**4. Novel approaches in cytotoxicity studies**

#### **4.1. Phenotype arrays**

Phenotype is the effect exerted by molecules (e.g., drugs, nanoparticles, etc.) on a cell, tissue or whole organism; thus, the phenotype screening provides a holistic analysis that usually is more comprehensive than the sum of its parts. "*Phenomics*" is a part of complex technologies that also include transcriptomics, proteomics and metabolomics. PMs give a possibility to screen thousands of cellular phenotypes in real time [64].

Phenotype MicroArrays™ (PMs) are a combination of microplate reader (that can measure OD every 1 min over few hours and provide information about kinetics of carbon energy reactions in a selected cell model) and microscopic modules equipped with fluorescence, brightfield, colour brightfield and phase contrast microscopy (for scanning changes in cell morphology during experiments). Phenotypic assays deliver more information and provide better understanding of the metabolic and cytotoxic effect of test substances [65]. Multiplex arrays can generate information on the use of energy pathways (based on the application of different nutrition analyses, PM-M1 to M4), effects of ions (PM-M5), hormones, metabolic effectors (PM-M6 to M8) and anti-cancer agents (PM-M11 to M14) (**Table 4**), cell number, cell health (based on cell health monitoring using phase contrast microscopy and kinetic determination of cellular energy) and apoptotic induction (via cell subpopulation analysis—examination of the increase in circularity due to cell shrinkage and cytoplasm condensation and lower phase signal exhibition) [65]. PMs can be used in genotype/phenotype analyses, cell line characterisation, metabolic reprogramming, cellular phenotype stability, Warburg effect, cell differentiation or bioprocess development [64]. Well-characterised model cell lines (e.g., HepG2, C3A, Colo205, A549, PC-3, IMR90, HL-60 or CEM) with defined metabolic properties can be used with the PM system to determine specific effects of nanomaterials on selected cell lines and to accurately identify the mechanism involved in the NP effect (e.g., mitochondrial toxicology) on the living system [66]. Array wells coated with different substances and combined with the redox assay (MA or MB redox dyes to measure cell energy [NADH] changes) are used for phenotypic determination. Comparison of two cell lines is visualised by bioinformatic software that highlights differences in recorded phenotypes (**Figure 5**) [64, 67].

on nanoparticle surface. Moreover, the effect of "self-healing" defects between both Si─O─Si bonds of double-linked Si atoms explains the very low rate of dissolution at the point of zero

('contr' – control sample) and mSiO<sup>2</sup>

A similar effect of PSB incubation on mesoporous silica nanospheres was observed by Yamada

dation with the final shape deformation and collapse of structures [61]. Another study based on core-shell magnetic mesoporous silica nanoparticles presented comparable results. Silica mesoporous hollow shells immersed in PBS for 2 days displayed structure deformation and additional cavities, whereas 8-day incubation showed complete degradation and coagulation resulting in new structure formation [62]. The erosion of mesoporous silica nanosphere structure modified with titanium dioxide was also observed in contact with *Streptomyces* cells. After 24-h incubation, mesoporous silica shell structure was degraded with simultaneous appearance of agglomerates, which clearly demonstrated that nanomaterial structure and

Phenotype is the effect exerted by molecules (e.g., drugs, nanoparticles, etc.) on a cell, tissue or whole organism; thus, the phenotype screening provides a holistic analysis that usually is more

porous structure was degradable

incubated in PBS, for 24 (a),

displayed size and shape degra-

net proton charge (PZPC) of the surface [60].

**Figure 4.** Transmission electron microscopy of mSiO<sup>2</sup>

48 (b) and 96 h (c, d) [58].

190 Cytotoxicity

composition could be affected by living cells [63].

