Environmental Toxicology

*Corresponding author: Mohammad Ali Eghbal, Tel: +98 41 33372250-1, Email: [email protected], §: These authors contributed equally ©2015 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers.

Adv Pharm Bull, 2015, 5(4), 447-454 doi: 10.15171/apb.2015.061

http://apb.tbzmed.ac.ir

Advanced

Pharmaceutical

Bulletin

A Review of Molecular Mechanisms Involved in Toxicity of

Nanoparticles

Javad Khalili Fard1,2,3,4§, Samira Jafari4,5§, Mohammad Ali Eghbal2,3*

1 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 2 Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 3 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tabriz University of Medical Science, Tabriz, Iran. 4 Student Research Committee, Tabriz University of Medical Science, Tabriz, Iran. 5 Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Science, Tabriz, Iran.

Introduction

Nanotechnology advancement in medical sciences led to

the design and synthesis of nanostructures for biomedical

applications. Due to unique properties of NPs such as

small size (1-100 nm in diameter) and the greater surface

area to volume ratio as well as different electronic,

magnetic, optical and mechanical properties and also

particle shape, these particles hold great interests in the

various fields.1-6

It may seem that NPs do not have toxic effects.

However, the greater surface area to volume ratio of

these particles causes their higher chemical reactivity and

results in increased production of reactive oxygen

species (ROS). Indeed, the NPs surface area is a key

factor in their intrinsic toxicity because of the interaction

of their surfaces with biological system.7-10

ROS formation is one of the mechanisms of NPs toxicity

which could cause oxidative stress, inflammation and

consequent damages to the proteins, cell membrane and

DNA. Therefore, assessment of nanoparticles toxicity is

necessary in biomedical applications including drug

delivery systems, gene delivery and therapeutic

applications.11-14

Prooxidants are chemicals that induce oxidative stress

through either creating reactive oxygen species or

inhibiting antioxidants. NPs react with cells and induce

their prooxidant effects via intracellular ROS generation

involving mitochondrial respiration and activation of

NADPH-dependent enzyme systems.15-17

NPs can activate the cellular redox system specifically in

the lungs where the immune cells including alveolar

macrophages (AM) and neutrophils act as direct ROS

inducers. Professional phagocytic cells of the immune

system including neutrophils and AMs induce

remarkable ROS upon internalization of NPs via the

NADPH oxidase enzyme system.16,18

In this review, we have focused on introducing in vitro

toxicity assays for cytotoxicity assessment of

nanoparticles. We have also reviewed toxic effect of

several nanoparticles such as carbon nanotubes, titanium

dioxide NPs, quantum dots, gold NPs and silver NPs.

Cytotoxicity assays of nanoparticles

Cytotoxicity assays are classified as in vivo and in vitro

tests. In vivo toxicity assays (cell-based assay) are time-

consuming and expensive and involve ethical issues but

in vitro toxicity tests (cell cultured-based assay) are

faster, convenient, less expensive and devoid of any

ethical issues. Due to these advantages, in vitro assays

are the first choice for toxicity assessment of most

nanomaterials.19

In vitro methods include approaches for assessment of

integrity of the cell membrane and the metabolic activity

Article info

Article History:

Received: 4 February 2015 Revised: 20 August 2015

Accepted: 25 August 2015

ePublished: 30 November 2015

Keywords:

 Oxidative stress

 Reactivity oxygen species

 Cytotoxicity

 Nanoparticles

 Mechanism

 Prooxidant effects

Abstract In recent decades, the use of nanomaterials has received much attention in industrial and

medical fields. However, some reports have mentioned adverse effects of these materials on

the biological systems and cellular components. There are several major mechanisms for

cytotoxicity of nanoparticles (NPs) such as physicochemical properties, contamination with

toxic element, fibrous structure, high surface charge and radical species generation. In this

review, a brief key mechanisms involved in toxic effect of NPs are given, followed by the in

vitro toxicity assays of NPs and prooxidant effects of several NPs such as carbon nanotubes,

titanium dioxide NPs, quantum dots, gold NPs and silver NPs.

Review Article

448 | Advanced Pharmaceutical Bulletin, 2015, 5(4), 447-454

Khalili fard et al.

of viable cells. Evaluation of cell membrane integrity is

one of the most common approaches to measure cell

viability. It is based on the leakage of substances such as

lactate dehydrogenase (LDH) that normally reside inside

cells to the external environment and the measurement of

LDH activity in the extracellular media. Alternatively,

membrane integrity can be determined by penetration of

dyes such as trypan blue and neutral red into the

damaged cells and staining intracellular components.

These dyes cannot enter living cells. Metabolic activity

of viable cells could be determined through colorimetric

assays, such as the MTT and MTS assays.20-23

Bioluminescent methods including methods using

luciferase, which catalyzes the formation of light from

adenosine triphosphate (ATP) are also commonly used as

cell viability assays in which the number of surviving

cells is determined by measuring the uptake and

accumulation of neutral red dye and trypan blue after

exposure to the toxicant.24-26 Among in vitro methods,

LDH, MTT and MTS assay are most widely used for

assessment of nanoparticles cytotoxicity (Table 1).27

LDH test

In general, LDH test is a colorimetric assay that

quantitatively measures LDH, a marker of cell membrane

integrity, released from damaged cells into the culture

media. This assay is a fast, simple and reliable method for

determining cellular toxicity.28

MTT assay

MTT assay is another candidate assay for measurement

of cytotoxicity of NPs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-

Diphenyltetrazolium Bromide, (MTT), is a yellow

substance which reduces to purple insoluble formazan

crystals by mitochondrial succinate dehydrogenases in

viable cells. This method is directly related to the

number of viable cells.29

MTS assay

In the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-

tetrazolium) assay, viable cells will convert tetrazolium

salt into a colored soluble formazan product by

mitochondrial dehydrogenase enzymes. Indeed, in MTS

assay, similar to MTT assay, a colorimetric product is

formed. The formazan produced is directly proportional

to the number of living cells in the culture.30

Toxicity mechanisms of nanoparticles

Physicochemical reactivity of NPs lead to the formation

of free radicals or ROS including superoxide radical

anions and hydroxyl radicals direct or indirect through

activation of oxidative enzymatic pathways result in

oxidative stress (Figure 1).31-36 In general, there are

several sources for oxidative stress:

