oryzae cells. SDS-PAGE analysis (I) of whole proteins following growth in the presence of 0 (lane 2), 15 (lane 3), 35 (lane 4), 70 (lane 5), 130 (lane 6), and 250 mM (lane 7) of Au (III). Arrows indicate suppressed and induced proteins. (Adapted with permission from Das, S. K., Das, A. R., Guha, A. K., 2010. Microbial synthesis of multishaped gold nanostructures. Small. 6(9), 1012e1021, copyright John Wiley and Sons, 2010. Reprinted with permission from Das, S. K., Liang, J., Schmidt, M., Laffir, F., Marsili, E., 2012. Biomineralization mechanism of gold by Zygomycete fungus Rhyzopus oryzae. ACS Nano 6 (7), 6165e6173, copyright American Chemical Society 2012.)

groups incorporated in a very precise and controlled manner. Slocik et al. (2004) reported using cowpea chlorotic mottle viruses of unmodified SubE (yeast), (HRE)SubE engineered with interior HRE peptide epitopes (AHHAHHAAD), and wild type as viral templates for the potentiated reduction of gold nanoparticles.

Synthesis of Nanostructured Materials by Plant Extracts Plants represent one of the most versatile and abundant life forms on Earth. As such, the plant tissues, sap, extract, etc. have been utilized for the reduction of metal salts, thereby producing biocompatible nanostructured material. As the process utilizes plant biomass and its extracts as reducing agent, they do not cause pollution

in the environment and is deemed to be one of the most environment friendly methods of nanostructural material formation. However, the cost of tissue culture acts as formidable limitation for widespread application of such techniques. Tripathy et al. (2008) reported the biomimetic synthesis of silver nanoparticles by the aqueous extract of Azadirachta indica (Neem) leaves. They observed that the morphology and the size of the nanoparticles were strongly dependent on the process parameters. In the 4-h interaction period, nanoparticles below 20 nm size with just about spherical shape were produced. Kalaiyarasan et al. (2017) reported the formulation of silver nanoparticles using sea buckthorn leaf extract as a reducing agent. These leaf extracts are a rich source of organic acids, and inorganic compounds may be used

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for the formation of nanostructured materials. The asprepared spherical nanostructured material was also demonstrated for antibacterial efficacy.

NANO-BIO INTERFACE: A NEW FRONTIER OF POSSIBILITIES Unprecedented growth of nanostructured materials and their wide application is increasing the probability of engineered nanomaterials coming in contact with the living cells (human or nonhuman). The consequences are multifarious. Interaction with the nanomaterials leads to formation of particle wrappings, protein coronas, intracellular uptake, and biocatalytic processes that could have biocompatible or bio-adverse outcomes. Size, shape, surface-chemistry, surface coatings, and roughness of the nanomaterials determine the type of impact they exert on the various biological systems as and when they come in contact. Probing these various interfaces can prove beneficial in predicting the impact of a particular nano-bio interface and thus account for the safe use of nanostructured materials. Fig. 5.4 schematically represents the various facets of nano-bio interface and their myriad interactions. The composition of the particle surface (for example its hydrophobicity, charge, size, radius of curvature, coatings that exert steric or electrostatic effects) determines as to which biomolecule interact with the particles, thereby mediating its access to cells (Fig. 5.4A) (Nel et al., 2009). Once the nanostructured material gains entry into the cytoplasm, its interaction with nucleic acid/ cytosolic components/organelles determines its impact on the biological system (Fig. 5.4B). Composed of proteins, cholesterol, and lipopolysaccharides, the phospholipid bilayer separates the cell from the rest of the biological environment. This membranous barrier serves as a protection barrier for the cell interior from the external environment, thereby allowing the maintenance of controlled conditions in the cell cytosol. As such, they serve as the communication interface between the cell and the outer environment. Smaller/nonpolar entities such as oxygen can pass across the membrane via simple diffusion. Larger/polar molecules require a much enhanced system of protein mediation for passage into the cellular milieu. However, it has been demonstrated that nanoparticles can traverse the cell membrane without being involved in any receptor-mediated interaction (Kettler et al., 2014). Interaction of nanomaterials with the biomolecules (DNA, RNA or protein) ensures the targeted effect of the nanomaterials on the cells. Cellular responses to nanostructured materials in a biological medium reflect

