IEEE TRANSACTIONS ON MAGNETICS, VOL. 40, NO.

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Directly Labeling Ferrite Nanoparticles With Tc-99m Radioisotope for Diagnostic Applications
Chao-Ming Fu, Yuh-Feng Wang, Yu-Chiang Chao, Shih-Hung Hung, and Ming-Da Yang
AbstractWe report a novel approach of directly labeling the radioisotope Tc99m with magnetite (Fe3 O4 ) nanoparticles for diagnostic applications. The magnetite nanoparticles were first synthesized from aqueous solutions of Fe(II) and Fe(III) chloride, with addition of ammonium hydroxide. Subsequently, the radioisotope Tc-99m were mixed directly into the reaction solution with ferrite precipitation. The labeling efficiency, of ferrite nanoparticles bound with Tc-99m, is above 99%. As prepared radioactive ferrite nanoparticles were intravenously injected into rats. The biodistribution of radioactivity, monitored by gamma-camera, has shown the uptake of radiomicrospheres can be restrained and transferable in vivo in the living animals by applying an external NdFeB magnet. Final fate of the radiomicrospheres is to be related with the surface charge of particles. Modification of surface charge of the radiolabeled ferrite nanobeads for clinical targeted diagnosis is discussed. Index TermsFerrite, nanoparticles, radioisotope.

nanoparticles for diagnostic applications. In this approach, the radioisotope Tc-99m was mixed directly into the solution in which ferrite particles are synthesized. This direct labeling of ferrite nanoparticles is superior to the alternative methods, without requiring preprocess of chemical modification, yet achieving high labeling efficiency. Moreover, without bulking volume due to chemical modification, the direct labeled ferrite particles remain in nanosize, being of great advantage as contrast agent for in vivo imaging. Applicability of such directly radiolabeled ferrite nanobeads for targeted diagnosis will be demonstrated. II. MATERIALS AND METHODS The ferrite Fe O nanoparticles used in this study were synthesized from a reaction solution of FeCl (0.05 M) and FeCl (0.05 M) and a pH adjusting solution of NH OH (50 ml), which were simultaneously dropped into an aqueous solution in an open vessel (200 ml in volume), at room temperature. During the syntheses, the amount of the added NH OH solution was controlled so that the pH value of the aqueous solution would pH . Since the synbe near the neutral condition thesis was performed in open air, the air oxidized the Fe ions to Fe , and formed Fe O particles in aqueous solutions. X-ray diffraction analysis of the synthesized particles has shown typical peaks of spinel crystalline structure of Fe O . From the X-ray peak, an average diameter of 15 nm was deduced by Langevins formulation. The deduced particle size is comparable to TEM analysis of the particles synthesized by the same chemical process [6]. The magnetization curve, measured by vibration sample magnetometer, has shown nearly superparamagnetic behavior, with the saturation magnetization of 62 emu/g and the coercivity of 11 Oe. For labeling ferrite nanoparticles with the radiotracer, Technetium-99m (Tc-99m) with a radioactivity of 1.11 GBq (30 mCi) was prepared. As prepared Tc-99m was injected into a sterile, vacuuming collecting vial (5 ml in volume). Subsequently, a stennous solution of 0.5 ml was injected into the same vial, and stirred for 10 s. The stennous solution, which mainly contains stennous fluoride, is a reducing agent that reduces surface charge of Tc-99m to a suitable condition for labeling with pharmaceuticals. Meanwhile, a diluted ferrite nanoparticles solution was prepared by extracting 0.1 ml solution containing ferrite nanoparticles and diluting it with 0.9 ml pyrogen-free isotonic saline. Then, a diluted ferrite nanoparticles solution of 0.5 ml was added into the vial containing Tc-99m. This was kept at room temperature for 2 min, in order to have an entire labeling of radiotracer with ferrite nanoparticles.

I. INTRODUCTION

AGNETIC microspheres for biomedical applications have been exploited for many decades. The magnetic nanospheres have many applications [1][4], such as targeting agents for directing the chemotherapeutic drug to a localized disease site, magnetic immunoassay for the rapid detection of virulent bacteria, hyperthermia treating for cancer by applied ac magnetic field, in vivo contrast agents for magnetic resonance imaging (MRI), etc. In addition, the magnetically directed transport of radionuclides by magnetic nanospheres has being an innovative field of interest. Magnetic radioactive nanospheres have the advantage of being able to deliver high concentrations of radioactivity to the target area, without damaging normal surrounding tissue. For example, magnetic poly(lactic acid) microspheres, containing magnetite nanoparticles, as carriers for radioactive yttrium, have shown great promise for regional and intracavitary radiotherapy [5]. Up to now, syntheses of nanosized magnetic spheres have been well established [6][10]. In general, the biomolecule immobilization or radionuclides labeling onto the magnetic particle requires complicated chemical modifications of the surfaces. We report in this paper a novel method by directly labeling the radioactive Tc-99m with magnetite Fe O
Manuscript received October 15, 2003. This work was supported in part by the Taiwan National Science Foundation under Grant NSC-91-2112-M-017-004 and Grant NSC-93-2112-M-017-001. C.-M. Fu, Y.-C. Chao, S.-H. Hung, and M.-D. Yang are with the Physics Department, National Kaoshiung Normal University, Kaoshiung, Taiwan, R.O.C. (e-mail: chaomingfu@yahoo.com.tw). Y.-F. Wang is with the Department of Nuclear Medicine, Buddhish Dalin TzuChi General Hospital, Taiwan, R.O.C. Digital Object Identifier 10.1109/TMAG.2004.834199

