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Targeting Neural Stem Cellswith Titanium Dioxide

Nanoparticles Coupled toSpecific Monoclonal Antibodies

GEMA ELVIRA ,1 BERTA MORENO,2 IGNACIO DEL V ALLE,1,3

JOSE A. G ARCIA –S ANZ,1 M ARI A C ANILLAS,2 E VA CHINARRO,2

JOSE R. JURADO2

AND A UGUSTO SILVA 1,*

1Cellular and Molecular Medicine Department, Centro de Investigaciones

Biolo gicas CSIC 28040 Madrid, Spain 2 Instituto de Ceramica y Vidrio, CSIC, 28049 Madrid, Spain

3 Molecular Embryology Department, Max-Planck-Institute of Immunobiology

Freiburg, Germany

ABSTRACT: Aiming to characterize the use of biomaterials in cancer therapy,we took advantage of the n-type semiconductor properties, which uponirradiation excite their electrons into the conduction band to induce photoelec-trochemical reactions generating oxygen reactive species (ROS). Indeed,photoactivated TiO2 nanoparticles have been shown to kill in vitro eitherbacteria or tumor cells in culture following UV irradiation, as a consequence of the ROS levels generated; the killing was highly effective although devoid of specificity. In this report, we have directed the TiO2 nanoparticles to particular

targets by coupling them to the monoclonal antibody (mAb) Nilo1, recognizing a surface antigen in neural stem cells within a cell culture, to explore thepossibility of making this process specific. TiO2 nanoparticles generated withparticular rutile/anatase ratios were coupled to Nilo1 antibody and thecomplexes formed were highly stable. The coupled antibody retained the abilityto identify neural stem cells and upon UV irradiation, the TiO2 nanoparticleswere activated, inducing the selective photokilling of the antibody-targeted cells.Thus, these data indicate that antibody-TiO2 complexes could be used to

*Author to whom correspondence should be addressed. E-mail: [email protected] 2 and 3 appear in color online: http://jba.sagepub.com

JOURNAL OF BIOMATERIALS APPLICATIONS Volume 00—2010 1

0885-3282/10/00 0001–21 $10.00/0 DOI: 10.1177/0885328210393294ß The Author(s), 2010. Reprints and permissions:http://www.sagepub.co.uk/journalsPermissions.nav

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specifically remove target cell subpopulations, as demonstrated with neural stemcells. The possible applications in cancer therapy are discussed.

KEY WORDS: monoclonal antibody coupled nanoparticles, cell recognition,apoptosis, cancer cell targeting, TiO2.

INTRODUCTION

T itanium based metals are widely used as surgical implants inorthopedics and dentistry due to their mechanical properties and

high bioaffinity. In particular it is thought that part of the biocompat-ibility is due to the acquisition of a titanium dioxide (TiO2) coating [1,2].

TiO2 nanoparticles have a wide-range of applications such as photo-catalysis [3–5]. This has been used for organic compound photodegrada-tion [4–6] or photokilling of bacteria or eukaryotic cells [7–15], through a mechanism involving oxidized chain reactions and generation of reactiveoxygen species (ROS) [5,10,11,14,16–18].

TiO2 is a semiconductor, when it absorbs a photon with an energygreater than its valence band gap, an electron from the valence band canbe excited to the conduction band creating an electron–hole pair (e /hþ)[19]. Under such photoexcited state, the conduction band electron is

reactive as a reducing agent, whereas the concomitantly formed valencehole can participate as a potent oxidizing agent. Thus, the electron andhole can then reduce or oxidize species to yield reactive oxygen species(ROS) such as hydroxyl radicals, super oxide anions and hydrogenperoxides in aqueous solution [8,15,16,20,21].

The photocatalytic activity of TiO2 depends on parameters, such ascrystallinity, impurities, surface area and density of surface hydroxylgroups, where the crystal structure is determinant [22–24]. TiO2 can besynthesized in two structures; rutile or anatase characterized by band

gaps of 3.06 and 3.23 eV, respectively [22,25]. Anatase generally hasmuch higher photocatalytic activity than rutile [22,26]; however,commercial TiO2 particles consisting of rutile and anatase structure(4/1 w/w) display an increased activity as compared to pure anatase [27].

