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Cisplatin-loaded carbon-encapsulated iron nanoparticlesand their in vitro effects in magnetic fluid hyperthermia

Arthur Taylor a,b,*, Yulia Krupskaya b, Kai Kramer a, Susanne Fussel a, Rudiger Klingeler b,Bernd Buchner b, Manfred P. Wirth a

a Department of Urology, Medical Faculty, Dresden University of Technology, Fetscherstr. 74, 01307 Dresden, Germanyb Institute for Solid State and Materials Research (IFW), Helmholtzstr. 20, 01069 Dresden, Germany

A R T I C L E I N F O

Article history:

Received 7 December 2009

Accepted 6 March 2010

Available online 10 March 2010

0008-6223/$ - see front matter � 2010 Elsevidoi:10.1016/j.carbon.2010.03.009

* Corresponding author: Address: DepartmeDresden, Germany. Fax: +49 351 4585771.

E-mail address: arthur.taylor@uniklinikum

A B S T R A C T

Iron nanoparticles encapsulated by carbon are protected from reactions with their environ-

ment avoiding oxidation in ambient conditions and thus, preserving their magnetic prop-

erties. Such particles are good candidates for magnetic fluid hyperthermia. When graphite

shells are present, acidic treatments allow the formation of carboxylic groups on the nano-

particle surface. Those carboxylic groups can be used for further complexation with the

drug cisplatin. We show the possibility of loading cisplatin on such nanoparticles and that

the loading is dependent on the degree of surface functionalization. The drug release is

dependent on time and temperature, making it ideal for applications involving hyperther-

mia. We show the possibility of applying hyperthermia in vitro using these nanoparticles.

When loaded with cisplatin a stronger cytotoxic effect is observed. Such particles could

be potentially used as multimodal anti-cancer agents for therapies based on the synergistic

effect of chemotherapy and hyperthermia.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Magnetic nanoparticles have found a diverse range of potential

applications in medicine which include their use as contrast,

drug delivery, and hyperthermia agents [1]. The use of iron

oxide nanoparticles as heating probes for hyperthermia can

be traced back to the experiments of Gilchrist et al. in 1957

[2]. Clinical trials using such particles however, have only re-

cently started [3,4]. Lately, considerable research has been con-

ducted on the development of materials with increased

specific absorption rates (SAR) [5] and tuned Curie tempera-

tures [6] that allow a smart switch to avoid overheating. In com-

parison to iron oxides, metallic iron would be a good candidate

for increased SAR given its higher saturation magnetization. Its

application as a nanoparticle is however hindered by its quick

oxidation in ambient conditions. Feasibility of other metallic

er Ltd. All rights reserved

nt of Urology, Medical F

-dresden.de (A. Taylor).

magnets as cobalt or nickel might be limited by their potential

toxicity. One way to overcome these limitations is to have a

protective coat over the metallic nanoparticle hindering its

reaction with the environment. Methods for the synthesis of

such structures have already been developed making it feasible

to obtain core–shell nanostructures in which the metallic core

is encapsulated by a shell of precious metals, polymers, silica

or carbon [7]. The shell prevents their oxidation and thus, al-

lows the preservation of their magnetic properties.

Carbon-encapsulated magnetic nanoparticles (CEMN)

which have so far been synthesized consist of several graph-

ite layers that cover metals such as cobalt, iron and nickel [8–

11]. We have recently shown that carboxylic functionalities

can be introduced on the surface of such particles by means

of acidic treatments [12]. The presence of those functional-

ities allows the conjugation of biological molecules by means

.

aculty, Dresden University of Technology, Fetscherstr. 74, 01307

2328 C A R B O N 4 8 ( 2 0 1 0 ) 2 3 2 7 – 2 3 3 4

of diimide-activated amidation. Such molecules can be of

interest for improved biocompatibility, targeting and drug

transport, for example.

