*Corresponding author. Tel.: #49-30-45057073; fax: #49-30-45078979.

E-mail address: andreas.jordan@charite.de (A. Jordan)

Journal of Magnetism and Magnetic Materials 201 (1999) 413}419

Invited Paper

Magnetic #uid hyperthermia (MFH): Cancer treatment withAC magnetic "eld induced excitation of biocompatible

superparamagnetic nanoparticles

Andreas Jordan*, Regina Scholz, Peter Wust, Horst FaK hling, Roland Felix

Department of Radiation Oncology (WE 07), University Clinic Charite& , Medical Faculty of the Humboldt Universita( t zu Berlin, CampusVirchow-Klinikum, SFB 273, Augustenburger Platz 1, 13353 Berlin, Germany

Received 18 May 1998

Abstract

The story of hyperthermia with small particles in AC magnetic "elds started in the late 1950s, but most of the studieswere unfortunately conducted with inadequate animal systems, inexact thermometry and poor AC magnetic "eldparameters, so that any clinical implication was far behind the horizon.

More than three decades later, it was found, that colloidal dispersions of superparamagnetic (subdomain) iron oxidenanoparticles exhibit an extraordinary speci"c absorption rate (SAR [=/g]), which is much higher at clinically tolerableH

0f combinations in comparison to hysteresis heating of larger multidomain particles. This was the renaissance of

a cancer treatment method, which has gained more and more attention in the last few years. Due to the increasingnumber of randomized clinical trials preferentially in Europe with conventional E-"eld hyperthermia systems, the generalmedical and physical experience in hyperthermia application is also rapidly growing. Taking this increasing clinicalexperience carefully into account together with the huge amount of new biological data on heat response of cells andtissues, the approach of magnetic #uid hyperthermia (MFH) is nowadays more promising than ever before. The presentcontribution reviews the current state of the art and some of the future perspectives supported by advanced methods ofthe so-called nanotechnology. ( 1999 Elsevier Science B.V. All rights reserved.

Keywords: Magnetic #uids; Hyperthermia; SAR; Nanoparticles; Cancer; Biomedical applications

1. Basic principles of hyperthermia

Heating of certain organs or tissues to temper-atures between 413C and 463C preferentially for

cancer therapy is called &Hyperthermia'. Highertemperatures up to 563C, which yield widespreadnecrosis, coagulation or carbonization (dependingon temperature) is called &thermo-ablation'. Bothmechanisms act completely di!erent concerningbiological response and application technique. The&classical' hyperthermia induces almost reversibledamage to cells and tissues, but as an adjunct it

0304-8853/99/$ } see front matter ( 1999 Elsevier Science B.V. All rights reserved.PII: S 0 3 0 4 - 8 8 5 3 ( 9 9 ) 0 0 0 8 8 - 8

enhances radiation injury of tumor cells andchemotherapeutic e$cacy. Modern clinical hyper-thermia trials focus mainly on the optimization ofthermal homogeneity at moderate temperatures(42}433C) in the target volume, a problem whichrequires extensive technical e!orts and advancedtherapy and thermometry systems.

Heating to the target temperatures causes mod-erate cellular inactivation in a dose-dependentmanner. Although thermal dose}response curveslook quite similar to radiation or drug dose re-sponse curves, the critical target of thermal inac-tivation in the cell is not known yet. The mostprobable reason for this situation is that there is noindividual cellular target of hyperthermia, in con-trast to the well known DNA damage after irradia-tion [1,2]. Most of the biomolecules, especiallyregulatory proteins involved in cell growthand di!erentiation and the expression of certainreceptor molecules (involved in signal transductionpathways) are therefore largely in#uenced byhyperthermia.

Today, many cellular e!ects are known to beimportant for thermal inactivation. New insightsfrom molecular biology have shown that a fewminutes after hyperthermia, a special class of pro-teins is expressed in the cell, the so-called heatshock proteins (hsp). They protect the cell fromfurther heating or subsequent thermal treatmentsand lead to an increase of cell survival after pre-heating, an e!ect called thermotolerance [3]. Addi-tionally, the activity of certain regulatory proteins,kinases or cyclins is in#uenced by hyperthermia,causes alterations in the cell cycle and can eveninduce apoptosis, the cell death driven by the cellregulatory system itself [4}7]. Current research isalso trying to characterize the interactions of ther-mal tolerance and multidrug resistance [8]. Thecombined e!ect of radiation and hyperthermiatakes place at the cellular level and is mainly due tothe heat-induced malfunction of repair processesafter radiation-induced DNA damage. They areless e!ective when heat is given either before orafter irradiation, and a well-de"ned time intervalhas been described for the two modalities [9]. Fur-ther e!ects were observed on the tissue level such aschanges of microvasculature, blood #ow, energyand oxygen status [10]. Interestingly, heat treated

cancer cells may be better recognized by the hostimmune system due to alterations of some cellsurface receptor molecules which are then recog-nized by natural killer (NK) cells and inactivate thecancer cells, as has been recently demonstrated invitro [11]. The joint action of all the molecularmechanisms involved in hyperthermia is still underinvestigation.

