The influence of surface functionalization on the enhanced internalization of magnetic

nanoparticles in cancer cells

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Nanotechnology 20 (2009) 115103 (9pp) doi:10.1088/0957-4484/20/11/115103

The influence of surface functionalizationon the enhanced internalization ofmagnetic nanoparticles in cancer cellsAngeles Villanueva1,4, Magdalena Canete1, Alejandro G Roca2,Macarena Calero1, Sabino Veintemillas-Verdaguer2,Carlos J Serna2, Marıa del Puerto Morales2 and Rodolfo Miranda3

1 Departamento de Biologıa, Universidad Autonoma de Madrid, C/ Darwin 2, Cantoblanco,28049 Madrid, Spain2 Instituto de Ciencia de Materiales de Madrid, CSIC. C/ Sor Juana Ines de la Cruz 3,Cantoblanco, 28049 Madrid, Spain3 Departamento de Fısica de la Materia Condensada, Universidad Autonoma de Madrid andIMDEA Nanociencia, Madrid, Spain

E-mail: angeles.villanueva@uam.es

Received 10 November 2008, in final form 23 January 2009Published 24 February 2009Online at stacks.iop.org/Nano/20/115103

AbstractThe internalization and biocompatibility of iron oxide nanoparticles surface functionalized withfour differently charged carbohydrates have been tested in the human cervical carcinoma cellline (HeLa). Neutral, positive, and negative iron oxide nanoparticles were obtained by coatingwith dextran, aminodextran, heparin, and dimercaptosuccinic acid, resulting in colloidalsuspensions stable at pH 7 with similar aggregate size. No intracellular uptake was detected incells incubated with neutral charged nanoparticles, while negative particles showed differentbehaviour depending on the nature of the coating. Thus, dimercaptosuccinic-coatednanoparticles showed low cellular uptake with non-toxic effects, while heparin-coated particlesshowed cellular uptake only at high nanoparticle concentrations and induced abnormal mitoticspindle configurations. Finally, cationic magnetic nanoparticles show excellent properties forpossible in vivo biomedical applications such as cell tracking by magnetic resonance imaging(MRI) and cancer treatment by hyperthermia: (i) they enter into cells with high effectiveness,and are localized in endosomes; (ii) they can be easily detected inside cells by opticalmicroscopy, (iii) they are retained for relatively long periods of time, and (iv) they do not induceany cytotoxicity.

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1. Introduction

Magnetic nanoparticles are attracting widespread attentionbecause of their prospective medical applications, either ascontrast agents for magnetic resonance imaging (MRI), heatingmediators for cancer therapy by intracellular hyperthermia,or as drug delivery carriers [1–5]. The efficiency of thesetechniques is strongly dependent on the cell–nanoparticleinteraction and the ability to label, with relatively largeamounts of magnetic material, cells of interest [6, 7].

4 Author to whom any correspondence should be addressed.

However, the in vivo use of nanoparticles can also exertsome toxic effects in the human organism which are an issueof vital importance [8, 9]. Several reviews have presented asummary of the in vitro cytotoxicity data currently availableon nanoparticles [10–12], and on magnetic nanoparticles inparticular [13–15], though there is a lack of consensus due tothe variable methods, materials, and cell lines which makes thecomparison difficult [12]. In this study a systematic approachto testing the cytotoxicity of magnetic iron oxide nanoparticleswith different coatings on HeLa cells has been followed. Wehave attempted to determine the nanoparticle internalization

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Nanotechnology 20 (2009) 115103 A Villanueva et al

extent, the intracellular location, and the nanoparticle effect oncell viability and on the microtubules’ cytoskeleton.

