Magnetic properties and heating effect in bacterial magnetic nanoparticles

ARTICLE IN PRESS Journal of Magnetism and Magnetic Materials 321 (2009) 1521–1524

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Magnetic properties and heating effect in bacterial magnetic nanoparticles Milan Timko a,, Anezka Dzarova a, Jozef Kovac a, Andrzej Skumiel b, Arkadiusz Jo´zefczak b, Tomasz Hornowski b, Hubert Gojz˙ewski c,d, Vlasta Zavisova a, Martina Koneracka a, Adriana Sprincova a, Oliver Strbak a, Peter Kopcansky a, Natalia Tomasovicova a a

Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 040 01 Kosˇice, Slovakia ´ , Poland Institute of Acoustics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan c ´ University of Technology, Nieszawska 13A, 60-965 Poznan ´ , Poland Institute of Physics, Poznan d Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany b

a r t i c l e in fo

abstract

Available online 21 February 2009

A suspension of bacterial magnetosomes was investigated with respect to structural and magnetic properties and hyperthermic measurements. The mean particle diameter of about 35 nm was confirmed by transmission electron microscopy (TEM), X-ray and magnetic analysis. The X-ray powder diffraction peaks of magnetosomes fit very well with standard Fe3O4 reflections. The found value for specific absorption rate (SAR) of 171 W/g at 5 kA/m and 750 kHz means that magnetosomes may be considered as good materials for the biomedical applications in hyperthermia treatments. Moreover, they have biocompatible phospholipid membrane. & 2009 Elsevier B.V. All rights reserved.

Keywords: Magnetospirillum sp. strain AMB-1 Biomineralization Biological nanoparticle Magnetosome Magnetic characterization Hyperthermia SQUID magnetometer X-ray diffraction

Application of magnetic materials for hyperthermia treatment of biological tissues with the goal of tumor therapy has been known in principle for more than four decades [1]. The heating effect depends strongly on the magnetic properties of the particles, which may vary appreciably depending on their size and microstructure. The magnetite particles can be either ferromagnetic or superparamagnetic, and the magnetic moment of single-domain particle relaxes through either Brownian or Neel relaxation depending on the anisotropy and size of the particle [2,3]. It was shown by previous investigations that specific loss power (SLP) depends strongly on the mean particle size as well as the width of the size distribution [4–6]. The strong monotonous rise of SLP, nearly two orders of magnitude, from 15 up to 900 W/g with increasing particle core size from 7 up to 18.4 nm in magnetite, was described. This rise was clarified in the frame of the classical Debye theory of dispersions [5]. For the biocompatible magnetic iron oxides magnetite and maghemite, a maximum SLP was found above typical sizes of superparamagnetic particles but below the size of typical multidomain particles. On the other hand the SLP determined from hysteresis loops, susceptibility spectra and calorimetry with a maximum value of 960 W/g at 410 kHz and field amplitude of 10 kA/m was found for biological particles—magnetosomes with a mean size of about 35 nm [7]. However, the magnetic and structural properties of

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E-mail address: [email protected] (M. Timko). 0304-8853/$ - see front matter & 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jmmm.2009.02.077

magnetosomes have been insufficiently characterized up to now, mainly because of the unavailability of significant amounts of material. Magnetosomes, which can be obtained by a biomineralization process in magnetotactic bacteria (MTB), consist of magnetic mineral crystals magnetite or greigite [8] enveloped by a biological membrane that contains phospholipids and specific proteins [9]. The magnetosome membrane is not only critical for the control of crystal size and morphology, but also prevents the aggregation of extracted magnetosomes and thus stabilizes magnetosome suspensions. Magnetosome crystals of MTB are typically from 30 to about 140 nm in diameter, i.e. within the single-magnetic-domain size range, which maximizes the efficiency of the particle as a permanent magnetic carrier [10]. In many magnetotactic bacterial types, the magnetosome are characterized by narrow particle size distributions. However, whereas, the magnetite particles in magnetosome chain are usually oriented so that [111] crystallographic axis of each particle lies along the chain direction, the greigite particles in a magnetosome chain are usually oriented in [1 0 0] crystallographic axis. Whether the mineral particles are magnetite or greigite, the chain of magnetosome particles constitutes a permanent magnetic dipole fixed within the bacteria [11]. The remanent moment is generally close to its saturation value. Normally it is sufficiently larger than background thermal energy so that it, and consequently the bacteria, is oriented along geomagnetic field lines as it swims, causing the bacterium to migrate along the field lines.

