Nanotoxicology, 2012; Early Online, 1–12© 2012 Informa UK, Ltd.ISSN: 1743-5390 print / 1743-5404 onlineDOI: 10.3109/17435390.2012.741724

Comparative study of ZnO and TiO2 nanoparticles: physicochemicalcharacterisation and toxicological effects on human coloncarcinoma cells

Isabella De Angelis1, Flavia Barone1, Andrea Zijno1, Loreline Bizzarri1, Maria Teresa Russo2, Roberta Pozzi3,Fabio Franchini4, Guido Giudetti4, Chiara Uboldi4, Jessica Ponti4, Francois Rossi4, & Barbara De Berardis1

1Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299 Roma, Italy, 2NationalCenter for Chemicals, Istituto Superiore di Sanità, Rome, Italy, 3Department of Technology and Health, Istituto Superiore diSanità, Viale Regina Elena 299 Roma, Italy and 4European Commission, Joint Research Centre (JRC), Institute for Health andConsumer Protection, Nanobiosciences Unit, Ispra (VA), Italy

AbstractDespite human gastrointestinal exposure to nanoparticles (NPs),data on NPs toxicity in intestinal cells are quite scanty. In thisstudy we evaluated the toxicity induced by zinc oxide (ZnO) andtitanium dioxide (TiO2) NPs on Caco-2 cells. Only ZnO NPsproduced significant cytotoxicity, evaluated by two differentassays. The presence of foetal calf serum in culture mediumsignificantly reduced ZnO NPs toxicity as well as ion leakage andNP-cell interaction. The two NPs increased the intracellularamount of reactive oxygen species (ROS) after 6 h treatment.However, only ZnO NPs increased ROS and induced IL-8 releaseboth after 6 and 24 h. Experimental data indicate a main role ofchemical composition and solubility in ZnO NPs toxicity.Moreover our results suggest a key role of oxidative stress in ZnONPs cytotoxicity induction related both to ion leakage and to cellinteraction with NPs in serum-free medium.

Keywords: nanomaterial characteristics, Caco-2 cells, uptake,cytotoxicity, reactive oxygen species production

Introduction

Nanotechnology has become one of the leading technologiesof the past 10 years. According to a recent survey, over1000 nanotechnology-based products are available to con-sumers in the world (Project on Emerging Nanotechnologies2009). This is due to unique physico-chemical properties ofnanomaterials, which make them desirable for industrial andbiomedical applications.

The rapid development of the NPs-based products is amatter of concern among scientific community due to theirpotential human health effects. Some studies suggest that theincreased chemical reactivity of materials at nanoscale couldlead to unpredictable interactions with cellular system

interfering with proteins, DNA, lipids, membranes, orga-nelles and biological fluids.

Although the mechanisms underlying the NPs toxicity arenot yet elucidated, recent studies have indicated that thephysico-chemical characteristics of NPs (size, shape, surfacearea, chemical composition, solubility, surface charge) play acritical role in understanding the induced biologicalresponse (Yang et al. 2009; Bai et al. 2010; Xu et al. 2010;Pujalté et al. 2011).

It has been suggested that NPs can enter the mitochon-dria of cells, inducing oxidative stress and apoptosis, or thatthe large surface areas of NPs can induce greater productionof reactive oxygen species (ROS), potentially damaging DNA(Xia et al. 2007; Hussain et al. 2009). Other studies havesuggested that surface charge can influence particle uptake,ROS generation and genomic damage (Bhattacharya et al.2009; Zhang & Monteiro-Riviere 2009). Moreover, someauthors have also shown that soluble NPs might releaseions that undergo chemical reactions to form ROS (Brunneret al. 2006; Pujalté et al. 2011).

ZnO and TiO2 nanoparticles (ZnO NPs and TiO2 NPs) areamong the most used metal oxides nanoparticles in com-mercial products such as in electronic materials, rubbermanufacture, cosmetics and pharmaceuticals, paints anddental cements. Due to their antimicrobial and antifungalproperties, ZnO NPs were used in textiles, foods, cosmeticsand food packaging. Recent studies on applications as bio-markers for cancer diagnosis and on drug delivery show thatZnO NPs are versatile platforms for biomedical applicationsand therapeutic interventions (Shen et al. 2008; Guo et al.2008). TiO2 NPs have shown promise as photosensitizer forthe photodynamic therapy of human colon carcinoma cells(Zhang and Sun 2004).

Several in vitro toxicity studies of ZnO NPs and TiO2 NPsthrough pulmonary exposure are available (Bhattacharya

Correspondence: Barbara De Berardis, Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299 Roma, Italy.Tel: +390649902896. E-mail: barbara.deberardis@iss.it

(Received 11 January 2012; accepted 16 October 2012)

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et al. 2009, Xia et al. 2008; Kim et al. 2010; Huang et al. 2010;Heng et al. 2010; Hsiao & Huang 2011; Karlsson et al. 2009;Park et al. 2008). Moreover, in recent years there has beengrowing information on toxicity of these particles in humanepidermal cells (Sharma et al. 2009; Kocbeck et al. 2010;Shukla et al. 2011). Few data are available about the toxicityand cellular responses of intestinal cells exposed to ZnO NPsand TiO2 NPs. Koeneman et al. (2010) showed that TiO2 NPsdid not induce cell death, but at concentrations of 10 mg/mLand above they were able to cross the epithelial lining of theintestinal cells by transcytosis, penetrate into the cells with-out disrupting junctional complexes and induce a rise inintracellular-free calcium. Exposure to ZnO NPs for 24 hinduced a significant decrease of cell viability, apoptosis,increased generation of superoxide and depolarisation ofthe inner mitochondrial membrane on human colon carci-noma cells, RKO (Moos et al. 2010) and LoVo cells(De Berardis et al. 2010).