**4.1. Phenotype arrays**

**4. Novel approaches in cytotoxicity studies**

and co-workers [61], as these authors found that the mSiO<sup>2</sup>

after 2-day incubation in PBS. After 3-day incubation, mSiO<sup>2</sup>

For example, Phenotype MicroArrays™ (PM-M TOX1 Plate Energetic Substrate Assay, 96-well microplate coated with eight different oxidisable carbon sources—each of the eight nutrition sources coated on one of eight rows on a microplate) give the possibility to screen cell-based energetic phenotype in a target cell model, for example, the MDA-MB-231 RFP breast cancer cell line, using different cellular nutrition sources (e.g., α-D-glucose, inosine, D-galactose, D-glucose-1-phosphate, xylitol, α-ketoglutaric acid, D,L-β-hydroxybutyric acid or pyruvic acid). This kind of multiplex analysis provides information on cell morphology, metabolic activity (metabolic pathway activity), sensitivity in response to particular energetic additives and the final cellular genetic background characterisation. The addition of an apoptotic agent (e.g., oridonin), chemical inhibitor or stimulator provides an opportunity to evaluate the potential mechanism regulating the energy pathway [65, 66]. Another example of PM application was presented by Bochner et al. [68]. Based on four phenotypic assays (PM-M1 to M4, containing 367 substrate nutrients), different human cancer cell lines, including HepG2/C3A, HepG2, Colo 205, A549, PC-3, HL-60 and CCRF-CEM and two murine white and brown adipocyte cell lines were analysed to determine energy-producing pathways. The results showed that human cancer cell lines exhibited distinct metabolic activity profiles. Moreover, white and brown adipocyte cell lines also had different profiles of energetic activity; metabolic fingerprints were established in all cell lines [68]. Similarly, human endothelial cells from the coronary artery


**Phenotype MicroArrays™** **Feature Substrates/agents**

PM-M3 (Biolog) Energetic substrate array Carbon and energy sources/nitrogen sources (dipeptides):

PM-M4 (Biolog) Energetic substrate array Carbon and energy sources/nitrogen sources (dipeptides):

α-D-glucose

IL-2, IL-6, IL-8

PM-M5 (Biolog) Ions: NaCl, ammonium chloride, sodium selenite,

PM-M6 (Biolog) Hormone and metabolic effectors: dibutyryl-cAMP,

PM-M7 (Biolog) Hormone and metabolic effectors: insulin, resistin,

Gly-Gly, Gly-His, Gly-Ile, Gly-Leu, Gly-Lys, Gly-Met, Gly-Phe, Gly-Pro, Gly-Ser, Gly-Thr, Gly-Trp, Gly-Tyr, Gly-Val, His-Ala, His-Asp, His-Glu, His-Gly, His-His (c), His-Leu, His-Lys (d), His-Met, His-Pro, His-Ser, His-Trp, His-Tyr, His-Val, Ile-Ala, Ile-Arg (b), Ile-Asn, Ile-Gln, Ile-Gly, Ile-His, Ile-Ile, Ile-Leu, Ile-Met, Ile-Phe, Ile-Pro, Ile-Ser, Ile-Trp, Ile-Tyr, Ile-Val, Leu-Ala, Leu-Arg (b), Leu-Asn, Leu-Asp, Leu-Glu, Leu-Gly, Leu-His, Leu-Ile, Leu-Leu, Leu-Met, Leu-Phe, Leu-Pro, Leu-Ser, Leu-Trp, Leu-Tyr, Leu-Val, Lys-Ala (d), Lys-Arg (b), Lys-Asp, Lys-Glu, Lys-Gly, Lys-Ile (b), Lys-Leu (b), Lys-Lys, Lys-Met (e), Lys-Phe, Lys-Pro, Lys-Ser, Lys-Thr, Lys-Trp (b), Lys-Tyr (b), Lys-Val (d), Met-Arg (b), Met-Asp, Met-Gln, Met-Glu, Met-Gly, Met-His, Met-Ile, Met-Leu, Met-Lys (e), Met-Met, Met-Phe, Met-Pro, Met-Thr, Met-Trp, Met-Tyr, Met-Val, Phe-Ala, Phe-Asp, Phe-Glu, α-D-glucose

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Phe-Gly, Phe-Ile, Phe-Met, Phe-Phe, Phe-Pro, Phe-Ser, Phe-Trp, Phe-Tyr, Phe-Val, Pro-Ala, Pro-Arg (b), Pro-Asn, Pro-Asp, Pro-Glu, Pro-Gln, Pro-Gly, Pro-Hyp, Pro-Ile, Pro-Leu, Pro-Lys (b), Pro-Phe, Pro-Pro, Pro-Ser, Pro-Trp, Pro-Tyr, Pro-Val, Ser-Ala, Ser-Asn, Ser-Asp, Ser-Glu, Ser-Gln, Ser-Gly, Ser-His (b), Ser-Leu, Ser-Met, Ser-Phe, Ser-Pro, Ser-Ser, Ser-Tyr, Ser-Val, Thr-Ala, Thr-Arg (f), Thr-Asp, Thr-Glu, Thr-Gln, Thr-Gly, Thr-Leu, Thr-Met, Thr-Phe, Thr-Pro, Thr-Ser, Trp-Ala, Trp-Arg, Trp-Asp, Trp-Glu, Trp-Gly, Trp-Leu, Trp-Lys (e), Trp-Phe, Trp-Ser, Trp-Trp, Trp-Tyr, Trp-Val, Tyr-Ala, Tyr-Gln, Tyr-Glu, Tyr-Gly, Tyr-His, Tyr-Ile, Tyr-Leu, Tyr-Lys, Tyr-Phe, Tyr-Trp, Tyr-Tyr, Tyr-Val, Val-Ala, Val-Arg, Val-Asn, Val-Asp, Val-Glu, Val-Gln, Val-Gly, Val-His, Val-Ile, Val-Leu, Val-Lys, Val-Met, Val-Phe, Val-Pro, Val-Ser, Val-Tyr, Val-Val,

potassium chloride, calcium chloride, manganese chloride, zinc chloride, copper (II) chloride, cobalt chloride, iodine, sodium phosphate, sodium sulphate, sodium molybdate, sodium tungstate, sodium orthovanadate, potassium chromate, sodium pyrophosphate, sodium nitrate, sodium nitrite, lithium chloride, ferric chloride, magnesium chloride