the adsorbed biomolecule layer, rather than the material itself. Hence, it is now understood that more than the nanomaterial, it is the interaction between the biomolecules and the nanomaterial under consideration that determines the effect exerted by the nanomaterials on the biological cells (Lynch and Dawson, 2008). Evaluation of protein adsorption of bovine serum albumin (BSA), myoglobin (Mb), and cytochrome c (CytC) onto self-assembled monolayers of mercaptoundecanoic acid (MUA) on Au nanoparticles using the quartz crystal microbalance (QCM) demonstrates that all three proteins form adsorption layers consisting of an irreversibly adsorbed fraction and a reversibly adsorbed fraction (Kaufiman et al., 2007). Tang et al. (2015) reported that repulsive electrostatic interaction added upon with the membranemediated repulsion tends to make the nanoparticles wrapped by the membrane independently. Membrane wrapping is a unique process whereby the membrane integrity is not altered and the nanoparticle is either ingested or excreted out. Spontaneous wrapping occurs if the adhesive interaction between the nanoparticles and the membrane is sufficiently strong to compensate for the cost of membrane bending (Bahrami et al., 2014). Membrane wrapping is an interesting phenomenon whereby the membrane bipolar layers wrap itself around the nanostructured material (Fig. 5.5AeG), thereby facilitating its entry into the cellular lumen. Nanomaterial coating also determines the impact of the nanomaterial on the biological entities. As demonstrated by Devi et al. (2014), coating of silver nanoparticles with b-hydroxy propyl cyclodextrin (b-HPCD-AgNP) did not elicit hemoglobin lysis; whereas, coating with borohydride (BH4eAgNP) leads to lysis of hemoglobin as shown in Fig. 5.5H. It can thus be concluded that the nano-bio interface and its working mechanisms is directly proportional to the composition, structure, and surface properties of the nanostructured materials. These in turn determines the behavior of various biomolecules it meets and consequently exerts its specific impact of the biological entity.

APPLICATIONS OF SUSTAINABLE NANOSTRUCTURED MATERIALS IN BIOLOGICAL CELLS AND BIOMEDICAL FIELDS Application drives innovation. As such, the development of a cell culture procedure based on sustainable nanomaterials without a definite application will amount to wastage of effort and investment. Nanostructured materials have wide applications for the benefit of

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FIG. 5.4 Schematic representation of (A) myriad interactions at nano-bio interface and (B) schematic representation of the processes by which the nanostructured material exerts its effect on the cell. ((A) Part of the figure is reproduced with permission from Nel, A. E., Madler, L., Velegol, D., Xia, T., Hoek, E. M., Somasundaran, P., Klassif, F., Castranova, V., Thompson, M., 2009. Understanding biophysicochemical interactions at the nano-bio interface. Nat. Mater. 8 (7), 543e557, copyright Nature Publishing Group 2009.)

humankind, and this section discusses the exclusive use of nanoparticles on biological cells and systems for various functions.

Plant Growth-Promoting Factor Investigation of nanostructured materials and their derived formulations are in high demand in agricultural sector to replace the synthetic agrochemicals. Owing to its small size to large surface area, nanomaterials can penetrate various lines of barrier and enter the intracellular milieu to exert its unique effect. The use of such

nanostructured materials for the use of plant growth is a lucrative arena of research for the plant biotechnologists who can probe, investigate, and validate various concoctions of nanostructured materials for their applications in agriculture. Biocompatible nanostructured materials in this case would provide for an assured advantage over the conventional chemical fertilizers. Sonkar et al. (2012) reported formulation of watersoluble carbon nano-onions and their application as growth-promoting factor for gram (Cicer arietinum) plants. Under laboratory conditions, treatment of

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FIG. 5.5 The effect of particle linker density on the membrane wrapping state. Fluorescence signal (A) of a nonwrapped particle (green) and a membrane (magenta). The separate fluorescence signals of the membrane (B) and particle (C). In (DeF), the wrapped state is displayed analogously. The scale bar is 1 mm. Fraction of wrapped particles (G) as a function of linker density on floppy membranes (blue triangles, membrane tension s < 10 nN m
1) and tense membranes (red circles, s > 1 mN m
1). The solid line is the analytic model at s ¼ 0 derived from Equation 1 via the Boltzmann factor. Horizontal error bars show the spread (one standard deviation) in linker density. Effect of b-HPCD-AgNP and BH4eAgNP (H) on the secondary structure of Hb and biocompatibility toward RBC. (Adapted and reproduced with permission from Wel, C. V. D., Vahid, A., Saric, A., Idema, T., Heknrich, D., Kraft, D. J., 2016. Lipid membrane-mediated attraction between curvature inducing objects. Sci. Rep. UK, 6, 32825e32835, copyright Nature Publishing Group 2016. Reprinted with permission from Devi, L. B., Das, S. K., Mandal, A. B., 2014. Impact of Surface Functionalization of AgNPs on binding and conformational change of hemoglobin (Hb) and hemolytic behaviour. J. Phys. Chem. C 118 (51), 29739e29749, copyright American Chemical Society 2014.)