Titanium dioxide surfaces have a net negative charge at physiologicalpH values encountered in animal tissues and cell cultures due to its pK value of 4.0 [28]. It is well established that titanium dioxide surfacesbind cations, and that this binding is based on electrostatic interactionswith O- [28,29]. Additionally, its ability to bind several molecules and

proteins including antibodies has also been described [28,30], focusing on the study of dental and surgical titanium and hydroxyapatiteimplants [31,32]. Cellular cytotoxicity through TiO2 photoactivation is

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a complex process that involves two stages, an initial oxidative damagethat takes place on the cell surface membrane, making it somewhatpermeable, where cell viability is not compromised and a second stage

that implies the direct attack of TiO2 to intracellular componentsleading to cell death [14]. In addition, TiO2 nanoparticles attached to thecell membrane can enter the cytoplasm via phagocytosis [8,26,33]where, upon photocatalytic activation, the generated ROS can triggercell death.

Stem cells are functionally defined as self-renewing, multipotent cells,exhibiting multilineage differentiation potential [34–36]. Neural stemcells constitute a scarce cell population, localized in specific regions of the adult brain, able to differentiate into the three different cellular

types of adult brain: neurons, oligodendrocytes, and astrocytes. Themouse neural stem cells used in this study could be maintainedundifferentiated in vitro in suspension cultures growing as neurospherescomposed by the neural stem cells, early precursors and neuroblast cells,where the stem cell population still represents a small fraction of thecells within the neurosphere [37].

We took advantage of a monoclonal antibody referred to as Nilo1(neural identifying lineage from olfactory bulb), recently generated inour laboratory that identifies a cell surface antigen specific of neural

stem cells [38], to ascertain whether once coupled to TiO2 could directthe nanoparticles towards its specific targets and could give specificityfor the killing of the target cells upon TiO2 photoactivation. Our data shows that directing TiO2 with a specific monoclonal antibody allowstargeting the photocatalytic effect to the desired cell population,minimizing non-selective cell death. These data represent a proof-of-principle, for the selective in vitro depletion of minor populationstargeted with specific TiO2 coupled antibodies.

MATERIALS AND METHODS

Animals

All experiments described involving animals have been performed incompliance with the European Union and Spanish laws on animal carein experimentation (Council Directive 86/609/EEC). Animal methods,manipulation and experimental methods have been analyzed andapproved by the Committee of Animal Experimentation of the CSIC

and all efforts were made to minimize the number of animals used andtheir suffering. FVB and C57Bl/6 mice were originally obtained fromIFFA-Credo (St Germain sur l’Arbresle, France). Animals were bred

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and housed under standard laboratory conditions in our animalfacility.

TiO2 Powders Preparation

TiO2 nanopowders were synthesized by the precipitation route [39].Briefly, Ti (IV) isopropoxide (495% purity, Aldrich, St. Louis, MI,USA) was dissolved in absolute ethanol and heated to 608C for 24hunder constant stirring. Afterwards, the solution was poured in a glasstray, so that TiO2 powders hydrolyze precipitating within seconds,these powders were then dried in a heater at 1008C to remove theremaining solvent, and finally sieved though a 100 mm mesh obtaining

the nanocrystalline TiO2, referred below to as ‘as-prepared particles’.The as-prepared nanoparticles undergo calcination from 1008C to10008C during 12 h to promote phase transition from anatase to a mixture of anatase/rutile. Two different TiO2 particles of preparedpowders were selected, the as-prepared powders and these calcinedat 6008C.

TiO2 Nanoparticle Characterization

To characterize the phase and structure from TiO2 nanoparticles, X-ray diffraction analyses were performed on powder employing a Siemens D-5000 difractometer (Bruker AXS GmbH, Karlsruhe,Germany) operated at 50kV and 30 mA, with Cu K a radiation andNi-filter, the 2 range selected was 10–70. The scanning step was 0.058,the time/step 1.5 s and the rotation speed used was 15 rpm. Thecrystalline phase percentage was calculated from the XRD patternfollowing the method described by Zachariah [40]. Narrow-scananalysis was conducted within the 2 range of 20–308 as the main

peaks of anatase–TiO2 and rutile–TiO2 are contained within this range.Particle size was measured with the powders dispersed in ethanol bylaser analysis with a Mastersizer (Malvern Instruments Ltd, Malvern,UK). Specific surface area and powder density were measured by theB.E.T. method in a Monosorb Analyzer MS-13 (QuantaChromeInstruments, Boynton Beach, FL, USA). Thermal behavior wasfollowed by DTA-TG in a Netzsch STA 409/C (Netzsch, Gelb,Germany) in air up to 10008C with a heating and cooling rate of 58C min-1. Crystallite size and morphology of the as-prepared and

calcined powders were estimated by transmission electron microscopy(TEM), two different assays were prepared in organic media (ethanol)using a JEM-2000FX (Jeol, Tokyo, Japan) at 125 kV, and in an

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aqueous solution. For this experiment, 6 mg TiO2 particles (as-prepared,100% anatase composition) equilibrated in TBS buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl) were applied during 1 min to a carbon-

coated Cu–Pd grid without glow-discharge. After negative staining with 2% uranyl acetate for 40 s, samples were observed using a JEOL1230 transmission electron microscope operated at 100 kV.Micrographs were recorded at a magnification of 12,000Â.