3-isobutyl-1-methylxanthine, caffeine, epinephrine, norepinephrine, L-leucine, creatine, triiodothyronine, thyroxine, dexamethasone, hydrocortisone, progesterone, β-estradiol, 4,5α-dihydro-testosterone, aldosterone

glucagon, ghrelin, leptin, gastrin, exendin-3, hGH (somatotropin), IGF-I, FGF-1 (aFGF), PDGF-AB, IL-1β,


**Phenotype MicroArrays™**

192 Cytotoxicity

PM-M TOX1 (Biolog) Effect of a tested factor

on energy production (mitochondrial toxicity)

**Feature Substrates/agents**

PM-M1 (Biolog) Energetic substrate array Carbon and energy sources (simple sugars,

acid

Eight different carbon source: α-D-glucose, inosine, D-galactose, D-glucose-1-phosphate, xylitol,

polysaccharides, carboxylic acids): cyclodextrin, dextrin, glycogen, maltitol, maltotriose, D-maltose, D-trehalose, D-cellobiose, gentiobiose, D-glucose-6 phosphate, D-glucose-1-phosphate, L-glucose, D-glucose, 3-O-methyl-D-glucose, methyl-D-glucoside, D-salicin, D-sorbitol, N-acetyl-D-glucosamine, D-glucosaminic acid, D-glucuronic acid, chondroitin-6-sulphate, mannan, D-mannose, methyl-D-mannoside, D-mannitol, N-acetylβ-D-mannosamine, D-melezitose, sucrose, palatinose, D-turanose, D-tagatose, L-sorbose, L-rhamnose, L-fucose, D-fucose, D-fructose-6-phosphate, D-fructose, stachyose, D-raffinose, D-lactitol, lactulose, α-D-lactose, melibionic acid, D-melibiose, D-galactose, α-methyl-D-galactoside, N-acetyl-neuraminic acid, pectin, sedoheptulosan, thymidine, uridine, adenosine, inosine, adonitol, L-arabinose, D-arabinose, β-methyl-D-xylopyranoside, xylitol, myo-inositol, meso-erythritol, propylene glycol, ethanolamine D,L- α-glycerol-phosphate, glycerol, citric acid, tricarballylic acid, D,L-lactic acid, methyl D-lactate, methyl pyruvate, pyruvic acid, α-keto-glutaric acid, succinamic acid, succinic acid, mono-methyl succinate, tricarballylic acid, L-malic acid, D-malic acid, mesotartaric acid, acetoacetic acid (a), γ-amino-N-butyric acid, α-keto-butyric acid, α-hydroxy-butyric acid, D,L-βhydroxy-butyric acid, γ-hydroxy-butyric acid, butyric acid, 2,3-butanediol, 3-hydroxy-2-butanone, propionic

acid, acetic acid, hexanoic acid

derived nutrients, primarily amino acids, dipeptides): Tween 20, Tween 40, Tween 80, gelatin, L-alaninamide, L-alanine, D-alanine, L-arginine, L-asparagine, L-aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid, L-glutamine, glycine, L-histidine, L-homoserine, hydroxy-L-proline, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, D-serine, L-threonine, D-threonine, L-tryptophan, L-tyrosine, L-valine, Ala-Ala, Ala-Arg, Ala-Asn, Ala-Asp, Ala-Glu, Ala-Gln, Ala-Gly, Ala-His, Ala-Ile, Ala-Leu, Ala-Lys, Ala-Met, Ala-Phe, Ala-Pro, Ala-Ser, Ala-Thr, Ala-Trp, Ala-Tyr, Ala-Val, Arg-Ala (b), Arg-Arg (b), Arg-Asp, Arg-Gln, Arg-Glu, Arg-Ile (b), Arg-Leu (b), Arg-Lys (b), Arg-Met (b), Arg-Phe (b), Arg-Ser (b), Arg-Trp, Arg-Tyr (b), Arg-Val (b), Asn-Glu, Asn-Val, Asp-Ala, Asp-Asp, Asp-Glu, Asp-Gln, Asp-Gly, Asp-Leu, Asp-Lys, Asp-Phe, Asp-Trp, Asp-Val, Glu-Ala, Glu-Asp, Glu-Glu, Glu-Gly, Glu-Ser, Glu-Trp, Glu-Tyr, Glu-Val, Gln-Glu, Gln-Gln, Gln-Gly, Gly-Ala, Gly-Arg, Gly-Asn, Gly-Asp,