 Oxidant-generating properties of particles themselves

as well as their ability to stimulate generation of ROS

as a part of cellular response to nanoparticles

 Transition metal-based nanoparticles or transition

metal contaminants used as catalysts during the

production of non-metal nanoparticles.

 Relatively stable free radical intermediates present on

reactive surfaces of particles.

 Redox active groups resulting from functionalization

of nanoparticles

The following briefly introduces cytotoxicity of some of

nanoparticles such as carbon nanotubes, titanium dioxide

NPs, quantum dots, gold NPs and silver NPs.

Figure 1. ROS generation induced by NPs and their cytotoxicity mechanism.

Cytotoxicity of carbon nanotubes

Carbon nanotubes (CNTs), fiber shaped nanostructures,

are allotropes of carbon which are categorized as single

wall carbon nanotubes (SWCNT) and multi wall carbon

nanotubes (MWCNT). In addition to industrial uses,

carbon nanotubes, due to their unique electrical, physical

and thermal qualities hold great interest in biomedical

applications.37-39

Numerous reports have shown that CNT could induce

the ROS generation in

multitudes of cell lines and activation of ROS-associated

intracellular signaling pathways in a dose-dependent

manner such as mitogen activated protein kinase

(MAPK), activator protein-1 (AP-1) and nuclear factor

kappa-light-chain-enhancer of activated B cells (NF-κB)

in mesothelial cells.40-43

It has been reported that MWCNT are able to stimulate

the release of the cytokines, IL-1β, TNF-α, IL-6 and IL-8

from mesothelial cells and macrophages. Murphy et al.

demonstrated that direct exposure to MWCNT causes to

length-dependent cytokine release from macrophages but

not mesothelial cells. However, treatment of the

mesothelial cells with conditioned medium from CNT-

treated macrophages led to increased secretion of

cytokines. In another study, MWCNT were revealed to

trigger the macrophages to produce TGF-β1 and platelet-

derived growth factor (PDGF) that promoted the

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Advanced Pharmaceutical Bulletin, 2015, 5(4), 447-454

transformation of lung fibroblasts to myofibroblasts, a

major factor in development of fibrosis.44

Cytotoxicity of TiO2 nanoparticles

Widespread applications of titanium dioxide

nanoparticles (TiO2 NPs) in consumer products including

cosmetic, paints, pharmaceutical preparations, food

additives and so on is a result of their ability to confer

opacity and whiteness.45,46 In recent years, the

photocatalytic killing effect of TiO2 NPs on cancerous

cells has received great attention.47-49

The potential mechanism of cytotoxicity induced by

these non-soluble metal oxide NPs are still controversial.

In some literature, these NPs are even considered as a

natural nanomaterial.50 Conversely, some reports have

pointed out the potential toxicity of TiO2 nanoparticles,

including their ability to induce oxidative stress,

genotoxicity and immunotoxicity.51,52 However, the

mechanisms of these toxic effects are still blurred but

cytotoxicity evaluation of these metal oxide NPs is

important for in vivo and in vitro studies. Despite other

NPs such as ZnO, quantum dots and so, TiO2 NPs do not

release toxic ions hence toxicity of these particles could

be attributed to the size-dependent interaction between

nanoparticles and intracellular biomolecules adsorbed

onto nanoparticles.53-55 These interactions result in

generation of ROS, mitochondrial depolarization, plasma

membrane leakage, intracellular calcium influx and

cytokine release.56-59

In a study, Xiong et al. investigated size influence of

TiO2 NPs on their phototoxicity. Results showed that

there was a converse relationship between phototoxicity

and the size of these particles; as, the mortality of the

cells treated with 10 nm TiO2 NPs after photoactivation

by UV light was significantly higher than that of the cells

treated with larger particles (20 and 100 nm particles).

Furthermore, cytotoxicity of non-photoacivated 10, 20

and 100 nm NPs was not inconsiderable for cells treated

with them. In addition, the treated cells with 10 nm

photoactivated particles demonstrated a higher

generation of mitochondrial superoxide in comparison to

20 and 100 nm particles.

