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Keywords:Magnetic nanoparticles

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with the strong companies drive in the fast delivery of the new therapy to the patient. Four samples areunder study with variable hydrodynamic diameters from two companies (Micromod and Chemicell)consisting of iron-oxide magnetic nanoparticles. The tunable magnetic heating characteristics of the

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in cancer cells is proposed, by using double-effector nanoparticles

over, when hyperthermia may be applied in combination with

the applicabilityy assurance) ares will actually beto home-made

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particles, in an effort to see if beyond their actual modality if they

Contents lists available at ScienceDirect

.el

Journal of Magnetism an

Journal of Magnetism and Magnetic Materials 380 (2015) 360364http://dx.doi.org/10.1016/j.jmmm.2014.10.042common cancer treatments (e.g. chemotherapy), is proven to possess measurable heating efciency. Such a study outlines thepotential of market products and readily addresses the multi-functional role in modern theranostics by combination of initialrole with an additional heat-triggered action. Eventually, such

0304-8853/& 2014 Elsevier B.V. All rights reserved.

n Corresponding authorE-mail address: dsakel@physics.auth.gr (D. Sakellari).that generate reactive oxygen species (ROS) and heat [6]. More-In this work, we decided to initiate an examination of four

commercially available ferrouids, consisting of iron-oxide nano-due to their extreme localization at the nanoscale. Magnetic par-ticle hyperthermia (MPH) is a synergistic technique used in cancertreatment, along with chemotherapy or irradiation [3]. In MPH onecan take advantages of heat generated by MNPs when are exposedin an alternating (AC) magnetic eld [4]. The regional increase oftemperature, when applying inside a small region of the humanbody, may induce apoptosis or necrosis of the cancer cells, withoutdamaging the normal cells [5].

Recently, a new approach to achieve effective apoptotic results

aiming to the material-side properties (sample certy tuning) [9]. Meanwhile, issues concerning(formulation, reproducibility, toxicity and qualitusually postponed for the later stages when MNPintroduced to biomedical practice [10]. Contrarymagnetic nanoparticles, commercially producedrapidly pushed through the medical approvalcommercial drive and company resources are folivery of the new therapy to the patient as fast ananoparticles (MNPs) comprise a very promising candidate mainlyMost of the published works in the eld of biomedical appli-

cations of MNPs, utilize magnetic nanoparticles synthesized in-lab,ontrol and prop-1. Introduction

Biomedical exploration of novelenhanced individual and collectivefunctional range of applications is stgroups worldwide [1, 2]. Among varas drug-release, MRI contrast agenferrouids were correlated with particle, eld and colloidal solution features. Our work revealed a size-dependent magnetic heating efciency together with fast thermal response, features that are crucial foradequate thermal efciency combined with minimum treatment duration and show the potential of suchmaterials as multifunctional theranostic agents.

& 2014 Elsevier B.V. All rights reserved.

etic nanocarriers withres covering a multi-rsuit for many researchplication schemes suchbiomarkers, magnetic

enhance the effect of anticancer drug, thus promotes effectivetherapy schemes with reduced clinical dosage [7]. Beyond thestrictly therapeutic use of hyperthermia, it may also be exploitedin conjunction with other diagnostics techniques, such as MRI,taking advantages of the multifunctionality of the same magneticcarriers [8].Magnetic particle hyperthermiaExploring multifunctional potential of cmagnetic particle hyperthermia

Despoina Sakellari n, Stella Mathioudaki, Zoi KalpaxMakis AngelakerisDepartment of Physics, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece

a r t i c l e i n f o

Article history:Received 30 June 2014Received in revised form8 October 2014Accepted 9 October 2014Available online 18 October 2014

a b s t r a c t

In this work we examinecandidates for magnetic padditional heat triggered amay be rapidly pushed throaddressed successfully (i.e

journal homepage: wwwmercial ferrouids by

ou, Konstantinos Simeonidis,

election of commercially available magnetic iron oxide nanoparticles asicle hyperthermia applications combining their primary modality withns. Contrary to lab-made magnetic nanoparticles, commercial ferrouidsh the medical approval processes since their applicability has already beenrmulation, reproducibility, toxicity and quality assurance) in conjunction

sevier.com/locate/jmmm

d Magnetic Materials

systems may be further implemented in therapies, with fastersteps, since major tasks (e.g. biocompatibility and sustainability)have already been undertaken before these substances were re-leased in the market.

