Toxicology in Vitro 25 (2011) 1694–1700

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Toxicology in Vitro

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Assessment of the cytotoxicity of aluminium oxide nanoparticles on selectedmammalian cells

E. Radziun a,⇑, J. Dudkiewicz Wilczynska a, I. Ksia _zek a, K. Nowak a, E.L. Anuszewska a, A. Kunicki b,A. Olszyna c, T. Zabkowski d

a Department of Biochemistry and Biopharmaceuticals, National Medicines Institute, Chelmska 30/34, 00-725 Warsaw, Polandb Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Polandc Faculty of Materials Science and Engineering, Warsaw University of Technology, Woloska 141, 02-507 Warsaw, Polandd Military Institute of Medicine, Department of Urology, Szaserów 128, 04-141 Warsaw, Poland

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 April 2011Accepted 18 July 2011Available online 3 August 2011

Keywords:Aluminium oxide nanoparticlesCytotoxicityMammalian cellsL929 – murine fibroblast cell lineBJ – human fibroblast cell line

0887-2333/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.tiv.2011.07.010

⇑ Corresponding author. Tel.: +48 22 841 21 65x32E-mail address: eradziun@gmail.com (E. Radziun).

The rapid development of nanotechnology raises both enthusiasm and anxiety among researchers, whichis related to the safety use of the manufactured materials. Thus, the aim of this study was to investigatethe effect of aluminium oxide nanoparticles on the viability of selected mammalian cells in vitro. The alu-minium oxide nanoparticles were characterised using SEM and BET analyses. Based on Zeta (f) potentialmeasurements and particle size distribution, the tested suspensions of aluminium oxide nanoparticles inwater and nutrient solutions with or without FBS were classified as unstable. Cell viability, the degree ofapoptosis induction and nanoparticles internalization into the cells were assessed after 24 h of cell expo-sure to Al2O3 nanoparticles. Our results confirm the ability of aluminium oxide nanoparticles to penetratethrough the membranes of L929 and BJ cells. Despite this, there was no significant increase in apoptosisor decrease in cell viability observed, suggesting that aluminium oxide nanoparticles in the tested rangeof concentrations has no cytotoxic effects on the selected mammalian cells.

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1. Introduction

Nanotechnology is one of the fastest growing areas of advancedtechnology, thus being a source of hope for many branches of mod-ern industry, as well as for medicine and pharmacy. It is believedthat in the near future, properly designed nanoparticles of metaloxides will be used to impregnate clothing and medical devices,to combat viruses and bacteria, in new drug delivery systems orcancer therapy, as well as for cell labelling and in biosensors (Jengand Swanson, 2006; Lanone et al., 2009). Unfortunately, the enor-mous development of nanotechnology has increased the concen-tration of nanoparticles in the environment and humansurroundings, causing exposure to continuous, uncontrolled con-tact with nanomaterials. It has been proven that metal nanoparti-cles, which are inhaled or contact with the skin, may penetrate intothe bloodstream and may subsequently be transported to variousorgans of the animal and human body (Geiser et al., 2005; Shimadaet al., 2006; Sahoo et al., 2007). Despite the toxicological studiesconducted, it remains unclear which of the nanomaterials manu-factured on an industrial scale pose a danger to living organisms.So far there is a serious lack of information about the correlation

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between the effect of nanoparticles on organisms, and their size,structure and actual surface area, degree of aggregation, chemicalcomposition, degree of oxidation or crystalline phase. It is causedby a variety of toxicological methodology, which makes it difficultto compare the obtained results of reported studies (Lanone et al.,2009). For the medical science to fully benefit from the new nano-technology achievements, it is important not only to know the ef-fect of modifying the size of matter, but also to determine themechanisms of penetration of nanoparticles into the cells, theiractivity inside the cells, their metabolic pathways and the degreeof accumulation in different organs of living organisms.

