PAPER www.rsc.org/materials | Journal of Materials Chemistry

Carboxylic acid-stabilised iron oxide nanoparticles for use in magnetichyperthermia†

Luanne A. Thomas,a Linda Dekker,b Mathew Kallumadil,c Paul Southern,c Michael Wilson,b Sean P. Nair,b

Quentin A. Pankhurstc and Ivan P. Parkin*a

Received 23rd April 2009, Accepted 15th June 2009

First published as an Advance Article on the web 15th July 2009

DOI: 10.1039/b908187a

Iron oxide nanoparticles were made in the presence of three carboxylic acid functionalised organic

ligands (tiopronin, oxamic acid and succinic acid) using a co-precipitation method. The iron oxide was

a mixture of magnetite and maghemite with an average crystallite size less than 10 nm. The samples

were all dialysed prior to analysis to ensure high purity. Without the presence of a carboxylic acid, the

dialysis purification stage invoked complete precipitation and the sample was completely intractable.

The carboxylic acid stabilised particles could be dissolved in water to form a stable solution. The

samples prepared with tiopronin and succinic acid were close to neutral pH and were suitable for

magnetic fluid hyperthermia testing on Staphyloccocus aureus. Iron oxide produced with tiopronin was

able to achieve a 107-fold reduction in the viable count of the organism using a 2 � 2 minute exposure

to an AC magnetic field and this bactericidal effect could still be achieved using the same batch of

particles one week later. Oxidation of the samples did occur with aging or sonication and made the

heating response less effective after one month. The tiopronin stabilised nanoparticles were able to

achieve substantial kills of bacteria at concentrations between 6.25–50 mg/ml. This is, to our

knowledge, the first time magnetic hyperthermia has been used to kill bacteria. The heating rates

obtained from using an external magnetic alternating field on the iron oxide nanoparticle solutions

were four times greater than the best commercially available material. This novel method of killing

bacteria could form the basis of a new approach to the treatment of a variety of infectious diseases.

Introduction

The use of iron oxide in medicine is not new, as magnetite

(lodestone) was recorded by Hippocrates to be prescribed to

control bleeding and haemorrhage and treat arthritis and gout as

early as 460BC. In the 10th Century Avicenna used iron oxide

powder as an antidote to the swallowing of poisonous rust.1 In

contemporary times magnetic iron oxide particles are in devel-

opment for, or currently used for, many applications such as

magnetic separation, drug delivery, MRI contrast enhancement

and magnetic hyperthermia.2 We have an ongoing interest in the

formation of nanoparticles and the synthesis of complex oxide

materials by unconventional routes.

By exploitation of the magnetic properties of the nanosized

iron oxide it has been shown that magnetic hyperthermia can be

used for the destruction of tumour cells.3–5 When a magnetic

particle is placed into an AC magnetic field the magnetic

moments align in accordance to the field (B). The energy received

from the AC magnetic field is that which is within the boundaries

of the hysteresis loop and whilst subjected to this field the

aMaterials Chemistry Research Centre, Department of Chemistry,University College London, 20 Gordon Street, London, WC1H OAJ, UKbDivision of Microbial Diseases, UCL Eastman Dental Institute, UniversityCollege London, 256 Gray’s Inn Road, London, WC1X 8LD, UKcDavy Faraday Research Laboratory, The Royal Institution, 12 AlbermarleStreet, London

† Electronic supplementary information (ESI) available: Further XRD,FT-IR and Raman spectra. See DOI: 10.1039/b908187a