In general, surface charge plays an important role innanoparticle toxicity, with cationic surfaces being moretoxic than anionic ones, and neutral surfaces being themost biocompatible [16]. In the case of iron oxide, barenanoparticles showed some toxic effects, while particlescoated with polymer such as PEG (polyethylene glycol) arerelatively non-toxic due to their lack of cell uptake [13].When using smaller molecules such as DMSA, nanoparticlesshow dose-dependent cytotoxicity with internalization of thenanoparticles, affecting the cell function in the case ofneurons [17] but not in other cases [18–20]. The preciseinfluence of the surface charge of iron oxide nanoparticleson cellular internalization remains unclear, and there is verylittle information available concerning cationic iron oxidenanoparticles [21, 22]. Likewise, only a few studies haveexamined the effect of nanoparticles on the microtubuleorganization during mitosis, with conflicting results. Thus,dextran-coated iron oxide nanoparticles internalized intohuman dermal fibroblasts induce several alterations in tubulinand other cytoskeletal proteins [23], while gold nanoparticleshave been reported not to interfere with the cytoskeletalorganization (F-actin and microtubules) in a similar cellline [24].

We report here on the synthesis of iron oxide nanoparticlessmaller than 10 nm with similar aggregate size, but surfacemodified with carbohydrate molecules with neutral, cationic,and anionic charge. We have used similar nanoparticles fordesigning a delivery system for cytokine targeting to a tumoursite with promising results [4]. Now, a systematic study ofthe biological effects of these nanoparticles on a standardhuman tumour cell line (HeLa) has been carried out. Humanderived cells have been used to better predict human toxicity.After cell incubation with nanoparticles, the nanoparticle–cellinteractions were assessed by analysis of the efficiency of cellinternalization at different doses and exposure time, cellularlocation, cytotoxicity, and changes in microtubule organizationto explore the cellular signalling alterations behind the toxicity.

2. Materials and methods

2.1. Synthesis of nanoparticles and characterization

Magnetite nanoparticles were synthesized by the coprecip-itation method. A mixture of 10.01 g of FeSO4·H2O(Mw = 278.02 g mol−1) and 8.08 g of Fe(NO3)3·H2O(Mw = 404 g mol−1) was dissolved in 50 ml of water previ-ously bubbled with nitrogen to eliminate oxygen to keep theFe2+/Fe3+ ratio at 1:1.8. The iron salt solution was slowlyadded to a deoxygenated 400 ml basic solution of NaOH(pH 12) magnetically stirred and kept for 5 h under a nitrogenflow. The precipitate was then centrifuged at 9000 rpm toeliminate impurities and large aggregates.

Samples D and AD consisted of magnetite nanoparticlescoated with dextran and aminodextran, in particular DEAE-dextran, respectively. The coating was incorporated intothe NaOH solution (4.32 g of Mw = 10 000 g mol−1) before

the iron salt addition. Sample H consisted of magnetitenanoparticles coated by heparin after the particle synthesis.First of all, 100 mg of magnetite nanoparticles was dispersedby sonication in water at pH 5 and then 38.5 mg of heparinsodium salt was added to the dispersion. The dispersion wassonicated for 15 min and centrifuged to eliminate the excess ofheparin that is unbound to the magnetite nanoparticles surface.

For sample DMSA, hydrophobic magnetite nanoparticleswere synthesized by decomposition of iron acetylacetonate inphenyl ether [25]; in a second step, the nanoparticles weresurface modified with DMSA to remove the oleic acid andrender a negative charge at pH 7 [26].

All the samples were subjected to centrifugation andfiltration through a 0.22 μm pore membrane syringe anddialyzed to eliminate free coating molecules that could betoxic [12].

The particle size and size distribution were studied bytransmission electron microscopy (TEM) using a 200 keVJEOL-2000 FXII microscope. TEM samples were preparedby placing one drop of a dilute suspension of magnetitenanoparticles in water on a carbon-coated copper grid andallowing the solvent to evaporate at room temperature. Theaverage particle size (DTEM) and distribution were evaluatedby measuring the largest internal dimension of 300 particles.

The hydrodynamic size and evolution of the zeta potentialversus the pH were evaluated in a ZETASIZER NANO-ZSdevice (Malvern Instruments). The hydrodynamic size wasmeasured from a dilute suspension of the sample in water ina plastic cuvette at pH 7. The zeta potential was also measuredfrom a dilute suspension of the sample in water in a specialzeta-potential cuvette with 0.01 M concentration of KNO3 atdifferent pH.