Indeed, the higher cytotoxicity induced by smaller

particles is related to their higher surface area and hence

contain a larger number of surface-exposed TiO2

molecules. Phototoxicity of these NPs could be

decreased via surface coating with chitosan or PEMA

because of the prevention of biomolecule adsorption and

hydroxyl radicals (.OH) production in the

photoactivation process.54

In another study, size-dependent toxicity of both TiO2

and PLGA was investigated. Findings revealed that

biomedically used PLGA nanoparticles did not show

strong cytotoxic effect in comparison to TiO2

nanoparticles. However, the smaller PLGA nanoparticles

have the potential to trigger the release of TNF-α. 200

nm PLGA nanoparticles could not trigger any negative

response from cells. Higher cytotoxic effect was

observed in cells treated with TiO2 nanoparticles,

especially at concentrations higher than 100μg/ml. The

size-dependent cytotoxicity of both PLGA and TiO2

nanoparticles could be attributed to the smaller size and

larger specific surface area and thus exposure of more

molecules on the surface that led to the adsorption of

more biomolecules such as proteins in the environment.60

Cytotoxicity of quantum dots

Quantum dots (QDs), colloidal semiconductor

nanoparticles, are a promising type of NPs which possess

exceptional optical properties including high fluorescent

quantum yield, broad absorption, narrow emission and

high photostability. These properties make QDs an

attractive candidate for in vivo imaging instead of

fluorescent dyes.61

Similar to other NPs, cytotoxicity of QDs depends on

parameters including size, shape, concentration, charge,

redox activity, surface coatings and mechanical stability of

these particles. Toxicity of uncoated core CdSe or CdTe-

QDs have been investigated in some literature. Two major

mechanisms are involved in the toxicity effects of these

inorganic nanoparticles are as follows:62-65

1) Cd+2 ions existing in QDs structure:

These toxic metal ions cause toxic effects through

several routes such as interference with DNA repair

and substitution for physiologic Zn. Cd+2 ions

increase oxidative stress but they cannot directly

generate free radicals.

2) Free radical formation:

QDs of CdSe and CdTe are highly reactive, thus,

photoactivation of these QDs via visible or UV light

leads to their oxidation. Indeed, a photon of light

could excite the QD and consequently generates an

excited electron that transfers to molecular oxygen,

forming singlet oxygen. Reaction of singlet oxygen

with water/other biological molecules results in

production of free radicals.

Kauffer et al. recently demonstrated that variation in core

compositions and surface chemistries of QDs, CdSe QDs

vs. CdS QDs, lead to their different cytotoxicity. The

former produced •OH radicals immediately after light

activation, while the latter required extensive irradiation

to generate an equivalent amount of radicals. Therefore,

the toxicity observed for CdSe QDs could be directly

related to •OH radicals produced. Indeed, cytotoxicity of

colloidal NPs can be controlled and relieved by choosing

appropriate materials for QD core and suitable irradiation

condition.66

Cytotoxicity of gold nanoparticles

Gold nanoparticles (GNPs), are one of the promising

inorganic (NPs) that have attracted scientific and

technological interests due to their ease of synthesis,

chemical stability and excellent optical properties.67-69

These unique properties of GNPs, make them appealing

tools for cancer diagnosis and treatment.70-72

Most of in vitro studies have indicated that these NPs are

nontoxic for cells. Evaluation of GNPs cytotoxicity is

essential because of broad spectrum application of GNPs

450 | Advanced Pharmaceutical Bulletin, 2015, 5(4), 447-454

Khalili fard et al.

in biomedical sciences. In the most of literature

investigations have demonstrated that these inorganic

nanoparticles are nontoxic. Cytotoxicity of GNPs

depends on their size, shape and surrounding ligands.73,74

Anisotropic GNPs have more potential oxidation than the

isotropic ones due to their highly exposed surface areas

and defects. Also, in some literature investigations

exhibited that spherical GNPs are suitable for biomedical

application.75-77

Recently, the cytotoxicity effects of 5 and/or 15 nm

GNPs 5 and 15 nm in vitro on Balb/3T3 mouse

fibroblasts have been investigated. In order to understand

the observed differences in cytotoxicity of two sizes of

GNPs, Gioria et al. examined the uptake and the

intracellular distribution of the NPs. The results indicated

cytotoxicity effects only for the cells treated with 5 nm

GNPs but no toxicity was revealed on Balb/3T3 for 15

nm GNPs. This observation is due to high number of 5

nm GNPs taken-up by cells in comparison to the larger

particles (15 nm particles).78

Cytotoxicity of silver nanoparticles

Antimicrobial properties of silver nanoparticles (AgNPs)

cause to the use of these NPs in a broad spectrum of

consumer products including cosmetics, electronics,

household appliances, textiles, and food products.79,80 In

the recent decade, AgNPs have been used in medical

fields such as drug delivery, designing biosensors, and

imaging contrast agents etc.81-83 Thus, toxicity assay is an

important factor to be considered in their application for

biomedical purposes. Cytotoxicity of these NPs is related

to comfortable oxidation AgNPs to Ag+ ions which are

very toxic for biological systems and cellular

components.84-87

Compton and coworkers in a study showed that AgNPs

in aqueous system are more toxic compared to the bulk

Ag is more toxic due to the presence of dissolved

oxygen, its reduction on NPs and then the release of

H2O2 from AgNPs. Also, results demonstrated that ROS

generation from nanoparticulated Ag are greater than that

of macro (bulk) silver.88

Recently, in a report the size- and coating-dependent

toxicity of thoroughly characterized AgNPs was

investigated following exposure to human lung cells. The

results revealed that only the cytotoxicity of the 10 nm

particles was independent of surface coating. In contrast,

all AgNPs tested caused an increase in overall DNA

damage after 24 h which suggests independent

mechanisms for the cytotoxicity and DNA damage.

However, there was no increased production of

intracellular ROS; therefore, the toxicity observed was

related to the rate of intracellular Ag release. Interaction

with thiol and amino groups of biomolecules and

appearance of the toxicity effect on cellular components

were a result of sliver release. Thus, AgNPs with higher

Ag release are more toxic.89

Table 1. Some in vitro assays with type of NPs and cell types.