Carboxylic functionalities have also been exploited for

conjugation of the anti-cancer compound cisplatin [cis-

diamminedichloroplatinum(II)] (CDDP) to polymers [13–15].

The complexation is based on the replacement of the chloro

ligands of the drug with the carboxylic functionalities of the

polymer. The method reported in those studies opens the

possibility of conjugating cisplatin to CEMN containing car-

boxylic functionalities. This would allow such particles to be

used for magnetic drug delivery or bi-modal treatments based

on hyperthermia and chemotherapy. The last case is of spe-

cial interest since a synergistic effect when heat and drugs

are combined has been shown for several drugs, including

the potential to reverse chemoresistance [16].

In this work, we investigate the possibility of exploring car-

boxylic functionalities on the shell of carbon-encapsulated

iron nanoparticles (FeNPs) to load the anti-cancer agent cis-

platin and its in vitro implications for hyperthermia therapies.

2. Materials and methods

2.1. Materials

FeNPs with an average particle diameter of 25 nm (as stated

by the supplier) were obtained from Sun Innovations Inc. (Fre-

mont, USA). Acidic treatments were performed using reagent

grade acids from Merck (Darmstadt, Germany). CDDP, OPDA

(o-phenylenediamine) and functionalization reagents MES

(2-(N-morpholino)ethanesulfonic acid), EDC (1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide) and methylamine were

obtained from Sigma (Taufkirchen, Germany).

Fig. 1 – Schematic representation of drug loading on the nanop

generate carboxylic groups on their surface. The carboxylated n

amino containing molecules (FeNP–MA) or directly loaded with

nanoparticles are used: directly loaded with CDDP or functional

CDDP).

2.2. Loading and release of cisplatin

FeNPs were treated in two steps for purification and genera-

tion of carboxylic groups [12]. At first, the nanoparticles were

immersed in an 1 M HCl solution, sonicated for 5 min in a

bath sonicator and heated for 15 min at 120 �C. In a second

step, the FeNPs were treated with 5 M HNO3 for 3 h at 65 �C.

After each step, the nanoparticles were thoroughly washed

with distilled water. FeNPs that underwent these treatments

were denominated FeNP–COOH.

A second set of samples with a lower number of carboxylic

functionalities was prepared by conjugating the FeNP–COOH

with methylamine as previously described [12]. Conjugation

with methylamine consumes the carboxylic groups thus

reducing its density at the particle surface. This set of nano-

particles was denominated FeNP–MA.

An aqueous solution of CDDP was mixed with AgNO3 in a

1:2 M ratio and placed overnight on a platform rocker pro-

tected from light. This procedure allows the abstraction of

the chloro ligands from the drug and its precipitation in the

form of AgCl. The solution was then centrifuged for 30 min

at 3000g to concentrate the precipitate. The supernatant

(aquated CDDP) was used.

For CDDP complexation, FeNP–COOH or FeNP–MA powder

was suspended in a solution containing 100 lg/ml of aquated

CDDP. The weight ratio of nanoparticle used was of 10:1 in re-

spect to the drug. The suspension was protected from light

and placed on a platform rocker for 24 h. After this period,

the nanoparticles were washed twice with distilled water to

remove unbound CDDP. Washing was accomplished by con-

centrating the nanoparticles at the bottom of the flask with

a commercial NdFeB magnet followed by removal of the

supernatant and resuspension in distilled water. After drug

articles. The FeNP shells are oxidized with HNO3 in order to

anoparticles (FeNP–COOH) can be further functionalized with

aquated cisplatin (FeNP–CDDP). Here, two types of loaded

ized with methylamine and loaded with CDDP (FeNP–MA–

C A R B O N 4 8 ( 2 0 1 0 ) 2 3 2 7 – 2 3 3 4 2329

loading the FeNP–COOH and FeNP–MA were designated FeNP–

CDDP and FeNP–MA–CDDP respectively (Fig. 1).