gram plants with up to 30 mg mL
1 of as-prepared nanoonions over a period of 10 days led to significant growth of the plant biomass. To ascertain the growthpromoting activity of carbon nano-onions in gram plants, 10 days treated and untreated plants were transplanted into soil with standard carbon and nitrogen

composition. Plants treated with nanostructured carbon nano-onions showed higher growth rate and yielded more grams in comparison to its untreated counterpart. This observation therefore augments the positive effect of nanostructured material in promoting plant cell growth.

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Choudhary et al. (2017a,b) reported copper (Cu)chitosan nanostructured material-mediated reinforcement of defense responses and growth in maize (Zea mays L.). Cu-chitosan nanostructured material-treated maize plants exhibited significant defense response against Curvularia leave spot disease through heightened activity of antioxidant (superoxide dismutase and peroxidase) and defense enzymes (polyphenol oxidase and phenylalanine ammonia-lyase). Significant growth and disease resistance was observed at 0.04%e0.16% of Cu-chitosan nanoparticles application in pot and 0.12%e0.16% of nanoparticles in field conditions. Plant growth was assessed using parameters such as plant height, stem diameter, root length, number of roots, and chlorophyll content, which registered significant increase in nanoparticle-treated plants as in comparison to the untreated control.

Tissue Engineering Nanostructured materials provide a large platform for customized synthesis of various scaffolds intended for use in tissue engineering. In addition, certain nanostructured materials have been reported for promotion and proliferation of biological cells in vitro, thereby exhibiting the immense potential of nanostructured materials for use in the growth regulation of cells in vitro. Cunha et al. (2012) reported a formulation of multiwalled carbon nanotubes with magnetite (Fe3O4) nanoparticles. Iron oxide particles were deposited on multiwalled carbon nanotube surfaces by the deposition-precipitation method using salt precursors (Fe3þ/Fe2þ) in the basic solution. The as-prepared hybrid nanocomposite was analyzed in vitro by incubation with mesenchymal stem cells for a week. An analysis of cell proliferation by MTT assay (as described ahead) demonstrated that introduction of magnetite into multiwalled carbon nanotubes led to increase in cell proliferation and enhanced the biocompatibility of carbon nanotubes. Nerve regeneration is a potential field of research for obvious biomedical applications. Xiao et al. (2017) reported a novel application of gold nanoparticles modified with 6-mercaptopurine and neuron-penetrating peptide as a neuropathic agent for the promotion and proliferation of neurite growth of human neuroblastoma cells (SH-SY5Y). Upon treatment with the nanostructured conjugates, the cells showed higher metabolic activity in comparison to the control. The nanocomposite was demonstrated to attach and internalize the cells, thereby exerting its growth-promoting potencies. In addition, freezing and subsequent thawing of as-treated cells shows significant recovery within a very short span of time, indicating biocompatibility