This journal is ª The Royal Society of Chemistry 2009

material resistance causes it to give out thermal energy—induc-

tive heating. This has potential therapeutic use as magnetic fields

can penetrate the body and heat only in the vicinity of a magnetic

nanoparticle. The premise being that the iron oxide nanoparticles

could be surface-functionalised by cancer-specific binding

agents. These agents could be used in the body to surround

a tumour site, the application of the external field then heats the

nanoparticles and through thermal shock destroys the cells in the

vicinity of the tumour.6

The thermal energy dissipated from iron oxide nanoparticles

can be used to enhance/facilitate existing therapies such as

chemotherapy or radiotherapy. This is achieved due to increased

susceptibility of carcinogenic cells in temperature environments

of 42–45 �C, ultimately leading to apoptotic reactions. Hyper-

thermic treatment at 46 �C and above can cause necrosis in

tumour tissue, and theoretically could be used as a stand-alone

treatment method.7

Iron oxide nanoparticles have been made by a co-precipitation

method in the presence of a surfactant, often tetramethy-

lammonium hydroxide (TMAOH), to prevent aggregation and

precipitation. It is thought that the hydroxide anion is attached

to the surface of the nanoparticle and the [N(CH3)4]+ cation

preserves charge balance. This cationic atmosphere ensures

repulsion between other particulate environments.8 The problem

with this is that it creates a pH which is not desirable for bio-

logical applications. Reducing the pH to 7 by washing lowers this

ionic environment and causes the particles to coagulate and

precipitate.

J. Mater. Chem., 2009, 19, 6529–6535 | 6529

The coatings used to stabilise the iron oxide nanoparticles for

use in magnetic hyperthermia in humans must also aid the

circulation lifetime inside the body. This is done by increasing the

resistance of the nanoparticles to opsonization; opsonized

particles are easily identified by Kupffer cells in the liver and thus

are removed from the body. The proteins which adhere to foreign

bodies bind much quicker and easier to hydrophobic particles.9

Many coatings of iron oxide nanoparticles have been made using

organic molecules such as carboxylates, siloxanes, sulfates,

phosphonates and phosphates.10,11 The purpose of this was to

find a coating that is hydrophilic to encourage a stable colloidal

suspension in a biocompatible medium such as water. In the case

of carboxylic acids it is believed that the acid reacts with the iron

around the surface of the nanoparticle and is chemisorbed onto

the surface. A variety of carboxylic acid coatings have been

produced including oleic acid, folic acid and lauric acid.12–14

Other common coatings for iron oxide nanoparticles are

hydrophilic polymers, particularly dextran, polyethylene glycol

(PEG) and polyethylene oxide (PEO). These coatings promote

stability in aqueous media and lend themselves to be able to

associate further molecules.15

In this paper we present the first data that show the total

thermoblation of the bacterium Staphylococcus aureus using

superparamagnetic iron oxide formed in the presence of N-(2-

mercaptopropionyl)glycine (tiopronin). We also investigate and

characterise the iron oxide nanoparticles synthesised in the

presence of aminooxoacetic acid (oxamic acid) and butanedioic

acid (succinic acid). We show that the iron oxide nanoparticles

made with tiopronin as the ligand show significantly superior

heating characteristics to commercially available samples. To our

knowledge, this is the first reported application of magnetic

hyperthermia to kill bacteria. Such a process could find appli-

cation in the treatment of a range of infectious diseases.

Experimental

Materials

All chemicals were purchased from Sigma Aldrich and were used

as received with no further purification; iron(III) chloride

hexahydrate (99%), iron(II) chloride tetrahydrate ($99%), tet-

ramethylammonium hydroxide (25% in water w/w) (TMAOH),

N-(2-mercaptopropionyl)glycine (tiopronin) ($98%), amino-

oxoacetic acid (oxamic acid) ($98%) and butanedioic acid

(succinic acid) (99+%). Resovist was received from Bayer

Schering and Nanomag D100 was kindly donated by Micromod.

All water used was distilled.