2. Clinical hyperthermia

State-of-the-art radiofrequency (RF-) hyperther-mia systems, e.g. annular phased array systems(APAS) for regional hyperthermia of deep seatedtumors, are still limited by the known heterogeneityof tissue electrical conductivities or high perfusedtissues, which makes selective heating of those re-gions with such E-"eld dominant systems very di$-cult. Further application techniques are wholebody hyperthermia (WBH, with water-"ltered in-fra-red irradiation), local hyperthermia (e.g. withcurrent sheet applicators) and interstitial hyper-thermia (requires implantation of microwave- orRF-antennas or self-regulating thermoseeds).A nearly unsolved problem are the bone of thepelvis or the scull, which &shield' deep tissues in thecavity of bones, which often result in &hot spot'phenomena, which are di$cult to predict in certaindi!erent locations of the body. The overall e!ect isthermal underdosage in the target region, whichoften yields recurrent tumor growth. Despite theseuncertainities, several (preferentially European)randomized trials demonstrated therapeutic bene"tof &state-of-the-art' (RF-) hyperthermia, so far: Vander Zee and his co-worker [12] reported higherlocal control after three years follow-up with thecombination of hyperthermia and radiation (RHT)with advanced rectal carcinoma, bladder and cervixcarcinoma. With the ladder tumor entity evena survival bene"t was observed. Our group in Be-rlin (Rau et al., [13]) has found higher responserates with RHT of advanced rectal carcinoma ina pre-operative approach. Valdagni and his co-worker in Italy [14] found higher local control ofadvanced lymph node metastases after RHT. TheDanish group of Jens Overgaard [15] had successwith malignant melanoma, i.e. increase of local

414 A. Jordan et al. / Journal of Magnetism and Magnetic Materials 201 (1999) 413}419

control (2 years follow-up). Claire Vernon with hergroup in London [16] obtained higher responserates of recurrent mammary carcinoma when ir-rradiation was combined with local hyperthermia.Penny Sneed and her group (USA) had success withglioblastoma multiformae in a randomized trial ofbrachytherapy boost plus interstitial hyperthermiawhich yielded an overall survival bene"t in thehyperthermia arm [17].

Summarizing all these clinical studies, it is main-ly accepted that preferentially the problems ofphysical power deposition still limit the clinicaloutcome. This includes not only thermal underdos-age of critical regions, there are also large limita-tions on the body target sites, which are toodi$cult to treat, like brain tumors. Conclusivelythere is a large demand on alternative physicalconcepts, which may o!er deep seated power de-position in almost every region of the body.

3. Much more than simple particle heating:the biological concept of magnetic 6uidhyperthermia (MFH)

In the early 1960s, a few US groups were the "rst,who tried to perform hyperthermia with magnetiz-able microparticles, which were heated by anexternally applied AC magnetic "eld. The use ofH-"eld dominant systems together with powerabsorbing material instead of power steering ofE-"eld dominant systems is therefore an oldidea. However, before the early nineties, the statusof this research was di!use and clinical applicationwas unthinkable. Poor de"ned animal systems [18]or ex vivo tissue samples were used to test an intra-tissue heating up to the present [19]. In contrast,solid and comparable in vivo tumor growth dataare needed including all the important controls,precise on-line temperature monitoring and patho-logical tissue inspection. Those studies have beenrarely performed, so far.

For the "rst time, our group could demonstratein 1993 [20], which H-"eld amplitudes and fre-quencies (i.e. speci"c absorption rate, SAR) are tol-erable in humans. Based on the Brown and NeeH lrelaxivity, it was shown, that subdomain particles(nanometer in size) absorb much more power at

tolerable AC magnetic "elds than is obtained bywell known hysteresis heating of multidomain (mi-crons in size) particles. The SAR of magnetic #uidsis iH2

0f, where i is a material constant for a given

H0

f combination.This was the renaissance of a cancer treatment

method, which has gained more and more attentionin the last ten years.

Systematic in vitro studies were presented byChan and co-worker (1993) [21] and in a moreextended study by our group in 1996 [22], whichboth show consistently, that inactivation of cancercells with AC magnetic "eld excited nanoparticles isequal to the best homogeneous heating, i.e. waterbath heating, for a given time temperature sched-ule. This result was not self-evident, because a largenumber of single particles, but each acting in prin-ciple as a hot source surprisingly yield a temper-ature homogeneity, which was comparable towater, containing much more excited moleculesthan particles existing in a magnetic #uid. Accord-ing to these encouraging in vitro results, a homo-geneous cell or tissue inactivation was expected invivo, too, if the #uid could be administered almosthomogeneously throughout the target region.

