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Biomaterials 35 (2014) 2981e2986

Contents lists avai

Biomaterials

journal homepage: www.elsevier .com/locate/biomater ia ls

Poly-ε-caprolactone tungsten oxide nanoparticles as a contrast agentfor X-ray computed tomography

Anshuman Jakhmola a,b, Nicolas Anton a,b,*, Halina Anton a,c, Nadia Messaddeq d,François Hallouard e,f, Andrey Klymchenko a,c, Yves Mely a,c, Thierry F. Vandamme a,b

aUniversity of Strasbourg, Faculty of Pharmacy, 74 Route du Rhin, 67401 Illkirch, FrancebCNRS 7199, Laboratoire de Conception et Application de Molécules Bioactives, équipe de Pharmacie Biogalénique, 67401 Illkirch, FrancecCNRS 7213, Laboratoire de Biophotonique et Pharmacologie, 67401 Illkirch, Franced Institute of Genetics and Molecular and Cellular Biology (IGBMC), CNRS/INSERM/Collège de France, Illkirch, FranceeUniversité Lyon-1, F-69622 Villeurbanne, FrancefCNRS UMR 5007, Laboratoire d’Automatique et de Génie des Procédés (LAGEP), F-69622 Villeurbanne, France

a r t i c l e i n f o

Article history:Received 19 November 2013Accepted 13 December 2013Available online 3 January 2014

Keywords:NanoparticlesTungsten oxideContrast agentMicro-CTX-ray imagingPreclinical imaging

* Corresponding author. University of Strasbourg, Fdu Rhin, 67401 Illkirch, France.

E-mail address: nanton@unistra.fr (N. Anton).

0142-9612/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2013.12.032

a b s t r a c t

Inorganic nanomaterials based on heavy elements represent a new class of contrast agents for X-raycomputed tomography (CT). Recent advances have shown that these materials are highly suited for CTimaging due to their high density and X-ray absorption capabilities. In this contribution, we demon-strated that tungsten oxide (WO3) nanoparticles coated by poly-ε-caprolactone (PCL) can be used asefficient contrast agent for CT imaging. The obtained particles were characterized by electron microscopy(TEM and SEM), and dynamic light scattering (DLS). We also validated their use for enhanced in vivoimaging, since these nanoparticles were observed to display high X-ray attenuation properties and cir-culation time (up to 3 h), permitting blood pool imaging.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Modern X-ray contrasting agents approved for clinical use arebased on small hydrophilic iodinated molecules that are effectiveand considered as safe at low dose. However, these contrast agentsexhibit limitations such as rapid renal clearance, vascular perme-ation and low specificity [1,2]. Moreover, their fabrication, pro-cessing and purification require complex stringent chemistry, andmulti-step methodologies.

Nano-engineered smart targeting contrast agent based oninorganic nanomaterials have recently emerged as unique variantsof these traditional X-ray iodinated contrast agents [1,3e5]. Theseinorganic nanoparticles (NPs) contain heavy atoms, and thusstrongly absorb X-ray radiation, increasing imaging contrast in CTby many folds even at low X-ray doses [4,6]. This reduces theexposure time and thus the hazards associated with highly ionizingradiations. The strength of inorganic NPs also lies in their simplerfabrication process as well as in the possibility to easily tailor their

aculty of Pharmacy, 74 Route

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properties and morphology by adjusting the reaction conditions,and to modify their surface by ligands and antibodies in order totarget them to specific biomarkers of disease. Inorganic materials ofvery different nature are able of absorbing X-rays and give signifi-cant contrast enhancement in vivo. However, it is only during thelast decade that research in this field has really emerged, with mainapplications in structural imaging, targeted imaging, ortheragnostics.

