**General Cytotoxicity and Its Application in Nanomaterial Analysis Nanomaterial Analysis**

**General Cytotoxicity and Its Application in** 

DOI: 10.5772/intechopen.72578

Magdalena Jedrzejczak-Silicka and Ewa Mijowska Additional information is available at the end of the chapter

Magdalena Jedrzejczak-Silicka and Ewa Mijowska

Additional information is available at the end of the chapter

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

#### **Abstract**

The recent increasing interest in the use of different nanoparticles in biological and medical applications encouraged scientists to analyse their potential impact on biological systems. The biocompatibility analyses of novel materials for medical applications are conducted using quantitative and qualitative techniques collected by the International Standards Organization (ISO). The well-known assays, such as tetrazolium-based assays used for mitochondrial function monitoring, LDH for membrane permeability determination and neutral red uptake (NRU) describing lysosome function, need to be optimised due to specific properties of wide range of nanomaterials. Physicochemical properties of nanoparticles (NPs) such as size, composition, concentration, shape and surface (e.g., charge, coating, aspect ratio), as well as the cell type play a crucial role in determining the nanomaterial toxicity (also uptake pathway(s) of NPs). Different nanomaterials exhibit different cytotoxicity from relatively non-toxic hexagonal boron nitride to rutile TiO<sup>2</sup> NPs that induce oxidative DNA damage in the absence of UV light. Finally, the results of the nanomedical analysis can be enriched by holographic microscopy that gives valuable information about the doubling time (DT), cell segmentation, track cell movement and changes in cell morphology. The results can be also completed by phenotype microarrays (PMs) and atomic force microscopy (AFM) techniques that fulfil experimental data.

**Keywords:** general cytotoxicity, nanomaterials, AFM analysis, holographic analysis, phenotype microarrays

### **1. Introduction**

This chapter is dedicated to selected methods used to analyse the biocompatibility/cytotoxicity of different nanomaterials. The effect of nanomaterials on cellular metabolism and relative viability can differ according to their properties and experimental design. As shown by Frewin et al. [1],

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

biocompatibility analyses of novel materials for medical applications are conducted using quantitative and qualitative techniques. These techniques have been collected by the International Standards Organization (ISO) 10,993<sup>1</sup> (ISO-10993-1, 2009; ISO-10993-5, 2009; ISO-10993-6, 2007).

Nanotechnology and nanobiotechnology have focused scientists' attention in the last few years on their application in biomedical research, such as detecting and monitoring systems of cells within the body, delivery systems for various drugs, hyperthermia treatment, photodynamic therapy and tissue engineering [2]. The term 'nano' may be considered a different state of aggregation of matter in all its states—solid, liquid, gas and plasma [3].

The phenomenon of nanoparticles is based on their different physical (optical and electromagnetic), chemical (catalytic) and mechanical properties that depend on particle size, as well as surface and quantum effects. The surface effects manifest as scaling of physical properties (increased atomic fraction on particle surface compared to the interior), which includes increased chemical reactivity and reduced melting point of nanoparticles compared to larger particles of bulk material. NPs have a very large surface area in comparison to microparticles and larger materials, making this large surface area available for chemical reactions [4]. In addition, nanoparticles can be classified according to their composition (inorganic and organic, lipid-based and polymeric NPs), dimensionality, morphology, uniformity and agglomeration [**Table 1**] [3, 5]. Another classification divides nanomaterials into three groups: zero-dimensional materials (quantum dots), varying in shape and diameter; one-dimensional materials (nanorods and nanowires) and twodimensional materials (nanobelts, nanodisks and nanosheets) [2].

The toxicity and cytotoxicity of nanomaterials are complex and depend on various factors, such as chemical composition, crystalline structure, size (at the nanolevel, the basic physicochemical properties of materials can change with variation in size) or aggregation. Nanomaterial composition determines its chemical interaction with cells, cellular uptake mechanisms and intracellular localisation. Chemical composition may also induce oxidative stress. For example, silver nanoparticle aggregates are more toxic than asbestos; CNTs are highly toxic and evoke much more damaging effect to the lungs than carbon black or silica NPs, but titanium oxide, iron oxide and zirconium oxide NPs are less toxic than asbestos [3, 6].