Due to the increasing consumer exposure and the signi-ficant lack of data about the toxicological properties of ZnONPs and TiO2 NPs on gastrointestinal tract, in this study weevaluated the toxicological effects of ZnO NPs and TiO2 NPson Caco-2 cell line. It is a well-known in vitro intestinalmodel used in many pharmaceutical studies to determineintestinal permeability of iron, cadmium, copper, calciumand zinc (Zhou and Yokel 2005), and it has been indicatedas immortalised cell line to characterise the potential humanhealtheffect fromoral exposure tonanomaterials (Oberdörsteret al. 2005). Moreover, we attempted to relate the role of thephysico-chemical characteristics of ZnO NPs and TiO2 NPsto the toxic effects ensuing from NPs-cell interaction. Wecharacterised the NPs in terms of size, surface area, numberparticles, agglomeration state, surface charge, chemical com-position, impurities, ion-release, by dynamic light scattering(DLS), electron microscopy (SEM and TEM) and inductivelycoupled plasma-mass spectrometer (ICP-MS). The influenceof physical and chemical properties of ZnO NPs and TiO2 NPson observed biological effects was discussed.

Materials and methods

ParticlesZnO NPs, TiO2 anatase NPs were obtained from SigmaAldrich Company Ltd. The nominal sizes of particles aspurchased are 50–70 nm for ZnO NPs and <25 nm forTiO2 NPs. The manufacturer makes no statement on purityof ZnO NPs, whereas a purity of 99.7% for TiO2 anataseNPs is reported.

DLS characterisationNanoparticles were suspended both in ethanol and inserum-free culture medium to a final concentration of0.1 mg/mL in a glass scintillation vial, probe-sonicated inice for 20 min under temperature-controlled conditions, at100% amplitude (Hielscher UP200S, 200W).

The DLS measurements were performed on 1 mL samplevolume on a Zetasizer Nano Zs (Malvern Instruments, UK),following the protocols indicated by ISO 13321:1996 and22412:2008. After a 2 min equilibration step at 25�C, each

sample underwent three measurements. Read number andduration for each measurement were set on ‘automatic’ onthe Zetasizer control software. Only intensity distributiondata were considered for the analysis. Values were obtainedby averaging the three measurements using Malvern’sproprietary software.

Surface chargeZeta potential measurements were conducted to check thecolloidal stability and surface charge of NP preparations inMilli-Q� water and in serum-free culture medium. Themeasurements were conducted in triplicate on 700 ml vol-ume samples on the Zetasizer Nano Zs (Malvern Instru-ments, UK), using the automatic measurement protocolsrecommended by Malvern.

Single particle characterisation by electron microscopyTwo to three mg of particles were weighted with a MettlerH54 AR analytical balance (precision 0.01 mg). The parti-cles were suspended both in ethanol and in cell culturemedium to evaluate the characteristics of particles in foodsimulators (EFSA 2011) and to mimic their exposurewith biological systems. Stock suspensions of particleswere probe-sonicated (Vibracell, Sonics & Materials Inc.,USA) and immediately after sonication 2 mL of suspen-sions were filtered through 0.05 mm pore polycarbonatemembranes.

For SEM analysis portions of filters with particles weremounted on stubs and coated with a thin gold film depositedby sputtering; for TEM analysis a thin carbon film wasevaporated on polycarbonate membranes with particles,then the polycarbonate was dissolved by chloroform.

SEM and TEM were used to characterise the single NPs.Morphological analysis of single particles was performed bya SEM FEI XL30 (FEI Company, the Netherlands) equippedwith Soft Imaging System. More than 1000 particles wereanalysed and particle number in the suspensions and surfacearea of particles were estimated (De Berardis et al. 2010).Morphology and primary size of the particles were alsodetermined by a TEM (FEI Company, the Netherlands).

Inductively Coupled Plasma Mass Spectrometry (ICP-MS)characterisation and ion leakageElemental impurities present in ZnO NPs and TiO2 NPs weremeasured by Inductively Coupled Plasma Mass Spectrom-etry (Agilent Technologies, 7700 series, Canada). ZnO NPs(3.4 mg) were analysed after dissolution in ultrapure nitricacid and microwave digestion.

TiO2 NPs (29.7 mg) were mineralised with hydrofluoricacid (HF) 39.5% RPE (Carlo ERBA SpA, Italy) by microwavedigestion (Explorer, CEM Corporation, USA) applying 10 minat 300 watt, 120 psi, 200�C. The solution was diluted inMilli-Q� water (18.2 MW*cm, Millipore Corp. USA).

The total amount of Zn and Ti (Zn or Ti as part of ZnO orTiO2 NPs) and the amount of Zn or Ti ions weremeasured byICP-MS. The NPs suspensions were incubated in completeand serum-free cell culture medium.

Two mL of NP suspensions were prepared and dividedinto two aliquots of 1 mL each. In one aliquot, the total

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amount of Zn or Ti was directly measured by ICP-MS,whereas the second aliquot was filtered by centrifugation(3000 rpm corresponding to 1810 G for 20 min) using a10 KDa, Amicon� Ultra-4, membrane (Millipore, Italy) toremove the NPs. The absence of NPs in the filtered solutionwas verified by DLS thus indicating the absence of artefactsin the measurement of Zn or Ti ion concentration.

The quantification of Zn or Ti present in the two aliquotswas done at time 0 and after 6 h of NPs incubation understandard cell culture conditions (37�C, 5% of CO2, 90% ofhumidity, HERAEUS incubator, Germany) by ICP-MSanalysis after sample digestion as described above.

Cell cultureCaco-2 cells (ATCC collection) were routinely cultured understandard cell culture conditions (37�C, 5% of CO2, 90% ofhumidity) in Dulbecco’s modified Eagle’s medium with highglucose (4.5 g/L), supplemented with 100 IU/mL penicillinand 100 mg/ml streptomycin, 4 mM/L glutamine, 1%non-essential amino acids, 10 mM HEPES and 10%heat inactivated foetal calf serum (FCS) (GIBCO BRL,Gaithersburg, MD).

Cell viabilityTo test the potential cytotoxicity exerted by TiO2 and ZnONPs on Caco-2 cells, Neutral Red Uptake (NRU) and ColonyForming Efficiency (CFE) assays have been performed.