The crystalline phase of the iron oxide particles wasidentified by powder x-ray diffraction. The patterns werecollected between 5◦ and 80◦(2θ) in a Phillips 1710diffractometer using Cu Kα radiation. The crystal size(DXRD) was calculated from the broadening of the (311)reflection of the spinel structure following standard procedures.Fourier transform infrared (FTIR) spectra of the iron oxidenanoparticles were recorded between 3600 and 400 cm−1 ina Nicolet 20 SXC FTIR spectroscope in order to confirm thephase of the iron oxide, the nature of the coating, and itsbonding to the surface. Samples were prepared by diluting theiron oxide powder in KBr at 2% by weight and pressing into apellet.

Magnetic characterization of the samples was carried outin a vibrating sample magnetometer (MLVSM9 MagLab 9 T,Oxford Instruments) at room temperature. Magnetizationcurves were recorded by first saturating the sample in a fieldof 3 T and then sweeping the field range between 3 and −3 Tat 0.5 T min−1.

2.2. Cell culture and nanoparticle internalization

HeLa (human cervical carcinoma) cells were grown as mono-layers in Dulbecco’s modified Eagles’s medium (DMEM) with50 units ml−1 penicillin, 50 μg ml−1 streptomycin, and supple-mented with foetal bovine serum (FBS), at a final concentration

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of 10%. All the media, sera, and antibiotics were providedby Gibco (Paisley, UK). Cell cultures were performed in a5% CO2 atmosphere at 37 ◦C and maintained in an incubator.For the experiments, the cells were seeded into 24 multiwellplates at an initial density of 3500 cells/well. Treatmentswere initiated three days after plating (approximately 70%confluence). Depending on the experiments, the cells wereseeded on 10 mm square glass coverslips placed into thewells. In order to analyse the internalization of nanoparticles,HeLa cells were grown on coverslips and incubated for 1–24 h with different concentrations of nanoparticles (0.05, 0.1,and 0.5 mg Fe ml−1 culture medium). After incubation, thecontaining medium was removed; the cells were washed threetimes with PBS and observed immediately under bright lightmicroscopy.

2.3. Intracellular localization of nanoparticles

To determine the intracellular localization of AD, H, andDMSA nanoparticles, the endocytic compartments of the HeLacells were labelled with the fluoroprobe LysoTracker RedDND-99 (50 nM, Molecular Probes, Eugene, Oregon) inthe culture medium at 37 ◦C for 30 min. Previously, thecells were incubated with nanoparticles (0.05–0.5 mg Fe ml−1)for 24 h. After labelling, the coverslips were washed withPBS, fixed with paraformaldehyde (4% wt/vol), and washedwith PBS. The cells in mounting media (Prolong, MolecularProbes) were observed in an Olympus BX61 epifluorescencemicroscope under bright light illumination or fluorescence(green excitation filter) to detect the internalized nanoparticlesand the emission of LysoTracker, respectively.

2.4. Prussian blue staining

The cells, preincubated with nanoparticles, were visualized byPrussian blue staining for iron detection. For this microscopictechnique, the cells were fixed in ice-cold methanol (5 min),stained with an equal volume of 2% hydrochloric acidand 2% potassium ferrocyanide trihydrate for 15 min, andcounterstained with 0.5% neutral red for 3 min. Thepreparations were then washed with distilled water, air dried,and mounted in DePeX (Serva, Germany). On the otherhand, AD release was microscopically analysed after severalperiods of post-incubation (24–48 h) in fresh culture medium.Microscopic observations and photography were performed inan Olympus BX61 under bright light illumination.

2.5. Evaluation of cell viability

The viability of HeLa cells was determined using astandard methyl thiazol tetrazolium bromide (MTT) assay(Sigma, St Louis, USA). Briefly, 24 h after incubationwith nanoparticles, MTT was added to each well (the finalconcentration of MTT in medium was 50 μg ml−1) for 3 h at37 ◦C. The formazan that formed in the cells was dissolvedadding 0.5 ml of DMSO in each dish, and the optical densitywas evaluated at 570 nm in a microplate reader (Tecan SpectraFluor spectrophotometer). Cell survival was expressed as thepercentage of absorption of treated cells in comparison with

that of control cells (not incubated with any nanoparticles).The results obtained are the mean value and standard deviation(SD) from at least six experiments.

