Iron Oxide Monocrystalline Nanoflowers for Highly EfficientMagnetic HyperthermiaPierre Hugounenq,‡,⊥ Michael Levy,†,⊥ Damien Alloyeau,§ Lenaïc Lartigue,† Emmanuelle Dubois,‡

Valerie Cabuil,‡ Christian Ricolleau,§ Stephane Roux,∥ Claire Wilhelm,† Florence Gazeau,*,†

and Rana Bazzi*,‡,∥

‡Laboratoire Physico-Chimie des Electrolytes, Colloïdes et Chimie Analytique, UMR 7195 CNRS/Universite Pierre et MarieCurie/ESPCI, 4 place Jussieu, F-75252 Paris Cedex 05, France†Laboratoire Matiere et Systemes Complexes, UMR 7057 CNRS/Universite Paris Diderot, 10 rue Alice Domon et Leonie Duquet,F-75205 Paris Cedex 13, France§Laboratoire Materiaux et Phenomenes Quantiques, UMR 7162 CNRS/Universite Paris Diderot, 10 rue Alice Domon et LeonieDuquet, F-75205 Paris Cedex 13, France∥Institut UTINAM, UMR 6213 CNRS/Universite de Franche-Comte, 16 route de Gray, 25030 Besancon, France

*S Supporting Information

ABSTRACT: Magnetic nanoparticles exhibit a high potentialto selectively treat cancer by hyperthermia provided that highheating capacity can be reached. In this work, we report anefficient synthesis of novel structures of magnetic iron oxide.The particles, obtained by applying a modified “polyol”protocol, present a particular shape: they look constituted ofsmaller grains of approximately 11 nm, assembled in a flower-shaped structure. These nanoflowers, dispersed in water atphysiological pH, present particularly interesting magneticproperties and a great capacity of heating. The value of thespecific loss power (SLP) of these nanoflowers is 1 order of magnitude higher than the SLP reported for conventional 11 nmsingle-domain maghemite nanoparticles in the same condition of field exposure.

■ INTRODUCTION

The development of efficient agents for cancer therapy rests ona crucial dilemma since these agents must exhibit both a highcytotoxic effect against the cancerous cells and a greatinnocuousness for healthy tissues. Such a paradoxical characteris difficult to gather within a single agent. The strategies whichare based on the remote control of the cytoxicity of thetherapeutic agents appear therefore very promising since theseagents are designed to become toxic only after activation by anexternal stimulus.1−4 This implies that the therapeutic agentsexhibit a safe behavior after administration. In other words,their accumulation is observed only in the diseased tissue andthe excess is removed by excretion (urine and feces). Incomparison to the case of the molecules, the biodistribution ofthe multifunctional nanoparticles after intravenous injection canbe more easily and accurately controlled as revealed by recentstudies.1,4−7 Moreover, the nanoparticles can be designed forcombining several complementary imaging techniques whichcan be useful for monitoring in real time their biodistribu-tion.1,4,6,7 For these reasons, nanoparticles which can berendered cytotoxic by an external stimulus have receivedmuch attention because therapeutic activity can be induced atthe most opportune moment, i.e., when the amount ofnanoparticles is both high in the tumor and low in the

surrounding healthy tissue. As a result, the tumor can beseriously altered with minor side effects. Among the differentphysical triggers, high frequency magnetic fields enable theconversion of magnetic energy into heat by nanomagnets andmediate localized hyperthermia that can be used for cancertherapy (thermal ablation), thermally activated treatments, ordrug delivery and release. Clinical phase II studies are underwayto evaluate the safety and therapeutic efficacy of nanoparticle-mediated magnetic hyperthermia after direct injection of ironoxide nanoparticles in glioblastoma and prostate carcinoma.8

This nanotherapy shows the advantages of being controlledremotely through a nonionizing stimulus, actuated and repeatedon-demand, and limited to the zones where nanomagnets areaccumulated. Nevertheless, in sites (e.g., metastasis) where onlylow numbers of nanoparticles may be specifically targeted, theapplicability of magnetic hyperthermia is limited by thedifficulty of obtaining a suitable increase in temperature.Therefore, a long-standing goal has been to optimize theintrinsic heating capacities of the nanoparticles while circum-venting the use of very high frequency or high amplitude

Received: March 16, 2012Revised: June 1, 2012Published: June 14, 2012

Article

pubs.acs.org/JPCC

© 2012 American Chemical Society 15702 dx.doi.org/10.1021/jp3025478 | J. Phys. Chem. C 2012, 116, 15702−15712

magnetic fields (for clinical use, the product of the frequencyand the magnetic field amplitude should be smaller than 5 ×109 A·m−1·s−1),9,10 which can induce nonlocalized temperatureincrease due to Eddy currents.11