Neutral Red Uptake (NRU) assayCaco-2 cells were plated into 24-multiwell plates (BectonDickinson, San Jose, CA, USA) at a density of 8 � 104

cells/well and treated after 4 days of culture, with 1, 2.5, 5,10 and 20 mg/cm2 of TiO2 and ZnO NPs for 6 and 24 h (threereplicates for each concentration). Both NPs were suspendedin culture medium (with or without FCS) and sonicated.A negative control (cells maintained in complete or serumfree medium) was run in parallel.

Neutral Red Uptake determination was performedaccording to Borenfreund & Puerner (1985). Results werenormalised to the control and expressed as percentage ofviability. No interference between NPs and neutral red dyewas observed.

No interference between NPs and neutral red dye wasobserved.

Statistical analysis was performed by one-way analysis ofvariance and by post hoc Least Significance Difference (LSD)test (SPSS Statistics 17.0).

Colony Forming Efficiency (CFE) assayOn Day 0, 500 Caco-2 cells were seeded in 3 mL of freshcomplete medium in each 60-mm Petri dish (three replicatesper concentration). After 24 h of incubation, the treatmentsuspensions of ZnO NPs, TiO2 NPs (1, 5 and 20 mg/cm2,corresponding to 7, 35 and 140 mg/mL) and ZnCl2 (1, 2.5, 5,7.5, 10, 15, 25, 50 and 100 mg/mL) (Sigma, Italy) wereprepared in fresh complete or serum-free cell culturemedium and added to the cells. A positive control(Na2CrO4�6H20; 10–3 M) was performed in parallel. Cellswere kept in contact with the treatment suspension for

6 and 24 h, only for 6 h in the case of ZnCl2, and thenthe test compounds were removed and replaced with com-plete fresh medium. At Day 9 after seeding, the colonies werefirst fixed using a solution of 4% (v/v) Formaldehyde (Sigma-Aldrich; St. Louis, USA) in PBS (Invitrogen; Carlsbad, CA,USA), then stained using 0.4% (v/v) of GIEMSA (Sigma-Aldrich; St. Louis, USA) in Milli-Q� water. After drying,colonies (composed of at least 50 cells) were automaticallyscored and counted using an automated cell colony counter(GelCount�; Oxford Optronix Ltd., Oxford, UK).

The results were normalised to the control (cells exposedto fresh complete or serum-free culturemedia) and expressedas % CFE ((average of treatment colonies/average of solventcontrol colonies) �100). The corresponding Standard ErrorMean was calculated for three independent experiments andat least three replicates for each experimental point(sem = (SD/Hnumber of replicates). The statistically signifi-cant difference for CFE values versus controls was calculatedby the one-way ANOVA analysis (GraphPadPrism4 statisticalsoftware, GraphPad Inc., CA, USA).

Cellular uptakeFor the uptake evaluation, 8 � 105 cells were seeded into25 cm2

flask (Corning Costar, Italy) in 5 mL of complete cellculture medium. After 24 h, cells were treated with 1, 5 and20 mg/cm2 of ZnO and TiO2 NPs (corresponding to 5, 25 and100 mg/mL) and with 5- 15- 25- 50- 100 mg/mL of ZnCl2(Sigma, Italy) prepared in complete and serum-free cellculture medium for 6 h.

After exposure, culture medium was removed and col-lected for each sample, the cells were then washed twice with5 mL of PBS and each was collected in a separate tube. Cellswere detached using 1 mL of trypsin and harvested with4 mL of complete culture medium. Cells were counted usinga Bürker chamber (Marienfeld, Germany) with trypan bluesolution (Sigma, Italy).

Cells suspension was centrifuged at 200 � g for 5 min at4oC; the supernatant was transferred into a new tube. Cellspellet was directly mineralised with Aqua Regia (HNO3: HCl1:3) and digested using microwave (two cycles at 950 W for20 min). The amount of Zn or Ti in cells (cellular uptake) wasmeasured by ICP-MS. Results are expressed as picograms ofZn/cell (pg/cell) and of Ti/cell (pg/cell).

Determination of reactive oxygen species (ROS)productionThe intracellular amount of ROS was measured using thechloromethyl derivative of dihydrofluorescein diacetate(CM-H2DCFDA, Invitrogen). Cells were plated at 1.2 �105 cells in 6-multiwell plates in triplicate and after 48 hof culture they were treated with increasing dose of nano-particles. After 6 and 24 h of treatment the monolayers werewashed with PBS, incubated for 30 min in the dark at roomtemperature with 5 mM CM-H2DCFDAs and suspended inPBS + EDTA. Fluorescence was measured using excitationand emission wavelengths of 485 and 535 nm, respectively,on a FACScan (Becton Dickinson). The values wereexpressed as mean fluorescence of the cell population.H2O2 (50 mM)-treated cells were used as positive control.

ZnO and TiO2 NPs toxicological comparative study

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The interference of NPs on CM-H2DCFDA signals wasevaluated in absence of cells. No influence of NPs onfluorescence levels respect to background was observed.

Measurement of proinflammatory mediator releaseTumour necrosis factor-a (TNF-a), interleukin-6 (IL-6) andinterleukin-8 (IL-8) cytokine release was measured in theculture media of Caco-2 cells with 1 or 2.5 mg/cm2 of ZnONPs or TiO2 NPs, using ELISA kit (Biotrak, Cellular Commu-nication Assays, AmershamPharmacia Biotech, Uppsala Swe-den). After 6 and 24 h of nanoparticles treatment, cellsupernatants were collected, centrifuged at 1200 rpm for5 min and frozen at –80�C until tested. After the addition ofchromogen, the absorbance at 450–550 nm for TNF-a and450 nm for IL-6 and IL-8 wasmeasured in amicroplate reader(Biorad, California). Statistically significant differencesbetween unexposed and NP-exposed cells were assessed byStudent’s paired t test. A p < 0.05 was considered significant.