PM-M2 (Biolog) Energetic substrate array Carbon and energy sources/nitrogen sources (protein-

α-D-glucose

α-ketoglutaric acid, D,L-β-hydroxybutyric acid, pyruvic


The profiling of human normal and cancer cells was also conducted by Parmar et al. [70]. HEK293, OV90, TOV112D, KLE, MES-SA and SKBR cell lines were selected to determine differences in response to anti-cancer agents using PM (PM-M11 to M14) and the effect of these agents on the mTOR signalling pathway by measuring S6 kinase (S6K) level. From a wide range of anti-cancer drugs, celastrol was found to inhibit the growth of SKBR, MESA-SA and TOV11D and target the mTOR signalling pathway [70]. In another study, Martinez-Reyes et al. [71] reported that mitochondrial metabolism was necessary for histone acetylation, hypoxiainducible transcription factor (HIF-1) activation and proliferation based on WT-POLG and

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The application of PMs in nanotechnology is only a matter of time, thanks to the efficient and rapid determination of precise sites and modes of action of the tested substances. PMs provide a possibility to compare specificities of the study agents (e.g., drug and nanomaterialdrug conjugates) and the effect of the agent and its side effects. Finally, the PM system can be

The limitations of large-scale phenotyping techniques, including PM analysis, are related to the characteristics of all cells. PMs will not reveal the phenotypes of all cells, because cells have many phenotypes that are dependent on their environments. Different cells are constantly adapting in various ways to culture (environment) changes by altering their gene-expression pattern, protein content, membrane and cytoskeleton constitution and surface receptors. Moreover, the PM system will likely not record phenotypes that specifically involve intracellular structures (e.g., cytoskeleton, organelles or surface structures). In addition, the effect of some genes might be cryptic and the function of those genes only occurs under highly specific conditions; thus, it cannot be always determined in conditions

Another approach to phenotypic screening is focused on microarray-based three-dimensional (3D) systems. 3D culture models may better mimic the *in vivo* cellular microenvironment and may be critical for cell phenotypes [74]. It should also be mentioned that cell migration, compound-mediated cytotoxicity, cellular adhesion, proliferation and differentiation can also be evaluated using non-invasive, labelled-free xCELLigence system. Electrical impedance monitoring is based on a set of gold microelectrodes fused to the bottom surface of a microtitre plate

used for drug interactions or drug-nanomaterial analysis [67, 72].

**Figure 5.** Schematic visualisation that highlights differences in recorded phenotypes [64].

DN-POLG-HEK293 cell lines [71].

provided by PM cultures [73].

**Table 4.** Array examples [65].

(HCAEC), umbilical vein (HUVEC) and normal lung fibroblasts (NHLFs) were selected for cellular metabolism monitoring also with the use of phenotypic assays (PM-M1 to M4). The results obtained in this study demonstrated that all three cell lines strongly utilised adenosine, inosine, D-mannose and dextrin. HCAEC also metabolised mannan, pectin, gelatine and tricarballylic acid, while the HUVEC cell line did not exhibit the ability to metabolise any other unique substrates. NHLFs were able to additionally utilise sugars and carboxylic acids [69].

**Figure 5.** Schematic visualisation that highlights differences in recorded phenotypes [64].

The profiling of human normal and cancer cells was also conducted by Parmar et al. [70]. HEK293, OV90, TOV112D, KLE, MES-SA and SKBR cell lines were selected to determine differences in response to anti-cancer agents using PM (PM-M11 to M14) and the effect of these agents on the mTOR signalling pathway by measuring S6 kinase (S6K) level. From a wide range of anti-cancer drugs, celastrol was found to inhibit the growth of SKBR, MESA-SA and TOV11D and target the mTOR signalling pathway [70]. In another study, Martinez-Reyes et al. [71] reported that mitochondrial metabolism was necessary for histone acetylation, hypoxiainducible transcription factor (HIF-1) activation and proliferation based on WT-POLG and DN-POLG-HEK293 cell lines [71].

The application of PMs in nanotechnology is only a matter of time, thanks to the efficient and rapid determination of precise sites and modes of action of the tested substances. PMs provide a possibility to compare specificities of the study agents (e.g., drug and nanomaterialdrug conjugates) and the effect of the agent and its side effects. Finally, the PM system can be used for drug interactions or drug-nanomaterial analysis [67, 72].

