Mat. Res. Soc. Symp. Proc. Vol. 676 © 2001 Materials Research Society Biomedical application of ferrofluids containing magnetite nanoparticles D.K. Kim 2, W. Voit 1,3, W. Zapka3, B. Bjelke4, M. Muhammed 2 and K.V. Rao 1 1 Engineering Materials Physics Division, and 2 Materials Chemistry Division, Royal Institute of Technology, SE-100 44 Stockholm, Sweden 3 XaarJet AB, SE-175 26 Järfälla, Sweden 4 MR Research Center, Dept. of Clinical Neuroscience, Karolinska Institutet, SE-171 76 Stockholm, Sweden ABSTRACT Ferrofluids containing superparamagnetic Fe3O4 nanoparticles have been prepared by a controlled co-precipitation method. The aggregation of the particles was prevented by using a polymeric starch network as a coating agent. Structural and magnetic measurements reveal a particle size of around 6 nm, with a clear evidence of a uniform particle coating. The ferrofluids have been used as a contrast agent for MR imaging in biological tissue. INTRODUCTION Superparamagnetic nanoparticles, conjugated to various monoclonal antibodies [1], peptides or protein [2], show potential applications for in vivo monitoring of brain inter-cellular communications system and target-oriented MR imaging in both animals and human brains. Magnetic nanoparticles as contrast agents in MR were used for localization and diagnosis of brain and cardiac infarcts, liver lesions or tumors, where the particles tend to accumulate at higher levels due to the difference in tissue fluidity and endocytotic process. There are several commercially available dextran coated superparamagnetic iron oxide for MR imaging [3,4]. However, these particles have a rather broad size distribution of 120-180 nm, and the size of these particles is much larger than the extracellular space (<50nm). Therefore, it is desirable to have nanosized magnetic cores surrounded by biocompatible outer shells. It is also necessary that these shell coated magnetic nanoparticles can be manipulated by external fields and at the same time forms a stable fluid without particle agglomeration. In this study, we present the preparation, characterization and application of a uniformly monodispersed colloidal ferrofluid containing superparamagnetic magnetite nanoparticles, which were produced by a controlled coprecipitation method. We demonstrate the use of this ferrofluid for biomedical purposes, especially as MR contrast agents for imaging the diffusion of superparamagnetic nanoparticles through capillaries in rat brain. The particles are coated with starch, a long-chain polymer of D-glucose, which provides a good biocompatibility, biodegradability and is not toxic. An investigation of the morphological and magnetic properties of the synthesized ferrofluid is included in this paper. Y8.32.1 SAMPLE PREPARATION AND EXPERIMENTAL METHODS Based on Massart’s methods [5], aqueous mixture of ferric and ferrous salts and NaOH as an alkali source were prepared as stock solutions. The synthesis of magnetite nanoparticles has been carried out via a controlled chemical coprecipitation approach, as described in detail in a separate paper [6]. During the synthesis process, N2 gas was flown in a closed system through the reaction medium, to prevent critical oxidation of Fe2+. Various amounts of starch were dissolved in 100 ml deionized water at 90°C. After the starch was thoroughly dissolved, the solution was placed immediately in a 60°C water bath until the starch solution temperature was decreased to the water bath temperature. Precursor solutions for the Fe source were poured into the prepared starch solution under vigorous stirring. 25 ml of the iron-containing starch source solution was added drop-wise into 200 ml of 1.0M NaOH under vigorous mechanical stirring (2000rpm) for 2hrs at 60°C. During boiling, approximately 50% of the water was evaporated and the remaining solution was cooled to room temperature for 12 hrs. The remaining gels were washed out with deionized water until the pH was less than 8.5. The starch coated iron oxide particles were dialyzed at 37°C for 2-3 days in the presence of adequate stirring. The structural properties of the obtained Fe3O4 particles were analyzed by X-ray diffraction (XRD) measurements using a Philips PW 1830 diffractometer. For the estimation of the average crystal size D, Scherrer’s formula was used. Transmission electron microscopy (TEM) images of the samples were performed on a JEOL-2000EX microscope. IR-spectroscopy data was measured on dried samples with a Bruker IFS 66 FT-IR. For the measurement of the zetapotential of the particles in 5mM and 10 mM NaCl background electrolyte solutions, a Zetasizer 2000 from Malvern Instrument Ltd. was used. The suspensions for the measurement had a fixed concentration and a given pH was adjusted by NaOH and HCl stock solutions. Magnetic investigations were carried out using a Quantum Design MPMS2 SQUID magnetometer and, for AC-magnetic measurements, a high sensitive laboratory-built AC susceptometer with a three-coil mutual inductance bridge. The AC-frequency was used from 29 to 433 Hz with a constant driving field of 10 Oe (rms) for these measurements. The MRI recordings were performed on a Biospec Avance 47/40 spectrometer (Bruker, Karlsruhe, Germany) with a 4.7 Tesla, 40 cm bore diameter magnet, equipped with a 12 cm self shielded gradient system capable of switching 200 mT/m in 250 µs. Animals were imaged using a purpose built RF coil. Two MR sequences were used. A gradient echo (GEFI) and a multi-slice MRI protocol with a Rapid Acquisition with Relaxation