Classical example of heavy atoms used for these NPs are gold,silver [7e12], lanthanides [13e16], tantalum, bismuth [17e23], andtheir combinations [24e26]. Gold-based NPs are the mostfrequently used [7e11] since they offer a good compromise in termsof safety, simple formulation and easy surface functionalization.However, gold colloids are expensive to produce in large scale andshow size related toxicity issues [27e29]. It therefore becomesnecessary to work on an alternative non-precious heavy metalhaving X-ray attenuation capabilities similar to gold butmuchmorecost effective [13e23]. Several previous studies on heavy metal likezirconium, yttrium, bismuth, tantalum displayed promising results[4], but herein tungsten was selected since it is the heaviestelement in living structures (Z ¼ 74) [30,31], so that in addition tohigh X-ray absorption, it should exhibit lower toxicity than other

A. Jakhmola et al. / Biomaterials 35 (2014) 2981e29862982

metallic elements. In the present study, we showed the formulationof efficient contrast agent for X-ray CT imaging based on tungstenthat overcome the limitations of the classical iodinated contrastagents. The NPs were formulated using tungsten oxide nanocrystals(WO3) coated with biodegradable polymer (poly-ε-caprolactone,PCL) and PEGylated surfactants, to stabilize them in aqueous me-dium. NPs around 100 nmwere obtained. The morphology and sizeof the NPs, as well as their X-ray contrasting properties, and toxicitywere characterized. Finally, in vivo imaging experiments performedin a micro-CT scanner in mice allowed the evaluation of theircontrasting properties, along with their pharmacokinetics in blood,liver, spleen and kidneys.

2. Materials and methods

2.1. Materials

Tungsten (VI) chloride (WCl6, 99.9%), anhydrous benzyl alcohol (BA, 99.8%),poly-ε-caprolactone (PCL, Mw 10,000) and the solvents (analytical grade) werepurchased from SigmaeAldrich (Saint-Louis, USA). Nonionic surfactant (CremophorELP�, PEG-35 ricinoleate) was a kind gift from BASF (Ludwigshafen, Germany).Phosphate buffered saline (PBS) was obtained from Eurobio (Courtaboeuf, France).

2.2. Synthesis of WO3 nanoparticles

Tungsten NPs were synthesized by the method of Niederberger et al. [32,33].Briefly, 400 mg of WCl6 was slowly dissolved in 20 mL of benzyl alcohol understirring. The reaction vessel was then covered and heated in an oil bath at 100 �C for2 days, the color of solution slowly changed from dark blue to yellow green withtime. Finally, after 48 h the suspensionwas centrifuged, washedwith ethanol severaltimes and dried.

2.3. PCL Coating of WO3 nanoparticles

Once dry, the WO3 NPs powder was weighted and placed in acetone containingPCL at 1 wt.% and surfactant. The amount of surfactant was varied from 1 to 5 wt.%,while the WO3/PCL weight ratio was varied from 50/50 to 80/20. The suspension ofNPs, polymer and surfactant wasmixed (Vortex�) and placed in a sonication bath for5 min to form a homogeneous mixture. Once homogenized, the organic phase isthen rapidly poured with gentle stirring into deionized water phase. The organicphase volume is kept at 1/20th of the final volume. PCL nanoprecipitation occursimmediately, forming stable PCL-coated WO3 nanoparticles. Next, acetone wasevaporated using a rotary evaporator (Büchi, Flawil, Switzerland) and the WO3 NPssuspension was dialyzed (cut-off 12e14,000 Da, Medicell International Ltd, London,UK) for 48 h against deionized water (volume ratio: 1/150, water changed every12 h) in order to remove the free surfactants, the residual compounds and thesolvent.

2.4. Physicochemical characterization

2.4.1. Dynamic light scattering (DLS)Size distributions and polydispersity indices (PDI) were measured by DLS with a

Malvern apparatus (NanoZS�, Malvern, Orsay, France). The helium/neon laser,4 mW, was operated at 633 nm, with the scatter angle fixed at 173� and the tem-perature maintained at 25 �C. DLS data were analyzed using a cumulant-basedmethod.

2.4.2. Scanning electron microscopy (SEM)Samples were diluted twice in deionized water, spread on 12mmdiameter glass

slides, and dried at room temperature. The samples were contacted with carbonpaste, then metallized with gold-palladium and analyzed with a SEM Philips SIRIONFEI apparatus.

2.4.3. Transmission electron microscopy (TEM)Since tungsten is a contrasting material for electrons, samples were used

without any staining agent. Samples were diluted (1/10) with deionized water. Adrop of the suspension was placed on a carbon grid (carbon type-A, 300 mesh,copper, Ted Pella Inc. Redding, U.S.A.), and dried at 40 �C. Observations were carriedout using a Philips Morgagni 268D electron microscope.