It has been reported that many attempts have already beenmade at determining the biocompatibility of nanometric aluminafor plant species, bacterial cells, animals and humans. Yang andWatts (2005) found that aluminium oxide nanoparticles shows aminor inhibitory effect on seed germination and root elongationin plants such as maize, cabbage and carrot, which is no longerseen after coating the nanoparticles with phenanthrene. In theiropinion the phytotoxicity of aluminium oxide nanoparticles isdue to the presence of reactive hydroxyl groups, which wereblocked after covering the nanomaterial surface with phenan-threne. Lin and Xing (2007) showed that the phytotoxic activityof nanometric Al2O3 is significantly lower compared to nanoparti-cles of zinc and zinc oxide. The high toxicity of zinc oxide

E. Radziun et al. / Toxicology in Vitro 25 (2011) 1694–1700 1695

nanoparticles was also confirmed by studies involving Escherichiacoli bacteria (Hu et al., 2009). Nanometric alumina (Al2O3) showedan average toxicity; the lethal dose for 50% of E. coli bacteria was326.1 mg/l. Tin (IV) oxide and titanium oxide nanoparticles werethe least toxic in this study.

The response of A549 human pneumocytes after exposure todifferent nanoparticles showed that aluminium oxide nanoparti-cles induce a minor cytotoxic effect compared to nanometric tita-nium dioxide and carbon nanotubes (Simon-Deckers et al., 2008).The cytotoxic activity of aluminium oxide nanoparticles from var-ious manufacturers was also estimated for pulmonary macro-phages derived from mice and rats (Soto et al., 2005; Wagneret al., 2007). Murine macrophages were found to be highly sensi-tive to the presence of aluminium oxide nanoparticles. At a con-centration as low as 10 lg/ml, after 48 hours of exposure to50 nm aluminium oxide nanoparticles, the viability of these cellsdecreased by half compared to the control culture (Soto et al.,2005). However, after 24 h of exposure to aluminium oxide nano-particles of an average grain size 40 nm, there was no changes inthe cell viability of rat alveolar macrophages (NR8383 line), evenat a concentration as high as 250 lg Al2O3/ml. Moreover, therewas no significant decrease in phagocytosis ability after 24 h ofexposure of macrophages to Al2O3 nanoparticles at concentration25 lg/ml (Wagner et al., 2007).

One of the more important uses of nanometre-sized alumina inmedicine may be its addition for orthopaedic/dental implants.According to the researchers, the most common cause of orthopae-dic implant failure is rejection resulting from aseptic inflammatoryreactions and loosening at the implant-bone tissue interface. Thecauses may include too large, micrometre grain size of conven-tional implants and their crumbling as a result of mechanical wear.Reducing the grain size of aluminium oxide not only mimics thephysiological bone cell size, but also increases the mechanicalresistance to wear. Additionally, increased specific surface area al-lows for better integration of the implant surface with bone. It isbelieved that the use of nanometric aluminium oxide could solvethe current problems associated with implantation by enhancingosseointegration and preventing graft rejection (Gutwein andWebster, 2002; Yamamoto et al., 2004). In the near future, nano-metric alumina could be also used for magnetodynamic therapy.In studies conducted by Giri et al. (2003), ferrimagnetic ‘‘thermo-grains’’ were coated with thermodynamically stable and biologi-cally inert nanometre Al2O3. Tests carried out with human whiteblood cells confirmed the improved biocompatibility and adhesionto the nanometric alumina-coated ferrimagnetic nanoparticles.Highly-developed specific surface of nanomatric aluminium oxidecoating will thus allow for selective treatment of mainly the tu-mour cells with an appropriate dose of heat, causing tumour necro-sis. Aluminium oxide nanoparticles may soon replace theadjuvants in antiviral vaccines (Frey et al., 1997, 1999); they maybe used to transfect mammalian cells, or for water treatment andpurification to remove the key pathogenic viruses such as HIV-1or AAV (Link et al., 2007).

The literature contains descriptions of many innovative meth-ods of synthesising nanometre-sized grains of aluminium oxide.In the method described by Kunicki et al. (2006), aluminium oxidenanoparticles are produced by thermal decomposition of a precur-sor obtained in the reaction of an aluminium organic compoundand aluminium alcoholate with oxygen and water vapour fromthe air.

The purpose of this study was to evaluate the effect of nanometricc-Al2O3, obtained using the method described above, on selectednormal mammalian cells. The in vitro studies were conducted onL929 cell line, recommended for the evaluation of cytotoxic activityof biomedical materials (PN-EN ISO 10993-5, 2009). In view of thepossible applications of the tested compound, experiments with

normal human fibroblasts (BJ cell line) were also carried out. TheEZ4U test was used to assess the cytotoxic effects of aluminiumoxide nanoparticles. Induction of apoptosis in cells treated withnanometric Al2O3 was evaluated using flow cytometry (annexin Vlabelled with fluorescein isothiocyanate (FITC) test and propidiumiodide). Penetration of nanometric aluminium oxide into mamma-lian cells in vitro was estimated based on the results of the determi-nation of aluminium content obtained using optical emissionspectrometry with inductively coupled plasma ICP-OES. In addition,before starting in vitro biological tests, we evaluated the stability ofaluminium oxide nanoparticles suspensions in sterile water and cellculture media on the basis of f potential measurements.