The limitations of large-scale phenotyping techniques, including PM analysis, are related to the characteristics of all cells. PMs will not reveal the phenotypes of all cells, because cells have many phenotypes that are dependent on their environments. Different cells are constantly adapting in various ways to culture (environment) changes by altering their gene-expression pattern, protein content, membrane and cytoskeleton constitution and surface receptors. Moreover, the PM system will likely not record phenotypes that specifically involve intracellular structures (e.g., cytoskeleton, organelles or surface structures). In addition, the effect of some genes might be cryptic and the function of those genes only occurs under highly specific conditions; thus, it cannot be always determined in conditions provided by PM cultures [73].

Another approach to phenotypic screening is focused on microarray-based three-dimensional (3D) systems. 3D culture models may better mimic the *in vivo* cellular microenvironment and may be critical for cell phenotypes [74]. It should also be mentioned that cell migration, compound-mediated cytotoxicity, cellular adhesion, proliferation and differentiation can also be evaluated using non-invasive, labelled-free xCELLigence system. Electrical impedance monitoring is based on a set of gold microelectrodes fused to the bottom surface of a microtitre plate

(HCAEC), umbilical vein (HUVEC) and normal lung fibroblasts (NHLFs) were selected for cellular metabolism monitoring also with the use of phenotypic assays (PM-M1 to M4). The results obtained in this study demonstrated that all three cell lines strongly utilised adenosine, inosine, D-mannose and dextrin. HCAEC also metabolised mannan, pectin, gelatine and tricarballylic acid, while the HUVEC cell line did not exhibit the ability to metabolise any other unique substrates. NHLFs were able to additionally utilise sugars and carboxylic acids [69].

**Feature Substrates/agents**

PM-M8 (Biolog) Hormone and metabolic effectors: (Arg8) – vasopressin,

salt

ellagic acid

PM-M11 (Biolog) Anti-cancer agents: solasodine, rotenone, aklavine

PM-M12 (Biolog) Anti-cancer agents: tamoxifen citrate, thioguanine,

PM-M13 (Biolog) Anti-cancer agents: monocrotaline, altretamine,

PM-M14 (Biolog) Anti-cancer agents: cepharanthine, 4′-demethyl

parathyroid hormone, prolactin, calcitonin, calcitriol (1α,25-dihydroxyvitamin D3), luteinizing hormone (LH), luteinizing hormone releasing hormone (LH-RH), chorionic gonadotropin human (HCG), adrenocorticotropic hormone human (ACTH), thyrotropic hormone (TSH), thyrotropin releasing hormone acetate salt (TRH), IFN-γ, TNF-α, adenosine, Gly-His-Lys acetate

hydrochloride, deguelin(−), celastrol, juglone, sanguinarine sulphate, dactinomycin, methylmethane sulfonate, azathioprine, busulfan, aclarubicin, chloramphenicol, chloroquine diphosphate, cyclophosphamide, diethylcarbamazine citrate, emetine, fluorouracil, hydroxyurea, mechlorethamine, mercaptopurine, quinacrine hydrochloride, streptozosin

acriflavinium hydrochloride, pentamidine isethionate, mycophenolic acid, aminopterin, berberine chloride, emodin, puromycin hydrochloride, neriifolin, 5-fluoro-5′-deoxyurldine, carboplatin, cisplatin, zidovudine (AZT), azacytidine, cycloheximide, azaserine, p-fluorophenylalanine, dimethylhydrazine hydrochloride, phenethyl caffeate (CAPE), camptothecin, amygdalin,

carmustine, mitoxantrone hydrochloride, urethane, thiotepa, thiodiglycol, pipobroman, etanidazole, semustine, gossypol, formestane, ancitabine hydrochloride, nimustine, aminolevulinic acid hydrochloride, picropodo-phyllotoxin, beta-peltatin, perillyl alcohol, dibenzoylmethane, 6-amino nicotinamide,

epipodophyllotoxin, miltefosine, elaidyl phosphocholine, podofilox, colchicine, methotrexate, acivicin, floxuridine, lefunomide, rapamycin, 13-cis retinoic acid, all-trans retinoic acid, piceatannol, (+)-catechin, mitomycin C, cytosine-beta-D-arabinofuranoside, daunorubicin hydrochloride, doxorubicin hydrochloride, etoposide, nocodazole, quercetin dihydrate, vinblastine sulphate

carmofur, indole-3-carbinol, rifaximin

**Phenotype MicroArrays™**

194 Cytotoxicity

**Table 4.** Array examples [65].

well. The magnitude of impedance is dependent on the number of cells, the size and shape of the cells and cell-substrate attachment quality; therefore, it gives the possibility to analyse the effect, for example, of nanomaterials on cell morphology, adhesion and biocompatibility [75].