Different mechanisms may be responsible for the thermalenergy generated by nanomagnets in an alternating magneticfield. These mechanisms are strongly related to the magneticproperties of nanoparticles and particularly to their magneticstate, being superparamagnetic or ferromagnetic monodomain,pseudomonodomain, or multidomain depending on their sizein a typical range of 5−100 nm. Thermal losses arise from thedelayed response of a nanoparticle’s magnetization to the time-dependent magnetic field. The process of magnetizationreversal is dominated by the displacement of domain wallsfor the multidomain particles, whereas thermal relaxations(either Neel or Brown relaxation) perturb the rotation of thesingle magnetic moment of superparamagnetic monodomainnanoparticles. Brownian relaxation also depend on the mobilityof the nanoparticles, which may change in a biologicalenvironment.12 In between these extreme cases correspondingto the largest (>60 nm) and to the smallest (<20 nm) particledimensions, the deterministic rotation of ferromagneticmonodomains or more complex processes of magnetizationreversal (such as vortex states) may occur for intermediate sizesof nanoparticles.13 Since the pioneering work of Jordan,14 amajority of studies have focused on nanoparticles in thesuperparamagnetic regime in an attempt to optimize theperformance of magnetically induced hyperthermia.15,16 Severaladvantages have been pointed out for 8−20 nm iron oxidenanoparticles, such as their high stability in colloidalsuspension, their easy administration in the body, theirconcomitant properties as biocompatible magnetic resonanceimaging (MRI) contrast agents, and a comprehensivetheoretical description of their magnetic behavior dominatedby Neel and Brown relaxation mechanisms predicting anoptimum diameter for heat generation.17 However, theexperimental results fail to meet the expectation of a largeheating power consistent with the theory, owing to the sizepolydispersity of real samples or their altered magneticproperties due to finite size effects. Remarkably, the bestheating capacity was obtained for nanoparticles in a larger sizerange (30−60 nm) but synthesized by bacteria, limitingtherefore their availability. Hergt et al.18 recently proposedthat the optimum conditions for nanoparticle mediatedmagnetic hyperthermia should be found beyond the super-paramagnetic size range.19 Clusters of magnetic nanoparticlessynthesized embedded in a polymer or organic coating (alsocalled multicore nanoparticles) have been proposed as efficientmediators for hyperthermia in cancer treatment.20−23 Thepeculiar structure of such nanosized clusters have been showntheoretically and experimentally to influence their magneticproperties.24−26 However, the relationship between magneto-structural properties and heating efficiency (depending on theexcitation field) is still not clear. Although more complicatedmagnetic behaviors may take place in particles with largerdimensions or with complex structures, their exploration seemspromising and motivated the present study which waspeculiarly focused on the synthesis and characterization ofmagnetic nanostructures arising from the assembly ofmonocrystalline maghemite grains.

■ EXPERIMENTAL SECTION

Chemicals. Iron(II) chloride tetrahydrate (FeCl2·4H2O,99%), iron(III) nitrate nonahydrate (Fe(NO3)3·9H2O, 98%),sodium hydroxide (NaOH, 99.99%), diethylene glycol (DEG,99%), N-methyldiethanolamine (NMDEA, 99%), sodiumcitrate tribasic dihydrate (98%), and nitric acid (HNO3, 70%)were purchased from Sigma-Aldrich (France). Iron(III)chloride hexahydrate (FeCl3·6H2O, 99%), ethanol, acetone,and ethyl acetate were obtained from VWR (France). Allreagents were used without further purification.

Synthesis of Flower-Shaped Maghemite Nanopar-ticles. Typically, 1.082 g (4 mmol) of FeCl3·6H2O and 0.398 g(2 mmol) of FeCl2·4H2O were completely dissolved in 80 g ofa liquid mixture of DEG and NMDEA with 1:0 and 1:1 (v/v)ratios. The solution was stirred for 1 h. Separately, 0.64 g (16mmol) of NaOH was dissolved in 40 g of polyols. This solutionwas added to the solution of iron chlorides, and the resultingmixture was stirred for another 3 h. Then, the temperature waselevated to 220 °C using a variety of temperature profiles(stepwise heating (10 °C every 5 min) or regular heating (2°C·min−1)). Once the temperature was set to 220 °C, thesolution was stirred for x hours (with x = 0.5, 2, 4, 12, and 24h), and then cooled slowly to room temperature by removingthe heating plate. Different quantities of NaOH (0.95, 1, and1.5 equiv) were investigated. The black sediments wereseparated magnetically and washed with a mixture of ethanoland ethyl acetate (1:1, v/v) several times to eliminate organicand inorganic impurities. Possible iron hydroxides wereremoved by treatment with 10% nitric acid. A 8.25 g sampleof iron(III) nitrate was then dissolved in 20 mL of water andadded to the nanoparticles. The resulting mixture was heated to80 °C for 45 min to achieve a complete oxidation of thenanoparticles. After another treatment with 10% nitric acid, theparticles were washed twice with acetone and diethyl ether andredispersed in water. At this stage, an aqueous dispersion ofmaghemite nanoparticles is stable in acid or basic conditionswith a point of zero charge near pH 7.3. In order to improvecolloidal stability at biological pH, citrate anions were grafted totheir surface by adding 0.3 mol of sodium citrate per 1 mol ofiron element. The citrate to iron molar ratio after magneticextraction of the nanoparticles from the excess of citrate wasequal to 0.2. The colloidal suspension was ready to use andstable at room temperature for months. The final iron contentwas checked by flame spectrometry.