2. Materials and methods

of hyperthermia curve (Temperature vs time). Generally, nano-particles as heating mediators should have the highest possibleSAR values resulting to the smallest amount of MNPs to be tar-geted to the tumor. Since heat mediation may occur also via non-magnetic pathways, in order to exclude thermal exchange with theenvironment and isolate the magnetic-origin heating contribution,we have incorporated in SAR calculation a set of adiabatic cor-rections (by subtracting the solvent signal prior to tting thecooling curve to provide the law of cooling), as described in detail

rouid saturation eld (i.e. our case). Thus, it provides a quanti-able measure of heating efcacy irrespective of the experimental

.

Product name Mean hydrodynamic particle Core type Shape Coa

DexcomDex

D. Sakellari et al. / Journal of Magnetism and Magnetic Materials 380 (2015) 360364 361size (nm)

Nanomags-D-spio 50110 Iron oxide Cluster-typed

FluidMAG-DX 50, 100, 200 Maghemite multi-domaincoreCommercially available ferrouids, consist of magnetic ironoxide nanoparticles of two different companies: a) Micromod [11]Nanomags-D-spio and b) Chemicell [12] FluidMAG-DX-SSS whereSSS corresponds to their mean hydrodynamic diameter, 50, 100and 200 nm. An overview of their features as provided with thecompanies datasheets is collected in Table 1. In order to measurethe iron concentration of each commercial ferrouid the atomicabsorption spectrophotometry was used, by a Perkin Elmer AA-nalyst 800 instrument by dissolving a quantity of particles dis-persion in HCl.

Structural and morphological characterization of commercialMNPs were carried out with X-Ray Diffractometer (XRD), using aSIEMENS D500 X-Ray diffractometer, and with TransmissionElectron Microscopy (TEM), using a JEOL 1010 operating at 100 kV.For the specimen preparation a drop of diluted solution was de-posited on a carbon coated grid. The mean particle size was esti-mated by applying the log normal function in the size-statisticaldistribution derived from a series of TEM images depicting morethan 300 MNPs.

Hyperthermia measurements were performed by a water-cooled induction coil machine, with varying eld amplitude (1624 kA/m) and constant frequency of 765 kHz. The frequency usedseems relatively high according to limit of applied AMFs (Alter-nating Magnetic Fields) in humans which derives from the productof Hf (our case: 152limit: 4.85108 A/m s). This threshold forpatient discomfort arises from subjecting regions of 15 cm to13.56 MHz and 35.8 A/m and acts up to date as a general extra-polation guideline to the maximum frequency and amplitude thatcan be applied in MPH. However, such an extrapolation should becautiously reconsidered especially in nanoscale applications byaccounting additional parameters that affect eddy heating in tis-sues, namely, the effect of diameter: it is evident that smallertissue regions could tolerate larger eld-frequency products[4].

For hyperthermia characterization solution samples were pre-pared from the initial commercial product. In each case we pre-pared six concentrations of 0.1, 0.2, 0.5, 1, 2, 3 mgFe/mL by addingdistilled water as solvent. Each experimental circle included a600 s heating and a 600 s cooling stage. Thermal efciency offerrouids is quantied by estimating the Specic Absorption Rate(SAR), which refers to the amount of energy converted into heatper time and mass of the magnetic material. SAR was calculated byusing the following equation [13]:

=

SAR cm

m t (1)f

Fe

where c the specic heat of the solution, mf and mFe the total so-lution mass and iron content respectively, /t the initial slope

Table 1Characteristics of commercial ferrouids as provided by the companies datasheetsinfrastructure and comparative evaluations between independentresearch groups may be conducted.