Results

Hydrodynamic size and surface chargeThe mean particle sizes evaluated by intensity, for 0.1 mg/mLNP suspensions in ethanol and in Caco-2 medium withoutserum after probe sonication, are reported in Table I.

The probe sonication was effective in improving samplemonodispersity for ZnO NPs both in ethanol and in cellculture medium, as shown by polydispersity index(PdI = 0.117).

On the other hand, the TiO2 NPs in ethanol after sonicationpersist in being polydisperse (PdI = 0.335). A main size peakaround 770 nm and a secondary around to ~150 nm wereobserved. Themonodispersity of TiO2 NPs in culturemediumsuspension improves, as indicated by lower values of PdI.

Remarkably, the mean sizes of ZnO and TiO2 NPs, oncedispersed in ethanol and culture medium, largely differ fromthe manufacturer declared ones, indicating an agglomera-tion state that even sonication steps cannot solve: this is aknown issue when dispersing dry powders into test disper-sants (Bihari et al. 2008; Taurozzi et al. 2011).

In addition, due to DLS technical limits (Calzolai et al.2011), the NPs size distribution in complete culture mediumshowed unsuitable results indicating NPs agglomerated/aggregated in suspensions.

The Z-Potentials of ZnO and TiO2 NPs were negativeeither in water or in DMEM as shown in Table I. Moreover,

the measurement of Z-potential indicated a lower stability ofTiO2 NPs in both dispersants (ethanol and cell culturemedium) as compared to ZnO NPs.

Electron microscopy characterisationTEM observations allowed us to recognise that ZnO NPswere present with two different morphologies: 1) spherulesranging from 45 to 60 nm in diameter, 2) “rod-like” particles,approximately up to 70 nm in width and 170 nm in length.Moreover agglomerates of spherules and rod-like particleswere observable (Figure 1).

Size distribution, obtained by SEM equipped with SoftImaging System for ZnO NPs suspended in ethanol, showedan average diameter equal to 199 ± 30 nm, as shownin Table I. Only 34% of particles possessed dimensionsbelow 100 nm, the remaining particles were agglomeratesranging from 100 to 800 nm. In serum-free DMEM, the sizedistribution for ZnO NPs showed an average diameter of128 ± 36 nm. Fifty-five percent of particles possessed dimen-sions below 100 nm, the remaining particles were agglom-erates ranging from 100 to 900 nm. In culture mediumcontaining serum, the average diameter of ZnO NPs sizedistribution was of 150 ± 31 nm and 43% of particlespossessed dimensions in the range of 50–90 nm; the remain-ing particles ranged from 100 to 1.15 mm.

TEM observations of TiO2 NPs showed two different typesofmorphologies: 1) single spherules approximately 20–60 nmindiameter, 2)particleswith irregularshapeup to40nmwidthand 60 nm length. Moreover large agglomerates of spherulesand chains of spherules were observable (Figure 2).

Size distribution of TiO2 NPs suspended in ethanolshowed an average diameter equal to 284 ± 43 nm(Table I). Only 16% of particles possessed dimensions below100 nm. Most of particles were agglomerates ranging from100 nm to 1.4 mm. When TiO2 NPs were suspended inDMEM without serum the size distribution showed anaverage diameter of 220 ± 68 nm and 33% of particlespossessed dimensions below 100 nm. The remaining parti-cles were agglomerates with size ranging from 100 nm to1.6 mm. In DMEM with serum the average diameter was of200 ± 44 nm, 39% of particles were in the size range of 40–90 nm and the remaining showed dimensions from 100 to2.5 mm.

Morphological data indicate that electron microscopycould not be able to evaluate significant differences in

Table I. Physico-chemical characteristics of ZnO and TiO2 nanoparticles.

NPsPolydispersity

IndexMean hydrodynamic

diameter (nm) Primary size (nm)Average

diameter (nm)Particle

number (part/g) Z-potential (mV)

ZnO 0.117a ± 0.040 340.20a ± 12.04 45–170$ 199^,a ± 30 3.5 � 1013^,b –27.8d ± 0.4

0.121b ± 0.118 941.60b ± 118.30 128^,b ± 36 (7.12dpH 25�C)150^,c ± 31 –15.6b ± 2.4

(7.74bpH 25�C)TiO2 0.335a ± 0.020 771.90a ± 110.00 20–60$ 284^,a ± 43 1.3 � 1013^,b –14.7d ± 0.4

0.143b ± 0.109 1080.00b ± 190.50 220^,b ± 68 (7.16dpH 25�C)200^,c ± 44 –9.2b ± 0.5

(7.78bpH 25�C)aDetermined in ethanol; bDetermined in serum-free culture medium; cDetermined in complete culture medium; dDetermined in Milli-Q water; $Measured by TEM;^Measured by SEM.

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agglomeration of NPs in complete culture medium, possiblydue to interference of proteins.

In Table I the particle numbers for ZnO and TiO2 sus-pended in culture medium are displayed. Our resultsshowed that particle number in TiO2 suspension was lowerthan the value obtained for ZnO; moreover, from the analysisof diameter and aspect ratio of NPs we estimated that about11% of TiO2 NPs were present as single particles with adiameter ranging from 20 to 60 nm. On the contrary, in ZnOsuspension, we observed about 30% of single nanoparticlesin the same granulometric range. Our data suggest that thelower particle number for TiO2 was probably due to higheragglomeration state of these NPs.

Inductively coupled plasma mass spectrometry analysisof nanoparticlesThe ICP-MS analysis indicated the presence of 0.47% of Cuand traces of Ni, and Pb in the ZnONP sample, whereas 4.0%of Sc, 0.6% of Sb and 0.5% of B are found in the TiO2 NPs.

The correspondence between real and expected concen-trationof ZnOandTiO2,measured as Zn andTi concentration

in the exposure suspensions of 1, 5 and 20 mg/cm2 (corre-sponding to 5, 25 and 100 mg/mL of ZnO and TiO2)by ICP-MS was good. In fact, we expected 5, 25 and100 mg/mL and we obtained 6, 27 and 119 mg/mL of ZnO(Table II) and 6, 32 and 94 of TiO2, respectively (Table III).