The crystalline structure effect of NP toxicity causes that some nanomaterials with a specific crystalline structure do not exhibit high toxicity, but other allotropes can strongly affect cell viability and exert an effect on human organism. Sato and co-workers [7] demonstrated that TiO2 allotropes exhibited different toxicity. Rutile TiO<sup>2</sup> NPs (200 nm) induced oxidative DNA damage in the absence of UV light and also TiO<sup>2</sup> NPs (10–20 nm) stimulated reactive oxygen species (ROS) production under corresponding conditions; in contrast, anatase NPs of the same size did not cause this effect [3, 8, 9].

Another factor that determines nanomaterial toxicity is the size of NPs. In most cases, smaller nanoparticles are able to pass through physiological barriers. Small-size nanoparticles can enter cells by phagocytosis and other mechanisms (e.g., micropinocytosis, receptor-mediated endocytosis (RME) pathways mediated by caveolae, clathrin and caveolae/clathrin-independent endocytosis) [10, 11]. NP ability to enter the cells determines adhesive interactions, such as van der Waals forces, steric interactions or electrostatic charges [3, 12, 13]. Moreover, NPs smaller than 100 nm are not phagocytized as opposed to larger nanoparticles, but they enter via RME pathways [2, 11]. NP uptake can also occur in the absence of specific cell surface receptors. Nanoparticles smaller than 50 nm can easily enter most cells (with greater cytotoxicity),

**Classes Types Structure Size Properties**

Single-walled CNT (SWCNTs)

Diameter between 0.4 and 100 nm; length between several nanometres up to centimetres

General Cytotoxicity and Its Application in Nanomaterial Analysis

From 0.1 up to 300 μm

Diameter between 2 and 10 nm

Sizes of 1–100 nm

Diameter between 1 and 100 nm in size

Sizes of 1 and 100 nm

Improved compressive strength, tensile bending strength, flexural strength, durability, piezoelectric response, sensing ability

179

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

Large area; its surface can be easily functionalized with functional groups; ideal for high drug loading via π-π stacking, hydrophobic or electrostatic interactions; exceptional mechanical

properties

stability

properties

properties

50–300 nm Porous structure and large

Large area, enhanced biocompatibility, good mechanical strength, surface functionality, colloidal

Absorb and scatter light; catalyst applications; anti-fungal, anti-microbial

Significant anti-microbial

Superparamagnetic properties; the surface area-to-volume ratio is significantly high; higher binding capacity and excellent dispersibility

surface area; chemical stability; surface functionality and biocompatibility

Multi-walled CNT (MWCNTs)

Hydrophobic twodimensional single monoatomic layers

Oxidised form of GF (GO)

Truncated octahedral

(GF)

structure

Colloidal gold, nanorods, nanowires

Colloidal silver, spherical silver nanoparticles, diamond, octagonal, thin sheets

Magnetite (Fe3

oxidised form maghemite (γ-Fe<sup>2</sup>

a porous, hexagonal, cubic and cage type

Nanosilica Solid material with

O4 );

> O3 )

Carbon-based nanoparticles

Inorganic nanoparticles

Mesoporous nanoparticles (MSN, MSNPs) Carbon nanotubes

Graphene (GF) and graphene oxide (GO)

Nanodiamonds

Gold nanoparticles (GNPs, AuNPs)

Silver nanoparticles

(AgNPs)

Iron oxide nanoparticles (IONPs)

**Table 1.** Types of nanomaterials [3, 5, 7].

(ND)

(CNTs)

<sup>1</sup> ISO-10993 ISO-10993-1 (ISO-10993-1 (2009) - Biological evaluation of medical devices — Part 1: Evaluation and testing within a risk management process; ISO-10993-5 (2009) - Biological evaluation of medical devices — Part 5: Tests for in vitro cytotoxicity; ISO-10993-6 (2007) – Biological evaluation of medical devices — Part 6: Tests for local effects after implantation


**Table 1.** Types of nanomaterials [3, 5, 7].

biocompatibility analyses of novel materials for medical applications are conducted using quantitative and qualitative techniques. These techniques have been collected by the International

Nanotechnology and nanobiotechnology have focused scientists' attention in the last few years on their application in biomedical research, such as detecting and monitoring systems of cells within the body, delivery systems for various drugs, hyperthermia treatment, photodynamic therapy and tissue engineering [2]. The term 'nano' may be considered a different

The phenomenon of nanoparticles is based on their different physical (optical and electromagnetic), chemical (catalytic) and mechanical properties that depend on particle size, as well as surface and quantum effects. The surface effects manifest as scaling of physical properties (increased atomic fraction on particle surface compared to the interior), which includes increased chemical reactivity and reduced melting point of nanoparticles compared to larger particles of bulk material. NPs have a very large surface area in comparison to microparticles and larger materials, making this large surface area available for chemical reactions [4]. In addition, nanoparticles can be classified according to their composition (inorganic and organic, lipid-based and polymeric NPs), dimensionality, morphology, uniformity and agglomeration [**Table 1**] [3, 5]. Another classification divides nanomaterials into three groups: zero-dimensional materials (quantum dots), varying in shape and diameter; one-dimensional materials (nanorods and nanowires) and two-