2. Materials and methods

2.1. Chemicals

EMEM medium with L-glutamine, fetal bovine serum (FBS), amixture of antibiotics: Penicillin – Streptomycin – AmphotericinB, trypsin-EDTA – 200 mg/l EDTA and 170,000 U/l trypsin (Lonza),phosphate buffer saline (PBS), and PBS without Ca2+ and Mg2+

(Institute of Immunology and Experimental Therapy), sterile water(Polfa Lublin), MTT (Sigma-Aldrich), 2-Propanol– (CH3)2CHOH P99.7% (Rathburn Chemicals), Easy For You EZ4U test (BiomedicaGmbH, Vienna Austria), human albumin (Biomed), Bradford re-agent (Bio-Rad Protein Assay), sodium hydroxide analytical grade(Chempur), redistilled water further purified in a Nanopure Deion-ization System (Barnstead), nitric acid (V) – HNO3 ca 65% analyticalgrade (POCH S.A.), FITC Annexin V Apoptosis Detection Kit I (Bec-ton Dickinson Biosciences).

2.2. Aluminium oxide nanoparticles characterization

Assessment of the biological activity was carried out for alumin-ium oxide nanoparticles, produced according to the described andpatented methods (Kunicki et al., 2006). Nano-sized aluminiumoxide was produced by thermal decomposition of a precursor ob-tained in a reaction of an aluminium organic compound Et3Aland aluminium alcoholate (iPrO)3Al with air. Calcination of theprecursor was performed at 700 �C for 48 h. In order to break upthe forming loose clusters of nanoparticles, the obtained nanopow-der was deagglomerated for three hours in boiling hexane. Alumin-ium oxide nanoparticles were characterised on the basis of SEMimages obtained using a LEO 1530 Scanning Electron Microscope.Al2O3 nanopowder specific surface area was determined usingthe BET method (Micromeritics ASAP 2020 M), consisting in themeasurement of nitrogen adsorption isotherm.

2.3. Size distribution and Zeta potential measurement

Aluminium oxide nanoparticles were suspended in sterile waterto make stock solution (10 mg/ml). The stock solution was mixedusing a vortex, then sonicated for 30 s to reduce agglomerationof nanoparticles. The resulting stock suspension was diluted 10-fold in EMEM medium supplemented with 1% antibiotics and inEMEM medium supplemented with 1% antibiotics and 10% FBS.The stability of aluminium oxide nanoparticles suspensions inthe cell culture media and sterile water was evaluated on the basisof measurements performed at room temperature: Al2O3 particlesize distribution in these dispersion media, and f potential of eachsuspension. Both parameters were determined using ZetaSizerNano-ZS particle size analyser (Malvern).

For cell exposure aluminium oxide nanoparticles suspensionswere prepared in the complete medium (EMEM + 10% FBS + 1%antibiotics) at concentrations: 10; 50; 100; 200 and 400 lg/ml.

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2.4. Cell cultures

The following cell cultures were used in our studies:

j L929 – murine fibroblasts, from ATCC.j BJ – normal human cells, skin fibroblasts, from ATCC.

Cells were grown as monolayers in T-25 or T-75 culture flask, inEMEM medium with L-glutamine, supplemented with 10% FBS andwith 1% antibiotics (Penicillin-Streptomycin-Amphotericin B). Forassays, the cells were plated in 96- or 6-well plates, at a densityneeded to reach at least 70% confluence. The cells were maintainedat 37 �C under a humidified atmosphere with 5% CO2.

2.5. Incubation with nanoparticles

After 24 h of incubation of L929 cells, and 48 hours of incuba-tion of BJ cells in the above-mentioned conditions, the culturemedium was removed and replaced with the medium with alu-minium oxide nanoparticles in concentrations ranging from 10 to400 lg Al2O3/ml. The incubation was continued for another 24 h.The culture of control cells was carried out under similar condi-tions in a culture medium without added nanoparticles. The cellswere observed by an inverted microscope Nikon Eclipse TS-100/F, in order to assess their morphology and to check their overallhealth after exposure to Al2O3 nanoparticles.