Characterization of Flower-Shaped Maghemite Nano-particles. Direct measurement of the size, the size distribution,and the colloidal stability of the nanoparticles was performedvia photon correlation spectroscopy (PCS) with a Zetasizer HS(Malvern Instruments). The colloids were 10-fold diluted andthe measurements were taken in water and culture medium.The structure of the samples was identified by X-ray

diffraction (XRD), and data were collected at room temper-ature using a D500 Siemens diffractometer equipped with aquartz monochromator.Transmission electron microscopy (TEM) experiments were

carried out on a JEM-2100F field emission electron microscopeoperating at 200 kV and equipped with a high-resolution polepiece and a 4k × 4k Gatan CCD Camera. The diameter of thenanoflowers has been determined from the TEM pictures usingthe software ImageJ and in counting at least 200 particles.Magnetization curves of the colloidal suspensions of

nanoflowers ([Fe] ∼ 10−2 M) were measured at 300 K as a

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function of the magnetic field in the range 0−104 G using alaboratory-made vibrating magnetometer.Temperature dependent magnetization experiments on the

colloidal suspension of nanoflowers in glycerol ([Fe] ∼ 10−2

M) were collected with a Quantum Design MPMS-5S SQUIDmagnetometer working in the temperature range 1.8−350 Kand the magnetic field range 0−5 T. The samples were dilutedin glycerol to avoid strain due to crystallization of the aqueousliquid carrier and to minimize magnetic dipole−dipoleinteractions. Zero field cooled (ZFC)/field cooled (FC)experiments consisted of measuring the static magnetizationfor a field of 50 G as a function of the temperature from 5 to300 K in samples cooled without a field (ZFC), or from 300 to5 K in the presence of a 50 G field (FC). The hysteresis loopswere performed at 200 and 5 K in ZFC samples.Specific Loss Power (SLP) Measurements. The labo-

ratory-made device used for the magnetic hyperthermiameasurements was previously described.15 It consists of aresonant RLC circuit with a 16 mm coil. The field amplitudecan be varied from 6.4 to 21.5 kA·m−1 (from 80 to 268 G), andthe magnetic field frequency can be varied from 300 to 900kHz. The coil was thermalized at 37 °C. The temperature was

probed with a fluorooptic fiber thermometer. The SLP wascalculated using the following formula:

=CVm

Tt

SLPdd

s

where C is the volume specific heat capacity of the sample(Cwater = 4185 J L−1 K−1, Cglycerol = 3086 J L−1 K−1), Vs = 300 μLis the sample volume, and m is the mass of iron oxide. The massof iron oxide is determined from the total amount of iron in thesample, measured by flame atomic absorption spectroscopy.The concentration in iron in all the samples was setapproximately to 5 × 10−2 mol·L−1.To obtain nanoparticles fixed in gelatin for the magnetization

curve and the SLP measurements, 1 mL of a solutioncontaining deionized water and 20% by mass gelatin washeated to 40 °C and added under mechanical stirring to 1 mLof solution containing the citrated nanoflowers. The resultingsolution was cooled at room temperature under stirring. Thenanoflowers in glycerol suspension were obtained by mixingcitrate coated nanoflowers with glycerol.

Figure 1. TEM and HRTEM micrographs and Fourier transformation of (A−C) flowerlike nanoparticles, (D−F) particles synthesized in DEG, and(G−I) particles synthesized by coprecipitation.

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■ RESULTS AND DISCUSSION

The polyol route is an efficient tool for the synthesis of oxidenanoparticles with tunable sizes and shapes that can be imposedby the temperature, the nature of the precursors, the choice ofthe solvents, and the duration of the reaction.27−32 The alkalinehydrolysis of FeCl3·6H2O and FeCl2·4H2O with stoichiometricratio in a mixture of polyols (diethylene glycol (DEG) and N-methyldiethanolamine (NMDEA), 1:1 w/w) at 220 °C(stepwise heating, 10 °C every 5 min) for 4 h yielded particleswith complex structure (Figure 1A).These particles look constituted of small grains of

approximately 11 nm which are assembled in a flower-shapedstructure (Figure 1B). The nanoflower size determined fromTEM observations follows a log-normal distribution with amean diameter of 42 nm and a polydispersity index value of0.16. The crystalline diameter of the flowers measured by XRDis found to be 11.3 nm, which is close to the TEM estimation ofthe granular substructure. Surprisingly, high resolution TEMimages (Figure 1B) reveals that, in most cases, the nanoflowersare single crystalline and constituted of small grains with thesame crystalline orientation. This is clearly indicated by theFourier transform of the HRTEM image characteristic of amonocrystal (Figure 1B) which is consistent with a maghemitestructure. This maghemite structure is confirmed by X-raydiffraction studies (Figure S1 in the Supporting Information).Actually, by taking a closer look at the images of monocrystal-line nanoflowers, we measure small crystal structure misalign-ments from 1 to 3° between the grains (Figure 1C). These