The toxicity and cytotoxicity of nanomaterials are complex and depend on various factors, such as chemical composition, crystalline structure, size (at the nanolevel, the basic physicochemical properties of materials can change with variation in size) or aggregation. Nanomaterial composition determines its chemical interaction with cells, cellular uptake mechanisms and intracellular localisation. Chemical composition may also induce oxidative stress. For example, silver nanoparticle aggregates are more toxic than asbestos; CNTs are highly toxic and evoke much more damaging effect to the lungs than carbon black or silica NPs, but titanium

The crystalline structure effect of NP toxicity causes that some nanomaterials with a specific crystalline structure do not exhibit high toxicity, but other allotropes can strongly affect cell viability and exert an effect on human organism. Sato and co-workers [7] demonstrated that

species (ROS) production under corresponding conditions; in contrast, anatase NPs of the

Another factor that determines nanomaterial toxicity is the size of NPs. In most cases, smaller nanoparticles are able to pass through physiological barriers. Small-size nanoparticles can

ISO-10993 ISO-10993-1 (ISO-10993-1 (2009) - Biological evaluation of medical devices — Part 1: Evaluation and testing within a risk management process; ISO-10993-5 (2009) - Biological evaluation of medical devices — Part 5: Tests for in vitro cytotoxicity; ISO-10993-6 (2007) – Biological evaluation of medical devices — Part 6: Tests for local effects after

state of aggregation of matter in all its states—solid, liquid, gas and plasma [3].

dimensional materials (nanobelts, nanodisks and nanosheets) [2].

allotropes exhibited different toxicity. Rutile TiO<sup>2</sup>

damage in the absence of UV light and also TiO<sup>2</sup>

same size did not cause this effect [3, 8, 9].

oxide, iron oxide and zirconium oxide NPs are less toxic than asbestos [3, 6].

(ISO-10993-1, 2009; ISO-10993-5, 2009; ISO-10993-6, 2007).

NPs (200 nm) induced oxidative DNA

NPs (10–20 nm) stimulated reactive oxygen

Standards Organization (ISO) 10,993<sup>1</sup>

178 Cytotoxicity

TiO2

1

implantation

enter cells by phagocytosis and other mechanisms (e.g., micropinocytosis, receptor-mediated endocytosis (RME) pathways mediated by caveolae, clathrin and caveolae/clathrin-independent endocytosis) [10, 11]. NP ability to enter the cells determines adhesive interactions, such as van der Waals forces, steric interactions or electrostatic charges [3, 12, 13]. Moreover, NPs smaller than 100 nm are not phagocytized as opposed to larger nanoparticles, but they enter via RME pathways [2, 11]. NP uptake can also occur in the absence of specific cell surface receptors. Nanoparticles smaller than 50 nm can easily enter most cells (with greater cytotoxicity), while nanoscale devices smaller than 20 nm can cross blood vessels and cumulate in tissues [2]. Particles with larger surface area display tendency to agglomerate in the liquid, interact with molecules, such as proteins and DNA and may cause oxidation and DNA damage [3, 4, 14].

using alternative cytotoxicity assays based on tetrazolium salts, e.g., XTT, WST-1, INT or other assays that complement the analysis, e.g., Alamar Blue (AB), neutral red uptake (NRU) assay, LDH leakage assay, flow cytometry, cell death analysis (using trypan blue or annexin V/propidium iodide), protein concentration measurements using the Bradford assay, measurements of mitochondrial membrane permeability (MMP) or loss of glutathione (e.g., GSH) and the