The release of cisplatin was studied in water or saline

solutions at 37 �C. For that purpose, 2 mg of freshly prepared

FeNP–CDDP or FeNP–MA–CDDP were suspended in 500 ll of

water or 0.9% saline solution. At different intervals the nano-

particles were concentrated at the bottom of the tube with a

magnet and the 500 ll of supernatant was taken for platinum

quantification. The nanoparticles were then resuspended in

500 ll of fresh solution and the procedure repeated at several

time points from which a cumulative drug release curve was

obtained.

The effect of heating on the drug release was studied by

suspending 500 lg of freshly loaded FeNP–CDDP in 500 ll of

saline. The suspension was incubated for 30 min in an ice

bath (4 �C), or on a heating block (37, 43 and 50 �C). After this

period, the nanoparticles were concentrated with a magnet

and the saline solution taken for platinum quantification.

2.3. Quantification of platinum

Cisplatin was quantified using a slightly modified colorimetric

assay based on the reaction of platinum with o-phenylenedi-

amine (OPDA) [17]. In brief, 500 ll of a CDDP containing solu-

tion was mixed with the same volume of 1.4 mg/ml OPDA

dissolved in dimethylformamide (DMF, Merck, Darmstadt,

Germany). The mixture was heated to 100 �C for 10 min and

then cooled in an ice bath for further 10 min after which it

was transferred to a poly(methyl methacrylate) cuvette

(Brand, Wertheim, Germany) for absorbance measurement

in an UV-Spectrophotometer (Ultrospec 3000, GE Healthcare,

Munich, Germany). Absorbance was measured against a

water blank at three points (600, 720 and 800 nm). The absor-

bance at 720 nm corresponds to the maximum peak of the

reaction product. Values at 600 and 800 nm were taken for

baseline correction. A calibration curve was constructed with

Fig. 2 – Time dependent cumulative drug release from FeNP–CDD

water or saline and at different time points, the nanoparticles we

the supernatant removed for platinum measurement. Immediat

resuspended in fresh solution. The curves show the cumulative

The values in the ordinate correspond to the amount of drug re

solutions with known concentrations of CDDP and the mea-

surement range was of 1–20 lg/ml.

2.4. Magnetic properties and hyperthermia studies

Magnetic measurements of the dry powders were conducted

in a Alternating Gradient Magnetometer (AGM 2900, Princeton

Measurement Corporation, Princeton, USA) at room tempera-

ture in magnetic fields up to B = 1 T. The heating effect of the

FeNP–COOH in alternating (AC) magnetic fields was measured

in a system consisting of high-frequency generator with an

impedance matching network and a water-cooled magnetic

coil system [18]. A coil with five turns, bore diameter of

30 mm and height of 40 mm was used for measurements.

The setup provides AC magnetic fields with a frequency

f = 128 kHz and magnetic fields strengths from 0 to 120 kA/

m. The temperature measurements were done by means of

a fiber optic temperature controller (Luxtron One, Luxtron)

with readings every 1 s. The heating effect at different mag-

netic fields was studied on a FeNP–COOH solution suspended

in distilled water containing 1% methylcellulose (4000 cP, Sig-

ma, Germany). SAR was calculated according to Eq. (1) where

the specific heat capacity of the sample (c) was considered to

be that of solvent (water, c = 4.118 J/g K).

SAR ¼ cdTdtj t! 0 ð1Þ

2.5. Cell culture studies

The prostate cancer DU-145 cell line (American Type Culture

Collection, Germany) was used for cell culture experiments.

Cells were routinely cultured in Dulbecco’s Modified Eagle’s

Medium (DMEM) supplemented with 10% Fetal Calf Serum,

1% HEPES buffer, 1% MEM non-essential amino-acids and

1% streptomycin/penicillin (all from Invitrogen, Karlsruhe,

Germany) at 37 �C in a 5% CO2 incubator.

P and FeNP–MA–CDDP. The nanoparticles were suspended in

re concentrated at the bottom of the tube with a magnet and

ely after removal of the supernatant the nanoparticles were

release of CDDP ± SD from three independent experiments.

leased per milligram of nanoparticle.

