Shape-engineered titanium dioxide nanoparticles (TiO₂-NPs): cytotoxicity and genotoxicity in bronchial epithelial cells

Highlights

• Cytotoxicity/genotoxicity evaluation of engineered TiO₂-NPs with different shapes (bipyramid, rods, platelets).

• TiO₂-NPs cytotoxicity was low, influenced by the shape and by light exposure.

• Genotoxicity was influenced by cellular-uptake and aggregation tendency of TiO₂-NPs.

• The presence of light enhanced the oxidative DNA damage.

• It seems that shape engineered TiO₂-NPs are safer than the commercial ones.

Abstract

The aim of this study was to evaluate cytotoxicity (WST-1 assay), LDH release (LDH assay) and genotoxicity (Comet assay) of three engineered TiO₂-NPs with different shapes (bipyramids, rods, platelets) in comparison with two commercial TiO₂-NPs (P25, food grade). After NPs characterization (SEM/T-SEM and DLS), biological effects of NPs were assessed on BEAS-2B cells in presence/absence of light. The cellular uptake of NPs was analyzed using Raman spectroscopy.

The cytotoxic effects were mostly slight. After light exposure, the largest cytotoxicity (WST-1 assay) was observed for rods; P25, bipyramids and platelets showed a similar effect; no effect was induced by food grade. No LDH release was detected, confirming the low effect on plasma membrane. Food grade and platelets induced direct genotoxicity while P25, food grade and platelets caused oxidative DNA damage. No genotoxic or oxidative damage was induced by bipyramids and rods. Biological effects were overall lower in darkness than after light exposure. Considering that only food grade, P25 and platelets (more agglomerated) were internalized by cells, the uptake resulted correlated with genotoxicity.

In conclusion, cytotoxicity of NPs was low and affected by shape and light exposure, while genotoxicity was influenced by cellular-uptake and aggregation tendency.

Introduction

Nanoparticles (NPs) are defined as particles having their three dimension in the range of 1–100 nm (ISO, 2015). Actually, many consumer products incorporates NPs. The technological, medical and economic benefits of NPs are considerable, but the presence of nanoparticles in the environment could cause adverse effects to humans. NPs have a greater surface area per mass unit, so they potentially have an increased biological activity compared to fine particles. Moreover, NPs size is comparable to the size of cellular structures, so NPs might potentially emulate biological molecules or interfere physically with biological processes (Magdolenova et al., 2012a).

TiO₂ is the oxide of titanium and it has different crystalline structures: anatase, brookite and rutile. Brookite is not produced by industry and is not incorporated in commercial products. In contrast, rutile and anatase are largely used in commercial products (Jovanovic, 2015). TiO₂ is one of the most frequently applied NPs and it is in the top five NPs used in consumer products (Shi et al., 2013). TiO₂-NPs produced are used primarily as a pigment owing to their brightness, resistance to discoloration and high refractive index. As a pigment, TiO₂-NPs are incorporated in paints, plastic materials, paper, foods, medical products and cosmetics. Due to its catalytic and photocatalytic properties, TiO₂ is also used as an antimicrobial agent and a catalyst for purification of air and water (Bonetta et al., 2013; Tomankova et al., 2015).

TiO₂-NPs could be engineered in terms of shapes and sizes by changing synthesis conditions such as raw material, temperature, acidic and alkaline conditions. Engineered TiO₂-NPs with various shapes (e.g. rods, dots and belts) have been prepared for different applications (Bernard and Curtiss, 2005; Sha et al., 2015; Wang et al., 2004). In particular, engineered fiber-shaped nanomaterials (i.e. nanowires, nanotubes) are very attractive because they showed higher activity and advantages in photocatalysis, charge transfer and sensing applications due to their structure (Hamilton et al., 2009). However, these new and enhanced properties may also induce higher toxicological effects upon exposure with biological tissues.

Humans can be exposed to TiO₂-NPs via three portals of entry: oral (mainly via food consumption), dermal (often through cosmetic and sunscreen applications) and inhalation (mainly under occupational and manufacturing conditions) (Warheit and Donner, 2015).

Based on the evidence that TiO₂ can induce lung cancer in rats, TiO₂-NPs were classified as possibly carcinogenic to humans (group 2B) by the International Agency for Research on Cancer (IARC, 2010). Indeed, the inhalation and instillation of rutile and anatase TiO₂-NPs induced lung tumors (Xu et al., 2010), broncho-alveolar adenomas and cystic keratinizing squamous cell carcinomas (De Matteis et al., 2016; Mohra et al., 2006). TiO₂-NPs were also classified as potential occupational carcinogens by the National Institute for Occupational Safety and Health (NIOSH, 2011; Chen et al., 2014).

Many in vitro studies showed cytotoxicity, genotoxicity and oxidative effects induced by TiO₂-NPs through oxidants generation, inflammation and apoptosis (Jugan et al., 2011; Karlsson et al., 2015; Park et al., 2008; Shi et al., 2010). The potential of NPs to cause DNA damage is an important aspect that needs attention due to possible mutations and carcinogenesis. Physico-chemical characteristics of NPs have an important role in toxicity. Different studies showed that biological effects can be influenced by crystalline structure, size, shape, exterior area, agglomeration/aggregation and surface properties (Bhattacharya et al., 2009; Johnston et al., 2009). Some studies revealed that crystalline structure probably influences the induced toxicity, in particular the anatase seems to be more reactive (Sayes et al., 2006) and induces more toxic, genotoxic and inflammatory effects, than the rutile (Falck et al., 2009; Petkovic et al., 2011; Xue et al., 2010). However, other studies gave contradictory results with rutile forms being more toxic than anatase (Gurr et al., 2005; Numano et al., 2014; Uboldi et al., 2016). The effect of agglomeration/aggregation of NPs on toxicity is not well understood yet. In recent studies, some authors demonstrated that agglomeration can influence NPs genotoxicity (Magdolenova et al., 2012b; Prasad et al., 2013).

Although physico-chemical properties of NPs can have an important role in the impact on their toxicity, only few studies on shape dependent TiO₂ toxicity has been conducted (Allegri et al., 2016; Hamilton et al., 2009; Park et al., 2013). Additional studies are needed to evaluate the role of shape on TiO₂-NPs toxicity in order to produce useful data for assessing the safety of engineered NPs.

To address this issue, the aim of this study was to investigate cytotoxicity (WST-1 assay), LDH release (LDH assay) and genotoxicity (Comet assay) of three types of engineered TiO₂-NPs of different shapes (bipyramids, rods and platelet NPs) in BEAS-2B (cells isolated from human bronchial epithelium) in comparison with two commercial types of TiO₂-NPs (P25 and food grade). Since the exposure to TiO₂-NPs mainly occurs through respiratory tract (occupational and manufacturing conditions), human cells of the respiratory system (such as BEAS-2B), were selected as a good cell model for in vitro toxicology tests. All the TiO₂-NPs in this study were first physico-chemically characterized, even in different culture media to study their agglomeration state, and then they were biologically evaluated. In order to take into account the photocatalytic properties of the TiO₂-NPs, we investigated the cytotoxicity and genotoxicity on BEAS-2B under light exposure and in darkness. Moreover, a modern application of Raman spectroscopy, the 3D confocal Raman imaging, was used to study the uptake of the NPs within the BEAS-2B cells, as the Raman spectra provide information about both organic molecules and solid NPs simultaneously (Ahlinder et al., 2013).