misalignments arise from the presence of crystalline defaults atthe grain interfaces, which could explain the small crystallinediameter obtained by XRD. The structure of the flowers alsopresent filling defect holes possibly containing traces of solventsused for the synthesis. This phenomenon could help inunderstanding the tiny misalignments in the flower crystallinestructure. It must be noted that 30% of the nanoflowers are stillpolycrystalline, meaning that their grains have completelydifferent crystallographic orientations. The role of the solventsin the formation of the complex flower-shaped structure seemsdecisive since isolated spherical maghemite nanoparticles(called SNP, DEG) with an average diameter of 6 nm areobtained when DEG was used instead of the mixture of polyols(DEG + NMDEA) (Figure 1D−F). As previously described inthe literature,33 the physical and magnetic properties of thenanoparticles have a critical influence on the specific loss power(SLP), i.e., the gauge of the thermal dissipation per gram ofmagnetic material. Therefore, measurements on the saturationmagnetization (Ms) and the initial magnetic susceptibility (χ)have been carried out on the nanoflowers (Figure 2A), the 6nm spherical nanoparticles obtained in DEG (SNP, DEG), andthe 11 nm sized maghemite nanoparticles obtained by acoprecipitation method (SNP, coprec) (Figure 1G−I).The 11 nm sized maghemite nanoparticles were synthesized

as a reference since the nanoflowers are constituted of 11 nmsized grains of maghemite. For the nanoparticles synthesizedvia the coprecipitation method, the Ms reaches a value of 60emu·g−1, which is slightly lower than the value obtained for the

Figure 2. (A) Magnetization curves of nanoparticles: synthesized via coprecipitation (SNP, coprec; green dotted line), synthesized in DEG (SNP,DEG; black solid line), and flowerlike nanoparticles (NF) synthesized in a mixture of DEG and NMDEA (red dashed line). (B) Specific loss power(SLP) of the three samples (external magnetic field of 21.5 kA·m−1 at a frequency of 700 kHz).

Table 1. Experimental Conditions for the Synthesis of the Nanoparticles and Their Characteristicsa

sample NaOH equiv heating slope heating duration at 220 °C (h) diam from TEM (nm) χ Msb (emu·g‑1) SLP (W·g−1)

SNP, DEG 1 steps, 10 °C/5 min 4 6 3 48 48SNP, coprec 1 − − 11 12 60 110NF(s,sh10/5,0.5) 1 steps, 10 °C/5 min 0.5 22 51 66 728NF(s,sh10/5,2) 1 steps, 10 °C/5 min 2 38 − − 787NF(s,sh10/5,4) 1 steps, 10 °C/5 min 4 42 128 69 1175NF(s,sh10/5,12) 1 steps, 10 °C/5 min 12 48 358 73 1240NF(s,sh10/5,24) 1 steps, 10 °C/5 min 24 55 575 82 1547NF(us,sh10/5,4) 0.95 steps, 10 °C/5 min 4 34 58 66 1230NF(os,sh10/5,4) 1.5 steps, 10 °C/5 min 4 50 109 70 1790NF(s,rh2/1,4) 1 2 °C/1 min 4 21 67 82 1523NF(s,rh2/1,12) 1 2 °C/1 min 12 24 82 82 1992NF(s,rh2/1,24) 1 2 °C/1 min 24 28 89 82 1944

aSLP under an external magnetic field of 21.5 kA·m−1 at a frequency of 700 kHz. bSaturation magnetization.

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bulk material (80 emu·g−1).34,35 This result is expected becauseof the surface disorder effects on the nanoparticles.36 The Msvalue of the particles synthesized in DEG is even lower (48emu·g−1), which was expected since they are smaller. However,the Ms value of the flower (69 emu·g−1) is closer to the value ofthe bulk maghemite. This could be explained by both their largesize and their high crystallinity. The initial susceptibility valuesare 12 and 3, respectively, for the nanoparticles synthesized viacoprecipitation and the others with the polyol process. Asmaller value is obtained for the smaller particles, as predictedby the Langevin theory. The most interesting value is the oneobtained for the flowers, which possess an initial susceptibilityas high as 128. This high value of χ allows expectation of highvalues of SLP for these particles. The SLP of these nanoflowersdispersed in water reaches 1175 ± 20 W·g−1 under an externalmagnetic field of 21.5 kA·m−1 at a frequency of 700 kHz(Figure 2B). This value is 1 order of magnitude higher than theSLP found for the particles synthesized in DEG (48 W·g−1) andfor conventional 11 nm single-domain maghemite nanoparticles(110 W·g−1) in the same condition of field exposure, showingthe added value of the flower structure. In an attempt to furtheroptimize the SLP by tuning the magnetic and structuralproperties of the nanoflowers, we varied some importantparameters of the synthesis such as the amount of NaOH, thetime of heating at 220 °C, and the heating ramp. Thenanoflowers described in Figure 1 (hereafter called NF(s,sh10/5,4), “s” for stoichiometric amount of NaOH, “sh10/5” forstepwise heating (increase of 10 °C every 5 min), and “4” forthe heating duration (4 h)) will serve as a reference (Table 1).Whatever the amount of NaOH, the nanoflower structure ispreserved, but the amount of NaOH exerts an influence on thediameter of the nanoflowers (Figure 3A−C). When thesynthesis is carried out in understoichiometric conditions, the