was added to the culture. The second experiment involved MC3T3 plated on transparent titanate nanotubes (TNT) surface and the impact on adhesion and spreading process of the cells was demonstrated using the HoloMonitor. The authors have concluded that holographic digital microscopy is a useful tool for cellular behaviour analysis, but some limitations have also been found. Peter et al. [78] observed that under certain thicknesses, some parts of the cells (e.g., parts of the thin lamellipodium) slicked into the background surface. It was caused

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In our study, the effect of the h-BN-Au nanocomposite on L929 and MCF-7 cell lines was analysed during 12-h incubation using the HoloMonitor™ M4. L929 cells did not show any significant differences in the presence of the nanocomposite and the doubling time (DT) value was similar to DT obtained in the control culture (**Figure 6**). The results obtained for the MCF-7 cell line incubated with h-BN-Au demonstrated a stronger effect on cells. The DT analysis using holographic technique indicated a high reduction of proliferation capacity (the DT value for the MCF-7 control sample was 25.95 h, whereas for experimental cultures, it was

**Figure 6.** The morphology of the L929 and MCF-7 cell lines incubated with the h-BN-Au nanoparticles. L929 culture

time-points at 0 h (A); at 12 h (B); MCF-7 culture time-points at 0 h (C); at 12 h (D) [83].

by the limited vertical resolution of the optical system [78].

469.9 h) [83].

#### **4.2. Digital holography (DH) microscopy**

Holographic (transmission) microscopy is a high-resolution imaging technique that provides label-free and non-invasive, non-phototoxic and non-destructive method for real-time live cell culture analysis [76]. This type of microscopy allows for quantitative and qualitative measurements of living cells (not only cultures of mammalian cells, but also protozoan, bacterial and plant cells) and collecting information about cell surface area, cell viability and morphological changes, such as differentiation, proliferation, motility, cell death, confluence or cell segmentation (calculated from a particular hologram) [77–80]. Traditional brightfield microscopy has some limitations, such as difficulties in visualising individual cells due to their low contrast properties, whereas DH microscopy provides possibility to determine cell number directly in cell culture vessels [81]. The size of the HoloMonitor™ M4 (Phase Holographic Imaging AB, Lund, Sweden) makes it possible to place it in a cell culture incubator, so that cell observations can be conducted over long periods of time without any changes in cell culture conditions [78]. Digital holographic microscopy also enables the formation of three-dimensional (3D) images of the observed objects.

The presented technique is based on the phase shift (ϕ) of the probing laser light (or other coherent light source) that can be reflected or transmitted through the monitored object. The illuminating light is split into two beams (differing in phase): an object beam and a reference beam [78, 81]. The reference beam remains undisturbed, while the object beam is shifted in phase by the object [79]. Next, the object beam is re-joined and interferes with the reference beam and creates a hologram that is recorded on a digital image sensor (CCD or CMOS) [77, 81]. The total phase shift can be translated into optical thickness (*L*) and depends on the physical thickness of the examined object, wavelength (λ) and refraction index (*n*). Optical thickness can be measured at nanometre resolution [78, 81].

Holographic phase imaging is an excellent tool for cell morphometric characterisation and cell migration studies. This technique has recently been applied in clinical diagnostics, e.g., screening for malaria infection of erythrocytes, cancer cell analyses or sperm quality [79]. Interest in the use of DH microscopy in research is constantly increasing. For example, Lajkó et al. [82] analysed the effect of a drug based on GnRH-III (gonadotropin-releasing hormone-III) on melanoma cells. Holographic phase imaging was used to visualise the migratory behaviour of melanoma cells in response to daunorubicin (Dau) coupled with GnRH-III and its derivatives (modified at position 4 with Lys(Ac) (conj1) or Lys(nBu) (conj2)). Cell migration analysis showed increased migration activity when cells were exposed to conj1, whereas conj2 decreased melanoma cell activity and exerted an immobilising effect on tumour cell spreading; thus, it was a better candidate for targeted tumour therapy [82]. Monitoring of HeLa cancer cells and MC3T3-E1 preosteoblast cells via holographic technique was also conducted by Peter et al. [78]. These authors evaluated cell movements and morphological parameters of cells in two experiments. In the first one, the HoloMonitor™ M4 was used to detect the effect of EGCg (green tea—epigallocatechin gallate) on HeLa cell motility. Time-lap images showed that migration, motility and the speed of motility were reduced after EGCg was added to the culture. The second experiment involved MC3T3 plated on transparent titanate nanotubes (TNT) surface and the impact on adhesion and spreading process of the cells was demonstrated using the HoloMonitor. The authors have concluded that holographic digital microscopy is a useful tool for cellular behaviour analysis, but some limitations have also been found. Peter et al. [78] observed that under certain thicknesses, some parts of the cells (e.g., parts of the thin lamellipodium) slicked into the background surface. It was caused by the limited vertical resolution of the optical system [78].

well. The magnitude of impedance is dependent on the number of cells, the size and shape of the cells and cell-substrate attachment quality; therefore, it gives the possibility to analyse the effect, for example, of nanomaterials on cell morphology, adhesion and biocompatibility [75].