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and excellent cellular tolerance of the nanomaterial. Choudhary et al. (2017a,b) demonstrated formation of reduced graphene oxide (rGO) via microbial reduction and its cell proliferative abilities on fibroblast (3T6) cells (Fig. 5.6). Fig. 5.6A demonstrated bioreduction of graphene oxide to reduced graphene oxide by Pleurotus sajor-caju cells, while Fig. 5.6BeD cyto- and hemocompatibility behavior of the synthesized reduced graphene oxide in the concentration range of 0e80 mg mL
1. Pentela et al. (2018) reported the preparation of microcapsules from functionalized polymers, carbon nanotubes, and silver nanoparticles for pHresponsive targeted drug delivery system to specifically kill the cancer cells. Fig. 5.6EeH revealed live/dead assay using Acridine Orange/Propidium Iodide (AO/ PI) for staining of A431 cancerous cells treated with microcapsules containing cisplatin at pH values of 7.0 and 5.4 at different time intervals of 7.5 and 15 h. Cisplatinincorporated microcapsules treated at pH 7.0 for 7.5 and 15 h, respectively, showed no significant killing of cancer cells (Fig. 5.6E and G). In contrast, cisplatinincorporated microcapsules treated at pH 5.4 for 7.5 and 15 h showed the anticancer effect with distinct observations of more DNA damage and cell death of the cancer cells. Plant tissue culture forms the core of plant biology (conservation, genetic manipulation, bioactive compound production, plant improvement, and mass propagation). Nanomaterials are known for their inherent antibiotic properties. Utilizing these properties, nanomaterials can be used for disinfecting the plant tissue culture media for callus induction, somatic embryogenesis, genetic transformation, somaclonal variation, secondary metabolite production, and organogenesis. Graphene, for example, can be brought into use for construction of culture vial surfaces, thereby ensuring aseptic conditions. Nanomaterials and nanoparticles have been reported for the use in plant micronutrition. Palchoudhury et al. (2018) reported the use of a-Fe2O3 nanoparticles as a fertilizer. The novelty of this approach was to soak the seeds into nanoparticle containing solution, thereby restricting excess release of nanoparticles into the nature. The researchers used the seeds of green pea (Pisum sativum L.), chick pea (Cicer arientnum), green gram or mung bean (Vigna radiate), and black and red beans (Phaseolus vulgaris). The authors reported that the iron oxide nanoparticles could enhance the root growth by 88%e366% at considerably low concentrations (5.54  10
1 Fe mg L
1 Fe). Fig. 5.7A presents a schematic representation of use of nanofertilizers for the plant growth and development (Morales-Díaz et al., 2017). Fertilizers of nanoscale dimensions have proved to be an efficient nutrient

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FIG. 5.6 Bioreduction of graphene oxide to reduced graphene oxide by Pleurotus sajor-caju cells (A). Cyto and hemocompatibility behavior of the synthesized reduced graphene oxide in the concentration range of 0e80 mg mL
1 (BeD). Phase contrast and fluorescent micrographs (C) of fibroblast cells. Fluorescent staining was done with calcein AM and propidium iodide (PI). Hemolysis assay (D) with different concentration of rGO (0e80 mg mL
1) using whole blood cells. Live/dead assay using Acridine Orange/Propidium Iodide (AO/PI) staining of A431 cancerous cells treated with released solution from cisplatin-incorporated microcapsules at pH 7.0 and 5.4, for 7.5 and 15 h (EeH). (Reproduced with permission from Choudhary, P., Parandhaman, T, Ramalingam, B., Duraipandy, N., Kiran, M. S., Das, S. K., 2017. Fabrication of nontoxic reduced graphene oxide protein nanoframework as sustained antimicrobial coating for biomedical application. ACS Appl. Mater. Interfaces 9, 38255e38269, copyright American Chemical Society 2017. Adapted and reproduced with permission from Pentela, N., Duraipandy, N., Nikhil, S. V., Parandhaman, T., Kiran, M. S., Das, S. K., Jaisankar, S. N., Samanta, D., 2018. Microcapsule from diverse polyfunctional materials: synergic interactions for sharp response by pH change. New J. Chem. 42, 8366e8373, copyright Royal Chemical Society 2018.)

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FIG. 5.7 Schematic representation of (A) use of nanofertilizers in crops. On the right side are the recorded nanomaterials containing essential elements under a controlled release as a function of time or environmental stimuli. On the left are the nanoparticles of essential elements that are applied directly to the soil, irrigation water, or the surface of plants, fruits, or seeds. Smooth and uninterrupted tissue culture process (B) in the presence of AgNPs into the medium. Future prospects (C) envisioned for nanotechnology in plant tissue culture. ((A) Available as open access format from Morales-Díaz, A. B., Ortiz, H. O., Maldonado, A. J., Pliego, G. C., Morales, S. G., Mendoza, A. B., 2017. Application of nanoelements in plant nutrition and its impact in ecosystems. Adv. Nat. Sci. Nanosci. Nanotechnol. 8, 013001e013015, copyright IOP Science 2014. (B) and (C) Available as open access format from Kim, D. H., Gopal, J., Sivanesan, I., 2017. Nanomaterials in plant tissue culture: the disclosed and undisclosed. RSC Adv. 7, 36492e36505, copyright The Royal Society of Chemistry 2017.)

