Delivery of Superparamagnetic Nanoparticles for Local Chemotherapy ...

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mitoxantrone were given intraarterially into the tumor- supplying artery of tumor-bearing rabbits (VX2 squamous cell carcinoma) and focused in the tumor region ...

ANTICANCER RESEARCH 27: 2019-2022 (2007)

Delivery of Superparamagnetic Nanoparticles for Local Chemotherapy after Intraarterial Infusion and Magnetic Drug Targeting CHRISTOPH ALEXIOU1, ROLAND JURGONS1, CHRISTIAN SELIGER1, OLIVER BRUNKE2, HEINRICH IRO1 and STEFAN ODENBACH2 1Department

of Otorhinolaryngology, Head and Neck Surgery, University Erlangen-Nürnberg; of Fluid Mechanics, Technische Universität of Dresden, Germany

2Institute

Abstract. Background: Superparamagnetic nanoparticles are currently used as contrast agents for magnetic resonance imaging. These particles can also be used as drug carriers for local chemotherapy, called magnetic drug targeting. Using an external magnetic field, colloidal nanoparticles can be directed to a specific body compartment (i.e. tumor). Materials and Methods: After magnetic drug targeting in an experimental rabbit model with a VX2 squamous cell carcinoma, tumor tissue was extracted and embedded in paraffin for histology and X-ray imaging. Results: The distribution of magnetic nanoparticles was detected holistically with X-ray imaging and in detail using Prussian blue staining of histological cross sections. Conclusion: The biodistribution of magnetic nanoparticles can be visualized with X-ray imaging and histologically confirmed. Superparamagnetic nanoparticles are used in medicine in vitro (1) and in vivo. For one of the first in vivo applications of magnetic particles in humans, Alksne et al. (2) performed experiments with carbon-coated iron combined with an external magnetic field for occluding intracranial aneurysms. The therapeutic efficacy was confirmed by X-ray investigations. Furthermore, superparamagnetic particles are used as contrast agents for magnetic resonance imaging (3). A new approach in local cancer therapy is magnetic drug targeting (MDT). Starch-coated magnetic nanoparticles labelled with the chemotherapeutic agent mitoxantrone were given intraarterially into the tumorsupplying artery of tumor-bearing rabbits (VX2 squamous

cell carcinoma) and focused in the tumor region with an external magnetic field. With this delivery system, total tumor remission without negative side-effects could be accomplished using only 20% and 50% of the regular systemic chemotherapeutic dosage (4). Radioactive 59Fenanoparticles showed 114 times more activity in the tumor region after MDT compared to the control without a magnetic field (5). Furthermore, it was shown that with this system a high and specific enrichment of the bound chemotherapeutic agent in a desired body compartment (i.e. the tumor) was possible. HPLC-analysis of the chemotherapeutic agent after MDT revealed a 75-fold higher concentration of the administered dose in the tumor region compared to the regular systemic administration (5, 6). The aim of the present study was to non-invasively investigate the distribution of the particles with a common imaging technique (X-ray).

Materials and Methods Tumor model. Experiments were performed on 8 tumor-bearing rabbits. A VX2 squamous cell carcinoma was placed at the medial portion of the hind limb. Two weeks after implantation, the tumors reached a size of about 2 cm3 and the nanoparticles were administered intraarterially (i.a.) into the femoral artery. Magnetic field. For the experiments, a powerful electromagnet with a magnetic field strength of 1.7 Tesla was used. The pole shoe of the magnet was focused over the tumor region during the infusion of the nanoparticles. Magnetic nanoparticles. The nanoparticles in this study consisted of iron oxides covered by starch polymers for colloidal stabilization. The hydrodynamic diameter was approximately 250 nm.

Correspondence to: Ch. Alexiou, Department of Otorhinolaryngology, Head and Neck Surgery, Friedrich Alexander University ErlangenNürnberg, Waldstr. 1, 91054 Erlangen, Germany. Tel: +49 9131 8533142, Fax: +49 9131 8534828, e-mail: [email protected] Key Words: Magnetic drug targeting, magnetic nanoparticles, cancer treatment, chemotherapy, ferrofluids.