Assay Type of NPs Type of cells (system) References

MTT assay QDs Human embryonic kidney cells 90

TiO2 Human erythrocyte/ lymphocyte cells 59

Natural red TiO2 NPs Zebrafish embryos 91

LDH test TiO2 NPs Human kidney cells

92 CNTs human pneumocytes cells

MTS assay Ag NPs mouse embryonic fibroblasts 93

Gold NPs Mammalian cells 94

Trypan blue Gold NPs mouse fibroblast 78

TiO2 NPs human lung epithelial cells 95

Conclusion

Despite the wide spread applications of nano-sized

materials in various sciences areas, there are numerous

reports about side effects of these materials on biological

systems and cellular compartments. In addition to

physicochemical properties, the production of toxic ions,

fibrous structure, high surface charge and generation of

radical species result in cytotoxicity by NPs including

carbon nanotubes, titanium dioxide NPs, quantum dots,

gold NPs and silver NPs. Both in vivo and in vitro assays

are used for toxicity assessment of NPs. In vitro assays

have received more attentions compared to in vivo

assays due to being faster, convenient, less expensive,

and devoid lacking any ethical issues.

Ethical Issues

Not applicable.

Conflict of Interest

The authors declare that they have no conflict of interest.

References

1. Farokhzad OC, Langer R. Nanomedicine: Developing

smarter therapeutic and diagnostic modalities. Adv

Drug Deliv Rev 2006;58(14):1456-9. doi:

10.1016/j.addr.2006.09.011

2. Roco MC. Nanotechnology: Convergence with

modern biology and medicine. Curr Opin Biotechnol

2003;14(3):337-46. doi: 10.1016/S0958-

1669(03)00068-5

| 451

Mechanisms of Nanoparticle Toxicity

Advanced Pharmaceutical Bulletin, 2015, 5(4), 447-454

3. Caruthers SD, Wickline SA, Lanza GM.

Nanotechnological applications in medicine. Curr

Opin Biotechnol 2007;18(1):26-30. doi:

10.1016/j.copbio.2007.01.006

4. Silva GA. Introduction to nanotechnology and its

applications to medicine. Surg Neurol

2004;61(3):216-20. doi:

10.1016/j.surneu.2003.09.036

5. Singh M, Singh S, Prasad S, Gambhir IS.

Nanotechnology in medicine and antibacterial effect

of silver nanoparticles. Dig J Nanomater Bios

2008;3(3):115-22.

6. Nie S, Xing Y, Kim GJ, Simons JW. Nanotechnology

applications in cancer. Annu Rev Biomed Eng

2007;9:257-88. doi:

10.1146/annurev.bioeng.9.060906.152025

7. Choi O, Hu Z. Size dependent and reactive oxygen

species related nanosilver toxicity to nitrifying

bacteria. Environ Sci Technol 2008;42(12):4583-8.

doi: 10.1021/es703238h

8. Hussain SM, Hess KL, Gearhart JM, Geiss KT,

Schlager JJ. In vitro toxicity of nanoparticles in brl 3a

rat liver cells. Toxicol In Vitro 2005;19(7):975-83.

doi: 10.1016/j.tiv.2005.06.034

9. Zoroddu M, Medici S, Ledda A, Nurchi V, Lachowicz

J, Peana M. Toxicity of nanoparticles. Current

Medicinal Chem 2014;21(33):3837-53. doi:

10.2174/0929867321666140601162314

10. Marie Curie Initial Training Network -

Environmental Chemoinformatics (ECO). Toxicity of

nanoparticles. Project report – ITN-ECO: 2012.

11. Ahamed M, Karns M, Goodson M, Rowe J, Hussain

SM, Schlager JJ, et al. DNA damage response to

different surface chemistry of silver nanoparticles in

mammalian cells. Toxicol Appl Pharmacol

2008;233(3):404-10. doi: 10.1016/j.taap.2008.09.015

12. Sharma V, Shukla RK, Saxena N, Parmar D, Das M,

Dhawan A. DNA damaging potential of zinc oxide

nanoparticles in human epidermal cells. Toxicol Lett

2009;185(3):211-8. doi: 10.1016/j.toxlet.2009.01.008

13. Wang F, Yu L, Monopoli MP, Sandin P, Mahon E,

Salvati A, et al. The biomolecular corona is retained

during nanoparticle uptake and protects the cells from

the damage induced by cationic nanoparticles until

degraded in the lysosomes. Nanomedicine

2013;9(8):1159-68. doi: 10.1016/j.nano.2013.04.010

14. Elsaesser A, Howard CV. Toxicology of

nanoparticles. Adv Drug Deliv Rev 2012;64(2):129-

37. doi: 10.1016/j.addr.2011.09.001

15. Regoli F, Giuliani ME. Oxidative pathways of

chemical toxicity and oxidative stress biomarkers in

marine organisms. Mar Environ Res 2014;93:106-17.

doi: 10.1016/j.marenvres.2013.07.006

16. Jomova K, Baros S, Valko M. Redox active metal-

induced oxidative stress in biological systems.

Transition Met Chem 2012;37(2):127-34. doi:

10.1007/s11243-012-9583-6

17. Chen H, Yoshioka H, Kim GS, Jung JE, Okami N,

Sakata H, et al. Oxidative stress in ischemic brain

damage: mechanisms of cell death and potential

molecular targets for neuroprotection. Antioxid

Redox Signal 2011;14(8):1505-17. doi:

10.1089/ars.2010.3576

18. Soenen SJ, Rivera-Gil P, Montenegro JM, Parak WJ,

De Smedt SC, Braeckmans K. Cellular toxicity of

inorganic nanoparticles: Common aspects and

guidelines for improved nanotoxicity evaluation.

Nano Today 2011;6(5):446-65. doi:

10.1016/j.nantod.2011.08.001

19. Mahmoudi M, Hofmann H, Rothen-Rutishauser B,

Petri-Fink A. Assessing the in vitro and in vivo

toxicity of superparamagnetic iron oxide

nanoparticles. Chem Rev 2012;112(4):2323-38. doi:

10.1021/cr2002596

20. Fischer J, Prosenc MH, Wolff M, Hort N, Willumeit

R, Feyerabend F. Interference of magnesium

corrosion with tetrazolium-based cytotoxicity assays.