Preparation of iron oxide nanoparticles

A 2:1:1 ratio of iron(III): iron(II): carboxylic acid functionalised

ligand was chosen. 2.703 g (10 mmol) of FeCl3.6H2O and either

0.816 g, 0.445 g or 0.590 g (5 mmol) of either tiopronin, oxamic

acid or succinic acid respectively was placed into a round

bottomed flask under nitrogen and dissolved in 10 ml of degassed

water. To this was added 2.5 ml from a stock solution of 3.987 g

of FeCl2.4H2O (5 mmol) in 10 ml of degassed water. The mixture

was mechanically stirred and heated to 70 �C. 21 ml of tetra-

methylammonium hydroxide (25% in water w/w) was added

dropwise to the stirred solution over a 5 min period. The solution

6530 | J. Mater. Chem., 2009, 19, 6529–6535

was then allowed to cool to room temperature with stirring. The

solution was made up to 50 ml with distilled water and trans-

ferred to a wet 20 cm strip of dialysis tubing (cellulose membrane,

M.W. 12 400, average flat width 76 mm) that was knotted and

clipped at each end. This was dialysed for 4 d in 5 L of 15 MU

distilled water (changed daily) with constant slow stirring. The

solution of nanoparticles was collected from the dialysis bag and

by magnetic separation the majority of solvent was removed.

From this the sample was placed in a �80 �C freezer, frozen and

placed under high vacuum to remove the last of the solvent

(freeze dried). This required 2–3 d for it to form a completely dry

fine black powder.

The powder was dissolved by taking 0.5 g of the freeze-dried

product and sonicating in 10 ml of deionised water. This gave

a pH of 6.96, 3.95 and 6.65 for the samples of iron oxide made in

the presence of tiopronin (T), oxamic acid (O) and succinic

acid (S).

Characterisation of iron oxide nanoparticles

Analysis was carried out using X-ray diffraction (XRD) on

a Bruker DX8 using Cu Ka radiation; Raman using a Renishaw

inVia Raman Microscope with a laser wavelength of 785 nm and

power of 1%; infrared (IR) spectroscopy using a Perkin Elmer

FT-IR Spectrometer Spectrum RX1; thermogravimmetric anal-

ysis (TGA) using a Netzsh STA 449C instrument with helium

gas; magnetometry on a quantum design magnetic property

measurement system (MPMS)-5T and photon correlation spec-

troscopy (PCS) on a Zetasizer 3000 (Malvern, UK) was used to

measure the hydrodynamic diameter of the nanoparticles in

a plastic cuvette in distilled water at 25 �C. Some preliminary

TEM measurements were attempted and on the images taken

averaged ca 9.0 nm particle size (sample T), however despite

being firmly attaching to a resin, some of the particles were

sucked up into the magnetic lens and further characterisation

was not attempted.

Measurement of heat induction and microbial testing

Bacterial strains. The organism used in this investigation was

Staphylococcus aureus NCTC 6571. It was maintained as a plate

culture and subcultured weekly from freezer stocks on Nutrient

Agar (Oxoid Ltd, Basingstoke, UK). For experimental purposes,

a single colony was inoculated into Nutrient Broth No. 2 (Oxoid)

and grown aerobically for 16 h at 37 �C with shaking. Cells were

harvested by centrifugation and resuspended in sterile phosphate

buffered saline (PBS) to an OD600 nm of 0.05. In some experi-

ments, the adjusted bacterial suspensions were further diluted

1 in 10 and 1 in 100 in PBS.

Hyperthermia system. The magnetic AC hyperthermia

(MACH) system (patent pending) was powered by 150 V at ca.

0.8 A. This in-house built system encompasses a 2 cm

water-cooled coil with 6 windings and provided a field of

around 12 kA/m at a frequency of 1.05 MHz.

Magnetic nanoparticles. The magnetic nanoparticles used were

codified as T (iron oxide formed in the presence of tiopronin) and

S (iron oxide formed in the presence of succinic acid). Dilutions

This journal is ª The Royal Society of Chemistry 2009

Table 1 The three controls that were used in the experiments conductedto test the hyperthermic response and bacterial kill of iron oxide nano-particles formed in the presence of tiopronin or succinic acid, usinga co-precipitation method at 70 �C. Ticks are used to indicate the use ofa particular control, reactions were conducted in PBS (phosphatebuffered saline)

Control PBS

Nanoparticlesand bacterialsuspension

Exposure toMACH system

1 3 32 33 3

were made in PBS to obtain the desired concentration. Sample O

(oxamic acid) was not used due to the low pH that formed on

dissolution.