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Histology. Histological investigations were performed to visualize the distribution of the ferrofluids in the tumor tissue. Light-microscopy. The excised tissue samples, taken 1 h after ferrofluid administration were fixed for 24 h in 4% PBS formaldehyde. After dehydration through increasing alcohol

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ANTICANCER RESEARCH 27: 2019-2022 (2007) concentrations and a xylol-step they were embedded in paraffin and slides of 5 Ìm-thick slices were made. Prussian blue stain. The Prussian blue stain provides the histochemical evidence of the presence of trivalent iron according to the following reaction: trivalent iron is visible as blue pigment while the remaining structures appear red in color. X-ray-imaging. For the visualisation of the particle distribution, paraffin-embedded tumor tissue was investigated with x-rays in 221 pictures around 360 degrees. The x-ray machine (Institute of Fluid Mechanics, Technical University of Dresden, Germany) consisted of a passive-cooled integral X-ray tube with an acceleration voltage of up to 50 kV at an electron emission current of 1 mA. The limit of resolution with these parameters was 40 Ìm. X-ray visualisation was performed with a thermoelectrically-cooled slow-scan CCD camera (1024 x 1024 pixels).

Results X-ray-tomography images show that the whole vascular system of the tumor can be reached by the nanoparticles with the influence of a focused external magnetic field (Figure 1) and this was confirmed with the corresponding macroscopic and histological examination of the tumor (Figures 2, 3). Histological cross-sections show the nanoparticles after intraarterial application in the vascular system of the tumor (Figure 3a). Sporadic occurrence of ferrofluids in macrophages was detected (Figure 3b).

Discussion For 30 years, several approaches and carrier systems for the site-specific transport of therapeutics were developed in medicine (7, 8). Magnetic albumin microspheres containing chemotherapeutic agent (Adriamycin) were injected into the ventral caudal artery of rats in the absence and presence of an external magnetic field. These studies showed that the application of the magnet increased the targeting efficacy of the carrier by a factor of 6, as well as that of the drug exposure (9). In our experiments, the particles consist of magnetite (Á-Fe3O4) coated with phosphated starch molecules of defined polymer size. For magnetic drug targeting, the chemotherapeutic agent mitoxantrone has been reversibly bound to the polymer. Previous studies revealed that in HeLa cell culture, particles which were targeted by a magnetic force placed under the cell culture well for one hour were internalised by an endocytotic pathway (10). Kohler et al. (11) used particles with the same core but a different coating. The particles were coated with a self assembled monolayer of (3-aminopropyl) trimethylsiloxane conjugated by amidation with the cytostatic agent methotrexate, resulting in a covalent drug bond on the surface. Despite the different particle characteristics, both described the same manner of particle internalisation in the

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Figure 1. X-ray image of the VX2-tumor tissue embedded in paraffin after MDT. Nanoparticles in the vascular system of the tumor can clearly be seen.

cell by endocytosis. This leads to the indication that this is a general pathway for particle internalisation in HeLa cell culture. In contrast to the in vitro observations in tumor cell culture (10), the present in vivo data show a different pattern. After intraarterial infusion of magnetic nanoparticles in tumor-bearing rabbits, the ferrofluids are accumulated mainly in the vascular system of the tumor. Histological crosssections of the tumor tissue showed the nanoparticles in the tumor vessels. Infrequently, nanoparticles were detected in the intracellular space of the cells. The proof of iron presence was seen in macrophages and endothelial cells of the tumor tissue (5) (Figure 3a). X-ray images confirm the ferrofluid delivery shown by histology. The vessel system of the examined tissue samples are detectable by x-ray examination. Due to the radiographic technique of consecutive imaging over 360Æ of the whole tumor, it is possible to reconstitute a three-dimensional picture of the tumor and the distribution of the particles. An important parameter for the effectiveness of a local chemotherapy is the resulting distribution of the respective therapeutic agent in the tumor region (12, 13). Using drug loaded nanoparticles, the biodistribution is usually studied via histological cross-sections of the examined tissue samples, a technique which provides only very local information about the overall distribution. X-ray microtomography is a promising tool for gaining

Alexiou et al: Delivery of Superparamagnetic Nanoparticles in Magnetic Drug Targeting

Figure 2. a) Macroscopic view of paraffin-embedded VX2-tumor tissue after MDT. b) Cross view of a histological slide stained with Prussian blue. The nanoparticles are visible as blue pigment in the vessels of the tumor.