2.6. Cell viability evaluation (EZ4U assay)

The EZ4U assay was performed according to the protocol set outby the manufacturer. After 24 h of cell incubation with nanoparti-cles, 20 ll of previously prepared EZ4U reagent was added to eachwell, and the cells were incubated for 4 h at 37 �C in 5% CO2. Theabsorbance of the obtained solutions was measured at two wave-lengths: k = 492 and k = 620 nm. The absorbance measurement atk = 620 nm wavelength was recommended by the manufacturerto enable correcting the results for background from accidentallyleft fingerprints or cellular debris. Thus, the absorbance values ob-tained at 492 nm were reduced by the absorbance value obtainedat k = 620 nm. The absorbance of the solutions was measured spec-trophotometrically using an IEMS Reader MF plate reader(Labsystems).

The EZ4U test was performed in three independent experi-ments. Cell viability after treatment with Al2O3 nanoparticles wascalculated by referring the average absorbance values obtainedfor a different concentration of the nanoparticles to the averageabsorbance value of control cells, after reduction of the reagentbackground absorbance. Mean absorbance values obtained for con-trol cells were taken as 100% cell viability.

2.7. Determination of apoptosis induction by flow cytometry (test withannexin V – FITC and propidium iodide)

After 24 h of incubation with aluminium oxide nanoparticles,the culture medium and the cells previously detached using thetrypsin-EDTA solution, were collected into labelled centrifugetubes. Afterwards cells were centrifuged at 1500 rpm for 7 min,and the resulting pellets were suspended in PBS and centrifugedagain. After removing the supernatant, the cells were resuspendedin cold PBS, in the amount corresponding to approximately1 � 106 cells/ml. Then 100 ll of the prepared cell suspensions weretransferred into each cytometric tube. 2.5 ll of annexin V – FITCand 5 ll of propidium iodide were added to each tube; then thecontents were mixed and incubated in the dark at 25 �C for15 min. After incubation, 400 ll of the binding buffer, diluted10-fold with redistilled water, was added to each tube. The cells

number (viable, in early apoptosis, in late apoptosis, and necrotic)in the prepared samples were determined using a FACS Caliburflow cytometer (Becton Dickinson), equipped with CellQuestsoftware.

2.8. Determination of the degree of penetration of Al2O3 nanoparticlesinto the cells using the ICP-OES method

After 24 h of incubation with aluminium oxide nanoparticles,control cells were treated with medium with aluminium oxidenanoparticles at the highest (400 lg Al2O3/ml) for 10 min, to cor-rect data obtained by ICP-OES measurements for nanoparticles thatonly attached to the cell surface. The cells in the wells werewashed 6 times with PBS without Mg2+ and Ca2+ ions to removethe nanoparticles attached to the cell surface and the wells. The de-gree of nanoparticle washing was monitored under a microscope.Subsequently, the cells were detached using trypsin-EDTA solu-tion, transferred to centrifuge tubes and centrifuged at 1500 rpmfor 7 min. The resulting cell pellets were resuspended in PBS with-out Mg2+ and Ca2+ ions and centrifuged again. The supernatant wasremoved, and the obtained cell precipitates were resuspended in1 ml of redistilled water. 0.5 ml of each obtained cell suspensionwas transferred to Teflon vessels. Samples mineralization was car-ried out in nitric acid (V). Clear solutions after mineralisation weretransferred quantitatively into 25 ml flasks and diluted with redis-tilled water. Aluminium was determined using the standard curvemethod involving an optical emission spectrometer with induc-tively coupled plasma (ICP-OES) INTEGRA XL (GBC ScientificEquipment).

The remaining 0.5 ml of aqueous cell suspension was used todetermine total protein content using the Bradford method, to beused as a correction factor for the determination of the actual alu-minium content in the cells. For this purpose, the cells were lysedwith 0.5 ml of 1 M NaOH. After thorough mixing 20 ll of lysate wastransferred into each well of a 96-well plate, in sixfold repetition.In parallel, the standard curve for albumin was prepared in doublerepetition for the concentrations range from 0 to 10 lg/ml. Then180 ll of the Bradford reagent diluted fourfold was added to eachwell. The absorbance was measured at k = 600 nm wavelengthusing a Multiscan Plus spectrophotometer type 314 (Labsystem).The total protein content in the samples was calculated on the ba-sis of the determined standard curve for albumin, taking the totalprotein content in control cells as 100%.