2330 C A R B O N 4 8 ( 2 0 1 0 ) 2 3 2 7 – 2 3 3 4

Concentration dependent cytotoxicity of the FeNP–COOH

and FeNP–CDDP was probed using the cell Proliferation Re-

agent WST-1 (Roche, Mannheim, Germany). Cells were seeded

overnight in 96-well plates with a density of 7500 cells per

well. On the next day the culture medium was removed and

replaced with 100 ll of medium containing 1% methylcellu-

lose and the nanoparticles at different concentrations rang-

ing from 1 to 1000 lg/ml. Methylcellulose was added to

increase the medium viscosity thus avoiding significant sedi-

mentation of the nanoparticles. After 72 h the medium was

aspired from the wells, the cells were washed twice with

200 ll of PBS and incubated with 100 ll of fresh medium.

WST-1 reagent was added to the cells (10 ll per well) and color

development was followed for up to 60 min. Absorbance was

measured in an ELISA reader at 450 and 620 nm (the later as

reference). Measurements were performed in quadruplicate

and normalized to control (untreated) cells. A sigmoidal curve

was fitted to the data using commercial software (Origin,

Originlab, Northampton, USA) from which the half maximal

inhibitory concentration (IC50) values were calculated.

In vitro effects of hyperthermia treatments were evaluated

by measuring the cell ability to form clonogenic colonies. A

total of 20 · 103 cells were harvested and suspended in

300 ll of culture medium containing 1% methylcellulose and

FeNP–COOH or FeNP–CDDP at a concentration of 5 mg/ml.

Each of the obtained suspensions was divided into three sam-

ples. The control sample was kept at 37 �C for 30 min. The

other two samples were subjected to an alternating magnetic

field with H = 60 kA/m and f = 128 kHz in order to increase

their temperature to 43 or 48 �C. Once the treatment temper-

ature was attained the magnetic field was controlled in order

to keep the temperature stable during the treatment period

(30 min). After the treatment 2 ll of each sample (equivalent

to about 150 cells) was added to six well plates in triplicate

and the cells were cultured for 8 days to allow the formation

of colonies. After this period, cells were washed with PBS,

fixed and stained with Giemsa (Merck, Darmstadt, Germany).

Macroscopic colonies were counted and results were normal-

ized to control (untreated) cells. Statistical significance be-

tween control and treated cells was assessed with a two

tailed paired Student’s t-test.

Fig. 3 – Temperature dependent drug release from FeNP–

CDDP. Freshly prepared nanoparticles were suspended in

saline and immediately incubated at 4, 37, 43 or 50 �C for

30 min. After this period, nanoparticles were concentrated

at the bottom of the tube with a magnet and the supernatant

was taken for cisplatin measurement. The graph shows the

release of CDDP ± SD from three independent experiments.

3. Results and discussion

3.1. Loading and release of cisplatin

In this work, FeNPs are explored as multifunctional agents

that can be applied simultaneously as a drug carrier and as a

heating agent for magnetic hyperthermia. The drug loading

is based on the complexation of CDDP with carboxylic groups

on the surface of the particles. In order to obtain –COOH func-

tionalized particles (FeNP–COOH), FeNPs were treated with ni-

tric acid [12]. As a control condition to show that the density of

carboxylic groups affect the amount of drug loaded, part of the

FeNP–COOH was conjugated with methylamine. The resulting

nanoparticles (FeNP–MA) have less free carboxylic groups

(Fig. 1) and thus, a smaller loading of CDDP is expected.