Figure 3. Cross section of VX2-tumor tissue after magnetic drug targeting. Stain: Prussian blue; magnification: x400. a) Ferrofluids in the vascular system of the tumor. b) Proof of iron seen as blue pigment in a macrophage, marked with a circle.

information about the overall distribution of the particles in the tumor region. It is a strong and non-invasive procedure and could also be very useful to control the effectiveness of magnetic drug targeting in vivo.

Acknowledgements These studies were supported by DFG (Deutsche Forschungsgemeinschaft, Al 552/2) and by Wilhelm-SanderStiftung, Munich, Germany (2006.084.1).

References 1 Bilkenroth U, Taubert H, Riemann D, Rebmann U, Heynemann H and Meye A: Detection and enrichment of disseminated renal carcinoma cells from peripheral blood by immunomagnetic cell separation. Int J Cancer 92: 577-582, 2001. 2 Alksne JF, Fingerhut A and Rand R: Magnetically controlled metallic thrombosis of intracranial aneurysms. Surgery 60: 212218,1966.

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ANTICANCER RESEARCH 27: 2019-2022 (2007) 3 Taupitz M, Wagner S, Hamm B, Dienemann D, Lawaczeck R and Wolf KJ: MR Lymphography using iron oxide particles. Detection of lymph node metastases in the VX2 rabbit tumor model. Acta Radiol 34: 10-15, 1993. 4 Alexiou Ch, Arnold W, Klein R, Parak F, Hulin P, Bergemann C, Erhardt W, Wagenpfeil S and Lübbe AS: Locoregional cancer treatment with magnetic drug targeting. Cancer Res 60: 6641-6648, 2000. 5 Alexiou Ch, Jurgons R, Schmid RJ, Bergemann Ch, Henke J, Erhardt W, Huenges E and Parak FG: Magnetic drug targetingbiodistribution of the magnetic carrier and the chemotherapeutic agent mitoxantrone after locoregional cancer treatment. J Drug Targeting 11: 139-149, 2003. 6 Alexiou C, Jurgons R, Schmid R, Erhardt W, Bergemann C and Parak F: Magnetisches drug targeting – ein neuer Ansatz in der lokoregionären tumortherapie mit chemotherapeutika. HNO 53: 618-622, 2005. 7 Gupta PK: Drug targeting in chemotherapy: a clinical perspective. J Pharm Sci 79: 949-962, 1990. 8 Torchilin VP: Drug targeting. Eur J Pharm Sci 11: 81-91, 2000. 9 Gupta PK and Hung CT: Comparative disposition of adriamycin delivered via magnetic albumin microspheres in presence and absence of magnetic fields in rats. Life Sci 46: 471-484, 1990.

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10 Alexiou Ch, Jurgons R, Schmid RJ, Hilpert A, Bergemann Ch, Parak FG and Iro H: In vitro and in vivo investigations of targeted chemotherapy with magnetic nanoparticles. J Magn Magn Mater 293: 389-393, 2005. 11 Kohler N, Sun C, Wang J and Zhang M: Methotrexatemodified superparamagnetic nanoparticles and their intracellular uptake into human cancer cells. Langmuir 21: 8858-8864, 2005. 12 Alexiou Ch, Jurgons R, Seliger C, Kolb S, Heubeck B and Iro H: Distribution of mitoxantrone after magnetic drug targeting – fluorescence microscopic investigations on VX2 squamous cell carcinoma cells. Z Phys Chem 220: 235-240, 2006. 13 Collins JM: Pharmacological rationale for regional drug delivery. J Clin Oncol 2: 498-505, 1984.

Received December 13, 2006 Revised February 23, 2007 Accepted March 5, 2007

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