2.5. Biological evaluation

2.5.1. Cytotoxicity assaysHeLa cells were seeded in 96-well plates at a concentration of 104 cells per well

in 100 mL of medium (Dulbecco’s Modified Eagle’s Medium, DMEM) containing 10%fetal bovine serum,1wt.% of commercial solution of penicillin and streptomycin. TheHeLa cells were then incubated overnight at 37 �C under a controlled atmosphere(5% CO2 and 95% air). Next, contrast agents were incorporated, by substituting theculture medium for a similar one containing variable concentrations of WO3 NPs

(see details below). After incubation for 24 h, the mediumwas removed and the cellswere washed with PBS. Then, the wells were filled with cell culture medium con-taining MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide),incubated for 4 h at 37 �C, and the formazan crystals formed were dissolved byadding 100 mL of 10% of sodium dodecylsulfate (SDS), 0.01 M hydrochloric acid so-lution. U.V. absorbance was measured at 570 nm by spectrophotometry with amicroplate reader. Experiments were carried out in triplicate, and expressed as apercentage of viable cells compared to the control group.

2.5.2. Micro-CT imagingThe experiments were performed in agreement with the Committee of Animal

Research and Ethics of the University of Lyon-1.

2.5.2.1. In vitro experiments. The X-ray attenuation properties of the WO3 NPssuspensionwere evaluated at various concentrations with a micro-CT scanner (1076Skyscan�, Kartuizersweg, Belgium). Experimental parameters were: X-ray: 49 keV,129 mA; resolution: 35 mm; pitch: 0.4�; aluminum filters: 0.5 and 632 ms.

2.5.2.2. In vivo experiments. In vivo experiments were performed with a micro-CTscanner (INVEON�, Siemens, Munich, Germany). The experimental X-ray parame-ters were: 50 keV, 500 mA; resolution: 111.25 mm; pitch: 2�; aluminum filters: 0.5and 900 ms. The acquisitions were performed on Swiss mice (n ¼ 3). Beforeacquisition, mice were anesthetized with isoflurane. Then, PCL-coated WO3 NPs at0.1 Mwere intravenously injected (using a catheter) in the tail-vein, with an injectionvolume corresponding to 10% of the blood volume (i.e. 7.3 mL of nanoparticles pergram of mouse). Scans were performed before administration, immediately afterinjection, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h and 24 h. The Micro-CT raw data were treatedwith OsiriX viewer, to establish 2D maximum projection slices, and to quantify thesignal by placing the regions of interest (ROI) in the heart, liver, spleen and kidneys.

3. Results and discussion

3.1. Formulation and characterization of the W03 NPs

A first critical issue in this study was to prevent the WO3nanocrystal to aggregate when dispersed in aqueous medium. Thisnatural tendency of the nanocrystals is of course incompatible withtheir intravenous injection inmice, since large aggregates can causedeath within seconds after injection. To avoid aggregation, theWO3nanoparticles were coated with a polymer (PCL) and a surfactantthat inhibit the attractive inter-particle interactions, and thus, self-aggregation. PCL was selected as a coating agent as it is biode-gradable and used in a variety of applications such as drug deliverysystems, surgical implants or tissue engineering.

PCL-coated WO3 nanoparticles were formulated by a two-stepprocess (see above). Comparative morphological characterization ofboth coated and uncoated particles was carried out by electron mi-croscopy (TEM and SEM) and DLS. Representative TEM and SEMmi-crographs of uncoated bare tungsten oxide nanoparticles reveal thattheparticles formsquare, “quasi” two-dimensionalplatelets (Fig.1(a))with sizes ranging from30 to 100 nmand thickness around 5e10 nm.

PCL-coatedWO3 NPs are shown in Fig.1(b) and (c) with differentPCL concentrations. Since theWO3nanocrystals aredensematerials,they appear dark in transmissionmicroscopy (left column) whereasthe PCL shell appears light gray. Compared to uncoated WO3 NPs(Fig. 1(a)) with sharp edges and corners, the polymer-coated NPsshow smoother surface morphology (rounding of sharp tips andcorners of the nanoplatelets). Furthermore, the surfacemorphologyof the particles can be monitored by scanning microscopy (rightcolumn). Therefore, presenting TEM and SEM in vis-à-vis allowsrevealing the polymer coating and thus, the differences induced bythe different polymer concentrations. The polymer shell appearsthicker for a WO3/PCL weight ratio of 50% than for a ratio of 80%(corresponding to higher and lower polymer content, respectively).