2.6. Immunofluorescence staining of α-tubulin

HeLa cells were incubated with nanoparticles (0.5 mg Fe ml−1)for 24 h and then immunostained for α-tubulin. Briefly,cells grown on coverslips were fixed in ice-cold methanol for5 min, washed three times for 5 min with PBS, permeabilized5 min with 0.5% Triton X-100 (Sigma) in PBS, and laterincubated with the primary monoclonal mouse anti-α-tubulinantibody (1:100 in PBS/BSA, Sigma) for 1 h at 37 ◦C ina humidified chamber. The cells were washed with PBSthree times for 5 min, and incubated with the secondarygoat anti-mouse antibody fluorescein isothiocyanate (FITC)-labelled (Sigma) at a dilution 1:50 for 1 h at 37 ◦C. Finally, thecells were washed three times with PBS, and counterstainedwith the fluorochrome to DNA, Hoechst 33258 (0.05 mg ml−1

in distilled water, 3 min), washed with distilled water andmounted in Prolong (Molecular Probes). The fluorescence ofFITC (green) and Hoechst 33258 (blue) was observed underan Olympus BX61 epifluorescence microscope, equippedwith ultraviolet and blue exciting filter sets and an OlympusDP50 digital camera. The photographs were processed usingAdobe Photoshop CS software. The mitotic index (MI) wasevaluated in immunofluorescence processed samples (controland treated), in order to obtain quantitative data and expressthem as the percentage of cells in mitosis. A distinctionwas made between normal and abnormal mitotic cells, whichincluded cells with altered chromosome distribution andcells with multipolar spindles. Each experimental valuewas obtained by averaging the results of three independentexperiments after counting 4000 cells per experiment.

3. Results and discussion

3.1. Magnetic nanoparticles

Figure 1 shows a representative transmission electron mi-croscopy image of the nanoparticles generated by coprecipi-tation and by decomposition at high temperature (samples Dand DMSA); the particle size is in all cases lower than 10 nmand the standard deviation is in the monodispersed range(<0.20). Average sizes of 6 nm were obtained when thecoprecipitation was carried out in the presence of the polymer,as for nanoparticles coated with dextran and aminodextran(samples D and AD). It should be noted that sample D issimilar to commercial contrast agents such as Feridex [27].Larger particles with an average size of 10 nm were obtainedwhen the polymer was added after the nanoparticle formationto avoid its degradation, as for the heparin-coated samples(sample H). Finally, particles prepared by decomposition andfurther modified with DMSA (sample DMSA) had an averagediameter of 4.5 nm.

The nanoparticles synthesized were identified as mag-netite or maghemite by x-ray diffraction (XRD). No tracesof other iron oxide phases were found. The average crystalsize deduced from the broadening of the (311) reflection

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a b

Figure 1. (a) Transmission electron microscopy image of iron oxide nanoparticles prepared by coprecipitation in the presence of dextran(sample D) and (b) by decomposition in organic media and further surface modification with DMSA (sample DMSA). Scale bar: 50 nm.

Figure 2. Magnetization curve at room temperature for iron oxidenanoparticles coated with dextran (D).

peak in the XRD pattern was similar to the mean particlesize obtained from TEM images. The magnetic behaviourat 300 K was reversible for all the samples, as expectedfor particles around 10 nm in size [28] and illustrated infigure 2 for dextran-coated particles. This confirms thatthey are superparamagnetic at room temperature, anotherimportant requirement for biomedical applications [29]. Themagnetization curve does not change substantially with thedifferent coatings, indicating that the nature and the strength ofthe bond between the coating molecules and the nanoparticlesdoes not affect their magnetic properties.