2.9. Statistical analysis

Each experiment was carried out at least in triplicate. The EZ4Uassay was performed in three independent experiments. Other twotests (to assess the apoptosis and the penetration) were repeated infour independent experiments. All data were expressed asmean ± standard deviation (SD). Statistical analysis was performedusing the SYSTAT 13 software (ver. 13.00.05; Systat Software Inc.,USA). F-Snedecor’s test for equality of variances and unpaired Stu-dent’s t-test adjusted for both equal and unequal variances to esti-mate the equality of means were applied for groups comparison atsignificance level a = 0.05.

3. Results

3.1. Stability of aluminium oxide nanoparticles suspensions

The c-Al2O3 nanopowder with spherical shape of particles,average grain size 50–80 nm, average agglomerate size 230–550 nm, and highly developed specific surface area of more than203 m2/g, was obtained in accordance with the procedure de-

Fig. 2. Size distribution of aluminium oxide nanoparticles suspensions. Particle sizemeasurements were carried out for the initial aqueous suspension (after 5-minsonication), and two suspensions obtained by diluting the initial suspension in aculture medium (EMEM + 1% antibiotics) with or without the addition of 10% FBS.

E. Radziun et al. / Toxicology in Vitro 25 (2011) 1694–1700 1697

scribed by Kunicki et al. (2006). The representative SEM image ofAl2O3 nanoparticles is shown in Fig. 1. As given by literature data,suspensions of aluminium oxide nanoparticles in different dispers-ing media may have different stability. In particular, serum addi-tion to the culture media used for cell culture may affect thestability of suspensions and promote the agglomeration of ceramicnanoparticles (Jeng and Swanson, 2006; Wagner et al., 2007; Si-mon-Deckers et al., 2008; Lanone et al., 2009) Therefore, beforeproceeding to determine the biological activity of nanometricAl2O3, stability tests were performed for suspensions preparedin: sterile water, the culture medium (EMEM + 1% mixture of anti-biotics), the culture medium supplemented with 10% FBS, at se-lected concentrations.

Al2O3 particle size distribution was determined in order to esti-mate the degree of agglomeration and the size of aggregatesformed in the tested suspensions (Fig. 2). The average grain sizein the suspensions is summarized in Table 1. It should be notedthat it is the grain size of Al2O3 with a dispersion medium layeradsorbed on their surface. For the suspension of aluminananoparticles in sterile water, the smallest grain size observedwas 52–95 nm, similar to the particle size values obtained fromstereological analysis of SEM images of nanometric alumina. Theagglomerates visible on the SEM images (Fig. 1) were broken upto nanoparticles as a result of 5-min sonication of the suspensions.The suspension prepared in the culture medium (EMEM + 1% anti-biotics) supplemented with 10% bovine serum showed comparableparameters. However, in addition to the nanoparticles fraction of59–91 nm, the chart also indicates an agglomerates fraction of106–220 nm. Its content is about 10%. Contrary to expectations, asignificant increase in agglomeration of nanometric alumina wasseen in the EMEM medium supplemented with 1% antibiotics,without serum. In this dispersing medium, the agglomerates sizewas 255–396 nm.

The stability of the tested suspensions were estimated based onthe performed measurements of the electrical potential that occursat the interface between the adsorption (particles) and diffusion(medium) phases. The suspensions near the isoelectric point withvalues of the f potential of �5 to 5 mV, were characterised by astrong agglomeration of particles. The suspension stability in-creases with the increase in the absolute value of the f potential.The term ‘‘stable suspension’’ refers to a suspension with good dis-persing of nanoparticles and a low degree of agglomeration. Theperformed measurements showed that all of the prepared alumin-ium oxide nanoparticles suspensions were in the range of unstablesuspensions (Table 1). The highest absolute value of the f potential,equal to 11.4 mV at pH 7.75, was recorded for the Al2O3 nanopar-ticle suspension in sterile water. For suspensions of nanoparticlesin the culture medium (EMEM + 1% antibiotics), and in the culturemedium supplemented with 10% FBS, the absolute values of the fpotential were 8.43 at pH 7.97, and 10.6 mV at pH 8.34,respectively. The nanoparticles suspension in the FBS-free medium