On the basis of this extended physical and biolo-gical knowledge, advanced AC magnetic "eld ap-plicators were constructed for animal experiments.New studies were started with the isogenic C3Hmammary carcinoma of the mouse [23], which wastransplanted into the right hind leg. The animalswere treated for 30 min at 473C (intratumoralsteady state temperature) including several controls(untreated, coating substance and magnetic #uidalone). In this approach, the temperature was in-credibly high in order to test unconventionally (fora hyperthermia study) the potential of MFH asa mono-therapy in order to reach local tumor con-trol. Surprisingly, this was obtained in 44% of theanimals (Fig. 1). The magnetic #uid was given in-tralesionally without anaesthesia. Before AC mag-netic "eld treatment, magnetic #uid depots in thetarget region were observed as expected, but afterthe "rst MFH session, this distribution had beenhomogenized, which has been termed now as &ther-mal bystander e!ect'. This e!ect o!ers broad per-spectives not only in hyperthermia, but also in drugtargeting, gene and immune therapy. Moreover, the

A. Jordan et al. / Journal of Magnetism and Magnetic Materials 201 (1999) 413}419 415

Fig. 1. C3H mouse mammary carcinoma growth curves of untreated (control), dextran, dextran-ferrite and MFH treated animals. Note,that tumor growth remains nearly una!ected until AC magnetic "eld-induced heating yields 44% tumor control 30 days after treatment.

results suggest, that MFH might become a newminimal invasive modality for regional selectiveheat treatment on the microscopic level, which isnot possible by any other method, so far.

4. Recent results

In order to proceed from the encouraging resultswith the mammary carcinoma of the mouse, more

animal experiments are required to "x the currentstate of MFH as an almost site-speci"c modality,which allows regional heating in di!erent locationsof the body. As a precondition, the technology ofAC magnetic "eld application is currently underdevelopment [24]. If an almost regional AC mag-netic "eld application could be realized, migrationof any ferro#uid to distant locations could be ne-glected. Many aspects have to be considered, likeE-"eld shielding of the &patient', heat loss of the

416 A. Jordan et al. / Journal of Magnetism and Magnetic Materials 201 (1999) 413}419

Fig. 2. Prototype applicator for regional AC magnetic "eldadministration at a frequency of 500 kHz and "eld amplitudes ofup to 10 kA/m. Depending on body cross-section and tissueconductivity lower frequencies should be used with humans, i.e.50}100 kHz. The applicator site has a gap height of 20 mm,which is suitable for treating mice or other small animals.

core material, mechanical stress of the overall con-struction and more. A prototype AC magnetic "eldapplicator for regional MFH of small animals isshown in Fig. 2.

The second component of MFH }, the magnetic#uid is currently optimized, too. Since we knowfrom theoretical estimations, that for a given excita-tion frequency lH an &ideal' core size dH exists[25,26], which yields maximum SAR, an almostsharp core size distribution is required in order tominimize the therapeutic metal oxide mass requiredfor a given target volume.

The second aspect is the shell of the nanopar-ticles in a magnetic #uid. It supports colloidal sta-bilization, but it has also a contribution to thepower absorption due to the Brownian relaxationprocess. From the theoretical point of view, it islargely interesting how the core interacts exactlywithin the shell in an AC magnetic "eld. If the coreexhibits an oscillation within the shell dependingon excitation frequency and core magnetization,some optimization parameters related to the vis-cosity and structure of the shell material are ex-pected. Alternatively, if the whole particle isoscillating in the "eld, far less optimization wouldbe possible, because the necessity of aqueous non-toxic dispersion media for medical purposes wouldnot allow any hard changes to e.g. hydrocarbon-

related substitutes. Therefore, more physical stud-ies are required to exactly describe nanoparticleoscillation with respect to power absorption in ACmagnetic "elds around 50}100 kHz, using di!erentshells of di!erent hydrodynamic behaviour. Addi-tionally, modi"cations of core magnetization couldfurther enhance the speci"c power absorption, butagain, biocompatibility rules out many of thepromising elements, e.g. a cobalt dotation.

Besides this potential of SAR optimization, an-other large "eld of research has been recentlyopened: the transition from site-selectivity to tissueand cell speci"city. The shell is able to supportfunctionalized surfaces which can be coupled toa wide variety of biomolecules.

Two di!erent ferro#uids were used in our recentstudy: magnetite particles with aminosilan typeshell (dBU48, core diameter 10 nm/hydrodyn.30 nm) with largely positive surface charges and ofa magnetite with dextran type shell (dP6, corediameter 3 nm/hydrodyn. 70 nm) with a neutral tonegative surface charge.