The amount of Zn and Ti ions in NP suspensions of 1,5 and 20 mg/cm2 was measured at time 0 and after 6 h ofincubation under standard cell culture condition in completeand serum-free cell culture media. The results reportedin Table II show that at time 0 the 80% of ZnO NPs aredissolved in serum-free cell culture medium for 1 mg/cm2,59% for 5 mg/cm2 and 16% for 20 mg/cm2. In complete cellculture medium the amount of Zn ions was 20%, 36% and11% for 1, 5 and 20 mg/cm2, respectively.

After 6 h of incubation the amount of Zn remains constantfor all the tested concentrations showing no time-dependention leakage (Table II).

Our results demonstrate that the presence of serumprotein inhibits the complete ZnO NPs dissolution at thelower tested concentrations.

In the case of TiO2, we did not observe significant differ-ences in ions leakage after 6 h of incubation or in the twoconsidered cell culture conditions (with or without serum)(Table III).

Cytotoxicity induced by ZnO and TiO2 NP treatment onCaco-2 cellsCell viability was measured by Neutral Red (NRU) assayand Colony Forming Efficiency (CFE) assay. A dose-dependent decrease of cell viability was observed byNRU assay in presence of ZnO NPs after 6 and 24 h oftreatment with an increasing of statistically significantresults (p < 0.05 and p < 0.01, respectively). The presenceof FCS strongly reduced ZnO NP toxic effects, proba-bly through protein interaction with the NP surface(Figure 3A). A slight significant effect was observed onlyafter 24 h of treatment with 20 mg/cm2 (p < 0.01). No effecton Caco-2 cell viability was reported after treatmentwith TiO2 NPs at all the concentrations tested either inpresence or in absence of FCS (Figure 3B).

To evaluate the role of surface area and particle number inthe cytotoxicity inducedonCaco-2,NRUassaywasperformedexposing cells to several surface area and particle numberdosesof the two types of particles examined (Figure 4AandB).Regardless of metric doses (mass or surface area or particlenumber), the dose-viability curves were identical and onlyZnO NPs induced a significant cell death.

Figure 5A shows the cytotoxicity, assessed by CFE,induced by ZnO NPs on Caco-2 cells after 6 and 24 h ofexposure. A statistically significant cytotoxic effect inducedwas observed for both exposure times to 5 mg/cm2 (p < 0.01,6 h; p < 0.001, 24 h) and 20 mg/cm2 (p < 0.001, 6 and 24 h) inabsence of serum. Lower cytotoxicity was observed after 24 hof exposure to ZnO NPs in presence of serum (p < 0.01) asdisplayed by NRU assay.

Statistically significant toxicity was observed, by CFE, after6 h of exposure to ZnCl2 15 mg/mL exposure in presence ofserum (p < 0.05) and in its absence (p < 0.001), while 25,50 and 100 mg/mL induced complete cell death (Figure 5B).

Figure 2. TEM images of agglomerates of spherules observed forTiO2 NPs.

Figure 1. TEM images of spherule and rod-like agglomerates observedfor ZnO NPs.

ZnO and TiO2 NPs toxicological comparative study

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Figure 5C shows no cytotoxicity for TiO2 on Caco-2 cellsafter 6 and 24 h of exposure independently to the presence orabsence of serum.

The CFE assay is usually a more sensitive one in com-parison with others such as NRU or MTT (Ceriotti et al. 2007;Ponti et al. 2009; Ponti et al. 2010), but in this work NRU andCFE assays showed similar results, in particular in the case ofcell exposure under serum-free conditions.

ZnO and TiO2 NPs cell interactionThe ZnO and TiO2 NPs cell interaction wasmeasured by ICP-MS after 6 h of Caco-2 exposure to 1, 5 and 20 mg/cm2 of ZnOor TiO2 NPs, under complete and serum-free cell cultureconditions.

Our results showed higher cell interaction for cellexposed to ZnO NPs in serum-free culture medium(1.90 ± 0.16; 2.36 ± 0.10; 4.75 ± 0.34 pg Zn/cell for 1,5 and 20 mg/cm2, corresponding to 5, 25 and 100 mg/mL,

respectively) than in complete cell culture medium (1.14 ±0.11; 0.45 ± 0.04; 1.32 ± 0.11 pg Zn/cell for 1, 5 and 20 mg/cm2, corresponding to 5, 25 and 100 mg/mL, respectively)(Figure 6A).

Similar results were found after 6 h of Caco-2 exposure to15, 25, 50 and 100 mg/mL of ZnCl2. The observed Zn cellinteraction was higher after exposure in serum-free culturemedium (1.38 ± 0.2; 12.02 ± 0.10; 44.86 ± 0.25; 31.88 ± 0.31 pgZn/cell, respectively) than in complete cell culture medium,where the Zn in cells was not detectable because the Znconcentration was lower than the ICP-MS detection limit.However, under serum-free cell culture condition, compa-ring the same concentration of exposure, the Zn cell inter-action of ZnCl2 was higher than Zn contained in ZnO NPs(12.02 ± 0.10 vs. 2.36 ± 0.10; 31.88 ± 0.31 vs. 4.75 ± 0.34 pg Zn/cell in cells exposed to ZnCl2 and ZnO NPs, respectively),except in the case of 5 mg/mL for which the uptake

Table II. (A) ZnO and Zn concentration measured by ICP-MS in the suspensions prepared for cell treatment (treatment suspensions) expressed inmg/cm2 and mg/mL. (B) Concentrations of Zn ions measured in complete culturemedium and serum-freemedium. Results are expressed as mean ofthree experiments ± SD.A

ZnO and Zn concentration measured in treatment suspensions

Expected (ZnO) mg/cm2 Expected (ZnO) mg/mL Real (ZnO) mg/mL Expected (Zn) mg/mL Real (Zn)a mg/mL

1 5 6 ± 1 4 5 ± 1

5 25 27 ± 5 20 22 ± 8

20 100 119 ± 35 80 96 ± 11

B

Real [Zn] mg/mL measured after removal of NPs

Serum-free culture medium Complete culture medium

Real (Zn)a mg/mL Time 0 Time 6 hours Time 0 Time 6 hours

5 ± 1 4 ± 0.9 5 ± 1.7 1 ± 0 1 ± 0.6

22 ± 8 13 ± 1.2 14 ± 0.8 8 ± 0.3 8 ± 2.5

96 ± 11 16 ± 2.7 16 ± 0.2 11 ± 3.0 11 ± 2.6aTotal amount of Zn present in ZnO NPs suspensions (NPs + ions).