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material for the plants, as it can directly be taken up by the plants without much modification. However, the ultimate fate of these nanomaterials in the environment is yet to be ascertained in detail. Silver nanoparticles (AgNPs) is the most versatile of all nanoparticles, acting as disinfectant, as agent for differentiation and morphogenesis, for causing somaclonal variation, as secondary metabolite and as agent for genetic transformation. Zinc oxide (ZnO) nanoparticles have been implicated for use in disinfection and in causing differentiation and morphogenesis. Smooth and uninterrupted growth of shoot cultures of Vanilla planifolia was observed when incubated in Murashige and Skoog (MS) medium supplemented with 50e200 mg mL
1 of AgNPs (Fig. 5.7B) (Kim et al., 2017); however, media without AgNPs showed retarded growth of Vanilla planifolia due to microbial contamination. Equipped with such nanostructured materials, the future seems to be full of bright opportunities. Some of the fields such as tissue culture (Fig. 5.7C) can now go for continuous maintenance over an extended period of time without getting infected.

Stem Cell Applications Regenerative medicine targets to achieve functional rehabilitation of tissues or cells injured through disease, aging, or wound. Stem cells is a special class of cells that can self-renew and differentiate into specific types of functional cell (such as cardiac, pancreatic, and nerve cells), to be transplanted into the patient to replace damaged tissue, thereby addressing the basic principle of regenerative medicine. Contemporary practice of storing the stem cells of neonates from the umbilical cord is done in anticipation of future application of such stored stem cells for restoring the functioning of damaged tissue. It is a new realm of nanoparticles application, and the stakes are very high as it promises to deliver custom-based nanomedical solutions to pertinent questions of regeneration. Many lives are lost due to unavailability of organs in face of an accident. Organ transplantation is restricted by the availability of donors and high processing costs. In vitro nanotechnology-based organogenesis may pave a way for countering this issue by production of artificial organs. Tissue engineering using nanomaterials has been explored with success for more than a decade now. Ideally, stem cells are recovered from the healthy organs of the patient, cultivated in cell culture media for generating substantial number of viable stem cells. These cells are then seeded into the 3D porous scaffold containing growth factors, organ specific molecules, and nanostructured material,

providing for the structural integrity and tissue organization. Finally, the engineered tissue is then transplanted into the patient body. The scheme of nanostructured material-based organogenesis has been shown in Fig. 5.8. Neuronal regeneration from stem cells has always been an enigma for the investigators. The major challenge faced for neuronal regeneration is the short half-life period of the neuronal growth factors. For example, half-life time of brain-derived neurotropic factor (BDNF), basal fibroblast growth factor (FGF-2), and b-nerve growth factor (bNGF) in blood are 10, 1.5e3, and 30 min respectively. Conjugating these growth factors with iron oxide nanoparticles (IONPs) increases their stability and prolongs their activity. Ziv-Polat et al. (2015) reported the use of iron oxide nanoparticles in neuronal tissue engineering. They used thrombin-conjugated nanoparticles for the development of a novel magnetic fibrin hydrogel scaffold for the 3D culture of neuronal cells. Carbon nanotubes have been in place for some time now for the culture of stem cells. Garnica-Gutierrez et al. (2018) investigated the effect of citric acid functionalization of carbon nanotubes on mesenchymal stem cells in vitro. The modified nanotubes exhibited heightened intracellular incorporation, cell proliferation, absence of apoptosis, and lessened cytotoxicity as compared to those of unpolymerized carbon nanotubes. The effects of nitrogen-doped carbon nanotubes were also reported, showing that functionalized undoped carbon nanotubes present stronger stimulation of cell proliferation in comparison to functionalized and polymerized carbon nanotubes. Carbon nanotube doped with nitrogen was found to promote apoptotic behavior. Park (2017) illustrated the preparation of multimodal magnetic nanoclusters and their applications in tracking of stem cells, directed migration, and gene delivery. Wang et al. (2011) developed a novel fluorescent magnetite nanocluster (FMNC) by embedding individual magnetite nanoparticles (NPs) into a polystyrene scaffold coated with two layers of silica and a layer of rhodamine sandwiched within. Fig. 5.9 presents a schematic representation of high MRsensitive FMNC for stem cell tracking in ischemic mouse brain. Stem cell differentiation also is a major aspect of stem cell research. Multiple nanostructured materials have been reported for regulation of stem cell differentiation. Volkova et al. (2017) investigated the effect of different concentration of the 15 nm gold nanoparticles on the immunophenotype, synthesis of collagen type I, ability to direct differentiation,

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FIG. 5.8 Schematic representation of the concept of tissue engineering. Viable cells are isolated (A) and then cultivated (B) to increase the number of viable cells. Thereafter, they are seeded into the 3D scaffold (C) mimicking organ-specific environment. Following organogenic signals (as delivered by organ specific small molecules), the cells now organize into tissue (D), which are then transplanted to the patient under treatment. (Adapted with permission from Dvir, T., Timko, B. P., Kohane, D. S., Langer, R., 2011. Nanotechnological strategies for engineering complex tissues. Nat. Nanotechnol. 6, 13e22, copyright Nature Publishing Group 2016.)