Bacterial magnetic hyperthermia. Aliquots (200 ml) of

a suspension of S. aureus in sterile PBS (approximately 107–108

colony forming units), were placed into Eppendorf tubes. To

each tube, 200 ml of the magnetic nanoparticle solution (made as

above) was added. Samples were vortexed and then exposed to

the hyperthermia heater for the desired length of time. A

constant air flow covered the sample chamber in the MACH

system to provide a 37 �C stable environment. The temperature

of each tube was recorded using a Luxtron Fibre Optic

Temperature measurement system (FOT Lab Kit, LumaSense�Technologies, CA, USA). Three controls were in place, these are

shown in Table 1. A further control was run whereby bacteria

were added after the particles had been exposed for 2 min to the

MACH.

Following exposure in the machine, 25 ml aliquots of the test

and control samples were taken and added to 225 ml of PBS.

Serial dilutions were made, and 20 ml aliquots (in duplicate) from

each dilution were plated on to nutrient agar, and grown over-

night at 37 �C to enumerate survivors.

Results

Preparation

All samples were synthesised using a co-precipitation technique

at 70 �C, whereby ferrous and ferric chloride salts were dissolved

alongside a carboxylic acid ligand; tiopronin, oxamic acid, or

succinic acid, in water and then reduced using excess tetrame-

thylammonium hydroxide. The products were purified by dial-

ysis and isolated as black powders by freeze-drying and labelled

as T, O or S (depending on the carboxylic acid used). The

resulting powder was readily soluble in water and PBS and

formed a strong ferrofluid that could be suspended upside down

by a magnet (see graphical abstract).

Fig. 1 TGA graphs and elemental analysis of 3 samples T, O and S; iron

oxide nanoparticles formed in the presence of tiopronin, oxamic acid

and succinic acid respectively, using a co-precipitation method at 70 �C.

There is no obvious correlation between the mass loss and the elemental

analysis.

Characterisation

X-Ray diffraction, Raman and FT-IR spectroscopy all

confirmed the samples were a magnetite/maghemite mix (see

ESI†). FT-IR and elemental analysis gave tentative evidence for

the presence of the carboxylic acids. The IR gave some weak

bands and the CHN combustion analysis showed the expected

This journal is ª The Royal Society of Chemistry 2009

ratios for the acids but the ligand was present at the 1–2 atom%

level. These facts indicate that the vast majority of the sample

was iron oxide.

TGA data in an inert atmosphere was obtained on each sample

to try and correlate the elemental analysis values but there was no

clear match (Fig. 1). However, there were mass losses occurring

at temperatures expected for organic material. In all three cases

there was a final mass loss around 460–480 �C that is likely to be

the irreversible phase transition from maghemite to hematite in

which oxygen is evolved.

The crystallite size of the nanoparticles was determined from

XRD by measuring the peak broadening and then applying the

Scherrer equation with a Gaussian fit. Measurements were

correlated to a LaB6 standard. The average crystallite was

calculated to be 9.4 nm, 7.4 nm and 7.9 nm for samples T, S and

O respectively.

The average hydrodynamic particle size of the three samples

when dissolved in distilled water (pH 7) was measured using

a zeta-sizer. This gave an average particle diameter of 135.2 nm

for T with a polydispersity index of 0.265. The hydrodynamic

J. Mater. Chem., 2009, 19, 6529–6535 | 6531

size measurements for the O and S gave larger results, with the

measurement for O being extremely polydisperse.

Fig. 2 shows the SQUID magnetometry data for the samples.

In all cases the samples were shown to retain little net magnet-

isation away from an external field which is desirable for

biomedical applications. T gave the greatest saturation magnet-

isation at 300 K. Fig. 3 displays the zero field cooling, and

field cooling curves for T, O and S.

Fig. 2 Hysteresis loops taken at 300 K and 10 K for 3 samples T, O and

S; iron oxide nanoparticles formed in the presence of tiopronin, oxamic

acid and succinic acid respectively, using a co-precipitation method at

70 �C. (A) The full hysteresis loop at 300K, (B) the full hysteresis loop at

10 K. The inserts provide more detail around the origin for clarity.

Fig. 3 Zero field cooling (ZFC) and field cooling (FC) curves for

3 samples T, O and S; iron oxide nanoparticles formed in the presence of

tiopronin, oxamic acid and succinic acid respectively, using a co-precip-

itation method at 70 �C.