The formulation process of these NPs was optimized by workingon two formulation parameters: (i) the surfactant amount and (ii)the WO3/PCL weight ratio. First, the effect of surfactant on the sizeof the pure PCL polymer NPs was monitored (in absence of WO3nanoparticles) (Fig. 2(a)). The concentration of PCL was kept con-stant at 1 wt.%, while the amount of surfactant was increased from1 to 5 wt.%. The hydrodynamic diameter of the PCL nanoparticles

Fig. 1. Electron microscopy of WO3 nanoparticles (surfactant concentration in acetone: 5 wt.%). Left column: TEM. Right column: SEM. (a) non-coated WO3 NPs. (b) PCL-coated WO3

NPs with WO3/PCL weight ratio ¼ 50%. (c) PCL-coated WO3 NPs with WO3/PCL weight ratio ¼ 80%.

A. Jakhmola et al. / Biomaterials 35 (2014) 2981e2986 2983

showed a steep decrease from 200 nmwithout surfactant to aroundto 120 nm at 4e5 wt.% of surfactant. The polydispersity index (PDI)was found overall invariant <0.2, indicating that the suspensionspresented a narrow size distribution.

Next, the effect of the WO3/PCL weight ratio was monitored onthe hydrodynamic diameter of the NPs. The amount of surfactantwas fixed at 5 wt.%, while the ratio of WO3/PCL was increased from50 to 80%. The particle size increased on adding WO3 NPs, from120 nm to 200 nm, along with a constant PDI of around 0.14.Moreover, a decrease in the polymer amount was observed todecrease the thickness of the coating layer (Fig. 1) and increase thesize of the NPs. Thus, the coated NPs exhibit a coreeshell structure,with a WO3 core surrounded by a thin layer of polymeric shell.Increasing the WO3 amount merely results in increasing the size ofthe core and thus the overall size of the NPs.

From Fig. 2, it appears that the minimal amount of polymersufficient to coat and stabilize the WO3 NPs in aqueous solution,corresponds to a WO3/PCL weight ratio of 80%.

3.2. Cytotoxicity of the NPs

We next performed in vitro cytotoxicity assays on model celllines (HeLa) to evaluate the acceptable limit dosage compatiblewith in vivo parenteral administration. The results are reported inFig. 3, and disclose a LD50 value around 0.01 M. Moreover, thein vitro toxicity profiles of uncoated and PCL-coated WO3 NPs arevery similar, indicating that the polymer coating do not modify theintrinsic cytotoxicity of the WO3 NPs. Since the injection volumein vivo is 10% of the total blood volume, this means that onadministrating a suspension at 0.1 M, we will finally reach a con-centration of WO3 in blood around 0.01 M. In contrast to the cyto-toxicity assays, in vivo experiments in living mice revealed markeddifferences between the nude and the PCL-coated NPs. Indeed,suspensions of uncoated particles were extremely toxic leading tomice death within few seconds likely due to the particle aggrega-tion in blood vessels and capillaries, leading to embolism andsubsequent clogging of blood vessels. In contrast, i.v.

Fig. 2. Hydrodynamic diameter of (a) PCL nanoparticles (without WO3) as a function ofthe surfactant concentration in acetone (i.e. surfactant concentration in the solventphase before the solvent-diffusion stage); and (b) WO3/PCL nanoparticles (at 5 wt.% ofsurfactant in acetone) as a function of the WO3/PCL weight ratio. PDI values are shownfor each point in the graph.

Fig. 3. Cytotoxicity MTT experiments. HeLa cells were incubated for 24 h with variousconcentrations of WO3 NPs.

A. Jakhmola et al. / Biomaterials 35 (2014) 2981e29862984

administration of PCL-coated particles did not induce any clinicalsign of toxicity, proving the important role played by the PCL layerto inhibit particle agglomeration and clogging of blood vessels.