Enhancement (RARE) sequence were used. The following MR parameters were used: Acquisition matrix 256x128 and reconstructed 256x256, slice thickness 800 µm, field of view 4 cm. Specifically for the GEFI sequence TR 144.2 ms, TE 10.0 ms, averages 16 and for the RARE sequence recovery time 2095 ms, time between refocusing pulses and phase encoding gradient incrementation to yield an effective echo time TE 46.2 ms, averages 8, rare factor 16. RESULTS AND DISCUSSION Fig. 1 shows a TEM image of the prepared magnetite particles coated with starch. The image indicates that the magnetite particles are distributed homogeneously in the polymeric matrix. The particles have a spherical shape, as the nucleation speed per unit area is isotropic at the interface between the single iron oxide particles. The analysis of the TEM image allows determining the Y8.32.2 mean diameter of the particles and the particle size distribution by using an equation based on a log-normal function. The particles size determined in this way is 72 Å with a standard deviation σd = 0.2, which is close to the crystal size calculated from XRD data (60 Å). Fig.2 shows the results from the microelectrophoresis measurements of a diluted naked and a starch coated superparamagnetic magnetite suspension. The sample was prepared at high pH (pH=13) and left stirring overnight before the pH titration was started. 0.05M HCl was added directly to the sample and dispersed with an ultrasonic vibrator. Fig. 3 displays an isoelectric point for the naked Fe3O4 nanoparticles with 10mM NaCl electrolyte concentration at pH 6.6. Relatively strong external force might be necessary to break up the agglomerations that are formed when the surface charge density is low (pH range is between 4 to 8). At the high and low pH range, the zeta potential shows the highest values because the hydrodynamic forces introduced by ultrasonic should be strong enough to separate weak bondings. The starch coated magnetic nanoparticles show negative values at the whole pH range with an isoelectric point of pHiep=2.1. The result implies that any electrostatic and steric repulsion induced by coated starch on iron oxide is not significantly strong. However, the colloidal suspension shows stable behavior even with lower zeta potential values. This means that the starch-grafted magnetite has a flat layer and longer polymeric chains. Natural starch, which is a branched hydrophilic and cross-linked polymer, contains amylose usually amounting up to about 15 to 30% of the mass per granule. Potato starch consists of a core of crystalline amylose, which is surrounded concentrically by a layered shell of amylopectin. The molecules of amylopectin are deposited as fringed folded micells, and are bound to their neighbors by hydrogen bonds. The outer part of the amylopectin layers of a starch granule is swollen in some degree, and some of the molecules are extended toward water and loosely connected with other amylopectin molecules. Thus, the swollen starch granules form a threedimensional scaffold structure. This three-dimensional polymeric matrix keeps a constant 50 naked starch 40 30 20 9 P O D L W Q H W R S  D W H = 10 0 -10 -20 -30 -40 2 4 6 8 10 12 14 pH Figure 1. TEM micrograph of Fe3O4 nanoparticles swollen in starch polymeric matrix. Figure 2. Microelectrophoretic measurements of a dilute naked and starch coated superparamagnetic magnetite suspension at 10mM NaCl background electrolyte strengths. Y8.32.3 distance between individual iron oxide nanoparticles during nucleation. The iron oxide particles are usually nucleated from metal iron embedded in the polymer matrix. At a certain critical size rn, the increase in free energy due to the increase of the surface area is just balanced by the decrease in * due to the energy difference between the crystal and its liquor. This stage represents the end of nucleation and beginning of growth [7]. FT-IR spectral pattern of starch coated magnetite particles shows four peak at 3300, 1610, 1350 and 1000 cm-1 (Fig.3). The absorption regions at 3300 and 1610 cm-1 were due to water molecule and that at 1350 cm-1 to bending modes of O-C-H, C-C-H, and C-O-H angles. The absorption region at 1000cm-1 relates to C-C and C-O stretching modes of the polysaccharide backbone. The magnetic crystal size and the geometrical standard deviation σ of the size distribution were estimated using room temperature magnetization data. A model for monodomain, non-interacting particles was used for these calculations [8], as the investigated ferrofluid can be considered as a low density, week interacting magnetic fluid. Using the initial susceptibility χi and the bulk saturation magnetization of the particles per unit volume MS, the mean crystal volume diameter DV of the log normal volume distribution can be calculated by:  18k BT DV =   πM S χi 3ε M S H 0     1 3 (1) In this case, 1/H0 is the point where the high-field linear extrapolation of M versus 1/H crosses the abscissa, and ε is the crystal volume fraction. The standard deviation σ of DV is given by: 1   3χ H σ =  ln i 0 3  εMS      1 2 (2) With a Ms of 181.6 emu/cm3 from the room temperature SQUID magnetization measurement on the uncoated, dried Fe3O4 powder, an initial susceptibility χi of 2.79 × 10-3 and a volume fraction ε of 0.008 for the magnetite content in the ferrofluid, we can calculate a mean crystal volume diameter of 52 Å and a standard deviation σ of 0.33. This standard deviation is slightly larger than the value obtained from the TEM measurements. This