Acta Biomater 2010;6(5):1813-23. doi:

10.1016/j.actbio.2009.10.020

21. Rabolli V, Thomassen LC, Princen C, Napierska D,

Gonzalez L, Kirsch-Volders M, et al. Influence of

size, surface area and microporosity on the in vitro

cytotoxic activity of amorphous silica nanoparticles

in different cell types. Nanotoxicology 2010;4(3):307-

18. doi: 10.3109/17435390.2010.482749

22. Kumbıçak Ü, Çavaş T, Çinkılıç N, Kumbıçak Z,

Vatan Ö, Yılmaz D. Evaluation of in vitro

cytotoxicity and genotoxicity of copper-zinc alloy

nanoparticles in human lung epithelial cells. Food

Chem Toxicol 2014;73:105-12. doi:

10.1016/j.fct.2014.07.040

23. Fotakis G, Timbrell JA. In vitro cytotoxicity assays:

Comparison of ldh, neutral red, mtt and protein assay

in hepatoma cell lines following exposure to

cadmium chloride. Toxicol Lett 2006;160(2):171-7.

doi: 10.1016/j.toxlet.2005.07.001

24. Crouch SPM, Kozlowski R, Slater KJ, Fletcher J.

The use of atp bioluminescence as a measure of cell

proliferation and cytotoxicity. J Immunol Methods

1993;160(1):81-8. doi: 10.1016/0022-

1759(93)90011-U

25. Schiewe MH, Hawk EG, Actor DI, Krahn MM. Use

of a bacterial bioluminescence assay to assess toxicity

of contaminated marine sediments. Can J Fish Aquat

Sci 1985;42(7):1244-8. doi: 10.1139/f85-154

26. Benton MJ, Malott ML, Knight SS, Cooper CM,

Benson WH. Influence of sediment composition on

apparent toxicity in a solid-phase test using

bioluminescent bacteria. Environ Toxicol Chem

1995;14(3):411-4. doi: 10.1002/etc.5620140309

27. Asare N, Instanes C, Sandberg WJ, Refsnes M,

Schwarze P, Kruszewski M, et al. Cytotoxic and

genotoxic effects of silver nanoparticles in testicular

cells. Toxicology 2012;291(1-3):65-72. doi:

10.1016/j.tox.2011.10.022

28. Korzeniewski C, Callewaert DM. An enzyme-release

assay for natural cytotoxicity. J Immunol Methods

452 | Advanced Pharmaceutical Bulletin, 2015, 5(4), 447-454

Khalili fard et al.

1983;64(3):313-20. doi: 10.1016/0022-

1759(83)90438-6

29. van Meerloo J, Kaspers GJ, Cloos J. Cell sensitivity

assays: The MTT assay. In: Cree IA, editor. Cancer

cell culture. Hatfield: Springer; 2011. P. 237-45.

30. Malich G, Markovic B, Winder C. The sensitivity

and specificity of the mts tetrazolium assay for

detecting the in vitro cytotoxicity of 20 chemicals

using human cell lines. Toxicology 1997;124(3):179-

92. doi: 10.1016/S0300-483X(97)00151-0

31. Cho WS, Duffin R, Thielbeer F, Bradley M, Megson

IL, MacNee W, et al. Zeta potential and solubility to

toxic ions as mechanisms of lung inflammation

caused by metal/metal oxide nanoparticles. Toxicol

Sci 2012;126(2):469-77. doi:

10.1016/10.1093/toxsci/kfs006

32. Donaldson K, Poland CA, Schins RP. Possible

genotoxic mechanisms of nanoparticles: Criteria for

improved test strategies. Nanotoxicology

2010;4(4):414-20. doi:

10.3109/17435390.2010.482751

33. Stern ST, Adiseshaiah PP, Crist RM. Autophagy and

lysosomal dysfunction as emerging mechanisms of

nanomaterial toxicity. Part Fibre Toxicol

2012;9(1):20. doi: 10.1186/1743-8977-9-20

34. Li Y, Zhang W, Niu J, Chen Y. Surface-coating-

dependent dissolution, aggregation, and reactive

oxygen species (ros) generation of silver

nanoparticles under different irradiation conditions.

Environ Sci Technol 2013;47(18):10293-301. doi:

10.1021/es400945v

35. Nishanth RP, Jyotsna RG, Schlager JJ, Hussain SM,

Reddanna P. Inflammatory responses of raw 264.7

macrophages upon exposure to nanoparticles: Role of

ros-nfκb signaling pathway. Nanotoxicology

2011;5(4):502-16. doi:

10.3109/17435390.2010.541604

36. Luna-Velasco A, Field JA, Cobo-Curiel A, Sierra-

Alvarez R. Inorganic nanoparticles enhance the

production of reactive oxygen species (ros) during the

autoxidation of L-3,4-dihydroxyphenylalanine (L-

dopa). Chemosphere 2011;85(1):19-25. doi:

10.1016/j.chemosphere.2011.06.053

37. Shao W, Arghya P, Yiyong M, Rodes L, Prakash S.

Carbon nanotubes for use in medicine: Potentials and

limitations. In: Suzuki S, editor. Syntheses and

Applications of Carbon Nanotubes and Their

Composites. Croatia: InTech; 2013. P. 285-311.