and spectroscopic characteristics of bone marrow mesenchymal stem cells. The gold nanoparticles showed no changes in the expression of CD 45, CD 90 and CD 73 at a concentration of 1.5e9 mg mL
1. At concentrations of 6 and 9 mg mL
1 a decrease in levels of CD 44 cells by 6% and 9% was recorded, respectively. From these observations, the authors concluded that the concentration of gold nanoparticles at the range of 1.5e6 mg mL
1 are beneficial for mesenchymal stem cells; whereas, an increase of concentration to 9 mg mL
1 may lead to cytotoxic effects.

Bioimaging and Labeling Imaging or labeling has been the fundamental procedure of biological analysis since time immemorial. Electron absorptive properties of nanoparticles have been exploited to generate contrast, thereby being useful in labeling and bioimaging. Tissue engineering by the use of human stem cells (SCs) holds promise in revolutionizing the treatment of numerous diseases. A pressing need is felt to comprehend the distribution, movement, and role of SCs once implanted onto scaffolds. Iron oxide and Gadolinium-based MNPs have

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FIG. 5.9 Schematic representation of high MR sensitive fluorescent magnetite nanocluster for stem cell tracking in ischemic mouse brain. (Adapted with permission from Wang, Y., Xu, F., Zhang, C., Lei, D., Tang, Y., Xu, H., Zhang, Z., Lu, H., Du, X., Yang, G., 2011. High MR sensitive fluorescent magnetite nanocluster for stem cell tracking in ischemic mouse brain. Nanomed-Nanotechnol., 7(6), 1009e1019, copyright Elsevier 2011.)

been reported to be used for labeling and tracking stem cells by MRI since this clinically available imaging modality has high spatial resolution (Hachani et al., 2013). Based on the type of surface (silica, polylysine, or triphenylphosphonium groups), the polystyrene nanosensors are aggregated differentially into the extracellular and intracellular matrices of the cell or near mitochondria. Cho et al. (2014) made rhodamine-dyed nanoparticles highly biocompatible by coating the surface with phosphocholine and lectin (Fig. 5.10A). Such particles have very high affinity for sialic acid as over expressed on the surface of tumor cells, thereby selectively dying the tumor cells. Observation of structural integrity of nanoparticles is essential in bionanotechnology, but it is not always straightforward to measure in situ and in real time. Fluorescent labels used for tracking intrinsically nonfluorescent nanomaterials generally do not allow simultaneous observation of integrity. As a consequence, structural changes such as degradation and disassembly cannot easily be followed in situ using fluorescence signals. Meder et al. (2016) showed that Thioflavin T (ThT), a fluorophore and molecular rotor known to

tag specific fibril structures in amyloids, can “label” the structural integrity of widely used and intrinsically nonfluorescent silica nanoparticles (SiNPs). Entrapment of ThT in SiNPs controls the fluorophore’s relaxation pathway and leads to a red-shifted fluorescence spectrum providing real-time information on SiNP integrity. The dynamic change of ThT fluorescence during degradation of doped SiNPs is found much higher than that of common labels fluorescein and rhodamine (Meder et al., 2016).

Antimicrobial Effects of Nanoparticles Antimicrobial application of nanoparticles has been the traditional forte of nanoparticles application. A plethora of nanomaterials have been indicated for antimicrobial activity (Fig. 5.10B) (Wang et al., 2017). Owing to its unique properties, nanoparticles can exert their impact on the microbial cells at all levels, breaching and breaking its cellular integrity, thereby causing microbial cell death. Singh et al. (2016) reported loss of outer membrane integrity in Gram-negative pathogenic bacteria (Shigella dysenteriae, Salmonella infestis, and Vibrio

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FIG. 5.10 Selective bioimaging (A) of tumor cells by sialic acid binding of lectin-coated rhodamine-dyed nanoparticles. Specific antimicrobial target (B) and types of nanoparticles used for antimicrobial activity. (Adapted with permission from Cho, J., Kushiro, K., Teramura, Y., Takai, M., 2014. Lectin-tagged fluorescent polymeric nanoparticles for targeting of sialic acid on living cells. Biomacromolecules 15, 2012e2018, copyright American Chemical Society, 2014.)

parahaemolyticus) by silver nanoparticles loaded with Camellia sinensis leaf phytochemicals. The authors reported biosynthesis of silver nanoparticles using tea leaves (Camellia sinensis) decoction and were characterized using standard protocols. Spectral changes confirmed as-prepared silver nanoparticles interaction with the hydrophobic moiety of lipopolysaccharide, the outer membrane component of most of the Gram-negative bacteria. The nanostructured material was also demonstrated to inhibit biofilm formation by the three strains of bacteria in silo or in synergies.