After the drug was loaded, its release from the nanoparti-

cles was studied in water and saline solutions. Fig. 2 shows

the cumulative release of CDDP at different time points up

to 26 days. It is observed that for both types of functionaliza-

tion, the release of CDDP is only expressive in saline solu-

tions. These results are expected considering that the

chloride ions can replace the hydroxyl groups on the aquated

drug bringing it to its original form. Similar results have been

seen for polymer complexation, where CDDP release was

higher in saline [14]. Furthermore, of the total drug released

during the 26 days, nearly 50% is released in the first 24 h,

whereas the remaining is slowly released in the following

days. The total amount of drug released from the FeNP–CDDP

was of 12.8 lg/mg whereas for FeNP–MA–CDDP it was of

5.8 lg/mg. Taken together, these results evidence that com-

plexation with carboxylic groups is likely to be the main

mechanism for drug loading, rather than simply adsorption.

The confirmation that FeNP–MA also contained CDDP in sig-

nificant amounts also opens the possibility for multifunction-

alized nanoparticles where instead of methylamine,

biological molecules of relevance could be attached. Those

could include for example, targeting antibodies or molecules

that might improve its biocompatibility.

When considering its use for hyperthermia, it is important

to note that such treatments often involve high concentra-

tions of nanoparticles. In clinical trials involving iron oxides,

for example, suspensions with concentrations above 100 mg/

ml of nanoparticles are applied [4]. If similar concentrations

would be applied here, drug concentrations on the order of

1280 or 580 lg/ml could be reached for the FeNP–CDDP or

FeNP–MA–CDDP respectively.

Given its potential application for hyperthermia, we also

investigated if temperature influences the release of drug.

For those studies, only the nanoparticles carrying the highest

C A R B O N 4 8 ( 2 0 1 0 ) 2 3 2 7 – 2 3 3 4 2331

amount of drug (FeNP–CDDP) were studied. Freshly prepared

FeNP–CDDP was incubated for 30 min at four different tem-

peratures and the amount of drug release was measured. This

time period was chosen in view on clinical hyperthermia

treatments which do not usually exceed the limit of 30–

60 min. The results imply a temperature dependent release

(Fig. 3). At 4 �C, less than 0.2 lg/mg of cisplatin was released.

At body temperature (37 �C), 0.84 lg/mg was released and this

value was nearly doubled (1.43 lg/mg) when hyperthermia

temperatures were applied (43 �C). The amount of drug re-

leased reached 2.20 lg/mg at thermoablative conditions

(50 �C). Those results are of interest in light of potential hyper-

thermia applications, allowing an increased drug concentra-

tion concomitant with hyperthermia temperatures. A

synergistic effect has already been shown when high temper-

ature and cisplatin are applied together in vitro, including the

possibility to overcome mechanisms of cisplatin resistance in

cells [16,19].

3.2. Cytotoxicity of the nanoparticles

In order to be effective in an anti-cancer therapy, the drug in

question must be still active after the loading and release pro-

cesses. For that purpose, the cytotoxicity of FeNP–COOH and

FeNP–CDDP was measured against the prostate cancer cell

line DU-145. Fig. 4 shows the decay in metabolic activity after

72 h incubation with different nanoparticle concentrations

ranging from 1 to 1000 lg/ml. In comparison to the FeNP–

COOH, the curve for the FeNP–CDDP is clearly shifted to lower

concentrations, indicating an increase in cytotoxicity. The

calculated IC50 values amount to 359 and 86 lg/ml for the

FeNP–COOH and FeNP–CDDP respectively, implying a fourfold

decrease for the cisplatin-loaded nanoparticles. At the IC50

value of 86 lg/ml, a drug concentration of 0.75 lg/ml would

be expected after 72 h as calculated from the unloading

curves (Fig. 2). This is in good agreement the reported IC50 val-

ues of cisplatin alone for this cell line, which amounts to

0.5 lg/ml after 72 h treatment [20]. Cisplatin acts by crosslink-

Fig. 4 – Metabolic activity of DU-145 cells after incubation with Fe

incubated with the nanoparticles for 72 h, washed with PBS and

shown are the mean of measurements performed in quadrupli

standard deviation.

ing DNA strands, interfering with mitosis and inducing apop-

tosis. The crosslinking process occurs in a similar manner to

the process used to load the nanoparticles. Upon entering the

cell, the lower chloride content of the cytoplasm leads to

aquation of the molecule that in turn, can interact with the

DNA bases generating the crosslinks [21]. Our results show

that after being released, the drug is still active being able

to undergo these processes and induce cell death.