Holographic (transmission) microscopy is a high-resolution imaging technique that provides label-free and non-invasive, non-phototoxic and non-destructive method for real-time live cell culture analysis [76]. This type of microscopy allows for quantitative and qualitative measurements of living cells (not only cultures of mammalian cells, but also protozoan, bacterial and plant cells) and collecting information about cell surface area, cell viability and morphological changes, such as differentiation, proliferation, motility, cell death, confluence or cell segmentation (calculated from a particular hologram) [77–80]. Traditional brightfield microscopy has some limitations, such as difficulties in visualising individual cells due to their low contrast properties, whereas DH microscopy provides possibility to determine cell number directly in cell culture vessels [81]. The size of the HoloMonitor™ M4 (Phase Holographic Imaging AB, Lund, Sweden) makes it possible to place it in a cell culture incubator, so that cell observations can be conducted over long periods of time without any changes in cell culture conditions [78]. Digital holographic microscopy also enables the formation of three-dimensional (3D) images of the observed objects.

The presented technique is based on the phase shift (ϕ) of the probing laser light (or other coherent light source) that can be reflected or transmitted through the monitored object. The illuminating light is split into two beams (differing in phase): an object beam and a reference beam [78, 81]. The reference beam remains undisturbed, while the object beam is shifted in phase by the object [79]. Next, the object beam is re-joined and interferes with the reference beam and creates a hologram that is recorded on a digital image sensor (CCD or CMOS) [77, 81]. The total phase shift can be translated into optical thickness (*L*) and depends on the physical thickness of the examined object, wavelength (λ) and refraction index (*n*). Optical

Holographic phase imaging is an excellent tool for cell morphometric characterisation and cell migration studies. This technique has recently been applied in clinical diagnostics, e.g., screening for malaria infection of erythrocytes, cancer cell analyses or sperm quality [79]. Interest in the use of DH microscopy in research is constantly increasing. For example, Lajkó et al. [82] analysed the effect of a drug based on GnRH-III (gonadotropin-releasing hormone-III) on melanoma cells. Holographic phase imaging was used to visualise the migratory behaviour of melanoma cells in response to daunorubicin (Dau) coupled with GnRH-III and its derivatives (modified at position 4 with Lys(Ac) (conj1) or Lys(nBu) (conj2)). Cell migration analysis showed increased migration activity when cells were exposed to conj1, whereas conj2 decreased melanoma cell activity and exerted an immobilising effect on tumour cell spreading; thus, it was a better candidate for targeted tumour therapy [82]. Monitoring of HeLa cancer cells and MC3T3-E1 preosteoblast cells via holographic technique was also conducted by Peter et al. [78]. These authors evaluated cell movements and morphological parameters of cells in two experiments. In the first one, the HoloMonitor™ M4 was used to detect the effect of EGCg (green tea—epigallocatechin gallate) on HeLa cell motility. Time-lap images showed that migration, motility and the speed of motility were reduced after EGCg

thickness can be measured at nanometre resolution [78, 81].

**4.2. Digital holography (DH) microscopy**

196 Cytotoxicity

In our study, the effect of the h-BN-Au nanocomposite on L929 and MCF-7 cell lines was analysed during 12-h incubation using the HoloMonitor™ M4. L929 cells did not show any significant differences in the presence of the nanocomposite and the doubling time (DT) value was similar to DT obtained in the control culture (**Figure 6**). The results obtained for the MCF-7 cell line incubated with h-BN-Au demonstrated a stronger effect on cells. The DT analysis using holographic technique indicated a high reduction of proliferation capacity (the DT value for the MCF-7 control sample was 25.95 h, whereas for experimental cultures, it was 469.9 h) [83].

**Figure 6.** The morphology of the L929 and MCF-7 cell lines incubated with the h-BN-Au nanoparticles. L929 culture time-points at 0 h (A); at 12 h (B); MCF-7 culture time-points at 0 h (C); at 12 h (D) [83].

#### **4.3. Atomic force microscopy (AFM)**

Atomic force microscopy (AFM) is based on a laser reflected off a cantilever onto a scanning surface of the examined object and quantitative information about surface morphology and cell spread is collected.