Table III. (A) TiO2 and Ti concentration measured by ICP-MS in the suspensions prepared for cell treatment (treatment suspensions) expressed inmg/cm2 and mg/mL. (B) Concentrations of Ti ions measured in complete culture medium and serum-free medium. Results are expressed as mean ofthree experiments ± SD.A

TiO2 and Ti concentration measured in treatment suspensions

Expected (TiO2) mg/cm2 Expected (TiO2) mg/mL Real (TiO2) mg/mL Expected (Ti) mg/mL Real (Ti)a mg/mL

1 5 6 ± 2 3 4 ± 1

5 25 32 ± 7 15 19 ± 4

20 100 94 ± 12 60 56 ± 7

B

Real (Ti) mg/mL measured after removal of NPs

Real (Ti)a mg/mL Complete culture medium Serum-free culture medium

Time 0 Time 6 h Time 0 Time 6 h

4 ± 1 0.04 ± 0.03 0.16 ± 0.20 0.04 ± 0.04 0.12 ± 0.06

19 ± 4 0.07 ± 0.03 0.08 ± 0.02 0.03 ± 0.03 0.04 ± 0.03

56 ± 7 0.08 ± 0.07 0.04 ± 0.05 0.03 ± 0.05 0.04 ± 0.04aTotal amount of Ti present in TiO2 NPs suspensions (NPs + ions).

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Figure 3. Effects of different concentrations of (A) ZnONPs and (B) TiO2 NPs on cellular viability of Caco-2 cells determined by NRU assay, after 6 and24 h of exposure in culture medium with or without FCS. Data are expressed as mean ± SD of three independent experiments, performed in triplicate.Statistical significance calculated by one-way analysis of variance and by post hoc Least Significance Difference (LSD) test.*p < 0.05; **p < 0.01.

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Figure 4. Dose-cell viability relationship: A) cell viability vs. surface area of ZnO NPs and TiO2 NPs per volume unit; B) cell viability vs. number ofparticles of ZnO NPs and TiO2 NPs per volume unit.

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measurement was lower than the ICP-MS detection limit(data not shown).

Higher NPs cell interaction was observed for cells exposedto TiO2 NPs in serum-free culture medium (4 ± 0.2; 22 ± 4;160 ± 17 pg Ti/cell for 1, 5 and 20 mg/cm2, respectively) thanin complete cell culture medium (3 ± 1; 12 ± 4;70 ± 426 pgZn/cell for 1, 5 and 20 mg/cm2, corresponding to 5, 25 and100 mg/mL, respectively) (Figure 6B).

Analysis of ROS production induced by NP exposureThe ability of ZnO and TiO2 NPs to induce intracellularROS production was measured by DCFDA (Figure 7).Values are expressed as relative increase with respectto the control. ROS production was assessed at 1 and2.5 mg/cm2 of ZnO NPs because cytotoxic effect indu-ced at higher doses hampered a reliable evaluation ofROS. The whole dose range of 1–20 mg/cm2 was tested forTiO2 NPs.

Our data show that ZnO NPs are able to induce a slightincrease in ROS level after 6 and 24 h of treatment, inde-pendently on NP concentration (Figure 7A). At 1 mg/cm2 theintracellular ROS levels increase of 1.5 times with respect tocontrol after 6 and 24 h treatment; at 2.5 mg/cm2 of ZnO NPs

the increase in ROS production was similar to the lowerconcentration.

Caco-2 cells treated with TiO2 NPs showed higher level ofROS in a dose-dependent manner after 6 h (Figure 7B). Infact the increase of intracellular ROS induced by 1 and2.5 mg/cm2 of TiO2 NPs was higher than the one producedby the same doses of ZnO NPs. Finally after 24 h of incu-bation with TiO2 NPs, no significant ROS productioncompared to the control was observed.

Effects of NPs on inflammatory mediator releaseTo investigate the inflammatory potential of ZnO NPs andTiO2 NPs on Caco-2 cells, the release of TNF-a, IL-6 andIL-8 at 1 and 2.5 mg/cm2 for 6 and 24 h of treatment wasanalysed. Only IL-8 release was observed after incubations toboth NPs.

Caco-2 cells exposed to ZnO NPs showed a dose-dependent IL-8 release at both time points (Figure 8). After6 h of treatment, ZnONPs induced a slight IL-8 production of52 ± 9 pg/mL (p < 0.05) at 1 mg/cm2 and a more pronouncedrelease of 197 ± 16 pg/mL (p £ 0.005) at 2.5 mg/cm2. After 24 hof incubation, ZnO NPs markedly induced IL-8 release at

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Figure 5. Colony Forming Efficiency assay on Caco-2 cells exposed to 6 and 24 h ZnO NPs (A), 6 h ZnCl2 (B) and 6 and 24 h TiO2 NPs (C), underserum-free and complete culture medium. Results are expressed as Colony Forming efficiency (CFE) percentage of the negative control. Statisticalsignificance calculated by one-way ANOVA: *p < 0.05; **p < 0.01; *** p < 0.001.

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both concentrations (350 ± 11 pg/mL, p £ 0.005 and 643 ±14 pg/mL, p £ 0.005 at 1 and 2.5 mg/cm2, respectively).

