A Review of Molecular Mechanisms Involved in Toxicity of Nanoparticles

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


Introduction
Nanotechnology advancement in medical sciences led to the design and synthesis of nanostructures for biomedical applications.][3][4][5][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).[9][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.][13][14] Prooxidants are chemicals that induce oxidative stress through either creating reactive oxygen species or inhibiting antioxidants.][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. 16,18n 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. 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.5][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. 28T 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

Toxicity mechanisms of nanoparticles
2][33][34][35][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.

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).][42][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 CNTtreated macrophages led to increased secretion of cytokines.In another study, MWCNT were revealed to trigger the macrophages to produce TGF-β1 and plateletderived growth factor (PDGF) that promoted the | 449

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][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. 50Conversely, some reports have pointed out the potential toxicity of TiO2 nanoparticles, including their ability to induce oxidative stress, genotoxicity and immunotoxicity. 51,52However, 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.][58][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. 54n 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. 61imilar 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
][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 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. 73,74nisotropic GNPs have more potential oxidation than the isotropic ones due to their highly exposed surface areas and defects.][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. 79,80In 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.][86][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. 88ecently, 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

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