Figure 3 shows Fourier transform infrared spectra fordifferently coated nanoparticles with new bands at frequenciesbetween 1000 and 1400 cm−1, associated to C–O and C–Cbonds due to the presence of the polymers on the surface. Extrabands, at 1100 cm−1 for sample AD and at 1230 cm−1 forsample H, can be assigned to C–N bonds of the aminodextranand sulfates from heparin, respectively. At 1630 cm−1, anabsorption band is observed in all spectra due to adsorbedwater, but it is more intense for sample DMSA, which alsopresents an extra band at 1140 cm−1 due to the C=O stretchingmode of the coating.

Figure 3. Fourier transform infrared spectra for iron oxidenanoparticles functionalized with four different types of surfacecoating: D (dextran), AD (aminodextran), H (heparin) and DMSA(dimercaptosuccinic acid).

The carbohydrate molecules adsorbed on the nanoparticlesurfaces stabilize the particles in aqueous suspension underphysiological conditions (pH and salinity) [30]. Sampleswere subjected to centrifugation and filtration processes toreduce the hydrodynamic size to below 200 nm. Thehydrodynamic size is the effective aggregate size of theparticles in solution and should be below 200 nm to fit oneof the requirements for intravenous injection and thereforein vivo biomedical applications [31, 32]. Surface coatingconfers not only steric repulsion but also different surfacecharge. Thus, neutral, positive, and negatively charged ironoxide nanoparticles were obtained by coating with dextran(sample D), aminodextran (sample AD) and heparin (sampleH), respectively. Figure 4 shows zeta-potential measurements

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Figure 4. Zeta-potential measurements at different pH for colloidalsuspensions prepared from coated iron oxide nanoparticles.

at different pH, taken for the four colloidal suspensions. Thesurface potential is obviously different for the various coatings.At pH 7, for instance, the surface potential is +26 mV for theaminodextran-coated sample (AD), −9 mV for the dextran-coated sample (D), −27 mV for the heparin-coated sample (H),and −45 mV for the DMSA-coated particles (DMSA).

3.2. Nanoparticles internalization and cytotoxicity

First, cells were observed under bright light microscopywithout being processed to avoid potential artefacts of cellfixation. Nanoparticles of sample D (low surface charge) werenot observed inside HeLa cells under any condition. However,nanoparticles from the cationic sample AD (positive surfacecharge) were detected inside the cells under all experimental

conditions. Figure 5 shows the evolution with time andconcentration of the AD nanoparticle–cell interaction. modeof the coating.

Nanoparticles aggregate outside the cell membrane inthe first place (figure 5(a)), and then they are incorporatedhomogeneously into the cells (figure 5(b)), at increasingamounts for increasing concentrations (figure 5(c)). Besides,it was observed that internalized AD nanoparticles weresplit between the daughter cells in each cell divisioncycle (figure 5(d)). This process was recorded by aliving cell video microscopy (see the movie availableat stacks.iop.org/Nano/20/115103). In a time lapse from 0 to12 h, HeLa cells show a normal behaviour in the cell cycleduring AD incubation (0.1 mg Fe ml−1).

The intracellular pattern distribution for sample ADconsists in brown cytoplasmic spots of different sizes, butalways outside the cell nucleus, which have been identifiedas endosomes. LysoTracker Red DND-99, a specific probefor acidic compartments (endosomes), was used to confirmthis point. As shown in figure 6, the intracellular punctualdistribution of AD nanoparticles overlapped predominantlywith that of the LysoTracker dye, indicating their co-localization in the acidic organelles. Samples H and DMSAinduced the same endosome pattern to a lesser extent due to thereduced internalization (data not shown). A similar subcellulardistribution has been described for other nanoparticles inother cellular types [18, 33], and seems to indicate that themechanism for which nanoparticles enter into the cell is dueto endocytosis.

Figure 7 confirms the intracellular uptake of iron oxidenanoparticles described above by using a classical method

a b

c d

Figure 5. HeLa cells incubated with AD and visualized by optical microscopy (bright field). (a) HeLa cells incubated for 1 h with0.1 mg Fe ml−1 AD. (b) DX nanoparticles internalized inside living HeLa cells, after 24 h incubation with AD 0.1 mg Fe ml−1. (c) HeLa cellsincubated 24 h with AD 0.5 mg Fe ml−1. (d) HeLa cells incubated with 0.1 mg Fe ml−1 AD after cell division. Scale bars: 10 μm.