resulting nanoflowers (NF(us,sh10/5,4)) exhibit a smallerdiameter than the one of the reference NF(s,sh10/5,4) (34nm vs 42 nm), whereas an increase of the diameter (50 nm) isobserved for NF(os,sh10/5,4) in overstoichiometric conditions(Figure 3D). However, the influence of the NaOHstoichiometry is less obvious on the magnetic properties.Despite their smaller size, NF(us,sh10/5,4) have almost thesame SLP as the reference nanoflowers, NF(s,sh10/5,4). Bycontrast, the increase of the size led to higher SLP, whichreaches 1790 W·g−1 for NF(os,sh10/5,4). The high value ofSLP renders NF(os,sh10/5,4) very attractive for hyperthermiaapplication provided that high colloidal stability in aqueoussolutions can be ensured. However, a postfunctionalization ofthe nanflowers by highly hydrophilic citrate anions does notrender the colloids formed from NF(os,sh10/5,4) sufficientlystable in cell culture medium because of a too-large diameter(50 nm) that prevents their use in biological applications.The modification of the heating time at 220 °C in the

reference protocol (4 h) which led to the synthesis ofNF(s,sh10/5,4) does not alter the nanoflower morphology,but it induces a variation of the size, the polycrystallinecharacter, and SLP values (Figure 4, Table 1).It appears that the mean diameter of the nanoflowers

NF(s,sh10/5,x) (with x = 0.5, 2, 4, 12, and 24 h) increases from22 to 55 nm with heating time while the fraction ofmonocrystalline nanoflowers, determined by HRTEM, in-creases from 48% to almost 85% (Figure 4E). Concomitantly,the SLP values show a considerable variation from 728 ± 45 to1547 ± 30 W·g−1 (Figure 4F).Figure 5 depicts the temperature dependent magnetization

measurements and hysteresis loops on NF(s,sh10/5,x) (with x= 2, 4, 12, and 24 h). The samples were diluted in glycerol(final concentration [Fe] ∼ 10 mM) to avoid strain due to

Figure 3. Influence of NaOH stoichiometry on mean flower diameter and SLP. TEM micrographs of flowers synthesized with respectively (A) 0.95,(B) 1, and (C) 1.5 NaOH equivalents. (D) SLP (black bars) and mean diameter (red squares) of NF(us,sh10/5,4) (understoichiometry),NF(s,sh10/5,4) (stoichiometry, reference), and NF(os,sh10/5,4) (overstoichiometry).

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crystallization of the aqueous liquid carrier and to minimizedipole−dipole interactions. The temperature dependent experi-ment (Figure 5A) consists of measuring the static magnet-ization for a field of 50 G as a function of the temperature from5 to 300 K, in samples cooled without a field (zero field cooled(ZFC)), or from 300 to 5 K in the presence of a 50 G field(field cooled (FC)). The hysteresis loops were performed at200 and 5 K (Figure 5B,C). The results are consistent withlarge nanoparticles having consequently a large anisotropyenergy. Their blocking temperature, corresponding to themaximum of the ZFC magnetization and above which mostnanoparticles prove to be superparamagnetic, is indeed foundto be above the glycerol melting temperature of 215 K. Boththe ZFC experiment and the hysteresis loop at 200 K reflect thenanoparticle size distribution difference between samplesconsistent with TEM observations (Figure 4A−E). The shorterthe synthesis heating time at 220 °C, the larger thesuperparamagnetic population at a given temperature, i.e., thesmaller the nanoparticles, and consequently the higher the ZFCmagnetization and the smaller the coercive field at 200 K (thesuperparamagnetic population does not contribute to thehysteresis). Assuming that none of the nanoparticles aresuperparamagnetic at 5 K, the hysteresis loop at such atemperature informs about the nanoparticle anisotropy.