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M. Timko et al. / Journal of Magnetism and Magnetic Materials 321 (2009) 1521–1524

Because of their unique characteristics, magnetosomes have a high potential for nano- and biotechnological applications, which require specially designed particle surface. Especially, in biotechnological applications, functionalized bacterial magnetosomes represent an attractive alternative to chemically synthesized iron oxide particles. Magnetosome particles have been applied in numerous applications that range from the extraction of magnetic resonance imaging (MRI), magnetic drug targeting, DNA and RNA to the highly sensitive detection and concentration of toxic substances and development of immunoassays [12]. One of the potential application areas of magnetosomes is magnetic particle hyperthermia (MPH) [13]. As was pointed out recently an enhancement of specific heating power is of importance for reducing useful dosage applied to the tumor. Previous investigations on the suitability of magnetic nanoparticles for MPH [5–7] have shown that for the biocompatible magnetic iron oxides a core size range above about 20 nm is advantageous with respect to large specific heating power. Therefore, magnetosomes are of particular interest for testing their suitability for application in MPH tumor therapy. In the present paper, the structural and magnetic properties of magnetosomes obtained by biomineralization process in magnetotactic bacteria Magnetospirillum sp. strain AMB-1 are reported. The heating effect of the magnetosome solution as a result of absorbing energy from the alternating magnetic field has shown that magnetosome suspensions are capable of delivering sufficient heating power which may be of interest for magnetic particle hyperthermia. Bacterial magnetosomes investigated in this contribution were synthesized by magnetotactic bacteria Magnetospirillum sp. strain AMB-1 in laboratory conditions. This bacteria is a Gram-negative a-proteobacterium that is more oxygen-tolerant and easier to grow on a large scale. The detailed description of cultivation of magnetotactic bacteria and isolation of magnetosomes is given in our previous contribution [14]. Techniques for the isolation and purification of magnetosome particles from Magnetospirillum sp. are based on combination of centrifugation and the magnetic separation. Typically, 2.6 mg bacterial magnetite could be acquired from a 1000-mL culture of Magnetospirillum sp. AMB-1. These isolation and purification procedures leave the surrounding membrane intact and magnetosome preparations are apparently free of contaminating materials. Owing to the presence of the enveloping membrane, isolated magnetosome particles form stable, well-dispersed suspensions in water solution of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Phase identification of the magnetic iron oxide of the bacterial magnetosomes was performed by X-ray diffraction (XRD), as described in detail in [14]. XRD powder diffraction peaks of magnetosomes well fit with standard Fe3O4 reflections. This fact reveals that the magnetic nanocrystal within the magnetosome consisted of magnetite with no other phases. The background noise and presence of broader peaks are characteristic of particles with nanometer dimension. Magnetosome size calculated from the line-broadening of 311 peak of XRD powder diffraction patterns using the Scherer’s equation was estimated to be as 37 nm. The examination by transmission electron microscopy (TEM) was done using a JEOL1200EX microscope normally operated at 120 kV and 80,000  magnification by the replication technique. For the interest, we have chosen very interesting electron micrograph of the magnetosomes shown in Fig. 1 which reveals the known fact that magnetosomes are in bacteria arranged in straight chains which after isolation have tendency to form closed loops and so to minimize their magnetic stray field energy. The reason for these phenomena is existence of lipid membrane surrounding magnetic core prevents them to stick together by electrostatic repulsion [15]. The mean size and

Fig. 1. Transmission electron micrograph of magnetosomes.

standard deviation estimated from TEM was 34 and 6 nm, respectively. The particle size obtained from XRD and TEM measurements for all used samples are consistent and very close. The magnetosomes size distribution of hydrodynamic diameter was studied by dynamic light scattering with a standard setup of ALV GmbH (Germany) consisting an ALV5000 multiple tau digital correlator. Experiment duration of 120 s was selected at room temperature. All measurements were performed using a 633 nm wavelength He–Ne laser with the goniometer set of 301, 601, 901, 1201 and 1501. Dynamic light-scattering measurements have been performed, since it is a perfect tool to describe a particle size distribution in solutions, and since magnetic measurements can treat only magnetic cores and atomic force microscopy measurements gives a pure statistics of particle size distribution. For the description of the dispersion of magnetite particles the lognormal distribution is usually used ! 2 1 ln ðr=r 0 Þ f ðrÞ ¼ pffiffiffiffiffiffi exp  , (1) 2 r b 2p 2b where r is the magnetic radius, and r0 and b are the parameters of the distribution function which can be extracted from the magnetization curve. On the basis of these parameters the mean magnetic radius /rS and standard deviation s of particle sizes can be determined from the expressions ! !qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi hri ¼ r 0 exp

b2 2

;

s ¼ r0 exp

b2 2

2

exp b  1.