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IEEE TRANSACTIONS ON MAGNETICS, VOL. 40, NO. 4, JULY 2004

III. RESULTS AND DISCUSSION To achieve an efficient in vivo diagnostic application of the magnetic radioactive nanoparticles synthesized by the directly labeling approach, the labeling efficiency of ferrite Fe O nanoparticles bound with Tc-99m has to be verified. Thus, an in vitro labeling efficiency test were performed by using the instant thin-layer chromatography (ITLC) method, since it is a well accepted and uncomplicated process to examine the radiopharmaceutical quality in nuclear medicine. At first, a droplet of solution, containing radiolabeled ferrite nanoparticles, was applied by injection onto the end of the strips (0.5 5.5 cm) for ITLC. Then, the strip was immediately placed into a chromatography development tank containing acetone solvent. Accordingly, the solvent was migrated gradually from one end to the other end of the strip, passing through the droplet spot. After that, the strip was cut into two parts: proximal part contenting the spot of labeled Tc-99m-ferrite, and the distal part of the unbounded Tc-99m pertechnetate. The radioactivity of each part was measured by a gamma counter. The labeling efficiency more than 99% was obtained for the ferrite nanoparticles bound with Tc-99m synthesized by the novel directly labeling approach. Further testing has shown that the labeling efficiency of 90% can be sustained over a lifetime. The result implies the Tc-99m labeled ferrite nanoparticles promises for in vivo targeted diagnosis. During the ferrite formation reaction in aqueous solution, hydrolyzed ions or oxidizing reagent were formed on the surface of ferrite particle [7]. The hydrolyzed ions or oxidizing reagent may serve as chemical bridges for bonding with the reduced Technetium ions, thus acting in response for the direct labeling and the observed high labeling efficiency. Advanced study of the chemical reaction of ferrite with Tc-99m in stennous solution is needed to explore labeling formation of radiolabeled ferrite. The knowledge and exploration of the organ distribution of in vivo injected particle is necessary for targeted diagnosis. Thus, in the next step, the radiolabeled ferrite nanoparticles were intravenously injection into the tail vein of Wistar rats (male, 200250 g). The scintigraphic images were monitored by planar imaging, using a gamma camera (GE, DST XLi) with parallel collimator. Fig. 1 displays the scintigraphic images, obtained after intravenous injection of the radiolabeled ferrite nanoparticles into a rat, without external applied magnetic field. The top graphs display the evolution of radiotracer from the tail into the entire body of the rat. The lower left is an enlarged scintigram taken 5 min after injection, and the lower right one was taken at 10 min after injection. The higher brightness in the scintigram represents the higher accumulation of radioparticles. As seen in the scintigrams, almost all of the injected dose is taken up by the liver and lungs, without applied magnetic field. In order to determine biodistribution of radioisotope nanospheres, rats were scarified in time sequence (5, 30, 60, and 180 min). The radioactivity in liver, heart, lungs, intestine, stomach, spleen, kidneys, and the rest of the rat was measured by a low background-counter (CATINTEC CRC-15R). The radioisotope count in each organs is shown in Fig. 2, in 5, 30, 60, and 180 min, respectively. Almost 80%90% of the injected dose is taken up by the liver within 5 min after injection. A

Fig. 1. The scintigraph obtained after intravenously injection of the radiolabeled ferrite nanoparticles into the tail vein of a rat. Lower left: enlarged image taken at 5 min after injection. Lower right: 10 min after injection.

Fig. 2. Biodistribution of radioactive nanospheres in organs of the rats after injection, without applied external magnet.

second large uptake is by lung, kidneys and spleen. Little uptake is found in the heart and brain. We have noticed that the joints accumulate a higher dose of radioactivity than that of the bladder (not in the figure).