6532 | J. Mater. Chem., 2009, 19, 6529–6535

Heating experiments

Samples T and S were both shown to be of a suitable pH for

biomedical applications. The samples were subjected to testing in

the MACH system which applies an alternating current magnetic

field to induce a heating response at a concentration of 50 mg/ml,

Fig. 4.

Sample T produced an outstanding heating curve. A

comparison of the heating characteristics of sample T with

a range of the best commercially available samples for magnetic

hyperthermia was conducted against Bayer Schering’s Resovist

and Micromod’s Nanomag D100 nm (both samples being

dextran stabilised) at equivalent iron concentrations which was

calculated using SQUID measurements. Table 2 shows these

results, with T proving to be over 4 times a more effective heater

by comparison of the initial heating rate. The comparisons were

achieved by calculating the specific absorption rate (SAR) and

the intrinsic loss power (ILP). The ILP is a parameter that can be

calculated from eqn (1).16

ILP ¼ SAR/(frequency � magnetic field2) (1)

Antibacterial evaluation

The antibacterial properties of a freshly prepared sample of

T against suspensions of S. aureus were determined using the

MACH system. In all of the controls, no kills of bacteria were

seen—killing was only obtained when iron oxide nanoparticles

Fig. 4 Heating curves for 50 mg/ml T and S (iron oxide nanoparticles

formed in the presence of tiopronin and succinic acid respectively using

a co-precipitation method at 70 �C) when placed in an AC magnetic field

on the MACH system (patented).

Table 2 Comparison of heating parameters of T (iron oxide nano-particles formed in the presence of tiopronin using a co-precipitationmethod at 70 �C) with Bayer Schering’s Resovist and Micromod’sNanomag 100 nm when placed in an AC magnetic field on an in-housesystem named MACH (patent pending). Specific absorption rate (SAR)and intrinsic loss power (ILP) are compared for all 3 samples. ILP isa parameter for the comparison of samples devised by Pankhurst,Southern and Kallumadil. ILP ¼ SAR/(frequency � magnetic field2)16

SAR (W/g) ILP (Hm2/kg)

T 1179 6.1 � 10�9

Resovist 279 1.5 � 10�9

Nanomag 100nm 263 1.4 � 10�9

This journal is ª The Royal Society of Chemistry 2009

Fig. 7 Graph displaying the viable counts (cfu/ml) of Staphylococcus

were present and only if these samples were exposed to the

magnetic field in the MACH system. The nanoparticles did not

kill the bacteria in the absence of a magnetic field, indicating that

they had no intrinsic antibacterial activity. The initial testing

took place 1 day after the particles had been prepared, the

concentration was 50 mg/ml and a total kill of over 107 cfu/ml

was seen. The bath heated to 100 �C in less than 30 s. Fig. 5

displays the results of testing six days after the initial test. The

experiment demonstrates the different kill rates achieved by the

50 mg/ml solution of T when subjected to the MACH for 2 min,

1 min, 3 � 20 s and finally 6 � 10 s. The results show a total kill

after a 2 min constant exposure, with reduced kills following

a reduced exposure time of 60 s regardless of whether this was

a single 60 s exposure or in shorter pulses. Fig. 6 shows the kills

achieved at various concentrations one week after the first test.

This again shows a kill of 100% when a concentration of

Fig. 5 Graph displaying the viable counts (cfu/ml) of Staphylococcus

aureus when exposed to the MACH for varying times and with 50 mg/ml

of T (block grey) and when not exposed to the MACH (block black); iron

oxide nanoparticles formed in the presence of tiopronin, using a

co-precipitation method at 70 �C, when placed in an alternating current

magnetic field 6 days after first test of the ferrofluid. Control samples

contained PBS in place of T and were either exposed (dotted light grey),

or not exposed (dotted dark grey) to MACH.