(2)

The logarithmic-normal function parameters and mean values of hydrodynamic radius, and its standards deviations are shown in Table 1. Fig. 2 presents the scattered light intensity weighed particle size distribution of magnetosomes extremely dilutes in HEPES solvent measured under different laser beam angle perspective. The presented distribution is fitted to logarithmic-normal function, which is usually used for description of such media and it is defined as [16,17]. The results in Fig. 2 have shown the noticeable differences in hydrodynamic radius distribution for each laser beam perspective. This is due to broad distribution of sizediversified chains of magnetosomes covered by a phospholipid shell. The mean hydrodynamic radius value in respect to all angles can be estimated to be of about 240 nm. This briefly confirms, considering mean hydrodynamic radius, that the mean number of

ARTICLE IN PRESS M. Timko et al. / Journal of Magnetism and Magnetic Materials 321 (2009) 1521–1524

Table 1 Values and parameters calculated from logarithmic-normal function fitted to magnetosomes radius distribution.

2500 Am-1 2000 1750 1500 1250 1000 750 500

35 ro (nm)

b ()

/rS (nm)

s (nm)

30 60 90 120 150

271.0 200.5 180.7 189.1 160.5

0.580 0.581 0.636 0.678 0.584

320.6 237.4 221.2 238.0 190.3

202.8 150.4 156.2 181.8 121.3

199.7 147.3 124.8 125.5 119.5

(nm)

T [°C]

DLS angle (1)

rmaxa

30

1523

magnetosomes f = 750 kHz

H = 2500 A/m

2000 A/m 1750 A/m 1500 A/m

25 a Maximum hydrodynamic radius taken from fitted logarithmic-normal functions.

1250 A/m 1000 750 500

20 0

300

600

900

t [s] Intensity of distribution [a.u]

0.4

60° Fig. 3. Temperature increase produced by magnetosomes for different values of an alternating magnetic field.

90°

0.3

120° 0.2

0.1

0.0 0

200

400 600 Hydrodynamic radius [nm]

800

1000

Fig. 2. Scattered light intensity weighed particle size distribution of magnetosomes in solution.

magnetosomes covered by a phospholipid shell in one chain is about 7 magnetosomes (with assumption that one magnetosome/ magnetite is of about 35 nm in size as was estimated from XRD and TEM measurements). Magnetization measurements of the prepared magnetosomes suspension were carried out by SQUID magnetometer of Quantum Design. From the hysteresis loop measurement at 290 K the saturation magnetization 61.4 emu/g, coercivity field 14 Oe and remanence magnetization 29 emu/g were estimated. The remanence ratio (Mr/Ms) was 0.47 is in excellent agreement with the theoretical value of 0.5 for a random dispersion of singlemagnetic-domain particles. Examination of some micrographs showed that the cells contained from 6 to 15 magnetosomes with an average magnetosome volume of 6.6  1018 cm3. Using the value for Fe3O4 as 480 emu/cm3 at 295 K, this yields an average value of magnetic moment of 0.32  1014 emu per magnetosome. The heating effect of a solution with magnetosomes is a result of absorbing energy from the alternating magnetic field and converting it into heat. This phenomenon can set two ways mainly: (1) hysteresis losses during reversal of magnetization and (2) relaxation losses accompanying demagnetization. Thermal energy from a hysteresis loss depends on the type of the remagnetization process. Over certain portions the magnetization curve is irreversible and energy of the magnetic field is dissipated into the medium with each flux-reversal cycle in the form of heat. It is known that hysteresis losses strongly depend on the size of magnetic particles. Second mechanism of the heating effect is associated with a lag between the field and magnetization due to the relaxation nature of the magnetization process in ferrofluid. There are two mechanisms by which the magnetization of a