38. De Volder MF, Tawfick SH, Baughman RH, Hart

AJ. Carbon nanotubes: Present and future commercial

applications. Science 2013;339(6119):535-9. doi:

10.1126/science.1222453

39. O’connell MJ. Carbon nanotubes: Properties and

applications. Boca Raton: CRC Press; 2012.

40. Shvedova AA, Pietroiusti A, Fadeel B, Kagan VE.

Mechanisms of carbon nanotube-induced toxicity:

Focus on oxidative stress. Toxicol Appl Pharmacol

2012;261(2):121-33. doi: 10.1016/j.taap.2012.03.023

41. Johnston HJ, Hutchison GR, Christensen FM, Peters

S, Hankin S, Aschberger K, et al. A critical review of

the biological mechanisms underlying the in vivo and

in vitro toxicity of carbon nanotubes: The

contribution of physico-chemical characteristics.

Nanotoxicology 2010;4(2):207-46. doi:

10.3109/17435390903569639

42. Campagnolo L, Massimiani M, Palmieri G,

Bernardini R, Sacchetti C, Bergamaschi A, et al.

Biodistribution and toxicity of pegylated single wall

carbon nanotubes in pregnant mice. Part Fibre

Toxicol 2013;10(1):21. doi: 10.1186/1743-8977-10-

21

43. Clift MJ, Endes C, Vanhecke D, Wick P, Gehr P,

Schins RP, et al. A comparative study of different in

vitro lung cell culture systems to assess the most

beneficial tool for screening the potential adverse

effects of carbon nanotubes. Toxicol Sci

2014;137(1):55-64. doi: 10.1093/toxsci/kft216

44. Murphy FA, Poland CA, Duffin R, Al-Jamal KT,

Ali-Boucetta H, Nunes A, et al. Length-dependent

retention of carbon nanotubes in the pleural space of

mice initiates sustained inflammation and progressive

fibrosis on the parietal pleura. Am J Pathol

2011;178(6):2587-600. doi:

10.1016/j.ajpath.2011.02.040

45. Yin ZF, Wu L, Yang HG, Su YH. Recent progress in

biomedical applications of titanium dioxide. Phys

Chem Chem Phys 2013;15(14):4844-58. doi:

10.1039/C3CP43938K

46. Weir A, Westerhoff P, Fabricius L, Hristovski K, von

Goetz N. Titanium dioxide nanoparticles in food and

personal care products. Environ Sci Technol

2012;46(4):2242-50. doi: 10.1021/es204168d

47. Sha B, Gao W, Han Y, Wang S, Wu J, Xu F, et al.

Potential application of titanium dioxide

nanoparticles in the prevention of osteosarcoma and

chondrosarcoma recurrence. J Nanosci Nanotechnol

2013;13(2):1208-11. doi: 10.1166/jnn.2013.6081

48. Wu Q, Guo D, Du Y, Liu D, Wang D, Bi H. UVB

irradiation enhances TiO2 nanoparticle-induced

disruption of calcium homeostasis in human lens

epithelial cells. Photochem Photobiol

2014;90(6):1324-31. doi: 10.1111/php.12322

49. Pierzchala K, Lekka M, Magrez A, Kulik AJ, Forró

L, Sienkiewicz A. Photocatalytic and phototoxic

properties of TiO2-based nanofilaments: ESR and

AFM assays. Nanotoxicology 2012;6(8):813-24. doi:

10.3109/17435390.2011.625129

50. PetkoviC J, Zegura B, StevanoviC M, Drnovšek N,

UskokoviC D, Novak S, et al. DNA damage and

alterations in expression of DNA damage responsive

genes induced by TiO2 nanoparticles in human

hepatoma hepg2 cells. Nanotoxicology

2011;5(3):341-53. doi:

10.3109/17435390.2010.507316

51. Gerloff K, Fenoglio I, Carella E, Kolling J, Albrecht

C, Boots AW, et al. Distinctive toxicity of TiO2

rutile/anatase mixed phase nanoparticles on Caco-2

| 453

Mechanisms of Nanoparticle Toxicity

Advanced Pharmaceutical Bulletin, 2015, 5(4), 447-454

cells. Chem Res Toxicol 2012;25(3):646-55. doi:

10.1021/tx200334k

52. Montiel-Dávalos Al, Ventura-Gallegos JL, Alfaro-

Moreno E, Soria-Castro E, García-Latorre E,

Cabañas-Moreno JG, et al. TiO2 nanoparticles induce

dysfunction and activation of human endothelial

cells. Chem Res Toxicol 2012;25(4):920-30. doi:

10.1021/tx200551u

53. Jin C, Tang Y, Yang FG, Li XL, Xu S, Fan XY, et al.

Cellular toxicity of TiO2 nanoparticles in anatase and

rutile crystal phase. Biol Trace Elem Res 2011;141(1-

3):3-15. doi: 10.1007/s12011-010-8707-0

54. Xiong S, George S, Ji Z, Lin S, Yu H, Damoiseaux

R, et al. Size of TiO2 nanoparticles influences their

phototoxicity: An in vitro investigation. Arch Toxicol

2013;87(1):99-109. doi: 10.1007/s00204-012-0912-5

55. Ivask A, Bondarenko O, Jepihhina N, Kahru A.

Profiling of the reactive oxygen species-related

ecotoxicity of CuO, ZnO, TiO2, silver and fullerene

nanoparticles using a set of recombinant luminescent

escherichia coli strains: Differentiating the impact of

particles and solubilised metals. Anal Bioanal Chem

2010;398(2):701-16. doi: 10.1007/s00216-010-3962-

7

56. Chen EY, Garnica M, Wang YC, Mintz AJ, Chen

CS, Chin WC. A mixture of anatase and rutile TiO2

nanoparticles induces histamine secretion in mast

cells. Part Fibre Toxicol 2012;9:2. doi:

10.1186/1743-8977-9-2

57. Scherbart AM, Langer J, Bushmelev A, van Berlo D,

Haberzettl P, van Schooten FJ, et al. Contrasting

macrophage activation by fine and ultrafine titanium

dioxide particles is associated with different uptake

mechanisms. Part Fibre Toxicol 2011;8:31. doi:

10.1186/1743-8977-8-31

58. Fu J, Rong G, Deng Y. Mammalian cell cytotoxicity

and genotoxicity of metallic nanoparticles. Adv Sci

Lett 2012;5(1):294-8. doi: 10.1166/asl.2012.1946

59. Ghosh M, Chakraborty A, Mukherjee A. Cytotoxic,

genotoxic and the hemolytic effect of titanium

dioxide (TiO2) nanoparticles on human erythrocyte

and lymphocyte cells in vitro. J Appl Toxicol

2013;33(10):1097-110. doi: 10.1002/jat.2863

60. Xiong S, George S, Yu H, Damoiseaux R, France B,

Ng KW, et al. Size influences the cytotoxicity of poly

(lactic-co-glycolic acid) (PLGA) and titanium dioxide

(TiO2) nanoparticles. Arch Toxicol 2013;87(6):1075-

86. doi: 10.1007/s00204-012-0938-8

61. Osiński M, Parak WJ, Jovin TM, Yamamoto K.

Colloidal Quantum Dots for Biomedical Applications

V, SPIE International Symposium on Biomedical

Optics BiOS 2010, Vol. 7575, Paper 75750Z San

Francisco, California, 23‐25 January 2010.

62. Chen N, He Y, Su Y, Li X, Huang Q, Wang H, et al.

The cytotoxicity of cadmium-based quantum dots.

Biomaterials 2012;33(5):1238-44. doi:

10.1016/j.biomaterials.2011.10.070

63. Hoshino A, Hanada S, Yamamoto K. Toxicity of

nanocrystal quantum dots: The relevance of surface

modifications. Arch Toxicol 2011;85(7):707-20. doi:

10.1007/s00204-011-0695-0

64. Zheng X, Tian J, Weng L, Wu L, Jin Q, Zhao J, et al.

Cytotoxicity of cadmium-containing quantum dots

based on a study using a microfluidic chip.

Nanotechnology 2012;23(5):055102. doi:

10.1088/0957-4484/23/5/055102

65. Singh BR, Singh BN, Khan W, Singh HB, Naqvi

AH. ROS-mediated apoptotic cell death in prostate

cancer LNCaP cells induced by biosurfactant

stabilized CdS quantum dots. Biomaterials

2012;33(23):5753-67. doi:

10.1016/j.biomaterials.2012.04.045

66. Kauffer FA, Merlin C, Balan L, Schneider R.

Incidence of the core composition on the stability, the

ROS production and the toxicity of CdSe quantum

dots. J Hazard Mater 2014;268:246-55. doi:

10.1016/j.jhazmat.2014.01.029

67. Dykman L, Khlebtsov N. Gold nanoparticles in

biomedical applications: Recent advances and

perspectives. Chem Soc Rev 2012;41(6):2256-82. doi:

10.1039/C1CS15166E

68. Tiwari PM, Vig K, Dennis VA, Singh SR.

Functionalized gold nanoparticles and their

biomedical applications. Nanomaterials

2011;1(1):31-63. doi: 10.3390/nano1010031

69. Yeh YC, Creran B, Rotello VM. Gold nanoparticles:

Preparation, properties, and applications in

bionanotechnology. Nanoscale 2012;4(6):1871-80.

doi: 10.1039/C1NR11188D

70. Lim ZZJ, Li JEJ, Ng CT, Yung LYL, Bay BH. Gold

nanoparticles in cancer therapy. Acta Pharmacol Sin

2011;32(8):983-90. doi: 10.1038/aps.2011.82

71. Jain S, Hirst DG, O'sullivan JM. Gold nanoparticles

as novel agents for cancer therapy. Br J Radiol

2012;85(1010):101-13. doi: 10.1259/bjr/59448833

72. Heo DN, Yang DH, Moon HJ, Lee JB, Bae MS, Lee

SC, et al. Gold nanoparticles surface-functionalized

with paclitaxel drug and biotin receptor as theranostic

agents for cancer therapy. Biomaterials

2012;33(3):856-66. doi:

10.1016/j.biomaterials.2011.09.064

73. Khlebtsov N, Dykman L. Biodistribution and toxicity

of engineered gold nanoparticles: A review of in vitro

and in vivo studies. Chem Soc Rev 2011;40(3):1647-

71. doi: 10.1039/C0CS00018C

74. Taylor U, Barchanski A, Garrels W, Klein S, Kues

W, Barcikowski S, et al. Toxicity of gold

nanoparticles on somatic and reproductive cells. In:

Zahavy E, Ordentlich A, Yitzhaki S, Shafferman A,

editors. Nano-biotechnology for biomedical and

diagnostic research. New York: Springer; 2012. P.

125-33.

75. Loumaigne M, Richard A, Laverdant J, Nutarelli D,

Débarre A. Ligand-induced anisotropy of the two-

photon luminescence of spherical gold particles in

solution unraveled at the single particle level. Nano

lett 2010;10(8):2817-24. doi: 10.1021/nl100737y

454 | Advanced Pharmaceutical Bulletin, 2015, 5(4), 447-454

Khalili fard et al.