Fig. 6 Graph displaying the viable counts (cfu/ml) of Staphylococcus

aureus using varying concentrations of sample T; iron oxide nano-

particles formed in the presence of tiopronin, using a co-precipitation

method at 70 �C, when placed in an alternating current magnetic field for

2 min (block grey) 1 week after first test of the ferrofluid, or not placed in

an alternating current magnetic field (block black). Control samples

contained PBS in place of T, and were either exposed (dotted light grey)

or not exposed (dotted dark grey) to MACH. Indicated above the bar

graph is the temperature that the water bath heated to in �C.

aureus using sample T; iron oxide nanoparticles formed in the presence of

tiopronin, using a co-precipitation method at 70 �C, when placed in an

alternating current magnetic field for 2 min (block grey), 19 days after

first test of the ferrofluid, or not placed in an alternating current magnetic

field (block black). This is tested at two concentrations; 50 and 25 mg/ml.

The concentration of bacteria in the suspensions was also varied: undi-

luted suspension (panel A), diluted 1:10 in PBS (panel B) and diluted

1:100 (panel C). Control samples were placed in PBS in place of T, and

were either exposed (dotted light grey) or not exposed to MACH (dotted

dark grey).

This journal is ª The Royal Society of Chemistry 2009

50 mg/ml of T was used but reduced kills when T was present at

lower concentrations. Also displayed in Fig. 6 is the maximum

temperature the water bath heated to for each concentration.

Fig. 7 shows the results obtained using 50 and 25 mg/ml

concentrations 19 days after the initial antibacterial testing and

after sonication. The figure shows the kills obtained using

3 suspensions of S. aureus. The first is using an undiluted

suspension, the second uses a dilution of 1:10 and finally the third

uses a dilution of 1:100 S. aureus:PBS buffer. In all cases,

compared with the T-free control suspension, a 3 log10 reduction

in the viable count of S. aureus was achieved using a concentra-

tion of 50 mg/ml when the magnetic field was applied. Reduced

kills were obtained when the concentration of T was 25 mg/ml.

Discussion

Analysis of the iron oxide powder

The data collected for all three samples showed similar results.

FT-IR presented evidence for ligand coordination with nC–N and

nO–H prevalent on all spectra. nFe–O stretches were strongly

evident. In all cases the elemental analysis showed that very low

concentrations of the organic ligand were attached to the iron

oxide surface. Cases where TGA analysis was obtained did not

correspond to any significant mass losses. This information

indicates that the carboxylic acids added interfered with the

particle size and properties but there were minimal amounts

coordinated to the nanoparticle. It is important to note that the

pH values for all the samples varied when the powder was resus-

pended in deionised water, particularly, that of sample O which at

a pH of 3.95 was significantly lower than the other samples and

strongly indicated the presence of the acid. If there was any excess

of the base TMAOH a high pH would have been seen. Without the

J. Mater. Chem., 2009, 19, 6529–6535 | 6533

presence of the carboxylic acids, it was not possible to collect

isolated nanoparticles as following the dialysis purification the

particles were noted to have precipitated out of solution.

The pH changes are explained by the presence of free unat-

tached molecules of tiopronin, oxamic acid or succinic acid.

However, they are attached at low concentrations or mass% in

comparison to the total mass of the relatively heavy nanoparticle

then their fingerprint is difficult to observe by IR and Raman

because the iron oxide signatures dominate.

Hydrodynamic size measurements gave an average particle

diameter of 135.2 nm for T with a polydispersity index of 0.265.

The hydrodynamic size measurements for the O and S samples

gave larger results, with the measurement for O being extremely

polydisperse. These hydrodynamic measurements are signifi-

cantly larger than the crystallite sizes observed by XRD line-

broadening

M–H curves produced using the MPMS at 300 K and 10 K

show that all three samples (T, O and S) have little remnant

magnetisation at 300 K. The zero field cooling (ZFC) curves all

demonstrated a peak around the Verwey transition temperature.