Choudhary et al. (2017) fabricated nontoxic reduced graphene oxide protein nanoframework as sustained antimicrobial coating for biomedical application. The as-synthesized reduced graphene oxide protein nanoframework exhibited dose-dependent antibacterial activity and potential of killing of 94% of Escherichia coli when treated with 80 mg mL
1 of reduced graphene oxide for 4 h. Buszewski et al. (2018) reported synthesis of silver nanoparticles using acidophilic actinobacteria strain of Streptacidiphilus durhamensis HGG 16n isolate. The as-

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prepared nanomaterial ranged in size from 8 to 48 nm and was predominantly spherical in shape. Antimicrobial assay of the nanostructured material against the pathogenic microbes resulted in highest antimicrobial activity in the order of Pseudomonas aeruginosa, Staphylococcus aureus, Proteus mirabilis, Escherichia coli, Klebsiella pneumonia, and Bacillus subtilis.

Nanoparticles as Antitumor Agents Biomedical applications nanotechnology has gained popularity in recent past. Gold nanoparticles are known to possess unique combination of properties that allow them to act as highly multifunctional anticancer agents. In addition to their use as targeted contrast agents for photochemical cancer therapy, they can serve as scaffolds for increasingly potent cancer drug delivery, as a transfection agent for customized gene therapy and as intrinsic antineoplastic agents (Dreaden et al., 2011). The strong light absorbing properties of AuNPs makes it suitable as heat mediating objects; the absorbed light energy is dissipated into the surroundings of the particles, leading to generation of an elevated temperature in their vicinity. This effect can be employed to open polymer microcapsules, for example, for drug-delivery purposes and even destroy the cancerous cells. Wu et al. (2014) reported formulation of gold nanorods and silica-containing gold nanoshells that demonstrated nanomaterial-mediated photo-thermal

therapeutic efficiency against A549 and HeLa cancer cell lines. They demonstrated that both the number of particles and photothermal conversion efficiency of the as-prepared nanomaterials were the determining factors for the amount of heat generated and photothermal therapeutic efficiency in both cell types. Hence, the establishment of threshold average dosage for different nanomaterials (the minimum dose at which the heat production is efficient enough to cause death of tumor cells) is key to application of such technology for cancer therapy. The nanoparticles are functionalized with antibody specific to the cancerous cells. The functionalized nanoparticles specifically bind with the targeting cells, which can then be killed by hyperthermal therapy through heating the particle-loaded tissue. However, for such in vivo applications, the potential cytotoxicity of the nanoparticles might become a limitation and should be investigated with care. The size of nanoparticles determines their penetration ability and persistence in the tumor cells. The moderate particle size (50e200 nm) leads to intratumor accumulation, while both the deep penetration into tumor and the quick removal from the body demand particle size to be as small as possible (generally less than 10 nm). Rana et al. (2014) reported the use of nanostructured material in targeted tumor cell killing by inductive heating. Fig. 5.11 illustrates a scheme for nanohybrid

FIG. 5.11 Schematic representation of nanostructured material-mediated targeted tumor cell killing using the principle of inductive heating.

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preparation and its application. Tumor-specific disintegrable nanohybrids containing nanoscale functional inorganic nanoparticles could achieve long blood circulation and extravagate after reaching the tumor site, and they could be triggered by stimuli to disintegrate into small particles in favor of their penetration into deep tumor tissue and their clearance by the body (Wang et al., 2018).