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as placing a magnet near the neck and injecting near the neck, etc., still to be performed to explore the mechanism for controlling ferrite beads. IV. CONCLUSION We have reported a novel approach of directly labeling the radioisotope Tc-99m with ferrite nanoparticles for diagnostic applications. In this approach, the reduced Tc-99m ions were mixed directly into the solution in which ferrite particles are formed, without complicated preprocess of chemical modification. Analyzed by the instant thin-layer chromatography, the labeling efficiency is above 90% and can sustain over a lifetime. The biodistribution of radioactivity shows high uptake by the liver, after intravenously injecting the radiolabeled ferrite nanoparticles into the tail vein of a rat. The radioactivity can be enriched to a specific site by applying an external magnet. The result implies the applicable of the directly labeling of Tc-99m on ferrite nanoparticles for targeting diagnosis. On the other hand, the phagocytic uptake of radiobeads is strongly related to the surface charge of particle. Reducing the surface charge of ferrite nanoparticles may improve phagocytic uptake in magnetic targeting diagnosis. Surface modification of the radiolabeled ferrite may be obtained by adjusting the pH value in the aqueous reaction solution, or adding with reducing reagents. REFERENCES
[1] U. O. Hfeli and G. J. Pauer, In vitro and in vivo toxicity of magnetic microspheres, J. Magn. Magn. Mater., vol. 194, pp. 7682, 1999. [2] U. O. Hfeli, S. M. Sweeney, B. A. Beresford, E. H. Sim, and R. M. Macklis, Magnetically directed poly(lactic acid) 90Y-microspheres: novel agents for targeted intracavitary radiotherapy, J. Biomed. Res., vol. 2, no. 8, pp. 901908, 1994. [3] S. Sieben, C. Bergemann, A. Lbbe, B. Brockmann, and D. Rescheleit, Comparison of different particles and methods for magnetic isolation of circulating tumor cells, J. Magn. Magn. Mater., vol. 225, pp. 175179, 2001. [4] Y. Haik, V. Pai, and C.-J. Chen, Development of magnetic device for cell separation, J. Magn. Magn. Mater., vol. 194, pp. 254261, 1999. [5] U. O. Hfeli, S. M. Sweeney, B. A. Beresford, B. A. Humm, and R. M. Macklis, Effective targeting of magnetic radioisotope 90Y-microspheres to tumor cells by an externally applied magnetic field. Preliminary in vitro and in vivo results, Nucl. Med. Biol., vol. 22, pp. 147155, 1995. [6] K. Nishimura, M. Hasegawa, Y. Ogura, T. Nishi, K. Kataoka, and H. Handa, 4 C preparation of ferrite nanoparticles having protein molecules immobilized on their surfaces, J. Appl. Phys., vol. 91, pp. 85558556, 2003. [7] M. Abe and Y. Tamaura, Ferrite-plating in aqueous solution: a new method for preparing magnetic thin film, Jpn. J. Appl. Phys., vol. 22, pp. 511513, 1983. [8] M. Abe, Ferrite plating: A chemical method preparing oxide magnetic films at 24100 C, and its applications, Electrochim. Acta, vol. 45, pp. 33373343, 2000. [9] D. S. Kim, N. Matsushita, and M. Abe, Synthesis of the ZnNi-ferrite plated NiFe microspheres from aqueous solution, in Proc. IUMRS 2004. [10] A. Kondo and H. Fukud, Preparation of thermo-sensitive magnetic microspheres and their application to bioprocesses, Colloids Surf. A: Physiochem. Eng. Aspects, vol. 153, pp. 435438, 1999.

Fig. 3. The scintigraphic image obtained after intravenously injection of the Tc-99m labeled ferrite nanoparticles into a mice. Right: with applying external magnet near right thigh before injection. Left: replacing the magnet to left thigh.

High accumulation of the injected dose by the liver is to be associated to the phagocytic uptake in the Kupffer cell of the liver. As in many reports [1][5], most of the intravenously injected particles are recognized by the body as being foreign, and are removed from blood circulation by the mononuclear phagocytic system (MPS). The phagocytic uptake of particles by the macrophages is very fast and is completed within 5 min after intravenous injection. The uptake of particles is related to the physiochemical characterization of the particle size, particle charge, surface hydrophobicity, and the chemical nature of the functional groups. In viewing the magnetic targeting effect of the directly Tc-99m labeled ferrite nanoparticles, a commercial available NdFeB magnet has been placed onto the skin of the right thigh of the rat before injection. The scintigraphic image obtained after intravenous injection has demonstrated an enrichment of radioactivity on the site by applying a magnet, as shown in Fig. 3 (right). Next, we replaced the magnet onto the skin of the left thigh of the rat. Then, the radioparticles traveled through the bloodstream and migrated into the new side. As shown in Fig. 3 (left), the enriched radioisotope brightness has been relocated to the left side where the magnet was replaced for around 2 min. Moreover, if the magnet is held at its original position longer, the radioactivity is to be increased to the site. As a consequence, the radioactivity is more likely to sustain at the site after the magnet is removed, and may be dependent on the surface charge of radioparticles. We have noticed that the external magnet has to be applied proximately to the injection site to obtain high efficiency of magnetic attraction of radiolabeled ferrite nanoparticles. In case of placing the magnet at the neck of rat, there were no apparent magnetic attraction effect. This may be related to very fast uptake of particles by the macrophages. Attraction of magnetic radiobeads have to be achieved within first few blood circulation, to complete with phagocytic uptake. Further experiments, such