Rheology of the powder in aqueous dispersion was measured againstpH with a Brookfield LVDV II rheometer (Brookfield Engineering Laboratories, Inc. Middelboro, MS, USA). For these determinations, theas-prepared TiO2 nanoparticles were dispersed in distilled water stirring overnight, the mass/liquid ratio was fixed to 1.75:1 considering the high

specific surface area of the synthesized powders. The initial pH for thedispersion was 4.35. This pH was increased slowly by adding NH4OH(a.c.s. Reagent, 28–30%) drop wise. The band gap of nanocrystallineTiO2 as prepared and with a mixture of rutile/anatase content wasdetermined from the absorption spectra of the powders obtained using the UV-visible spectrophotometer (UV-Vis-IRC Lambda 950, PerkinElmer, Waltham, MS, USA) operated in the diffuse reflectance (DR)mode, for the wavelength within the range of 200–1000 nm. The bandgap was calculated using Equation (1) [41] that relates the absorption

coefficient (a) and the photon energy (h).

ðvÞn ¼ Bð E À EgÞ, ð1Þ

where B is the constant related to the effective masses associated withthe valence and conduction bands, Eg is the band gap energy, E¼h isthe photon energy, and n¼ 1/2 or 2, depending on whether the transitionis indirect or direct, respectively.

TiO2 Nanoparticles: Toxicity Determination

TiO 2 nanoparticles synthesis and characterization is described in supplementary methods.

To discard any toxicity from the TiO2 particles used, 12 FVB micewere intravenously (i.v.) injected with 160, 213 and 320 mg of TiO2

(mouse weight, 20–25 g, four per group). Phosphate buffered saline(PBS) was used as vehicle. Animals were evaluated for changes in theirbehavior and mortality twice every week during 3 months. Visual

histological analysis of liver, lungs, intestine, kidney, and heart, did notshow differences in form, size, patches or color as compared withuntreated animals.







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Nilo1 Binding to TiO2 Nanoparticles

TiO2 powder was dispersed in de-ionized water to obtain a particle

concentration of 10 mg/ mL. On a typical experiment, 0.5 mg of TiO2 wasdecanted at room temperature to enrich on smaller particles, increasing effective surface. Particles were incubated with 0.5 mg of Nilo1 on a finalvolume of 0.5 mL during 2 hours at 48C on a rolling wheel. Free antibodywas eliminated by several centrifugation washes (13,000 rpm, 48C) in de-ionized water. Finally, Nilo1-coupled particles were equilibrated withPBS buffer at 16mg/ mL. Nilo1 antibody coupled to nanoparticles separatedby SDS-PAGE on 12% (w/v) acrylamide gels was quantified by western-blot and densitometry referred to a defined amount of purified Nilo1.

TiO2 Photoactivation

Trypsinized adherent CT-2A cells (105 cells) were suspended in PBS,5% BSA, 0.025% NaN3 and incubated with 8–240 mg of TiO2 nanopar-ticles (9.6–288 ng Nilo1 coupled to the nanoparticles) during 1 h at 4 8C.Cells were isolated and washed by centrifugation and cultured in 6-wellplates with 2 mL of growth media until full adherence, then treated withone dosage of 366 nm UV radiation for 30 min at 10 cm from a 55 W UV

lamp (Camag, Muttenz, Switzerland) and subsequently incubated for36 h to 7 days without refresh media to avoid ROS dilution. As control, a catalyst-devoid irradiated cell suspension was used. Nanoparticlephotoactivation of labeled cells from neurospheres for the cell viabilityassays are described below.

Cell Viability Assays

Single cells (3Â104 cells) from neurosphere cell cultures were

incubated with 5–30mg of Nilo1–TiO2 particles containing 1.2 ng Nilo1/ mgTiO2 during 1h at 48C. After washing, cells were seeded on96-well platted in a final volume of 100 mL and UV-irradiated for 30 minat 366 nm. The viability and metabolic activity of irradiated Nilo1–TiO2

labeled cells cultured in 96-well plates was ascertained after 72 h with anMTS assay kit [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-nyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt], where the reductionof MTS into formazan by viable cells is determined by absorbance at492 nm after a 3 h reaction using a microplate spectrophotometer

Bichromatic MultiskanÕ

(LabSystems, Helsinki, Finland). Absorbancemeasures at 492 nm are directly proportional to the number of living cells in the cultures. As controls, 366 nm UV-irradiated for 30 min cell

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cultures without catalyst, and cell cultures with catalyst but without UV irradiation were used.