Cells incubated with FeNP–COOH displayed a significant

decay in metabolic activity at the highest concentrations only,

being most prominent when the nanoparticle concentration

exceeds 200 lg/ml. At those extreme concentrations however,

part of the material sediments and accumulates over the cells

and the surface of the wells, what could inhibit an effective

cell proliferation during the 72 h period. We thus believe that

those results arise mainly as a consequence of particle sedi-

mentation and not as a result of intrinsic toxicity of the

nanoparticles.

3.3. Magnetic properties and SAR

The application of these nanoparticles for hyperthermia pur-

poses relies on their magnetic properties. Fig. 5 shows the

hysteresis loops of the raw material (FeNP) and that of

FeNP–COOH. The acidic treatments result in a decay of their

magnetic saturation (Ms), from 150 to 100 emu/g whereas

the coercivity does not change. We have previously shown

that the decay in Ms is mainly by loss of iron during the acidic

treatments and not by its oxidation [12]. This is relevant for

potential hyperthermia applications since the heating of the

nanoparticles under an AC magnetic field relies among other

properties, on its magnetic saturation. Although the decay is

significant, the magnetic saturation is still superior to the the-

oretical saturation magnetization of the commonly used iron

oxides (90 emu/g for Fe3O4 and 80 emu/g for Fe2O3) [22]. The

critical feature for the application of those nanoparticles,

however, is the amount of heat that is generated upon expo-

sure to an AC magnetic field. For that purpose, the SAR was

NP–COOH or FeNP–CDDP. Cells were seeded in 96-well plates,

incubated with the WST-1 reagent for up to 60 min. Results

cate and normalized to untreated cells. Error bars represent

Fig. 5 – Hysteresis loop of the raw FeNP and FeNP–COOH powder at room temperature.

2332 C A R B O N 4 8 ( 2 0 1 0 ) 2 3 2 7 – 2 3 3 4

estimated for a solution with a concentration of 5 mg/ml. A

quadratic dependence of SAR on magnetic field strength is

observed (Fig. 6) and a SAR of up to 240 W/gFeNP–COOH is ob-

served at the highest magnetic fields confirming their feasi-

bility for hyperthermia.

3.4. Combination of chemotherapy and hyperthermiain vitro

The in vitro efficacy of hyperthermia using FeNP–COOH or a

combination of hyperthermia and chemotherapy using

FeNP–CDDP was studied by exposing DU-145 cells to either

type of nanoparticles for 30 min at different temperatures

Fig. 6 – SAR of FeNP–COOH as function of the magnetic field

strength. Values were measured from a water suspension

with a nanoparticle concentration of 5 mg/ml. Inset:

temperature evolution at 40 kA/m as an example of a typical

heating curve from which the SAR value is calculated.

(37, 43 or 48 �C). Temperatures above body temperature were

reached by AC magnetic heating. The measured temperatures

during treatment were of 42.5 ± 0.3 �C and 47.5 ± 0.4 �C for

hyperthermia and thermoablation conditions, respectively.

Fig. 7 shows the colony formation by 150 cells seeded after

each of the treatments. Cells treated with the FeNP–COOH at

37 �C showed 29% decay in the number of colonies which is

not statistically significant. At the hyperthermia tempera-

tures (�43 �C), a significant reduction in the number of colo-

nies formed is observed, which is down to 33% in relation to

untreated cells. A complete induction of cell death at those

conditions is not expected for two reasons: at first because

hyperthermia was induced in a single session of 30 min only.