CURRENT COMMERCIAL DEMANDS The market potential of biologically active nanostructured materials is huge and promises delivery of products with unique characteristics such as biocompatibility and biodegradability. Unavailability of organs for transplantation has led to death of many in India. Over 75,000 livers, 200,000 kidneys, and 50,000 hearts are in demand across the hospitals in the country at any given point of time (Business Standard, 2015). However, the current availability through organ donation and cadaver transplants is around 1500 livers, 7000 kidneys, and only 50 hearts. This exposes the huge gap that exists between the demand and the delivery of organ transplants. This opportunity can be wisely utilized by means of nanostructured material-based scaffold formation and tissue engineering. The worldwide tissue engineering market was valued at around 5 billion USD in 2016 and is expected to grow at a healthy rate (Grand view research, 2018). Another major area of exploration for nanostructured materials is the field of antibiotics. Silver nanostructured materials of varying sizes and shapes have been reported for its antimicrobial activity. Presently, the worldwide antibiotic market is expected to register a growth rate of 5% over the forecast period (2018e23) (Mordor Intelligence, 2018). With the global attention focused on addressing the challenge of multidrug-resistant microbes, nanostructured materials with its unconventional killing procedures (causing physical insults, etc.) can be brought into use, thereby increasing the market potential of nanostructured materials. As per market research, antimicrobial coating market is projected to reach USD 4.19 billion by 2021, at a CAGR (Compound Annual Growth Rate) of 12.1% from 2016 to 2021 (Market and Markets, 2017). Medical device is an application arena of nanostructured materials promising high dividends. Such devices are categorized into six major categories, out of which diagnostics imaging constitutes the largest chunk from the global perspective, with a yearly sales estimate of 60 billion USD in 2015. The net worth of global medical devices was estimated at 228 billion USD in 2015.

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At its current growth forecasts, the market is expected to reach 332 billion USD mark by 2020 (Make in India, 2017).

CONCLUSION AND FUTURE PROSPECTS The conventional methods of nanostructure material preparation lead to harmful impact on the biological environment that led to the development of green chemical synthesis as a promising route of nanomaterial synthesis. However, the possible impact of asprepared nanomaterials on biological cells had to be ascertained before releasing the same for commercial use. Thus, multiple tests were developed to gauge the impact of nanostructured materials on various biological cells. However, each of the tests had certain limitations. One might give a quantitative data (such as Trypan blue), whereas other might provide a qualitative data (such as LDH test). Hence, goal-oriented assessment of cell viability following nanomaterial exposure is the present trend. Application of nanostructured materials is a user choice-based feature. Ideally, nanomaterials can be produced and programmed in any manner to suite the consumer demands. Investigator visualization with the nanostructured materials is the only limitation. As discussed earlier, nanomaterials have been used for manufacturing plant growth factors, tissue engineering, stem cell applications, bioimaging and labeling, antimicrobial effects, and antitumor activity. The future is vast and immense possibilities are waiting. Few of the future projections that can be implied in the present discussion is the formulation of a more robust test method for scoring biological impact of nanoparticles exposure in all fronts. Scoring death of biological cells owing to nanoparticle exposure has been investigated in detail. However, there are other parameters that may not lead to mortality, but cause morbidity in biological cells. Such tests for scoring morbidity must be explored for gaining more indepth information about the impact of nanomaterial application on biological cells. Furthermore, application of nanomaterials should be fine-tuned. Despite several reports of nanomaterialbased inhibition of tumor growth, very few of the products reach the consumers and therefore there is a dearth in the treatment procedures. Human disease and means of combating disease have perpetually been the most ancient and concurrent pursuit of humankind. Production of robust treatment modules based on nanostructured materials is now both the need and the possibility that the humankind look up with significant expectations.

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Dynamics of Advanced Sustainable Nanomaterials and Their Related Nanocomposites

ACKNOWLEDGMENTS M.D.D. gratefully acknowledges Department of Science and Technology (DST), Government of India for National Post-Doctoral Fellowship (NPDF).

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CHAPTER 5

Materials for Culturing of Various Biological Cells

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FURTHER READING Dvir, T., Timko, B.P., Kohane, D.S., Langer, R., 2011. Nanotechnological strategies for engineering complex tissues. Nat. Nanotechnol. 6, 13e22. epagov, 2000. N,N-Dimethylformamide. Retrieved from: https://www.epa.gov/sites/production/files/2016-09/ documents/n-n-dimethylformamide.pdf. sciencelabcom, 2013. Material Safety Data Sheet Tetrabutylammonium Hydroxide, 40% MSDS. Retrieved from: http:// www.sciencelab.com/msds.php?msdsId¼9925188. Sotiriou, G.A., Singh, D., Zhang, F., Chalbot, M.G., SpielmanSun, E., Hoering, L., Kavouras, I.G., Lowry, G.V., Wohlleben, W., Demokritou, P., 2016. Thermal decomposition of nano-enabled thermoplastics: possible environmental health and safety implications. J. Hazard Mater. 305, 87e95. Wel, C.V.D., Vahid, A., Saric, A., Idema, T., Heknrich, D., Kraft, D.J., 2016. Lipid membrane-mediated attraction between curvature inducing objects. Sci. Rep. UK 6, 32825e32835.