Neurosphere Preparation and Cell Cultures

Adult neurospheres were prepared from the subventricular zone(SVZ) of 6–8 week old FVB mice as described [42]. Micro-dissected cellsfrom the SVZ were suspended in DMEM/F12 media and mechanicallydissociated. Cells were resuspended and plated in six-well plates (5000cells/cm2) in DMEM/F12 media supplemented with 20 ng/mL EFG,20 ng/mL bFGF (a kind gift of Dr. G. Gimenez, CIB) and 25mg/mLinsulin. Cells were incubated at 378C, 5% CO2, 95% humidity and

cultured for 7 days until full neurospheres were observed.CT-2A mouse astrocytoma cell line [43,44] was obtained as a gift from

Prof. T.N. Seyfried (Boston, MA, USA). Tumor cells were grown inDMEM media supplemented with 10% (v/v) fetal bovine serum at 378C,5% CO2, 95% humidity.

Immunocytochemistry

For immunocytochemistry analyses, cells were grown on 12 mm glass

coverslips, either directly (CT-2A cells) or after pre-coating them withMatrigel Basement Membrane Matrix Growth Factor Reduced (BDPharmingen, Franklin Lakes, NJ, USA) diluted 1:20 in culture media (neurospheres). Cells were fixed with 4% paraformaldehyde in PBSbuffer for 15 min at RT. Quenching was done adding 0.1 M glycine pH7.4 15 min at RT. Blocking was performed, following three PBS washes,by incubating the coverslips with 10% mouse serum in PBS 1 h at RT.Fixed cells were incubated overnight with Nilo1-coupled TiO2 particlesat 4 8C. After three PBS washes, cells were incubated for 1 h at RT with a

fluorescent secondary antibody (anti-hamster-FITC 1:100, anti-ham-ster-Cy5.5 1:500). TUNEL analyses were performed according tomanufacturer instructions 6 days after UV irradiation. Coverslipscontaining fixed cells were mounted in Dapi/Mowioll and visualized ina Nikon Eclipse 80i fluorescence microscope and LEICA TCS-SP2-AOBSconfocal microscope.

Flow Cytometry

Single cell suspensions from the adherent CT-2A cells were obtainedfollowing treatment 5 min at RT with 1ÂPBS–1 mM EDTA. Single cellsuspensions from neurospheres were obtained by mechanical







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disaggregation. Unspecific binding of the antibodies was blocked withPBS, 5% (w/v) BSA (30 min at 48C). Nilo1 mAb was used as a hybridoma supernatant, added undiluted to the cell suspensions and incubated for

1 h at 48C. After three PBS washes, cell suspensions were stained withsecondary fluorescent antibody anti-hamster-FITC 1:100 diluted inPBS, 5% BSA. Following three additional PBS washes, cells wereresuspended in 300 mL PBS and maintained cold on ice until flowcytometry measurements were made (Epics XL, Coulter, Boca Raton,FL, USA). Propidium iodine (25 mg/mL) was added to each sample, todiscriminate between alive and dead cells.

RESULTS

Synthesis and Characterization of TiO2 Nanoparticles

TiO2 nanoparticles were synthesized by the precipitation route sincea phase transition from anatase to rutile could be induced. Calcinationof the samples at different temperatures allowed modifications of therutile/anatase content and a full control of particle size andphotocatalytic activity [45]. These structural changes were confirmedcomparing the diffraction patterns from the as-prepared powders with

calcined powders (6008C, 12 h). While in the first, broad bandsindicative of the low crystallinity of the anatase phase (nearlyamorphous) were observed, diffraction of calcinated powders revealedthe presence of peaks indicative of high crystallinity, ascribed to a mixture of anatase and rutile phases (Figure 1(A)), which stillremained nanostructured (see below). In addition, the lack of significant weight changes detected on the TG curve, together withthe exothermic variations detected on the DTA curve during calcina-tion (Figure 1(B)) corroborated the lack of organic contaminants in the

sample and indicates that the peaks on the diffraction pattern are dueto crystallinity changes of the material.

Further characterization of the TiO2 nanoparticles showed a changeon specific surface area around (297 m2g -1 in as-prepared versus16 m2g -1 in the 6008C, 12 h calcined powder), concomitant with a density increase (from 2.37gcm-3 in as-prepared TiO2 to 3.52 gcm-3 incalcined), compatible with the formation of smooth and porousagglomerates during synthesis and a decrease on powder porosity,concomitant with a particle size increase during calcination.