Secondly, it is recognized that temperature sensitivity of tu-

mors is not an intrinsic property of the tumor cells them-

selves but a condition which is related to the tumor

physiology. In malign tissues, conditions of hypoxia and low

pH are present; and this harsh microenvironment makes

the cells more sensitive to temperature [23]. In the in vitro

conditions that were applied here, however, cells are evenly

exposed to a balanced pH and oxygen tension and therefore,

are unlikely to show the same temperature sensitivity as an

in vivo tumor tissue. A temperature of 43 �C is thus not en-

ough to have a complete cytotoxic effect under the conditions

that were applied. At the thermoablative temperatures

(�48 �C), however, death by necrosis is expected, what is re-

flected in our results in which no cells survived to form

colonies.

Cells exposed to the drug loaded nanoparticles (FeNP–

CDDP) did not form any colonies in any of the treatment

groups. Our studies on the temperature dependence on re-

lease have shown that 30 min at 37 �C is enough to have a re-

lease of about 0.85 lgCDDP/mgnanoparticle (cf. Fig. 3). That would

mean that a 5 mg/ml FeNP–CDDP solution would lead to cis-

platin concentrations higher than 4 lg/ml at this tempera-

ture. This value is eightfold above the IC50 observed for 72 h

exposure. Although the exposure in this experiment was rel-

atively short (30 min), the results clearly show that at this

Fig. 7 – Colony formation by cells that underwent different thermotherapy treatments with FeNP–COOH or FeNP–CDDP.

Results are normalized to control (untreated) conditions. A total of 20,000 cells were treated for 30 min with a 5 mg/ml

solution of nanoparticles suspended in culture medium. Heating was achieved under an AC magnetic field with H = 60 kA/m

at f = 128 kHz. After the treatment,150 cells were plated in six well plates in triplicate. Following 8 days of culture the colonies

were stained and counted. Error bars represent standard deviation. Asterisk denotes statistical difference in relation to the

control group (p < 0.05).

C A R B O N 4 8 ( 2 0 1 0 ) 2 3 2 7 – 2 3 3 4 2333

high drug concentrations death is induced in all cells. Even

though the amount of drug released in these in vitro condi-

tions was enough to kill all cells at 37 �C the situation in an

in vivo setting would be certainly different. Depending on

the case, cisplatin dosage for systemic treatments in vivo

can be as high as 34–200 mg per day for a patient of average

height and weight. Besides that, cells are not evenly exposed

to the drugs as in in vitro conditions, making the treatment

less effective. Those considerations show that the typical

drug load achieved in this study probably would not be com-

pletely effective in vivo at 37 �C but would certainly benefit a

bi-modal treatment in combination with hyperthermia. In

vivo studies are however necessary to confirm this

hypothesis.

It is important to bring into discussion that the magnetic

fields applied in this study (60 kA/m) would probably be too

high for application in humans. Limiting fields appear to be

on the order of 15 kA/m in humans in order to avoid local dis-

comfort [24]. In light of our results, however, it seems that if

concentrations similar to those which are clinically applied

(100 mg/ml) would be applied, satisfactory heating could be

achieved at lower fields. The fields and concentrations used

in this study were adjusted for the model being used (cells

in vitro) rather than tissues or animals.

4. Conclusions

The use of carbon-encapsulated nanoparticles makes the

application of metallic iron as an agent for magnetic fluid

hyperthermia feasible. We have shown that the surface func-

tionalization with carboxylic functionalities allows their load-

ing with the drug cisplatin which is released in saline

solutions. Our in vitro results have shown that these particles

can be successfully applied as multimodal anti-cancer agents

being suitable for a concomitant therapy based on chemo-

therapy and hyperthermia. The use of these structures can

be of special interest when applied for drug resistant cells,

for example, where the simultaneous thermotherapy might

render them drug sensitive. Further experimental work is

necessary using tissue and animal models in order to estab-

lish the suitability of these structures for the proposed

applications.

Acknowledgements

Research was supported by the European Community

through the Marie Curie Research Training Network CARBIO

under Contract MRTN-CT-2006-035616.

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