ferrofluid may relax after removing the applied magnetic field: the Brown and the Neel one. When both mechanisms act simultaneously, the mechanism with the shortest relaxation time being dominant. To study heating characteristics of samples with magnetosomes the heating system consisted of sine-wave power oscillator, an induction coil (solenoid with length of 78 mm and selfinductance Lo ¼ 15.6 mH in air). Quality factor of this empty coil was Qo ¼ 121 at frequency f ¼ 750 kHz. The detailed description of measuring arrangement is given in our previous articles [18]. A glass tube containing the sample was thermally isolated by a layer of material from the solenoid winding supported on a plastic sleeve. The volume of sample equals about 0.8 cm3. The change of temperature in this time was recorded with the help of a thermocouple with accuracy of 0.01 K. Hyperthermic measurements were performed at a frequency of f ¼ 750 kHz vs. the ACfield amplitude in the range 0–2.5 kA m1 (Fig. 3). The slope of the curve T(t) is a measure of the power release in a unit volume. From the fitting of the function (DT/Dt) ¼ (H/a)n to the experimental data the parameters a and n were determined which depend on several factors such as particle permeability, conductivity, size, shape and distribution. The observed Hn-law-type dependence of the temperature increase rate, (DT/Dt)t ¼ 0, on the amplitude of the magnetic field indicates the presence of superparamagnetic and partially ferromagnetic particles in the magnetic fluids studied since n42. The small amount of ferromagnetic particles causes energy losses associated with hysteresis and superparamagnetic particles cause energy losses associated with relaxation. On the basis of the obtained relation (DT/Dt)t ¼ 0 ¼ (H/14063)2.31 the specific absorption rate (SAR) values were calculated. The SAR is defined as the amount of heat released by a unit weight of the material per unit time. It can be calculated from the expression SARsample ¼ C S



DT Dt

"

# mW , g sample

(3)

where CS is the specific heat of the sample. The results reported in [19] confirm an often-used rule that a heat deposition rate of 100 mWcm3 in tissue will suffice in most circumstances for hyperthermia therapy. The values for SAR showed that for using of magnetosome-based magnetic fluids in the hyperthermia therapy the magnetic field intensities, HAC, greater than 2.5 kA/m are needed. The SAR data normalized with respect to the magnetite mass contents in the samples, mFe can be calculated from the

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Table 2 Densities r0 and r of the carrier liquid and the magnetosome-based magnetic fluid, respectively, mass contents of magnetite in the samples, mFe, and the values of the parameters a and n determined from thermal measurements.

in 1 g of magnetic material (magnetosome) was obtained:

r0 (g cm3)

r (g cm3)

FV (%)

mFe (g cm3)

a

n

0.998

1.002

1.1

0.003144

12162

2.31

The dependence of SAR on magnetic field with respect to the magnetite mass content in the sample calculated according to Eq. (5) (at f ¼ 750 kHz) is shown in Fig. 4. The found values for the SAR of 171 W/g at 5 kA/m and 841 W/g at 10 kA/m are comparable to values found by Hergt [7] for similar samples. The existence of biocompatible phospholipid membranes around magnetosomes and the obtained SAR values show that magnetosomes may be considered as good materials for the biomedical applications in hyperthermia.

200

 SAR ¼ 1332

2:31 H 12162



 W . g Fe

(5)

SAR [W/gFe]

150 magnetosomes A f = 750 kHz

Acknowledgements The Slovak Academy of Sciences, in the framework of Projects VEGA Nos. 6166, 0077, 0051, Project SAV-FM-EHP-2008-01-01 and the Slovak Research and Development Agency, in the framework of Projects APVV Nos. 0173-06, 0509-07 and 99-026505, supported this work. This work was also supported by the Polish Ministry of Education and Science under the Grant no. 4 T07B 041 30.

100 ΔT/Δt = (H/121622.31) 50

0

References

0

1000

2000

3000

4000

5000

H [Am-1]

Fig. 4. SAR values for the sample at f ¼ 750 kHz calculated with the aid of Eq. (5).

expression   rC P DT SAR ¼ mFe Dt t¼0



 W , g Fe

(4)

where r is the density of the sample and CPffiCwater ¼ 4.18 (J K1 g1) is the sample-specific heat capacity. The values of mFe ¼ 0.003144 (gFe cm3 sample) listed in Table 2 were obtained for the concentration of the magnetite grains in the ferromagnetic fluids, fV, and for the known densities r (g cm3 sample) and r0 of the sample and carrier liquid, respectively. On the basis of the relation, (DT/Dt)t ¼ 0 ¼ (H/12162)2.31, and Eq. (4) the following expression for the power dissipated as heat

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