In addition, TiO2 particle viscosity analyses, over a broad pH range,indicated an increase in particle dispersion in mild acidic media, whereasphysiological pH led to mild particle aggregation (Figure 1(C)).

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TiO2 nanoparticles formed agglomerates both in organic or aqueoussolvents of 2–8 mm average size, as measured either by laser analysis (notshown) or by transmission electron microscopy data (TEM) (Figure1(D)). These agglomerates were constituted by small particles in the

range of 10–15 nm (as-prepared) or 18 nm (anatase/rutile), as deter-mined by the Debye–Sherrer equation. The band gap energy for bothsamples was calculated from the UV–Vis measurements, estimating a

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Figure 1. TiO2 nanoparticle characterization and analysis of Nilo1 coupled to TiO2

particles. (A) X-ray diffractograms of TiO2 samples as prepared (thick line) and calcined

(6008C, 12 h) (thin line). The pattern is formed by anatase (open triangle) and rutile (black

triangle). (B) DTA (thin line) and TG (thick line) curves of the powder calcined at 600 8C/

12 h. (C) Viscosity vs pH graph for a TiO2 as-prepared dispersion. (D) TEM characteriza-

tion of TiO2 aggregates in Tris buffer saline. Particles were deposited onto a carbon coated

Cu–Pd grid and negative stained with 2% uranyl acetate for 40 s. (E) Nilo1 coupled to

calcined TiO2 nanoparticles was separated by SDS-PAGE in 12% acrylamide gels under

reduction conditions and transferred to PVDF membranes (b-e correspond to 2, 4, 8, and

12mL of the Nilo1–TiO2 particle suspension). Anti-hamster IgG-HRP was used as

secondary antibody after blocking. The amount of the antibody coupled to the particles

was quantified by comparison to the band from 0.75mg purified Nilo1 (a).







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value of 3.3 eV for the as-prepared nanoparticles shifting to 3.0 eV for thecalcined powders; data were consistent with reported values [22,25]. Thedecrease in energy band gap has been ascribed to the increase on rutile

phase during the calcination step.

Nilo1 Monoclonal Antibody and TiO2 Nanoparticles: Coupling

and Functional Effects

The lack of TiO2 nanoparticle toxicity was investigated both in vivoand in vitro (see below). Mice i.v. injected with TiO2 nanoparticles didnot present significant behavior changes. Although no differences invisual anatomy analysis of different organs were observed, compared

with untreated animals, further studies on liver function and detailedhistopathology analyses would be necessary to determine the lack of toxicity of these TiO2 nanoparticles. A s-prepared TiO2 nanoparticles(anatase) or 6008C calcined TiO2 (rutile/anatase) were then incubatedwith purified Nilo1 monoclonal antibody in deionized water (with a pHaround 5), to allow coupling of the antibody to dispersed particles, whichhave an increased specific surface area as compared to physiological pH(Figure 1(C)). It has been described that antibodies or proteins ingeneral, are able to bind to TiO2 structures through electrostatic surface

charges [28]. Coupling of Nilo1 mAb to TiO2 nanoparticles wascorroborated by SDS-PAGE, and the amount of Nilo1 bound wasdetermined by western-blot. Under a large excess of antibody, 1.2 ng were coupled to each mg of TiO2 (Figure 1(E)). The Nilo1–TiO2

complexes had a mean size of microns and, therefore, we will referthem as particles.

To demonstrate that Nilo1 mAb retained its binding specificity aftercoupling to as-prepared or calcined TiO2 nanoparticles, Nilo1–TiO2

complexes were added to actively growing neurospheres or to fixed

neurosphere cells cultured on MatrigelTM-coated glass where the cellsgrew adherent although maintaining stem cell characteristics (self-renewal capacity and pluripotency). The specific binding of the Nilo1mAb within these complexes was revealed using either a Cy5.5-(Figure 2(A)) or a FITC-labeled mouse anti-hamster IgG antibody(Figure 2(B)) and analyzed by fluorescence confocal microscopy,obtaining a staining pattern similar to Nilo1 mAb alone. The resultsrevealed that these complexes identified Nilo1þ cells within theneurosphere, thus directing the TiO2 nanoparticles to Nilo1þ cells

(Figure 2(A)). These complexes were then internalized by the neuralstem cells, as revealed by microscopy where Nilo1–TiO2 particles werefound on the same confocal plane as the nucleus (Figure 2(B)).