A number of studies have been carried out to verify the effect of NPs on assay reagents [22]. Wörle-Knirsch and co-workers [23] indicated that CNT analysed using the MTT assay caused false-positive results due to the strong interaction between CNT and the insoluble formazan crystals [19, 23]. In the aforementioned study, SWCNTs were analysed on A549 (human alveolar epithelial cell line), ECV304 (endothelial cells derived from umbilical cord) and NR8383 (rat alveolar macrophage cell line) cell cultures and the results obtained in the MTT assay were verified by WST-1, LDH, mitochondrial membrane potential (MMP) and annexin V/PI analysis. The MTT assay indicated that SWCNTs affected cell viability, reducing it almost by 60% after a 24-h incubation period. Moreover, the decreased cell viability did not recover after longer incubation or higher concentrations of nanotubes. The results of the MTT assay were verified using WST-1, and no reduction in viability was detected. LDH and MMP assays confirmed WST-1 results, and flow cytometry using annexin V/propidium iodide showed lack of necrosis and/or apoptosis. Wörle-Knirsch et al. [23] concluded that nanotoxicological assays

nanoparticles on living models is crucial due to their utilisation in food, beauty care and phar-

properties: the ability to catalyse redox reactions of molecules adsorbed on the surface during light exposition (λ < 385 nm). Photocatalytic reaction may occur by direct charge transfer

formazan. Formazan formation was found to be proportional to titania NPs, and this process was enhanced by daylight exposure. Moreover, the results obtained in the experiment without cellular model were validated using three cell lines—V79-4, HeLa and B16. The results dem-

wavelengths) and inversely proportional to initial concentration of MTT. Moreover, reaction

Casey et al. [26, 27] proved that single-walled carbon nanotubes (HiPco®) interacted with indicator dyes applied in Coomassie Blue, AlamarBlue™, neutral red uptake, MTT and WST-1 assays. In all cases, nanotubes interacted with dyes, which resulted in the reduction of the associated absorption/fluorescent emission. A spectroscopic study demonstrated that SWCNTs interacted with Coomassie and reduced the absorbance for all concentrations tested

HPO<sup>4</sup>

reaction may be also mediated by reactive oxygen species (ROS), e.g., hydroxyl radicals (˙

) formed at the interface of TiO<sup>2</sup>

toxicity. The effect of TiO<sup>2</sup>

and water. Lupu and Popescu [24]

concentrations higher than 50 μgml−1

and UV radiations (at 312 and 365 nm

(phosphate concentration of 0.005 M for

OH)

nanoparticles are known to exhibit photocatalytic

General Cytotoxicity and Its Application in Nanomaterial Analysis

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

181

) generated by light on the surface of titania nanoparticles. The

nanoparticles induced transformation of noncellular MTT to


activation of proinflammatory cytokines (e.g., IL-6, IL-8 and/or TNF-α) [15, 19, 21].

needed standardising with regard to the tested nanomaterial

macology industries. Additionally, TiO<sup>2</sup>

) and holes (h<sup>+</sup>

O2 −

onstrated false viability that increased up to 14% for TiO<sup>2</sup>

maximum efficiency), which is the basic component of PBS [25].

(formazan production rate) was proportional to TiO<sup>2</sup>

efficiency was enhanced by the presence of Na<sup>2</sup>

of electrons (e−

or superoxide anions (˙

[24]. In addition, the TiO<sup>2</sup>

clearly demonstrated that TiO<sup>2</sup>

Lupu and Popescu [24] used the MTT assay to evaluate TiO<sup>2</sup>

It is known that the shape (aspect ratio) also determines cellular uptake efficiency and may affect cell viability. Additionally, surface chemistry of nanomaterials modulates the response of biological systems and distribution in the organism. Surface functionalisation (with Fe3 O4 , gold nanoparticles; type of bonding on the surface, e.g., covalent, noncovalent; dispersing agents, e.g., PEG) is a crucial factor that can significantly change the toxicity of NPs and prevent NPs from aggregating; it can also change their fate in biological systems [2, 3, 11]. For example, functionalization of MWCNTs with sodium sulfonic acid salt (─SO<sup>3</sup> Na or -phenyl-SO<sup>3</sup> Na) increased their biocompatibility in comparison with unfunctionalised or carboxylic acid–functionalized (─COOH) MWCNTs [15]. Cellular uptake depends on different factors, such as nanomaterial and cell type, but also on environmental properties and the complexity of culture media. These specific conditions determine the aggregation process, which makes the interpretation of data on nanoparticle biodistribution or uptake difficult [3]. NP agglomerates affect and limit the direct extrapolation of *in vitro* data (providing a basis for understanding the mechanism of NP cytotoxicity and their uptake at the cellular level) to *in vivo* exposure [11, 16].

Intercellular localization of NPs and their interaction with cell components, such as the membrane, mitochondria, lysosomes and/or nucleus, are essential [11]. NPs can affect cell and organelle membranes, induce oxidative stress (ROS), DNA damage and mutagenesis and evoke apoptosis and protein up-/downregulation [11]; they can also modulate immune response [3, 17].