3. Results and discussion

Fig. 1a shows a representative XRD pattern for sample Fluid-MAG-DX-100 nm, when the iron oxide spinel structure (magnetiteand/or maghemite since their distinction may not be performedwith XRD/TEM) is conrmed. By applying Scherrer equation thecrystallite size was calculated at 8.4 nm. This is the rst hint, thatcommercial particles are practically, forming aggregates composedof nanosized superparamagnetic particles, as already discussed inour previous work on a Chemicell product [17]. Fig. 1b showing alow magnication TEM observation reveals the morphology ofcommercial MNPs. As one can see, the primary iron oxide MNPshave almost spherical shape with a mean particle size of 10 nm,forming aggregates of variable size averaging at 100 nm. Com-monly, during TEM specimen preparation, specically duringdrying stage, articial agglomerates may form. The clusters in thesamples under discussion pre-exist even in liquid form in ac-cordance with the product datasheets (see Table 1) and codedunder the name multi domain cores as a result of equilibratingmagnetic, gravitational and electrostatic forces. Fig. 1c is the

ting Magnetization/Characteristics

Use

tran iron oxideposites

Hc0.77 kA/m Ms469 emu/g

Detection purposes in MRI, mag-neto-immuno assays

tran matrix Super-paramagnetic Cell separation, MRI-diagnostics,Magnetic drug-targetingin our previous works [14, 15]. The energy dissipation rate (asshown in Eq. 1) is a sensitive function of particle and solutionfeatures together with the amplitude (H) and frequency (f) of thealternating magnetic eld.

Due to the lack of standards for the alternating magnetic eldamplitude and frequency, each research group characterizes theirparticles under different eld conditions leading to a wide spec-trum of SAR values due to the different experimental setups. Toaddress this issue, we follow what Kallumadil et al. [10] in-troduced, i.e. the concept of intrinsic loss power (ILP), (see Eq. 2)where SAR is normalized against the frequency and magnetic eldstrength facilitating direct comparison of the heating potential ofMNPs studied in this work with systems measured in differentlaboratories.

=ILP SARH f (2)2

Despite the fact, ILP consideration has some validity constraintsbased on linear response theory, [16] yet is safe for frequencies upto few MHz and for applied eld amplitudes lower than the fer-

relaxation refers to the rotation of nanoparticle inside the medium(water in our case) when exposed to an external applied eld andits contribution to the total amount of resulted heat depends onthe ability to perform this movement. The measurements protocolfollowed in present work, is the widely accepted protocol for thehyperthermia measurements, starting with the measurements ofthe solution as described in detail in our previous publications[14,15,18]. Currently, we are examining selected high-performancecommercial samples in vitro prior to their in vivo evaluation, in aneffort to examine the decrease of SAR efciency when MNPs areimmobilized within cells, due to Brownian contribution attenua-tion [19]. This project is still under study and will be a futuremanuscript continuing the scope of the current manuscript whichis to propose the alternate use of commercially available ferro-uids as hyperthermia agents.

Since one of the major issues in clinical application is to use theminimum dose needed each time without sparing the heatingoutcome, it is always useful to optimize the thermal performanceagainst solution concentration. Generally, in all samples, the larger

Fig. 1. Structural characterization schemes: XRD pattern (a) and TEM image(b) along with the EDP (c) for sample FluidMAG-DX-100 nm.

D. Sakellari et al. / Journal of Magnetism and Magnetic Materials 380 (2015) 360364362corresponding Electon Diffraction Pattern (EDP) of the Fig. 1bconrming the iron-oxide spinel structure of MNPs in goodagreement with the XRD pattern.

Hyperthermia measurements were carried out under standardfrequency, varying eld amplitude and ferrouid concentration.An indicative set of initial heating/cooling curves is presented inFig. 2, for sample FluidMAG-DX-100 nm, showing both the heatingand cooling stages and reference signal recorded for solution sol-vent. The potential of this system as hyperthermia heating agent isstraightforward, since its heating response, not only reaches thedesired hyperthermia region (shaded band denoting the 4145 Ctemperature region that facilitates successful heat penetration ofcancer cells) but also surpasses it in quite short time (from 20 to200 s, depending on the concentration). SAR values varied from771 to 1044 W/g for 1 and 0.1 mgFe/mL respectively and comparerelatively well with obtained values in iron-oxide MNPs preparedin-lab [9].