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Having established that the antibody retained its binding specificity within the Nilo1–TiO2 complexes, allowing targeting the

TiO2 nanoparticles to the Nilo1 positive cell subpopulation withinthe neurospheres, we aimed to determine whether nanoparticlephotoactivation within the complexes was able to induce neurosphere

(A) (B)

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Figure 2. Nilo1–TiO2 functionality in SVZ-derived neurosphere cultures. (A) DIC

confocal microscopy of fixed neurospheres incubated with calcined Nilo1–TiO2 (left

panels); Nilo1 coupled to TiO2 particles was revealed with a secondary antibody anti-

hamster Cy5.5 (red) (central panel); merge (right panel). Control (upper panels)

corresponds to neurosphere cells treated with TiO2 nanoparticles incubated with Cy5.5-

labeled secondary antibody. (B) Confocal microscopy of neurospheres growing in

MatrigelTM. Immunocytochemistry was performed on cells incubated with Nilo1–TiO2

particles. Nilo1 was revealed after fixation with a secondary FITC-labeled (green) anti-

hamster antibody. Nuclei were stained with Dapi (blue). (C) Cell viability of neurospheres

treated with Nilo1–TiO2 complexes with (red) or without photoactivation (black) was

determined with a MTS assay, where the surviving cell fraction was plotted against mg/mLof Nilo1–TiO2 particles added to 3Â 104 cells growing in a 96-well plate. (D) Bright field

microscopy of neurospheres cultured either in the absence or presence of as-prepared or

calcined Nilo1–TiO2 particles before UV-irradiation (left column), one day (central) or 15

days (right column) after UV-irradiation.







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cell death (photokilling). Neurosphere cultures were incubated withNilo1–TiO2 particles and subsequently UV-irradiated. As negativecontrol, the same UV-irradiated cultures in the absence of Nilo1–TiO2

particles were used. In addition, neurosphere cultures incubated withNilo1–TiO2 particles, but without UV irradiation, did not show apoptoticsigns at low nanoparticle concentrations, but at high TiO2 concentra-tions (0.25 mg/mL) the cells died due to the described TiO2 cytotoxicity[46]. Cultures were followed up for 15 days and cell viability wasdetermined either by quantitative MTS assays at 72 h (Figure 2(C)) orby microscopic analyses after 1 or 15 days (Figure 2(D)). Viabilitychanges were quantified comparing nonirradiated with UV-irradiatedcultures either in the absence or presence of Nilo1–TiO2 particles

(Figure 2(C) and (D)). These data indicated that TiO2 is still functionalafter coupling to Nilo1 antibody.

Nilo1–TiO2 Complexes Recognized a Small Population of

Cancer Cells Inducing Cell Death of Nilo1 Positive CellsFollowing Photoactivation

CT-2A is a well-established mouse astrocytoma cell line [47], whichcan be either maintained in vitro as an adherent cell culture, or

transplanted in vivo to generate type IV astrocytomas after a few weeks[43]. Immunocytochemical staining with Nilo1 mAb revealed that theantibody recognized a small subpopulation of these cells (Figure 3(A)),indicating that it might represent an excellent model to analyze thecytolytic effects of the Nilo1–TiO2 particles on tumors. Indeed,incubation of CT-2A tumor cells with Nilo1–TiO2 particles resulted,upon UV-irradiation, in the death of a subpopulation of cells using eitheranatase or rutile/anatase TiO2 nanoparticles (Figure 3(B)). Conversely,CT-2A cultures UV-irradiated in the absence of Nilo1–TiO2 complexes

were devoid of any apoptotic sign (Figure 3(B)). Cell death wasaccomplished through apoptosis as indicated by TUNEL analyses of CT-2A cells after Nilo1–TiO2 incubation and photoactivation. As controls, UV-irradiated CT-2A cells either untreated or incubatedwith TiO2 devoid of Nilo1 mAb showed only a few cells TUNEL positivedue to UV-irradiation (Figure 3(C)).

Multiparametric analyses of CT-2A cells were carried out by flowcytometry, where in addition to size and complexity, the greenfluorescence from Nilo1 mAb allowed to determining whether a given

cell was positive or negative for this antibody and propidium iodine (PI)allowed determining the fraction of viable (impermeable) and dead(permeable) cells on the culture (Figure 4). The analyses were performed

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on relatively large samples (410,000 cells) between 1.5 and 7 daysfollowing UV-photoactivation. Using this type of analyses, the fraction

of dead cells within the Nilo1þ and Nilo1- populations following UV-photoactivation of the Nilo1–TiO2 particles was quantified.The specificity was determined by direct comparison of PI staining on

(A)

(B)

(C)

45μm45μm

Control Nilo1-TiO2 As-prepared Nilo1-TiO2

Calcined

Control Nilo1-TiO2TiO2

Figure 3. Nilo1–TiO2 functionality in cancer cell cultures. (A) Confocal microscopy of