The deviation from the literature value indicates that there has

been some oxidation of the Fe(II) to Fe(III), which is likely to

have occurred at the surface and not throughout the bulk. This

also implies that the particle size is small, thus having a large

surface area.17,18

Antibacterial evaluation

The samples T and S both responded to an AC magnetic field

producing an increase in temperature of the surroundings. The

strongest of these was T which was then compared to two

samples that are commercially available (Bayer Schering’s

Resovist and Micromod’s Nanomag 100 nm). It was found to be

over 4 times better a heater when placed inside the MACH

system, when comparing the specific absorption rate and the

intrinsic loss power. The presence of the tiopronin in solution

with the iron precursors limits the growth time after nucleation,

promoting smaller particles that have high stability, thus low

aggregation at neutral pH. This promotion of optimum sized

particles leads to the superparamagnetism desired and effective

response to the AC magnetic field.

This preliminary data meant T was the ideal candidate for

further investigation as it was able to cause a significant heating

effect that was potentially able to denature cells. The detailed

experiments showed that at concentrations of 25 mg/ml and

50 mg/ml it is possible to achieve killing of S. aureus by several

orders of magnitude after a 2 min exposure in the MACH system.

There was a direct correlation between the concentration of the

ferrofluid and the kills achieved. The rate of killing being depen-

dent on the temperature achieved. The initial testing showed that

at a concentration of 50 mg/ml and above a total bacterial kill was

seen and the sample was readily heated to 100 �C. A concentration

of 50 mg/ml was still producing the same results more than one

week after initial testing when the nanoparticles were stored at

4 �C. At the lowest concentration tested (6.25 mg/ml), the

temperature reached was only 45.5 �C and consequently no

reduction in the viable count was achieved. S. aureus is a meso-

phile and is known to tolerate short exposure (10 min) to

a temperature of 50 �C. After 19 days and sonication the

6534 | J. Mater. Chem., 2009, 19, 6529–6535

antibacterial effectiveness of the iron oxide nanoparticles was

reduced but at 50 mg/ml they were still achieving more than a 3 log

kill. This suggests that aging does have an effect on the compo-

sition and time and sonication oxidise the iron oxide. Indeed, there

was a colour change from black to brown of the particles that is

indicative of this. For longevity it would be best to keep the

particles in an inert atmosphere until they needed to be used,

however it is important to note that they will not degrade instantly

and this makes them useful for the commercial and biological

purposes they are intended for. The exposure time to the MACH is

also an important parameter which affects the bacterial kill, when

using a 50 mg/ml concentration it was shown that total kill of

a suspension containing 107 cfu/ml of bacteria was achieved after

a two minute constant exposure. At one minute there was less kills.

When bacteria were added to nanoparticles that had previously

been exposed to a magnetic field in the MACH system, no killing

was detectable. This finding suggests that the bactericidal effect

observed in the other experiments is attributable to the heating

effect of the particles rather than the heat-induced liberation of

toxic moieties from the nanoparticles. Functionalisation of the

nanoparticles with bacteria-targeting moieties would enable the

achievement of a localised heating effect and consequent bacterial

killing without an accompanying temperature rise in host tissues.

Following such a development, this novel means of killing

bacteria could form the basis of a new approach to the treatment

of infectious diseases.

Conclusion

The synthesis of iron oxide nanoparticles in the presence of three

carboxylic acid functionalised ligands changes their solubility

properties and particle size. The particles which gave the best

heating response to an alternating AC magnetic field were those

which were formed with the ligand N-(2-mercaptopropionyl)-

glycine (tiopronin). This gave a stable solution of iron oxide

nanoparticles suspended in water at neutral pH. This nano-

particle solution was shown to have an excellent response to an

AC magnetic field at various concentrations. The heating ability

of this solution was over 4 times better than that of the best

commercial samples; Bayer Schering’s Resovist and Micromod’s

Nanomag 100 nm. There was a direct correlation between

the nanoparticle concentration and the kills of S. aureus. At

50 mg/ml a total kill of over 107 cfu of the bacteria was reported.