According to the Stoner−Wohlfarth model for uniaxialnanoparticles isotropically distributed, the coercive field Bc atvery low temperature corresponds indeed to half the anisotropyfield Ba = 2K/Ms, where K is the uniaxial anisotropy constant.Then, evaluating Bc(5 K) ≈ 200 G (Figure 5B) and assumingthat Ms corresponds to the bulk value, we found K ≈ 8200J·m−3.Although both the highest crystalline quality and the highest

heating capacity are obtained for the 24 h heating time, in vivoapplication of the corresponding nanoflowers NF(s,sh10/5,24)to magnetic hyperthermia is likely to be impeded because theyhave rather large and polydisperse sizes ranging between 40 and120 nm (mean diameter 55 nm), rendering more challengingtheir stabilization in biological medium. In order to obtainnanoflowers with smaller size but with similar magneticproperties, we thus change the condition of the early stagesof their formation.Using a regular and slow heating of the solution to 220 °C (2

°C/min instead of the stepwise heating), followed bymaturation steps of 4, 12, and 24 h at this temperature,monodisperse nanoflowers NF(s,rh2/1,4) (“rh” for regularheating), NF(s,rh2/1,12), and NF(s,rh2/1,24) (polydispersityindex of respectively 0.17, 0.2, and 0.23) are obtained (Figure6A−C). It is obvious that the heating mode has no influence on

Figure 4. TEM micrographs of flower synthesized with a stepwise heating to 220 °C with heating times of (A) 0.5 h (NF(s,sh10/5,0.5)), (B) 4 h(NF(s,sh10/5,4)), (C) 12 h (NF(s,sh10/5,12)), and (D) 24 h (NF(s,sh10/5,24)). (E) TEM diameters (black squares) and percentage ofpolycrystalline nanoflowers (red circles) of nanoflowers synthesized at 220 °C from 0.5 to 24 h. (F) SLP (black bars) and mean diameters (redsquares) of corresponding samples.

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the morphology of the nanoparticles since nanoflowers wereobtained with regular continuous heating and with stepwiseheating. Moreover, the size of the cores which was determinedfrom HRTEM micrographs and from XRD data remains almostidentical (about 11 nm) whatever the heating mode, theheating duration, and the amount of NaOH. However, thecomparison based on the size and SLP values of nanoflowersshows differences which can be assigned to the heating modes(Figure 6D).

Whatever the heating duration, the size of nanoflowersobtained with a regular continuous heating (NF(s,rh2/1,x)with x = 4, 12, and 24 h) is largely smaller than the nanoflowersobtained with stepwise heating (NF(s,sh10/5,x) with x = 4, 12,and 24 h) (Figure 6D, Table 1).On the other hand, the regular continuous heating provides

nanoflowers which exhibit, for the same heating duration at 220°C, higher SLP values than those of nanoflowers synthesizedwith a stepwise heating (Figure 6D, Table 1). Moreover,NF(s,rh2/1,x) (x = 4, 12, 24 h) are characterized by a highcolloidal stability in biological medium when they are coated bycitrate anions. In particular, the flowers NF(s,rh2/1,12) presentthe best compromise between size and heating power. Asshown by Figure 7, their hydrodynamic diameter does notchange significantly after dispersion in cell culture medium.Moreover, NF(s,rh2/1,12) exhibit an SLP as high as 1992 ± 34W·g−1 (Figure 6D).The magnetic behavior of NF(s,rh2/1,y) was compared to

the one of NF(s,sh,10/5,x) (with x = 2, 4, 12, and 24 h and y =4, 12, and 24 h). The static magnetization curves at roomtemperature show no hysteresis as expected from non-interacting nanoparticles that are free to rotate in their liquidcarrier. In a similar way, the saturation magnetization increasestoward the bulk values of maghemite (80 emu·g−1 at 300 K).Remarkably, the monodisperse nanoflowers NF(s,rh2/1,12)(24 nm) possess a saturation magnetization very close to thebulk value and an outstanding heating capacity (Figure 8).Moreover, the quasi-linear variation of their heating capacity

with both the field amplitude and frequency makes themparticularly attractive for medical applications in the limit of aclinically acceptable amplitude × frequency product (Figure 9).Furthermore, the SLP is slightly reduced when these

nanoflowers are dispersed in a highly viscous carrier, likeglycerol, or even blocked in a gelatin matrix mimicking, to someextent, the local environment of nanomagnets entrapped withinintracellular compartments (Figure 9).12 Their relatively smalldiameter and citrate coating make them easy to stabilize in aphysiological medium and amenable to systemic administrationin the body.According to the results reported above, we can infer a

possible mechanism for the formation and maturation of ironoxide nanoflowers. We can clearly separate the role of the firstheating period (distinguishing stepwise or regular heating)from the role of the duration at the final plateau temperature.The fact that different sizes of nanoflowers are obtaineddepending on the heating route, stepwise or regular, suggeststhat the aggregation of seeds mainly occurs during this step: forstepwise heating, the transient high heating slope favors theaggregation of a large number of seeds, whereas a lower heatingslope (corresponding to the regular heating) better regulatesthe seed aggregation. As a consequence, large aggregates areobtained following the stepwise heating, while aggregates oflimited size result from regular heating. Similarly, increasing theamount of NaOH results in large-sized flowers due to the initialreduction of repulsion between seeds. In a second stage, thefirst two hours at the plateau temperature still allow the growthof aggregates. However, further heating at the plateautemperature (220 °C) mainly induces the coalescence andreorganization of the aggregated seeds: the longer the plateauduration, the more aligned are the crystal lattices of theindividual seeds. The longer plateau duration clearly favors theripening of monocrystalline nanoflowers. Consistent with sucha mechanism, the flowers obtained after regular heating show a