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Figure 6. Localization of AD and accumulation of LysoTracker Red in endocytic compartments. (a) Cells incubated with 0.5 mg Fe ml−1 ADfor 24 h in bright-field microscopy. (b) Localization of LysoTracker in the same cells. (c) Merged images. A substantial fraction of the redfluorescence from the LysoTracker dye co-localizes with the brown spots of internalized AD nanoparticles. Scale bar: 10 μm.

Figure 7. HeLa cells incubated for 24 h with differently functionalized nanoparticles and stained with Prussian blue reaction for iron oxidedetection. (a)–(c) Cells incubated with sample AD (0.05, 0.1, and 0.5 mg Fe ml−1 respectively). (d) Cells incubated with sample H0.5 mg Fe ml−1. (e) Intracellular iron detected after incubation with 0.5 mg Fe ml−1 of DMSA. (f) Nanoparticles covered with sample D.Scale bars: 10 μm.

of stain. To this end, HeLa cells preincubated with differentlycoated nanoparticles were stained with Prussian blue andvisualized. Figures 7(a)–(c) show the increasing uptakeby HeLa cells after 24 h of incubation with increasingconcentrations of cationic (AD) sample. Clearly, the internalamount of iron-containing cytoplasmatic vesicles depends onthe AD concentration. At a concentration of 0.5 mg Fe ml−1,the cytoplasm is found to be full of particles aggregated aroundthe nucleus but never inside the nucleus. The nanoparticlesinternalized from the AD samples stayed inside the cellsfor long periods of time, allowing their visualization even24–48 h after removing sample AD from the cell culturemedium. Figures 7(d) and (e) show that cells cultivatedwith the anionic samples (H and DMSA, respectively) present

nanoparticles inside only at the maximum concentrationemployed (0.5 mg Fe ml−1) and after 24 h of incubation,but the intracellular uptake was clearly less than the uptakefor AD-cultivated cells (e.g. compare with figures 7(a)–(c)).Figure 7(f) illustrates that no intracellular uptake of iron oxidewas detected at any of the concentrations used in this studyin cells treated for 24 h with sample D, and the blue stainedparticles are only present outside the cells.

From these results it is clear that the internalization ofnanoparticles inside HeLa cells depended on the nanoparticlecoating charge and nature, concentration, and incubationtime. In addition, this is the first time that these types ofnanoparticle have been detected directly inside the cells undera visible light microscope, while for other nanoparticles it

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is necessary to use complementary techniques (Prussian bluestaining, TEM, or fluorescent-labelled nanoparticles) for theirvisualization [6, 18, 20, 33, 34].

The degree of cell survival was evaluated by means ofthe standard methyl thiazol tetrazolium bromide (MTT assay).The analysis of cytotoxicity after incubation of HeLa cellswith the nanoparticles showed that viability of cell culture isnot significantly affected or modified by the presence of thenanoparticles after 24 h of treatment (90–100% viability inrelation to the control sample). Only sample H at the highestconcentration decreases the HeLa cell viability significantly, asthe mean percentage of absorbance values for 0.5 mg Fe ml−1

H treatment was 74.8 ± 2.3%.

3.3. Effects on microtubules

Microtubules (MTs) are highly dynamic fibres of thecytoskeleton with critical functions in eukaryotic cells such asintracellular transport, organization of intracellular structure,and cell division. We have checked the effect of nanoparticleinternalization on the MTs during interphase and mitosis,by indirect immunofluorescence analysis to α-tubulin (DNAcounterstained with Hoechst 33258). Figures 8(a) and (b) showfluorescence images of the MTs (green) and DNA (blue) forinterphase and metaphase HeLa control cells, respectively.