Total kills were also achieved after a week using 50 mg/ml. Log

magnitude bacterial kills were seen up to 19 days after initial

testing, although there was evidence that the solution did degrade

with time and sonication. The concentration of T and exposure

time and type of exposure (pulse or continuous) to the MACH

affected kill rates. We believe this to be the first time magnetic

hyperthermia has been used to kill bacteria. It is possible that this

approach may offer, following the development of a specific

targeting system, a novel approach for the treatment of a variety

of infectious diseases including systemic infections.

Acknowledgements

The authors would like to thank Ondine Biopharma, EPSRC

and the Royal Institution for financial support. IPP thanks the

Royal Society Wolfson Trust for a merit award.

This journal is ª The Royal Society of Chemistry 2009

References

1 R. V. Ramanujan, Proceedings of the First InternationalBioengineering Conference, Singapore, 2004, p. 174.

2 Q. A. Pankhurst, J. Connolly, S. K. Jones and J. Dobson, J. Phys. D:Appl. Phys., 2003, 36, R167.

3 F. K. Storm, H. W. Baker, E. F. Scanlon, H. P. Plenk,P. M. Meadows, S. C. Cohen, C. E. Olson, J. W. Thomson,J. D. Khandekar and D. Roe, Cancer, 1985, 55, 2677.

4 M. Shinkai, M. Yanase, M. Suzuki, H. Honda, T. Wakabayashi,J. Yoshida and T. Kobayashi, J. Magn. Magn. Mater., 1999, 194,176.

5 A. Jordan, R. Scholz, P. Wust, H. Schirra, T. Schiestel, H. Schmidtand R. Felix, J. Magn. Magn. Mater., 1999, 194, 185.

6 D. J. Dunlop, J. Geophys. Res., 1973, 78, 1780.7 R. Hergt, S. Dutz, R. Mueller and M. Zeisberger, J. Phys.: Condens.

Matter, 2006, 18, S2919.8 P. Berger, N. B. Adelman, K. J. Beckman, D. J. Campbell, A. B. Ellis

and G. C. Lisensky, J. Chem. Educ., 1999, 76, 943.9 S. S. Davis, Trends Biotechnol., 1997, 15, 217.

This journal is ª The Royal Society of Chemistry 2009

10 L. Burgos-Asperilla, M. Darder, P. Aranda, L. Vazquez, M. Vazquezand E. Ruiz-Hitzky, J. Mater. Chem., 2007, 17, 4233.

11 L. A. Harris, J. D. Goff, A. Y. Carmichael, J. S. Riffle, J. J. Harburn,T. G. St Pierre and M. Saunders, Chem. Mater., 2003, 15, 1367.

12 P. Pradhan, J. Giri, R. Banerjee, J. Bellare and D. Bahadur, J. Magn.Magn. Mater., 2007, 311, 282.

13 F. Sonvico, S. Mornet, S. Vasseur, C. Dubernet, D. Jaillard,J. Degrouard, J. Hoebeke, E. Duguet, P. Colombo andP. Couvreur, Bioconjugate Chem., 2005, 16, 1181.

14 K. Jain Tapan, A. Morales Marco, K. Sahoo Sanjeeb, L. Leslie-Pelecky Diandra and V. Labhasetwar, Mol. Pharmacol., 2005, 2, 194.

15 C. C. Berry and A. S. G. Curtis, J. Phys. D: Appl. Phys., 2003, 36,R198.

16 M. Kallumadil, M. Tada, T. Nakagawa, M. Abe, P. Southern andQ. A. Pankhurst, J. Magn. Magn. Mater., 2009, 321, 1509.

17 O. Ozdemir, D. J. Dunlop and B. M. Moskowitz, Geophys. Res. Lett.,1993, 20, 1671.

18 R. Prozorov, T. Prozorov, S. K. Mallapragada, B. Narasimhan,T. J. Williams and D. A. Bazylinski, Phys. Rev. B: Condens. MatterMater. Phys., 2007, 76, 054406/1.

J. Mater. Chem., 2009, 19, 6529–6535 | 6535