Figure 5. (A) ZFC/FC curves at 50 G between 5 and 300 K, andhysteresis loops at (B) 200 and (C) 5 K of the samples NF(s,sh10/5,2), NF(s,sh10/5,4), NF(s,sh10/5,12), and NF (s,sh10/5,24);respectively black diamonds, red squares, blue triangles, and greendots.

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reduced number of grains (size < 28.5 nm) with high orderingand heating power, whereas stepwise heating leads to largerflowers. Thanks to the investigation of synthesis parameters,iron oxide nanoflowers have been optimized to achieve largeheating efficiency while allowing their colloidal stability inculture medium.The very high SLP of iron oxide nanoflowers places these

nanostructures among the most efficient heating magneticnanomediators reported in the literature. Various strategieshave been tested to enhance the heating power of convention-ally used magnetic nanocrystals, mainly by varying their sizeand composition. Owing to their high saturation magnetization,pure iron nanoparticles have been proposed as highly efficientnanomediators. For example, 16 nm iron nanocubes present anSLP equal to 1690 W·g−1 at 300 kHz and 660 G.37 However,such iron nanoparticles are prone to oxidation and remain

difficult to disperse into biocompatible medium. The mostsuccessful approach was to substitute metallic ions in thestructure of iron oxide ferrite nanoparticles in order to tunetheir saturation magnetization and magnetocrystalline aniso-tropy. Hence Zn dopant substitution has been shown tocritically enhance the saturation magnetization compared toundoped iron oxide, leading to a 4-fold enhancement of theSLP.38 In addition, the magnetocrystalline anisotropy of mixedferrite core−shell nanoparticles could be also varied by takingadvantage of the exchange coupling between a magneticallyhard core (high anisotropy) and magnetically soft shell (lowanisotropy). The versatile combination of core−shell ferritecomponent (varying M in MFe2O4, M = Mn, Fe, Co, Zn)enabled the achievement of a very high heating efficiency,ranging from 1000 to 4000 W·g−1 for a magnetic field intensity

Figure 6. TEM micrographs of nanoflowers synthesized with a regular continuous heating to 220 °C (2 °C·min−1) and heating duration at 220 °C of(A) 4 h NF(s,rh2/1,4), (B) 12 h NF(s,rh2/1,12), and (C) 24 h NF(s,rh2/1,24). (D) SLP and mean size of flowers synthesized with a stepwiseheating (gray bars and gray dots, respectively) and with regular heating (black bars and black dots, respectively).

Figure 7. Size distribution for NF(s,rh2/1,12) in water (green curve(W)) and in cell culture medium (red curve (C)) determined by PCSexperiments.

Figure 8. Saturation magnetization Ms (empty circles or squares) andinitial susceptibility χ (filled circles or squares) of colloidal suspensionof NF(s,sh10/5,x) with x = 0.5, 4, 12, and 24 h (circles) andNF(s,rh2/1,y) with y = 4,12, and 24 h (squares).

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of 37.3 kA·m−1 (466 G) at a frequency of 500 kHz. However,this strategy implies the use of metallic doping species, such asZn, Co, or Mn, whose toxicity and fate in the organism has notbeen evaluated so far.In the present work, we propose another strategy which

relies on the control of nanoparticle shape and architecture tooptimize magnetic energy conversion. Such an approach isinspired from biomineralized iron oxide nanostructuressynthesized by magnetotactic bacteria. The so-called magneto-somes are rather large (30−50 nm) iron oxide facetednanocrystals surrounded by a biological membrane. Comparedto chemically synthesized spherical counterparts, magneto-somes have been shown to be very efficient heatingnanomediators39 and their SLP is modulated depending ontheir close environment (embedded or not in membrane and inthe bacteria) and on the organization of magnetosomes inchains.40 Although there is no theoretical framework describingthe role of the shape and organization of magnetic nanocrystalson their heating efficiency and more generally on theirmagnetic properties, there is clearly a need to investigatesuch parameters to optimize hyperthermia. Here we found thatflowerlike assembly of maghemite nanoparticles had a heatingefficiency comparable to that of magnetosome chains extractedfrom bacteria40 and to 19 nm cubic maghemite nanoparticles.41