Figures 8(c) (fluorescence) and 8(d) (bright field) showthat the large amount of AD-covered nanoparticles inside thecells did not alter the normal morphology and distribution ofinterphase microtubules. The distributions of mitotic spindlesand chromosomes were also similar to the metaphase of thecontrol cells (not shown). The percentage of cells in mitosis(mitotic index, MI) for HeLa cells incubated with D, AD, orDMSA was similar to that of the control cells (4.9 ± 0.1%)

under all the experimental conditions.However, 24 h after the incubation with anionic, heparin-

covered nanoparticles at a concentration of 0.5 mg Fe ml−1,a two-fold increase in MI was detected (8.5 ± 0.2%) due alarger number of HeLa cells in the metaphase, 93.1 ± 0.1%of which were aberrant. The most frequent modificationswere the presence of misaligned chromosomes (figure 8(e))and extra poles in mitotic spindles (multipolar metaphases),as shown in figure 8(f). In contrast, interphasic MTs didnot show any evident alteration with regard to the control.The metaphase arrest is associated with the ∼25% decreasein the viability of HeLa cells cultivated with the H sample,as detected by the MTT assay. This is the first time thata metaphase arrest induced by any type of nanoparticle hasbeen described, and it is in line with recent reports thathave described drugs with antiproliferative effects throughperturbation of the microtubule dynamics and mitotic spindleformation, inducing a metaphase arrest of the cell cycle andsubsequent cell death by apoptosis [35, 36].

In summary, the work presented here is expected to con-tribute to clarifying the controversy between positively [37, 38]or negatively loaded nanoparticles [15, 17] and cytotoxicity.It is clear that nanocytotoxicity depends not only on thesurface chemical group but also on the size and the type ofnanoparticles, as has been recently reviewed [12].

Figure 8. HeLa cells incubated for 24 h with 0.5 mg Fe ml−1 AD orH, stained with α-tubulin specific antibodies and DNAcounterstained with Hoechst 33258. Fluorescence photomicrographsare merged images of microtubules (green) and DNA (blue).(a) Interphase HeLa control cells. (b) Metaphase HeLa control cell.((c), (d)) Interphase cells treated with AD and observed byfluorescence and bright-field microscopy, respectively.((e), (f)) Abnormal metaphases induced by H in HeLa cells. Scalebars: 10 μm.

In the case of magnetic iron oxide nanoparticles, itseems that animodextran-coated nanoparticles would be idealfor hyperthermia anti-tumour therapy due to their capacityto accumulate very efficiently into the tumour cells withoutcytotoxicity effects. Our results, in particular the LysoTrackerRed DND-99 assay, showed that the internalization of thesenanoparticles was due to endocytosis, and the formation ofholes was never observed as in the case of cationic goldnanoparticles [39]. The application of a magnetic alternatingfield located in the tumour area containing the particles wouldproduce a local heating, killing only the tumour cells andavoiding undesirable secondary effects in the rest of theorganism. Further investigations are in progress to assessthe potential of AD nanoparticles for clinical applicationsin oncology. In this sense, these particles can be furtherfunctionalized in water by covalent bonding of the amineswith carboxyl groups to bind biomolecules such as specificantibodies [40].

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4. Conclusions

Our results indicate that cancer cell response to magneticnanoparticle exposure depends on the charge and the natureof the surface-functionalizing molecules. Cationic dextran-covered magnetite nanoparticles provide efficient HeLa celllabelling for relative long periods, can be easily detected byoptical microscopy, and do not alter the cell viability or the cellcycle, which means that they could be used for tracking in livecells for days with no signs of toxicity. Identical nanoparticleswith a negatively charged cover accumulate less efficientlyinside the cells, as in the case of DMSA-coated nanoparticles,or could be potentially toxic, as it has been shown for heparin-coated nanoparticles. Nanoparticles with low charged coverare not internalized inside cancer cells and therefore are idealfor imaging purposes.

Acknowledgments

This work was supported by the Comunidad de Madridunder project Nanomagnet S-0505/MAT/0194 and by theSpanish Ministry of Science and Innovation through projectsNAN2004-08805-C04-01, NAN 2004-08881-C02-01, FIS2007–61114, CONSOLIDER on Molecular Nanoscience CSD2007-00010, and MAT2005-03179.

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