It is well known that thermal response of MNPs solution de-pends on the properties of MNPs, such as their magnetic featuresand particle size, as far as on hyperthermia applicable parameters(eld amplitude and frequency) [18]. Hence depending on themagnetic prole of MNPs, whether are superparamagnetic or ferro(i)magnetic, the hyperthermia results, as expressed by the tem-perature rise, may be attributed to the relaxation mechanism(namely Nel or Brown reversal) or hysteresis losses. BrownianFig. 2. Time dependent temperature curves for sample FluidMAG-DX-100 nm, forvarious concentrations (0.13 mgFe/mL), under 24 k A/m eld amplitude. Shadedband denotes the desired hyperthermia levels.the concentration the larger the increase of temperature, achievedin shorter time.

As discussed previously, to provide a more universal approach,ILP values were estimated in all cases and maximum values aredepicted in Fig. 3. The sample with optimum thermal responseseems to be FluidMAG-DX-100 nm. The reference horizontalline corresponds to the work of Kallumadil et al. [10] for thesample FluidMAG-D with 100 nm hydrodynamic diameter andILP2.01 nHm2/kg, under 900 kHz and 5.66 kA/m eld amplitude.It is well known that coating of MNPs may signicantly affect theirarrangements, magnetic features and eventually magnetic heatingefciency [9]. The reference sample has starch as a matrix while inour case dextran is incorporated as a biocompatible coating.Dextran, is a typical polysaccharide used for coating of iron oxideMNPs, providing biocompatibility, and is an established coating forclinical applied contrast agents. Furthermore, dextran coating, inthe sense of thickness of its surrounding layer, affects magneticcharacteristics (saturation magnetization or magnetic interactions)and hence the SAR values of MNPs [20]. ILP values seem to bemore sensitive to MNPs size with the 50 nm yielding relativelyhigh values. On the other hand sample of 200 nm seem to possesssmaller heating efciency while the Nanomags-D-spio samplewith a much wider size distribution (denoted also from thecompany) seem to have the milder heating effect. This may becorrelated with the magnetic features i.e. higher magnetizationvalues as MNPs grow in size.

Fig. 3. ILP values vs. concentration, under 20 kA/m eld amplitude. Reference line

refers to sample FluidMAG-D from Ref. 10.

netic nanoparticle hyperthermia enhancement of cisplatin chemotherapycancer treatment, Int. J. Hyperth. 29 (8) (2013) 845851.

D. Sakellari et al. / Journal of Magnetism and Magnetic Materials 380 (2015) 360364 363On the other hand, the external hyperthermia eld should al-ways be in position to manipulate the MNPs coercive eld, al-lowing the transferring of the maximum heating power from theapplied led to the ferrouid, as recently discussed in a previousmanuscript [14]. This seems to be the case for the FluidMAG-DX-100 nm sample. Regarding the concentration effect to thermalresponse of ferrouid, from Fig. 3, it seems that for the con-centrations examined the ILP values practically remain unaltered.In our case, the heating efciency is actually driven by the intrinsicMNPs features when solution concentrations are relatively smallso as not to allow the dipolar interactions. Meanwhile for muchbigger concentrations (45 mg/mL) dipolar interactions may leadto MNPs arrangements, thus overrule individual particle con-tribution and occasionally lead to enhancement of heating ef-ciency [21].

Since the saturation magnetization, as far as the general mag-netic prole affect the heating losses mechanisms, which in turndepends on MNPs core sizes as far as the total hydrodynamicdiameter, the ILP variations with the hydrodynamic diameter arepresented in Fig. 4 for samples of 1 mg /mL. The regional zone of

Fig. 4. ILP values (presented as shaded bands including the error bars) for samplesFluidMAG-DX-50, 100, 200 nm, measured in concentration 1 mgFe/mL, under765 kHz and 20, 24 kA/m. The dotted line corresponds to Nanomags-D Spio.Fe

ILP values is approximated from the two sets of measurements atspecic eld amplitudes of 20, 24 kA/m for the samples understudy where the width of the zone corresponds to the 10% errorbar in estimated SAR values. Again analogous reference region forNanomags-D-spio is given for comparison. It appears, that the ILPregion of Chemicell extends in a region between 1 and2.5 nHm2/kg becoming pronounced around the hydrodynamicdiameter of 100 nm.