Fig. 4. In vitro radio-opacity of the WO3 NPs suspensions as a function of NP con-centration. The dashed-lines indicate the level of radio-opacity of the commonly usediodinate-based commercial product (Fenestra VC�).

3.3. X-ray imaging of the NPs

Based on these preliminary data, X-ray phantom images wereacquired using various concentrations of PCL-coated WO3 NPsdispersed in deionized water.

As expected, theX-rayattenuation, expressed inHounsfield units(HU) scale, increased linearlywith increasing concentrations ofWO3in the samples (see Fig. 4), giving values close to those observed forother heavy elements like bismuth [20e23] and gold [7e11,34e36].

For comparison, we also measured the attenuation of thecommercially available iodinated contrasting agent (Fenestra VC�).A concentration as lowas 0.1MofWO3NPs absorbednearly the sameamount of X-rays as the commercially available iodinated contrastagent having an iodine concentration around 0.44 M [37,38].

Finally, the efficiency and toxicity of PCL-coated WO3 NPs as CTcontrast agent were evaluated in vivo on mice. The NPs wereintravenously injected at a concentration of 0.1 M in a volumeequivalent to 10% of the total blood volume. The results shown inFig. 5 report the contrast enhancement in blood, liver, spleen andkidneys followed up to 24 h after administration. The change inopacity (DHU) at a given time after administration quantifies the

Fig. 5. In vivo imaging performed in mice by Micro-CT. Contrast enhancement (DHU) after i.v. administration of PCL-coatedWO3 nanoparticles (0.1 M) for regions of interest (ROIs) inthe left ventricle (Heart), the hepatic parenchyma (Liver), the spleen (Spleen) and the left kidney (Kidney). The evolution of X-ray attenuation was followed-up during 24 h afterinjection; n ¼ 3. Pictures show representative average intensity projection of vascular contrast enhancement immediately after injection (top) and of kidneys 1 h after adminis-tration (bottom).

A. Jakhmola et al. / Biomaterials 35 (2014) 2981e2986 2985

contrast enhancement induced directly by the contrast agent (WO3NPs).

Immediately after administration, as expected, the WO3 NPsincrease the contrast level in blood. After this initial contrastenhancement, a mono-exponential decay of DHU was observedwith time, indicating a simple mechanism of NPs elimination fromthe body, with a half-life of around 1 h. The time-dependence of thecontrast enhancement in liver and spleen are almost similar, and donot exactly follow the one of blood, indicating that contrasts inthese organs are not only due to their vascularization but corre-spond also to a slight retention of the NPs in these organs. Kidneysalso displayed a significant contrast enhancement, which indicatedan efficient renal excretion of these particles. This was confirmed bythe mean intensity projection images of kidneys, which show thepresence of WO3 NPs within the renal pelvis and the beginning ofurethra.

These studies indicate an efficient elimination and excretion ofall the systemically administrated tungsten oxide from the body,thus avoiding any potential adverse long-term toxic effect due toaccumulation of the NPs in the tissues. The promising results ob-tained with these PCL-coated WO3 NPs offer two key advantages.Firstly, their blood circulation retention time is quite long (up to 2 hwhile it is around 10 min with the iodinated contrast agent used inclinic), which makes them highly promising for angiography ap-plications. Secondly, they are excreted rapidly and did not accu-mulate, thus minimizing any unwanted long term toxic effects.

4. Conclusions

This study reports the use of tungsten as a contrast agent for X-ray imaging. Nanocrystals of tungsten oxide synthesized by asimple method have been successfully coated with a biodegradablepolymer (PCL). The resulting NPs were stable in physiologicalconditions. Moreover, these polymer-coated NPs were shown todisplay good contrast for CT imaging even at a concentration as lowas 0.1 M. In vivo evaluation showed that they were non-toxic, dis-played a significant contrast enhancement, and were excretedwithin few hours after administration. This work demonstrates thattungsten oxide NPs can serve as a starting material for preparationof new generation of X-ray contrast agents.

Acknowledgments

Experimental part (in vivo imaging) of this study was performedon CERMEP e imagerie du vivant, Bron, F-69677, France, imagingfacilities. The authors want to thank the technical staff of theplatform.

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