In comparison to magnetite particles prepared by coprecipita-tion and with high SLP values due to aggregation42,43 or tomulticore nanoparticles reported previously using othersynthesis methods,21,26 the nanoflowers resulting from ourmodified polyol protocol exhibit much higher heating power,even when extrapolated to low magnetic field and frequency.For instance, multicore nanoparticles synthesized by copreci-pation and coated by dextran show an SLP of 400 W·g−1 inwater (262 W·g−1 in gelatin) in a field of 25 kA·m−1 and 400kHz, whereas nanoflowers present an SLP of 500 W·g−1 inwater (360 W·g−1 in gelatin) at the same frequency but for alower field strength (11 kA·m−1). The outstanding heatingpower of flowerlike nanoparticles made by polyol synthesislikely relates to their peculiar magnetostructural properties.Although the assemblies are composed of 11 nm nanocrystals,they do not behave as isolated grains. The control of synthesisparameters reported above leads to a combined tuning of sizeand crystallinity of flower nanostructures, the majority of whichare composed of highly ordered nanocrystals. Irrespective ofthe flower size, the better the crystallinity the larger thesaturation magnetization and the larger their SLP. Enhancedheating capacity may also arise from a favorable tuning of themagnetic anisotropy. It has been highlighted that nanoparticles

with reduced anisotropy compared to bulk maghemite (in therange 0.5 × 104−4 × 104 J/m3) and relatively large size (10−30nm) could be in an optimal regime for energy conversion.44,45

From blocking temperature measurements (TB being close tothe room temperature) and hysteresis curves at low temper-ature, we can infer that the nanoflowers possess a lowermagnetic anisotropy than the spherical 11 nm maghemitenanoparticles which exhibit high surface disorder andsubsequently high surface anisotropy.46−49 A possible causefor the reduction of anisotropy in nanoflowers could be thecoalescence and crystalline ordering of individual grains whichmay reduce their surface disorder. However, this issue needs tobe further investigated by complementary magnetic measure-ments.Another critical advantage of the proposed nanoflowers is

that they are made of iron oxide only and are free frompotentially toxic inorganic elements or biogenic componentswhich could reveal immunogenicity. Furthermore, sincemaghemite is the fully oxidized state of iron oxide, the chemicaland magnetic properties of nanoflowers do not change overtime in both aqueous and culture media. Their colloidalstability in culture medium also warrants the persistence oftheir high heating properties by preventing the formation oflarge aggregates. Since iron oxide nanoparticles have thecapacity to be degraded and processed by iron metabolism inthe organism without toxic effects,50 iron oxide basednanostructures remain the best candidates for translatingmagnetic hyperthermia in humans.

■ CONCLUSION

In summary, we developed novel nanostructures beyond thesuperparamagnetic size range for highly efficient magnetichyperthermia. The alkaline hydrolysis of iron(II) and iron(III)chlorides provided nanoparticles with flowerlike structure whenthe synthesis is carried out in a mixture of DEG and NMDEA.The flowerlike structure of the nanoparticles results from theassembly of maghemite nanoparticles whose size is about 11nm whatever the experimental conditions. The synthesisprocedure allows modulating the size, the polycrystallinecharacter, and the magnetic properties of the nanoflowers,resulting in a huge enhancement of their heating power (highSLP values) compared to conventional approaches while usingonly nontoxic iron oxide. The study reveals that NF(s,rh2/1,12) which were synthesized in stoichiometric conditions for12 h at 220 °C with a regular and slow heating to 220 °C (2°C·min−1) appear as an interesting compromise between thehigh heating potential (SLP value 1992 ± 34 W·g−1) and size

Figure 9. SLP as a function of (A) field amplitude (for a frequency of f = 700 kHz) and (B) magnetic field frequency (for a field amplitude of H =137.5 G (11 kA·m−1)) for NF(s,rh2/1,4), in water (circles), glycerol (squares), and gelatin (triangles).

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(24 nm). Although the exact mechanisms for thermal losses inthese nanoflowers remain to be delineated, they represent avery attractive biocompatible nanoscale platform for a variety ofapplications that rely on magnetic hyperthermia, especiallycancer treatment and thermally assisted drug delivery. It mustbe pointed out that image-guided hyperthermia can beenvisaged with these nanoflowers since maghemite nano-particles behave as negative contrast for MRI.

■ ASSOCIATED CONTENT*S Supporting InformationX-ray diffraction studies and TGA of NF powders. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: florence.gazeau@univ-paris-diderot.fr (F.G.); rana.bazzi@univ-fcomte.fr (R.B.). Tel.: +33 157 27 62 03 (F.G.);+33 381 66 62 94 (R.B.). Fax: +33 157 27 62 11 (F.G.); +33381 66 55 04 (R.B.).Author Contributions⊥These authors contributed equally to this work.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work has been supported by a grant (P.H.) from theRegion Ile-de-France and by the European project Magnifyco(Contract No. NMP4-SL-2009-228622). We are grateful toRegion Ile-de-France for convention SESAME 2000 E1435, forthe support of the JEOL 2100F electron microscope installed atIMPMC (UMR 7590).

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