It seems that the arrangement of MNPs in uniform size ag-gregates appear to provide an additional degree of freedom to netune the ILP values as exhibited by the FluidMAG-DX series. In allsample cases the hydrodynamic diameters correspond actually notto individual MNPs but to aggregates, where the dipolar interac-tions in conjunction with the chemical environment govern thesize and its uniformity of these aggregates leading eventually totunable magnetic heating features as directly seen in Fig. 4. Thesendings coincide with our recent work for another commercialproduct from Chemicell [17]. Although, further characterizationschemes in more samples from different companies, should beperformed in order to manipulate aggregate formation in a ben-eciary way, the observed tunable heating efciency certies thatcommercial ferrouids may also play a role as promising candi-dates in multifunctional modern theranostics.

Nanoparticles: a Synergistic Approach to Apoptotic Hyperthermia, Angew.

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[8] D. Yoo, J. H. Lee, T.-H. Shin, J. Cheon, Theranostic magnetic nanoparticles, Acc.Chem. Res. 44 (2011) 863.

[9] C. Grttner, K. Mller, J. Teller, F. Westphal, Synthesis and functionalization ofmagnetic nanoparticles for hyperthermia applications, Int. J. Hyperth. 29 (8)(2013) 777789.

[10] M. Kallumadil, M. Tadac, T. Nakagawac, M. Abec, P. Southern, Q.A. Pankhurst,Suitability of commercial colloids for magnetic hyperthermia, J. Magn. Magn.Mater. 321 (2009) 15091513.

[11] http://www.micromod.de.[12] http://www.chemicell.com.[13] M.A. Verges, R. Costo, A.G. Roca, J.F. Marco, G.F. Goya, C.J. Serna, M.P. Morales,

Uniform and water stable magnetite nanoparticles with diameters around themonodomainmultidomain limit, J. Phys. D: Appl. Phys 41 (2008) 134003.

[14] K. Simeonidis, C. Martinez-Boubeta, Ll Balcells, C. Monty, G. Stavropoulos,M. Mitrakas, A. Matsakidou, G. Vourlias, M. Angelakeris, Fe-based nano-particles as tunable magnetic particle hyperthermia agents, J. Appl. Phys. 114(2013) 103904.

[15] A. Chalkidou, K. Simeonidis, M. Angelakeris, T. Samaras, C. Martinez-Boubeta,Ll Balcells, K. Papazisis, C. Dendrinou-Samara, O. Kalogirou, In vitro applicationof Fe/MgO nanoparticles as magnetically mediated hyperthermia agents for[4] B. Kozissnik, A.C. Bohorquez, J. Dobson, C. Rinaldi, Magnetic uid hy-perthermia: advances, challenges, and opportunity, Int. J. Hyperth. 29 (8)(2013) 706714.

[5] K.L. ONeill, D.W. Fairbairn, M.J. Smith, B.S. Poe, Critical parameters inuencinghyperthermia-induced apoptosis in human lymphoid cell lines, Apoptosis 3(1998) 369375.

[6] D. Yoo, H. Jeong, C. Preihs, J. Choi, T. Shin, J.L. Sessler, J. Cheon, Double-Effector4. Conclusions

This work deals with the potential exploitation of a series ofcommercial iron-oxide ferrouids, as magnetic hyperthermiaagents. Our results show an enhanced heating efciency that canbe tuned by eld amplitude and ferrouid parameters. Thus, ifproperly tuned, commercial ferrouids may be also play a role ashyperthermia mediators. More specically, SAR seems to be ag-gregate-size dependent and the ferrouid with the optimum SARvalue was the FluidMAG-DX-100 nm, while the concentrationsunder study did not drastically affect the heating efciency. Be-yond the relative high thermal efciency of the above possiblenano-agents, MNPs should also fulll other criteria such as bio-compatibility and colloidal stability, usually driven mainly by thecoating material. The choice of the coating material determinesthe nal hydrodynamic diameter along with the magnetic inter-action, and also signicantly seems to be a key parameter for theoptimum thermal response. In the quest of modern theranosticsmaterials, a novel pathway is opened by exploring the wide rangeof accessible features in commercial ferrouids.

Acknowledgments

Authors would like to express their gratitude to the companiesfor providing the samples.

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D. Sakellari et al. / Journal of Magnetism and Magnetic Materials 380 (2015) 360364364

Exploring multifunctional potential of commercial ferrofluids by magnetic particle hyperthermiaIntroductionMaterials and methodsResults and discussionConclusionsAcknowledgmentsReferences