observation is in correspondence with earlier observations [9], and is due to the influence of particle interactions on the magnetization data. The value for the mean diameter is lower than the values from TEM and XRD measurements, which can be contributed to a magnetically dead layer on the surface of the iron oxide particles. The superparamagnetic nature of the ferrofluid can be seen on the measurement of the temperature dependence of the initial susceptibility in the zero-field cooled (ZFC) and field cooled (FC) case (Fig. 4). The blocking temperature TB can be estimated with 98 K from the cusp in the zero-field cooled (ZFC) curve. Above the blocking temperature, the magnetic particles become thermally unstable and internal relaxation occurs. The magnetization of the particles follows the classical superparamagnetic model with remanent magnetization approaching to zero, as suggested by Néel [10]. Without coating the magnetite, particles will form clusters due to the large attractive forces as an effect of the large surface to volume ratio, and the interaction between the particles will yield in a higher blocking temperature. This is shown in Fig 4, where the ZFC and FC curves are compared for the ferrofluid and for the bare magnetite particles after chemical removal of the coating material and drying. Y8.32.4 2.0 0.009 1.8 0.008 1.6 0.007 Magnetisation (emu) Absorbance Units 1.4 1.2 1.0 0.8 0.6 0.4 0.006 0.005 0.004 0.003 0.002 0.2 0.001 0.0 0.000 -0.2 4000 3500 3000 2500 2000 1500 1000 -0.001 -1 Wavenumber (cm ) Figure 3. FT-IR spectra of iron oxide particles coated with starch. field cooled (particles) zero field cooled (particles) field cooled (ferrofluid) zero field cooled (ferrofluid) 0 50 100 150 200 250 300 Temperature (K) Figure 4. Zero-field cooled (ZFC) and field cooled (FC) magnetization curves for the starch coated ferrofluid, compared to bare magnetite The interest in the use of superparamagnetic nanoparticles for biomedical purposes, especially for the use as MR imaging agent, is based onto some basic considerations. When organic tissue is exposed to an external magnetic field, there is a difference in energy levels between protons that point parallel or anti-parallel and this results in a net magnetic charge in one direction. The magnitude of this magnetic charge will depend on the strength of the external magnetic field and the amount of protons in the sample. This accounts for the need to use very strong magnetic fields and the inherent insensitivity of magnetic resonance imaging, as the sample has to contain more than about 5% water or fat to get a signal. To compensate the reduced signal intensity caused by this kind of detection limits, external superparamagnetic magnetite particles are introduced as a MR imaging agents. The effect is used that a spinning magnetic particle will start precessing around when it is placed in a strong magnetic field, similar to a spinning top precessing around the vertical axis before falling. The fluid in rat brain can be detected by making the hydrogen nuclei present in the water molecules under strong magnetic field. The intensity difference in the energy radiation between the fluid in rat brain and the injected superparamagnetic Fe3O4 nanoparticles can be seen by different darkness in MR images. Fig.5 shows the rostrocaudal projection of coronal slices from the rat brain one hour after injection of a ferrofluid containing starch coated Fe3O4 nanoparticles. The distribution of the Figure 5. Rostrocaudal projection of coronal slices from the rat brain one hour after injection of starch coated magnetite particles. Y8.32.5 particles in the living brain is mainly observed in the ventricular system, the lateral ventricle (Lv) in panel ‘a’ as well as in the 3:e ventricle (3v) in panel ‘b’. The particles are well distributed within the dorsal hippocampus (dHip) and do not seem to penetrate the external capsule (ec). The site of injection is labelled by an arrow in panel ‘c’. For these measurements, a gradient echo (GEFI) sequence was used as it is more sensitive to the susceptibility changes caused by the superparamagnetic iron particles and will qualitatively show the presence of the nanoparticles. The distribution of the particles was characterized using a multi-slice MRI protocol with a Rapid Acquisition with Relaxation Enhancement (RARE) sequence. CONCLUSIONS Magnetic cores surrounded by a biocompatible outer shell were prepared by a controlled coprecipitation method. The polymeric starch coated superparamagnetic Fe3O4 nanoparticles have a homogeneous size distribution, which was confirmed by different methods. The initial experiments on anaesthetized rats have shown the feasibility of using Fe3O4 nanoparticles for in vivo MRI imaging of the rat brain. The magnetic nanoparticles were well distributed within the dorsal hippocampus (dHip) after injection into the rat brain and did not seem to penetrate the external capsule (ec). ACKNOWLEDGEMENTS This work has been supported by the Swedish Foundation for Strategic Research (SSF), the Swedish Research Council for Engineering Sciences (TFR) and the Swedish Agency for Innovation Systems (VINNOVA – former NUTEK). REFERENCES 1. L. G. Remsen, C. I. McCormick, S. Roman-Goldstein, G. Nilaver, R. Weissleder, A. Bogdanov, I. Hellstrom, R. A. Kroll and E. A. Neuwelt, American Journal of Neuroradiology 17, 411 (1996) 2. B. K. Schaffer, C. Linker, M. Papisov, E. Tsai, N. Nossiff, T. Shibata, A. Bogdanov, Jr., T. 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