Exposure to TiO2 NPs did not induce IL-8 release after 6 hof treatment; a slight release of 121 ± 6 pg/mL and 162 ±9 pg/mL (p < 0.05) at 1 and 2.5 mg/cm2, respectively, wasobserved after 24 h of incubation.

Discussion

In our study we used Caco-2 cell line to investigate theinteraction of ZnO NPs and TiO2 NPs with an in vitrointestinal model.

We characterised the NPs with DLS, SEM, TEM andICP-MS to understand the role of particle physico-chemicalcharacteristics (particle number, surface area, impurities,solubility) in the observed toxicological properties.

The characterisation indicates that ZnONPs and TiO2 NPsdiffer in size, agglomeration and shape. The mean sizes ofZnO NPs and TiO2 NPs in suspension never correspond tothe manufacturer declared ones and no significant levels ofmetal impurities were detected in NPs.

Moreover, the size distributions of particles, measured byDLS, showed larger values with respect to SEM and TEMmeasurements, probably due to aggregation in the liquidphase or to polydispersity of suspension.

Electron microscopy analysis showed that the particlenumber in TiO2 suspension was lower than the value

obtained for ZnO, although they have smaller primarysize. This is probably due to higher agglomeration state ofTiO2 NPs than of ZnO NPs. This different NP agglomerationstate may influence the interaction with Caco-2 cells andpartially explain the observed differences between the twoNPs concerning their ability to cause cytotoxicity.

The results obtained in this study are summarisedin Table IV.

As far as toxic effects we observed ROS production eitherfor ZnO and TiO2 NPs, whereas a significant decrease in cellviability was found only for ZnO NPs. The presence ofFCS strongly reduced zinc leakage, cell interaction and con-sequently its cytotoxic effects. This is probably due to thepresence of proteins in FCS that can be adsorbed on ZnO NPssurface and change the rate of uptake (Horie et al. 2009).Besides ZnO NPs induced higher and significant inflamma-tion on Caco-2 cells. Recent in vitro studies have revealed thatZnO NPs have a strong effect on IL-8 production from humanbronchial epithelial cells, aortic endothelial cells andhuman colon carcinoma cells (Xia et al. 2008; Park et al.2007; Wu et al. 2010; Gojova et al. 2007; De Berardis et al.2010).

In this study we tried to relate the measured physico-chemical characteristics of ZnO and TiO2 NPs (surface area,particle number, agglomerate state, surface charge, impuri-ties in the composition) with the different biological activitieson Caco-2 cells.

Electron microscopy characterisation showed that, at thesame NPs mass, cells were exposed to different surface areaand particle number due to differences in their sizes and

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Figure 7. Intracellular ROS content in Caco-2 cells exposed to ZnONPs(A) and TiO2 NPs (B) for 6 and 24 h. Values (means ± SD) represent theaverage of three independent experiments assayed in triplicate.

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agglomeration state. However the differences between thecytotoxic effects of ZnO and TiO2 NPs were unrelated tometric used to indicate doses.

Although ZnO NPs have larger primary size than TiO2

NPs, when the cells were exposed to the same surface area ofNPs the first ones caused greater cell death.

Besides a dose-dependent decrease of cell viability wasobserved only for ZnO NPs for Caco-2 cells exposed to thesame particle number doses and the distance between thetwo curves relative to TiO2 and ZnO NPs did not changesignificantly.

The surface area parameter has been largely presented asa suitable dose metric to assessing the toxicity of NPs;nevertheless controversies exist regarding the biologicaleffects of this physical characteristic. Some researcherssuggested that, compared with the effect of composition,the surface area might not be an important parameteraffecting cytotoxicity (Yang et al. 2009; Hsiao & Huang2011; Gojova et al. 2007). Our data suggest that in this studyin which we assess NPs with different composition andsolubility the surface area and the particle number do notseem to be the most important factors for the toxic effectobserved. Moreover, Hsiao found that contact area betweena single NP and a single cell (real surface area) was moreimportant than the total specific area of NPs in the cytotoxi-city induction. We suggest that single NPs of ZnO, havinglarger surface area and probably higher contact area with thecells, could cause more cell damage than TiO2 NPs withsmaller surface area.

In the same work, Hsiao showed that the shape of ZnONPs appears to dominate the toxicity induction. As morpho-logical analysis showed two principal shapes, spherules androd-like particles for ZnO NPs, present data do not enable

to conclude anything on the importance of this physicalproperty in cytotoxicity observed for this type of NPs.

Another parameter considered to be relevant in NPs cellsinteraction is the surface charge. Literature data indicateminor cytotoxicity of negatively charged nanoparticles com-pared to positive ones (Nan et al. 2008). Our data showed anegative value of surface charge for both NPs analysed. Thisresult suggests that the surface charge could not explain thedifferent biological response of ZnO and TiO2 NPs on Caco-2.

Dissolution of nanoparticles is considered important inthe cytotoxicity mechanism induction when the properties ofmaterials favour toxic ions release in suspended medium orbiological environment. Brunner et al. showed that solubleNPs, such as ZnO, caused higher cell death than insolubleNPs, such as TiO2, on human MSTO and rodent 3T3 cells.However, data in literature are discrepant because ofdifferent suspending medium, pH, ultrasonic parametersand size of NPs. Xia et al. showed that ZnO NPs toxicityon macrophage and epithelial cells was directly related toparticle dissolution and Zn2+ release in cell culture medium.Pujalté et al. and Song et al. (2010) attributed to the dissolvedzinc ions a role in the cytotoxic effect and ROS production byZnO NPs on human kidney cells and mouse macrophages,respectively. On the contrary, Moos et al. found that ZnONPsrequire a direct contact with human colon carcinoma cells toinduce cytotoxicity, and the effect was independent of theamount of soluble Zn. Finally, Hsiao et al. measured thedissolution of zinc ions in the same culture medium as theone used in our study, serum-free DMEM. They suggestedthat particle-cell interaction was dominating in cytotoxicityon human lung epithelial cells due to the low value of Zn2+ inthe solution.