Primary human glioblastoma cells (staining pos-itive for GFAP/S100) originated from in-traoperative material were maintained in the 0 to1st passage and nanoparticle uptake in vitro wascompared to the uptake of established normalneuronal cells of the cerebral cortex (HCN-2,CRL-10742, GFAP negative) and a "broblast(EBF) line. The cells grew in presence of 0.6 mgferrite per ml medium for 2}8 days and uptake ofeach cell type was compared to similar cells innormal medium (controls). The cells were investi-gated by light and electron microscopy and the ironcontent of the cells was determined quantitatively.

In a preliminary set of in vitro experiments,largely higher uptake of the positive chargedaminosilan particles in comparison to the dextranmagnetite (up to 1000-fold) was observed with theprimary glioblastoma cells. Also larger uptake ofboth particle types was observed into glioblastomacells compared with normal neuronal cells and"broblasts (about 500}2000-fold). If this observeddi!erential particle endocytosis could be validatedby further systematic series of experiments, thiswould be a new strategy of particle targeting, unlikeconventional methods, e.g. with monoclonal anti-bodies. Obviously, both particle species induce

A. Jordan et al. / Journal of Magnetism and Magnetic Materials 201 (1999) 413}419 417

Fig. 3. Scanning electron micrograph of a colon adenocarcinoma cell, which has been grown in a ferro#uid containing growth medium(modi"ed aminosilan shell magnetite, core diameter around 10 nm, 0.6 mg/ml) for 72 h. Note, that the particles are highly adhesive to thesurface of the cell, which was not or far less observed with normal cells.

di!erent cellular adhesion and internalization pro-cesses, which may become especially important inview of diagnostic and therapeutic purposes. How-ever, further pharmacological studies must show,how blood circulation time and scavenging activityof the reticuloendothelial system may limit thispromising approach in vivo. Further carcinomacell types are now under investigation such ascolorectal and mammary carcinomas. A samplescanning electron micrograph (SEM) of a colorec-tal carcinoma cell is shown in Fig. 3. Note, that themagnetite particles are adhesive on the naturalsurface of the cells without any changes of normalcell structures, like the microvilli as has been ob-served by comparable micrographs of normal cellsin regular culture (not shown).

5. Perspectives

It is still a fascinating concept, that tumor cellscould be loaded with thousands of particles, whichwould become activated } comparable to genes

} only by a speci"c signal, yielding the death of allparticle containing cells as soon as an AC magnetic"eld is applied. Our cellular observations so farindicate that a tumor, which has taken up theseparticles, will not be able to get rid of them. Daugh-ter cells from a particle containing parent cellsshould therefore contain up to 50% of the particleamount of the parent cell. Hence, particle loadedtumor cells may not only contain the &markers' fortheir own death, the descendants would still havea higher risk of dying from future AC magnetic "eldapplications. It is therefore an exciting challenge forfuture research to increase the biological e$cacyand particle SAR in order to achieve advancedmagnetic #uids of which only a few particles arerequired for selective tumor cell inactivation.

Future animal studies and further investigationson cells and tissues with a broad spectrum of func-tionalized nanoparticles will elucidate the overallpotential of MFH. Besides these preclinical studies,which may yield bene"ts not before many years ofresearch, today large e!orts are needed to start a cli-nical study with existing biocompatible ferro-#uids

418 A. Jordan et al. / Journal of Magnetism and Magnetic Materials 201 (1999) 413}419

and a clinical prototype of the AC magnetic "eldapplicator. Suitable tumor entities should then becarefully chosen embedded into actual oncologicalconcepts, i.e. tumors with bad prognosis and tumorswhich are di$cult to heat, like brain tumors andtumors of high perfused organs (kidney, liver, lung).

In conclusion, the generation of functionalizedsurfaces of those particles for cancer cell targeting isthe next challenge for the future. Nanotechnologywill o!er new strategies towards these functional-ized particle systems, which might be able tocircumvent the known scavenging e!ects of thereticuloendothelial system. The ensemble of biolo-gical strategies, clinical hyperthermia experience,the discovery of the &thermal bystander e!ect', com-bined with methods of interventional radiology,microsurgery and the use of precisely operatingnavigation systems, will give us a new weaponagainst cancer, which is called MFH.

Acknowledgements

The authors wish to thank Mrs. Lajoux and Prof.Schnoy of the Department of Electron Microscopyand Prof. Maier-Hau! of the Department of Neuro-surgery, both of the University Clinic ChariteH , forvaluable co-operation and helpful discussions. Thisproject is supported by the Deutsche Forschungs-gemeinschaft (DFG), Sonderforschungsbereich 273(TP A8), Bonn, Germany.

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