Fig. 1. SEM images of c-Al2O3 nanopowder obtained by thermal decomposition of a

showed the lowest stability; this was confirmed by the presence ofagglomerates seen for this suspension on the particle size distribu-tion chart (Fig. 2). The contact surface area of aluminium oxidenanoparticles with water promotes the binding of hydroxyl groups,which are protonated or de-protonated, depending on pH. In aque-ous suspensions, the pH value effects also on the f potential value(Singh et al., 2005). Changes in the pH of the suspensions of nano-metric alumina in the culture media, resulting from the presence ofadditives necessary for cell proliferation (amino acids, vitamins,inorganic salts, etc.) affect the surface charge of nanoparticles,and thus the value of the f potential. Hence the f potential valuesfor aluminium oxide nanoparticles suspensions in sterile water arepositive (protonation of the –OH groups), and they are negative forsuspensions in the culture media (dissociation of the –OH groups).

The f potential values and the particle size distribution curvesof the suspensions have not confirmed an increased degree ofagglomeration of alumina nanoparticles obtained in a culture med-ium with added serum. On that basis, it was decided to conductexperiments on mammalian cells with aluminium oxide nanopar-ticles suspended in a culture medium supplemented with 10% FBS.

3.2. Effect of aluminium oxide nanoparticles on the viability of selectedmammalian cells

To assess cytotoxicity of aluminium oxide nanoparticles onL929 and BJ cells, a commercial EZ4U test was used, in which thecell viability assessment is based on the measurement of activityof a mitochondrial enzyme, succinate dehydrogenase. The test is

precursor at 700 �C for 48 h and three hour deaglomeration in boiling hexane.

Table 1Particle size and f potential measurement of Al2O3 nanoparticles suspensions.

Dispersant Concentration (mg/ml) T (�C) pH f potential (mV) Average size (nm)

Sterile water 10 25 7.75 11.4 78Medium (EMEM + 1% antibiotics) 1 25 8.34 �8.43 320Medium + 10% FBS 1 25 7.97 �10.6 92

Fig. 3. The effect of aluminium oxide nanoparticles on the viability of selectedmammalian cells assessed with the EZ4U assay. The data are expressed asmean ± SD of three independent experiments. (⁄) indicates a statistically significantdifference from the control group (p < 0.05).

1698 E. Radziun et al. / Toxicology in Vitro 25 (2011) 1694–1700

based on the fact that viable cells, with the participation of dehy-drogenase, are capable of processing a tetrazolium salt (EZ4U re-agent), directly into a coloured and non-toxic compound, solublein the culture medium. The solution absorbance value obtainedfrom the difference in absorbance values measured at wavelengthsof 492 and 620 nm is proportional to the number of viable cells inthe culture.

The EZ4U test showed a minor (<10%) cytotoxic effect of alu-minium oxide nanoparticles on the tested BJ and L929 cells. A low-er survival rate of cells incubated with alumina nanoparticles wasobserved in the case of murine fibroblasts. At the concentration ashigh as 400 lg Al2O3/ml the average cell viability after incubationwith the tested compound was 96.51% for human fibroblasts, and91.53% for murine fibroblasts and the difference was statisticallysignificant (p < 0.05) (Fig. 3). At the concentration range10–200 lg Al2O3/ml, the decrease of the cell viability for both cell

Table 2Percentage distribution of various fractions of L929 (A) and BJ (B) cells in a culture after 24the test with annexin V – FITC and propidium iodide. Mean values ± SD (n = 4).

Al2O3 nanoparticles concentration (lg/ml) LL

(A) L929 cell line (%)Control 93.62 ± 1.4110 93.98 ± 1.6650 92.91 ± 2.09100 88.12 ± 1.85200 86.42 ± 1.50400 89.00 ± 1.54

(B) BJ cell line (%)Control 91.98 ± 1.5910 90.98 ± 1,8050 91.47 ± 1.73100 85.49 ± 4.53200 86.99 ± 1.36400 88.82 ± 2.11

The table identifies four cell populations: LL – living cells (not stained with any of the recells in the late stage of apoptosis (stained with both markers); and UL – necrotic cells

lines was not statistically significant compared to the control. Dueto the relatively low toxicity of the tested compound, it was notpossible to determine the concentration values of Al2O3 nanoparti-cles resulting in a 50% cell growth inhibition (IC50).