In our work we observed higher amount of Zn ions inserum-free culture than in complete culture media probablydue to a masking effect of serum that seems able to inhibitthe ions release.

We compared the cytotoxicity evaluated by CFE after 6 hexposure of Caco 2 cells to ZnO NPs with that obtained forZnCl2 at doses reported in Table II on Zn ions leakagemeasured in culture medium after removal of NPs. Underserum-free culture conditions we observed that at 5 mg/cm2,corresponding to a measured 13 mg/mL of Zn ions leakage,cytotoxicity for ZnO NPs is similar to that observed for ZnCl2

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Figure 8. Effect of nanoparticles on IL-8 production in Caco-2 cells. Cells were incubated with 1 and 2,5 mg/cm2 of ZnO NPs and TiO2 NPs for 6 or24 h. Values (means ± SD) represent the average of two independent experiments assayed in triplicate. *p < 0.05. The values obtained ofIL-8 production were compared to control within each time point.

Table IV. Summary of the observed effects caused by ZnO and TiO2

NPs.

ZnO NPs TiO2 NPs

Ion leakage + -

Cytotoxicity (NRU assay and CFE assay) + -

Intracellular ROS level (DCFDA dye) + +

IL-8 release + -

Cellular uptake + +

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(CFE ~ 50% for ZnO NPs; CFE ~ 40% for ZnCl2 at 15 mg/mL)(Figure 5A). At 20 mg/cm2, corresponding to a measured16 mg/mL of Zn ions leakage, the cytotoxicity of ZnO NPs ishigher than that of ZnCl2 (CFE ~ 20% for ZnO NPs;CFE ~ 40% for ZnCl2 at 15 mg/mL) (Figure 5B). These resultssuggest that under serum-free culture conditions, the cyto-toxicity at 5 mg/cm2 is mainly due to Zn ions, whereas at20 mg/cm2 it is due to both Zn ions and ZnO NPs.

Under complete culture medium conditions, the cytotox-icity of both ZnO NPs and ZnCl2 was lower or not observed(Figure 5A and B). Concerning the Zn uptake, based on ourresults we can conclude that it is higher on Caco-2 cells afterexposure to ZnCl2 than for ZnO NPs and lower in presence ofserum than in its absence, showing a protective effect ofserum that seems able to inhibit the ions effect.

In addition, the higher cellular uptake (Figure 6A) and Znion leakage (Table II) of ZnO NPs in serum-free conditioncan explain the higher toxicity observed.

TiO2 NPs were not cytotoxic and they induced a significantROS production in Caco-2 cells only after 6 h. Some studieshave shown low cytotoxicity of TiO2 NPs (Pujalté et al. 2011;Hussain et al. 2009; Bhattacharya et al. 2009; Karlsson et al.2009; Hussain et al. 2005), whereas others observed cell deathinduction (Park et al. 2008; Falck et al. 2009; Vamanu et al.2008). At last Shukla et al. showed a mild cytotoxic responseby NRU assay in human epidermal cells only after 48 h of TiO2

exposure. Pujalté et al. demonstrated that, despite ROS pro-duction, no oxidative stress or nuclear translocation ofNF-kB were observed when human kidney cells were exposedto TiO2 NPs. On the contrary the exposure of cells to ZnO NPsinduced a significant alteration in intracellular glutathionelevels and a strong NF-kB nuclear translocation.

Although the TiO2 NPs did not induce any cytotoxic effect,these NPs showed a higher cell interaction than ZnO NPs(Figure 6B). Other studies analysed the cellular uptake ofZnO NPs (Sharma et al. 2011; Roy et al. 2011) and TiO2 NPs(Chen et al. 2010; Shukla et al. 2011). A recent study showeda higher uptake of TiO2 NPs compared to that of ZnO NPs inbacterial cells (Kumar et al. 2011). We suggest that the highercell interaction of TiO2 NPs observed in our study could bedue to spherical shape of these NPs compared to ZnO NPsin which both spherical and rod shape were present(Chithrani et al. 2006).

Conclusions

In conclusion our data on the comparative analysis of theZnO and TiO2 NP cytotoxic effect seem to indicate thatchemical properties (composition and dissolution) playeda primary role in the cell death induction.

Both NPs are able to increase the intracellular ROS levelsafter 6 h treatment but only ZnO NPs increased the release ofthe proinflammatory IL-8 cytokine. This finding suggests akey role of oxidative stress related to ion leakage and to cellinteraction with ZnO NPs in serum-free medium. Oxidativestress is a well-documented mechanism for cytotoxic dam-age induced by metal oxide NPs. Several studies highlightedthe involvement of oxidative stress in the toxicity mechanismof ZnO NPs on the human cells of epithelial lung, liver and

intestinal tract (Lin et al. 2009; Ahamed et al. 2011;Sharma et al. 2011; De Berardis et al. 2010).

Besides our data show that after 24 h exposure to TiO2

NPs, the ROS levels were lower than the one observed after6 h. Our results suggest that the ROS produced by the cells asa consequence of the ZnO NPs and TiO2 NPs interactioninduces different cellular responses. We suggest that Caco-2cells were able to maintain their antioxidant potential afterTiO2 NPs exposure under our experimental conditions. Onthe contrary, the observed cytotoxicity induced by ZnO NPsin Caco-2 cells could be due to the accumulation of ROS andthen to the induction of oxidative stress, as previouslyreported on LoVo cells (De Berardis et al. 2010).

Further investigations are needed to better elucidate theoxidative stress induction and to evaluate the potentialoxidative damage and genotoxicity of ZnO and TiO2 NPs.

Acknowledgements

This work has been performed in the framework of a col-laborative agreements N� CCR.IHCP.CA31557 between theIstituto Superiore di Sanità and the Joint Research Centre(JRC), Institute for Health and Consumer Protection (EU).

Declaration of interest

The authors report no conflicts of interest. The authors aloneare responsible for the content and writing of the paper.

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