CT-2A astrocytoma cells. Immunocytochemistry was performed by incubation of fixed cells

with Nilo1 followed by a secondary Cy5.5-labeled anti-hamster antibody (red). Nuclei werestained with Dapi (blue). The shape of the cells was observed with DIC merged with

confocal fluorescence signals (left panel). (B) Bright field microscopy of CT-2A cultures

devoid of Nilo1–TiO2 particles (left) or incubated with as-prepared (middle) or calcined

(right) Nilo1–TiO2 particles after UV irradiation. (C) TUNEL analysis of CT-2A cells in the

absence (left) or presence of TiO2 (central) or Nilo1–TiO2 particles (right) 6 days after UV

irradiation.







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0

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Figure 4. Nilo1-TiO2 induces a specific cell death. Flow cytometry analysis of CT-2A cells

seven days after UV-irradiation in the absence (control) or presence of Nilo1–TiO2

particles. On the basis of Nilo1 expression (A), the population was subdivided into Nilo1–

and Nilo1þ cells. Each subpopulation was independently analyzed for permeability to PI(B, C), allowing to separate alive from dead cells. Percentages for each subpopulation are

shown. (D) Representative experiment (n¼ 6) showing the specific cell death calculated as

the percentage of Nilo1þ dead cells over the total dead cell fraction after subtracting the

basal cell death due to UV-irradiation in the absence of particles. Analysis was performed

with cells treated with TiO2 in the absence (dashed) and in the presence of Nilo1 antibody

coupled to TiO2 (continuous).

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the Nilo1þ or Nilo1- populations with UV-irradiated cells devoid of particles. As an additional control, cells were incubated with TiO2

nanoparticles devoid of antibody, UV-photoactivated and processed as

above.CT-2A cells were subdivided into two populations based on Nilo1

expression (Nilo1þ and Nilo1- cell populations, Figure 4(A)). Each of these populations was further analyzed for its permeability to PI,allowing determination of the fraction of cells alive and dead within theNilo1- (Figure 4(B)) and Nilo1þ (Figure 4(C)) cells. Following theaddition of TiO2 nanoparticles or Nilo1–TiO2 particle complexes similaranalyses were carried out at different nanoparticle concentrations, andthe percentage of specific cell death calculated for each concentration

(Figure 4(D)). The data obtained show a specific increase on Nilo1–TiO2

mediated killing on the Nilo1þ cell population at nanoparticleconcentrations up to 16mg (containing 1.2 ng of Nilo1 per mg of nanoparticle), under conditions where the particles devoid of antibodydid not produce any detectable killing. As expected, however, increasing the concentration of Nilo1–TiO2 complexes led to a decrease on thespecific killing, concomitant with an increase on the killing of TiO2

particles devoid of Nilo1 mAb. This effect was most obviousat nanoparticle amounts above 80mg following UV-irradiation

(Figure 4(D)).

DISCUSSION

Titanium has been used in human implants for the last decades due toits high biocompatibility, which has been associated to the formation of a thin TiO2 layer on its surface [1,2]. TiO2 has the ability to be lightactivated at particular wavelengths, depending on its structure,generating a strong oxidant activity able to split water molecules into

reactive oxygen species (ROS). This characteristic has been widelyapplied on biotechnological processes [3,5] including, organic compounddegradation [4,6] or photokilling of bacteria and eukaryotic cells [7,8,10–15,48]. This led to the use of TiO2 nanoparticles for the treatment of residual waters to remove bacteria by photocatalysis [4,7,49] and morerecently to envisage the possibility of killing tumor cell lines [8,13–15]. Itis believed that the photocatalytic activity of TiO2 nanoparticles takesplace upon its endocytosis, and thus the generated ROS following UV-irradiation directly affects the cell that endocytosed the TiO2

nanoparticles [8,26,33]. Supporting this notion, it has been demon-strated that TiO2-nanoparticle encapsulation in liposomes acceleratesphagocytosis and cell death following photoactivation [8,50]. In addition,







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on human tumor cell lines expressing the carcinoembryonic antigen(CEA) an increase on cell death has been demonstrated with thesenanoparticles coated with anti-CEA mAb following electroporation of

the samples [13], again suggesting that endocytosis of the TiO2

nanoparticles is crucial to increase the killing efficiency on eukaryoticcells. It is obvious, however, that these protocols, although useful todemonstrate the ability of photoactivated TiO2 nanoparticles to killeukaryotic cells cannot be used in vivo. Furthermore, large amounts(0.4 mg) of TiO2 nanoparticles reduced tumor mass up to 50% uponintratumoral injection on subcutaneous tumors [8]. However, even aftersecondary treatments, an exponential tumor growth was detected, andthere was no data available regarding killing specificity.