3.3. Assessment of the effect of aluminium oxide nanoparticles onapoptosis induction

Apoptosis induction was assessed using a test consisting in cellstaining with annexin V – FITC and propidium iodide. Flow cytom-etry analysis allows the visualization and differentiation of viablecells, cells in early and late apoptosis, and necrotic cells. One ofthe first stages of apoptosis (programmed cell death) is the trans-location of phosphatidylserine from the inner surface of the cellmembrane to the extracellular surface. annexin V – FITC specifi-cally binds to phosphatidylserine in the presence of Ca2+, so it isused as a marker of apoptosis. Progressive necrosis or advancedapoptosis leads to loss of cell membrane integrity, allowing thepenetration of propidium iodide and it’s binding to cellular DNA.

The proapoptotic activity of nanometric aluminium oxide inthe concentration range of 10–400 lg/ml was determined after24-h incubation of murine and human fibroblasts with the testedcompound. No statistically significant increase in the percentageof apoptotic or necrotic cells was observed in the cultures treatedwith aluminium oxide nanoparticles compared to the control(p = 0.370 for L929 cells and p = 0.108 for BJ cells) (Table 2). Thepercentages of L929 cells in early and late apoptosis did not ex-ceed 8.26% and 6.0%, respectively. The analysis of BJ cells yieldedsimilar results, with the percentage of cells in early apoptosisreaching 8.14%, and the percentage of cells in late apoptosis ofup to 6.67%. The percentage of necrotic cells ranged between0.38–0.75% and 0.25–0.85% for murine and human fibroblasts,respectively. The results of cell survival estimated by this methodare concordant with those obtained in the EZ4U assay, that allowsto conclude that aluminium oxide nanoparticles are of a relativelyinert nature and show low cytotoxic activity.

-h exposure to aluminium oxide nanoparticles, in the selected concentration range, in

LR UR UL

1.08 ± 0.78 4.55 ± 0.61 0.75 ± 0.541.25 ± 0.64 4.35 ± 1.49 0.43 ± 0.141.72 ± 1.21 4.86 ± 0.79 0.51 ± 0.285.47 ± 0.80 6.00 ± 1.07 0.41 ± 0.088.26 ± 2.35 4.95 ± 1.80 0.38 ± 0.116.33 ± 1.89 4.13 ± 0.98 0.54 ± 0.13

1.05 ± 0.56 6.67 ± 1.05 0.30 ± 0.182.44 ± 0.99 6.46 ± 1.41 0.25 ± 0.061.90 ± 1.15 6.11 ± 0.84 0.52 ± 0.108.14 ± 3.87 5.61 ± 0.79 0.76 ± 0.077.23 ± 1.83 5.31 ± 0.99 0.47 ± 0.064.84 ± 1.20 5.49 ± 1.20 0.85 ± 0.12

agents); LR – cells in the early stage of apoptosis (bound to annexin V – FITC), UR –(only stained with propidium iodide).

Table 3The degree of aluminium oxide nanoparticle penetration into L929 and BJ cells,determined using the ICP-OES method (expressed as aluminium content). Meanvalues ± SD (n = 4).

Al2O3 nanoparticles concentration(lg/ml)

ng Al/lg protein

L929 BJ

Control 0.19 ± 0.03 0.14 ± 0.0210 0.46 ± 0.10 0.31 ± 0.05100 1.29 ± 0.29 1.14 ± 0.18200 1.69 ± 0.38 1.17 ± 0.23

E. Radziun et al. / Toxicology in Vitro 25 (2011) 1694–1700 1699

3.4. The degree of penetration of aluminium oxide nanoparticles intothe cells

The capability of aluminium oxide nanoparticles to penetrateinto L929 and BJ cells was assessed using the ICP-OES method. To-tal protein necessary to standardise the results of penetration wasdetermined using the Bradford method, which uses the capacity ofbinding Coomassie Brilliant Blue G-250 dye by electrostatic inter-actions with the protein. The average aluminum content found inthe cells is shown in Table 3. Aluminum detected in the controlcells came from nanoparticles that were attached only to the cellsurface. A nanoparticle concentration-dependent increase of alu-minium content in both cell lines was observed. At the concentra-tion of 10 lg/ml, there was an approximately 3.7-fold increase inaluminium content in both examined cell cultures in relation tothe control cultures, and a eightfold increase in aluminium contentcompared to cultures treated with the highest used concentrationof 200 lg/ml. There were no significant difference in the ability ofnanometric alumina to penetrate the cells of both tested lines(p = 0.128).