AFM is a crucial technique for determining cell interactions on the surface of the tested material. If material exhibits high biocompatibility, the surface of the material will allow cells to attach (interaction between cell-surface integrin receptors) and adsorb extracellular matrix (ECM) proteins. Surface properties, such as wettability, roughness or surface charge, are important for cellular attachment and lamellipodium/filopodium formation. The AFM measurement provides information on cellular morphology changes and lamellipodium/filopodium permissiveness. The measurement of atomic force microscopy of living cells can be performed in PBS and provides information on cell height, total cell surface area, attachment angle and extension of lamellipodia/filopodia. It is also possible to measure fixed cell (in 4% paraformaldehyde) topography and examine filopodia and lamellipodia. An interesting example is the analysis of H4 and PC12 cell lines plated on different materials—glass, polystyrene (PSt), silicon (Si), nanocrystalline diamond (NCD) and cubic silicon carbide (3C-SiC). In the latter study, AFM analysis demonstrated that the type of the surface determined cell height/area, attachment angle and the reduction of the lamellipodium/filopodium area. Cell-substrate interaction was different for H4 and PC12 cell lines, e.g., for H4 cells; the most negative interaction was recorded for glass, the most positive for 3C-SiC, while PC12 cells had the most negative interaction with glass, but the best with 3C-SiC and PSt. The authors concluded that AFM analysis indicated that neural cell interactions with 3C-SiC resulted in the optimal cell viability, morphology and interaction of cells with 3C-SiC surface [1]. Frewin et al. [1] published the results of AFM analysis concerning cellular interaction on graphene. The experiments focused on cytoskeleton organisation and the determination of the number of contact sites, and AFM technology can provide valuable information on the mechanism of cellular adhesion and proliferation on graphene surface. Different methods of graphene preparation, for example, mechanical cleaving, chemical synthesis and chemical vapour deposition (CVD) on metals or epitaxial growth on SiC, not only give graphene different electrical, optical or morphological properties, but also different biocompatibility. For example, the biocompatibility of a single graphene layer produced by CVD on Cu was higher in comparison with SiO<sup>2</sup> /Si surfaces studied on human osteoblasts and mesenchymal stem cells [1, 84]. In another study, epitaxially grown graphene films on (0001) 6H-SiC substrates were evaluated in cellular response experiments using AMF analysis. It was found that HaCaT (human keratinocytes) after 72-h culture on graphene and 6H-SiC surfaces exhibited similar morphology to cells cultured on the PSt control. On the other hand, the MTT assay suggested better biocompatibility for 6H-SiC than for the graphene surface. Moreover, different preparation of graphene surfaces (first one without any further surface treatment, and the second one additionally disinfected by immersion in ethanol) resulted in more homogeneous and increased cell adhesion on ethanol-sterilised graphene surface [1]. Our study also confirmed the undeniable value of AMF analysis in the experiment involving the MAC-T cell line seeded on different surfaces (glass, glass coated with poly-D-lysine) (**Figure 7**). In the aforementioned study, surface analysis and cell height analysis clearly exhibited differences in cell growth on the two surface variants [85].

Another notable study used the AFM technique not only for cell analysis after nanoparticle uptake, but also after exposure to rotating magnetic field (RMF). Observations of MCF-7 cells after 1.5-h incubation in 40 mT magnetic field revealed changes in cell surface, which was

**Figure 7.** AFM analysis of MAC-T cells: cell height on glass surface (A); cell height on glass coated with poly-D-lysine (B);

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http://dx.doi.org/10.5772/intechopen.72578

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The present overview describes and compares widely used biocompatibility/cytotoxicity assays in nanomaterial studies. Due to the type of nanoparticles and their properties, applicability of popular assays used for engineered nanomaterial screening might be limited. The significant numbers of false-positive or false-negative signals are generated [16]. The tendency

rougher with many small pore-like structures in comparison to untreated cells [86].

3D image of cell growing on glass surface (C); 3D image of cell growing on poly-D-lysine (D) [85].

**5. Conclusions**

of nanoparticles to:

General Cytotoxicity and Its Application in Nanomaterial Analysis http://dx.doi.org/10.5772/intechopen.72578 199

**Figure 7.** AFM analysis of MAC-T cells: cell height on glass surface (A); cell height on glass coated with poly-D-lysine (B); 3D image of cell growing on glass surface (C); 3D image of cell growing on poly-D-lysine (D) [85].

Another notable study used the AFM technique not only for cell analysis after nanoparticle uptake, but also after exposure to rotating magnetic field (RMF). Observations of MCF-7 cells after 1.5-h incubation in 40 mT magnetic field revealed changes in cell surface, which was rougher with many small pore-like structures in comparison to untreated cells [86].