76. Arnida, Janát-Amsbury MM, Ray A, Peterson CM,

Ghandehari H. Geometry and surface characteristics

of gold nanoparticles influence their biodistribution

and uptake by macrophages. Eur J Pharm Biopharm

2011;77(3):417-23. doi: 10.1016/j.ejpb.2010.11.010

77. Pissuwan D, Niidome T, Cortie MB. The

forthcoming applications of gold nanoparticles in

drug and gene delivery systems. J Control Release

2011;149(1):65-71. doi:

10.1016/j.jconrel.2009.12.006

78. Coradeghini R, Gioria S, García CP, Nativo P,

Franchini F, Gilliland D, et al. Size-dependent

toxicity and cell interaction mechanisms of gold

nanoparticles on mouse fibroblasts. Toxicol lett

2013;217(3):205-16. doi:

10.1016/j.toxlet.2012.11.022

79. Chaloupka K, Malam Y, Seifalian AM. Nanosilver as

a new generation of nanoproduct in biomedical

applications. Trends Biotechnol 2010;28(11):580-8.

doi: 10.1016/j.tibtech.2010.07.006

80. García-Barrasa J, López-de-Luzuriaga JM, Monge

M. Silver nanoparticles: Synthesis through chemical

methods in solution and biomedical applications.

Cent Eur J Chem 2011;9(1):7-19. doi:

10.2478/s11532-010-0124-x

81. Wang Y, Newell BB, Irudayaraj J. Folic acid

protected silver nanocarriers for targeted drug

delivery. J Biomed Nanotechnol 2012;8(5):751-9.

doi: 10.1166/jbn.2012.1437

82. Zhou W, Ma Y, Yang H, Ding Y, Luo X. A label-

free biosensor based on silver nanoparticles array for

clinical detection of serum p53 in head and neck

squamous cell carcinoma. Int J Nanomedicine

2011;6(1):381-6. doi: 10.2147/IJN.S13249

83. Wang J, Song D, Wang L, Zhang H, Zhang H, Sun

Y. Design and performances of immunoassay based

on spr biosensor with au/ag alloy nanocomposites.

Sensors Actuators B: Chem 2011;157(2):547-53. doi:

10.1016/j.snb.2011.05.020

84. Mei N, Zhang Y, Chen Y, Guo X, Ding W, Ali SF, et

al. Silver nanoparticle-induced mutations and

oxidative stress in mouse lymphoma cells. Environ

Mol Mutagen 2012;53(6):409-19. doi:

10.1002/em.21698

85. Kim S, Ryu DY. Silver nanoparticle-induced

oxidative stress, genotoxicity and apoptosis in

cultured cells and animal tissues. J Appl Toxicol

2013;33(2):78-89. doi: 10.1002/jat.2792

86. Xin L, Wang J, Wu Y, Guo S, Tong J. Increased

oxidative stress and activated heat shock proteins in

human cell lines by silver nanoparticles. Hum Exp

Toxicol 2015;34(3):315-23. doi:

10.1177/0960327114538988

87. Yang X, Gondikas AP, Marinakos SM, Auffan M,

Liu J, Hsu-Kim H, et al. Mechanism of silver

nanoparticle toxicity is dependent on dissolved silver

and surface coating in caenorhabditis elegans.

Environ Sci Technol 2012;46(2):1119-27. doi:

10.1021/es202417t

88. Batchelor-McAuley C, Tschulik K, Neumann C,

Laborda E, Compton RG. Why are silver

nanoparticles more toxic than bulk silver? Towards

understanding the dissolution and toxicity of silver

nanoparticles. Int J Electrochem Sci 2014;9(3):1132-

8.

89. Gliga AR, Skoglund S, Wallinder IO, Fadeel B,

Karlsson HL. Size-dependent cytotoxicity of silver

nanoparticles in human lung cells: The role of cellular

uptake, agglomeration and ag release. Part Fibre

Toxicol 2014;11(1):11. doi: 10.1186/1743-8977-11-

11

90. Sanwlani S, Rawat K, Pal M, Bohidar HB, Verma

AK. Cellular uptake induced biotoxicity of surface-

modified cdse quantum dots. J Nanopart Res

2014;16:2382. doi: 10.1007/s11051-014-2382-6

91. Vicario-Parés U, Castañaga L, Lacave JM, Oron M,

Reip P, Berhanu D, et al. Comparative toxicity of

metal oxide nanoparticles (CuO, ZnO and TiO2) to

developing zebrafish embryos. J Nanopart Res

2014;16:2550. doi: 10.1007/s11051-014-2550-8

92. Pujalté I, Passagne I, Brouillaud B, Tréguer M,

Durand E, Ohayon-Courtès C, et al. Cytotoxicity and

oxidative stress induced by different metallic

nanoparticles on human kidney cells. Part Fibre

Toxicol 2011;8:10. doi: 10.1186/1743-8977-8-10

93. Lee YH, Cheng FY, Chiu HW, Tsai JC, Fang CY,

Chen CW, et al. Cytotoxicity, oxidative stress,

apoptosis and the autophagic effects of silver

nanoparticles in mouse embryonic fibroblasts.

Biomaterials 2014;35(16):4706-15. doi:

10.1016/j.biomaterials.2014.02.021

94. Chueh PJ, Liang RY, Lee YH, Zeng ZM, Chuang

SM. Differential cytotoxic effects of gold

nanoparticles in different mammalian cell lines. J

Hazard Mater 2014;264:303-12. doi:

10.1016/j.jhazmat.2013.11.031

95. Grabowski N, Hillaireau H, Vergnaud J, Santiago

LA, Kerdine-Romer S, Pallardy M, et al. Toxicity of

surface-modified plga nanoparticles toward lung

alveolar epithelial cells. Int J Pharm

2013;454(2):686-94. doi:

10.1016/j.ijpharm.2013.05.025