4. Discussion

The present study investigated the effect of nanometric alumin-ium oxide on the viability of selected normal mammalian cells.

Aluminium oxide nanopowder was produced using an innova-tive method consisting in thermal decomposition of a precursorobtained in a reaction of an aluminium organic compound and alu-minium alcoholate with oxygen and water vapour from the air(Kunicki et al., 2006). This method of synthesis is characterizedby good reproducibility and allows obtaining aluminium oxidenanopowder with reproducible characteristics. The size of alumin-ium oxide grains averages from 50 to 80 nm, and the size ofagglomerates is 230–550 nm. The undesirable phenomenon, whichis Al2O3 agglomeration, is caused by highly developed surface areaof the obtained nanopowder (P 203 m2/g) and intermolecularinteractions (electrostatic, van der Waals).

It was found that the addition of fetal bovine serum has littleeffect on the stability of suspensions of aluminium oxide nanopar-ticles in the culture medium. The degree of nanoparticles agglom-eration in the medium supplemented with 10% FBS was lowercompared to the serum free medium. Based on the obtained parti-cle size distribution in the suspensions, it was concluded that thebest dispersing medium is pure water. The absolute values of thef potentials for each aluminium oxide nanoparticles suspensiontested allowed classifying them as unstable suspensions.

Due to the potential use of aluminium oxide nanoparticles inmedicine, including in orthopaedic implants or magnetodynamictherapy (Gutwein and Webster, 2002; Yamamoto et al., 2004; Giriet al., 2003), in pharmacy as drug vehicles and antiviral vaccines(Frey et al., 1997, 1999), the tests were performed using murinefibroblasts (L929 line), recommended for cytotoxicity evaluationof compounds intended for use in biomedical materials (PN-EN

ISO 10993-5, 2009), and normal human fibroblasts (BJ line). Thisstudy demonstrated that nanometric alumina shows no significantcytotoxic activity against the mammalian cells tested in the con-centrations range 10–200 lg Al2O3/ml (p < 0.05). The cytotoxic ef-fect at the highest concentration 400 lg Al2O3/ml was slightlyhigher in murine fibroblasts cultures. However, over 90% cell via-bility was still found in cultures incubated with aluminium oxidenanoparticles at this level.

It was shown that nanometric alumina does not induce apopto-sis of BJ and L929 cells, although it can penetrate into cells, whichwas initially seen in microscopic observation, and then confirmedby ICP-OES. As expected, the increase of the predetermined concen-tration of aluminium oxide nanoparticles resulted in an significantincrease of determined aluminium content in the cells (p < 0.05).This is consistent with the results from recent reports on the abilityof nanometre-sized objects to freely penetrate and pass throughbiological barriers. The dynamics of penetration of Al2O3 nanoparti-cles from the surrounding suspension into the cells was comparablefor both cell lines. Current knowledge suggests that the process ofinternalization may be induced by different mechanisms includingphagocythosis, pinocythosis, or receptor-mediated endocytosis (Si-mon-Deckers et al., 2008; Di Virgilio et al., 2010), and the possiblecytotoxic effect is caused by propagation of inflammation or induc-tion of oxidative stress in the cells (Hussain et al., 2005; Jeng andSwanson, 2006; Veranth et al., 2007; Dey et al., 2008; Oesterlinget al., 2008). According to the previous reports (Wagner et al.,2007; Simon-Deckers et al., 2008), our studies confirmed the lowcytotoxic activity, and thus a relatively good biocompatibility ofaluminium oxide nanoparticles.

Aluminium oxide nanoparticles used in our studies, showed nocytotoxic activity on the tested mammalian cells. However ob-tained results should be confirmed by further biological researchusing in vitro and in vivo methods. Before introducing aluminiumoxide nanoparticles into the human body, it is also important tounderstand the mechanisms of penetration of the compound intothe cells, its activity inside the cells, metabolic pathways and thedegree of accumulation in various organs of living organisms.

5. Conflict of interest statement

None declared.

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