Aiming to determine whether TiO2 nanoparticles could be specificallydirected towards particular targets, and on this way to kill a particularcell subpopulation with high specificity, TiO2 nanoparticles weresynthesized by the precipitation route and subsequently coupled tomonoclonal antibodies (Figure 1). The precipitated powders haveadvantages when compared to commercial nanoparticles, including chemical purity, a better control of particle size and shape [39], as wellas crystallinity and rutile/anatase concentration ratio, which turned outto be essential for the photocatalysis efficiency. These characteristics did

not change upon coupling with the antibody. The particles did notdisplay any detectable toxicity in vitro – below 0.16 mg/mL even afterUV-irradiation (Figures 2(C) and 4(D)), nor in vivo after i.v. injection of up to 12 mg/kg, in agreement with previously published data [51–55].The fact that TiO2 nanoparticles could be targeted with Nilo1 antibodywould reduce significantly the amount of nanoparticles and, therefore,their possible toxic effect.

We took advantage of Nilo1, a mAb recently generated in ourlaboratory, which has been shown to recognize neural stem cells, both in

tissue sections as well as on neurosphere cultures [38]. Coupling of Nilo1to TiO2 nanoparticles was demonstrated by SDS-PAGE and immuno-cytochemistry (Figures 1 and 2), and took place most likely throughelectrostatic surface charges [28,29]. The antibody coupled to thenanoparticles was able to identify the same subpopulation of cells thanthe uncoupled antibody (Figures 2, 3(A), and 4(A)) whereas the TiO2

nanoparticles were still photoactivated and induce cell death onneurospheres (Figure 2).

Nilo1 mAb recognized a subpopulation of cells within a neurosphere,

but also a subpopulation of cells within the CT-2A astrocytoma (Figures 2 and 3). Thus, neurospheres were used as targets of theNilo1–TiO2 particles to set up the photoactivation conditions to detect

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apoptosis on Nilo1þ cells. In order to analyze the specificity of theprocess, the tumor cell line CT-2A growing in vitro was used. In thiscase, since only a fraction of the cells were positive for Nilo1 antibody,

we could compare the fraction of Nilo1þ and Nilo1- cells dying following photoactivation of the Nilo1–TiO2 particles. Specific killing of the Nilo1þ

cells was demonstrated (Figure 4), despite the low antibody/nanoparticleratio (1.2 ng/ mg). As predicted, with an excess of Nilo1–TiO2 complexesunspecific cell killing was detected, as demonstrated by the increase of apoptosis observed with the same concentrations of TiO2 nanoparticlesdevoid of antibody (Figure 4(D)).

Thus, these experiments demonstrate that mAbs complexed to TiO2

nanoparticles give specificity to the photokilling. The mAb Nilo1

recognizes neural stem cells and a subpopulation of cells within theneural tumor mass representing, most likely, the neural cancer stemcells [56]. These cells represent the fraction of the tumor cells with theability to self-renew and generate the heterogeneous lineages presentwithin the tumor mass. It is likely that Nilo1 might not be the optimalantibody to treat tumors in vivo, due to its ability to recognize bothnormal and cancer stem cells, but definitively allowed demonstrating that TiO2 nanoparticles could be specifically targeted to a particularcell subpopulation, directing ROS activity to specifically deplete cancer

stem cells.In conclusion, these data suggest that Nilo1–TiO2 complexes

can be used to specifically deplete in vitro cancer stem cells.Furthermore, TiO2 nanoparticles can be directed to a particular targetby coupling them to an appropriate monoclonal antibody recognizing cell surface molecules. The coupling would give specificity to theTiO2 photocatalytic-mediated killing since the antibody retainedits specificity within the complexes and upon UV-irradiation, the TiO2

nanoparticles were activated, inducing the selective removal of the

antibody-targeted cells.

ACKNOWLEDGMENTS

We are grateful to Prof. G. Gimenez (CIB-CSIC) for providing thebFGF used in this study. The work in the author’s laboratories has beensupported by grants: SAF2006-4826 from the Spanish Ministry of Science and Innovation; RD06/0010/1010 from the Spanish Ministryof Health; S-BIO-204/2006 from the Comunidad de Madrid; and

PIF200620F0041 from the Spanish National Research Council(to A.S.), NEST/STREP (FP6) 028473-2 from European Union; andPIF-200660F0103 from CSIC (to JRJ). We would like to acknowledge







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the valuable help of P. Lastres and M. Seisdedos for flow cytometryanalyses and confocal microscopy, respectively.

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