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Magnetic drug targeting: biodistribution and dependency on magnetic field strength
Journal of Magnetism and Magnetic Materials 252 (2002) 363–366
Magnetic drug targeting: biodistribution and dependency on magnetic field strength$ Ch. Alexioua,*, A. Schmidtb, R. Kleinb, P. Hulinb, Ch. Bergemannc, W. Arnoldb b
a Department of Otorhinolaryngology, Head and Neck Surgery, University of Erlangen-Nurnberg, Erlangen, Germany . Department of Otorhinolaryngology, Head and Neck Surgery, Klinikum Rechts der Isar, Technical University of Munich, Isamaninger Strasse 22, 81675 Munich, Germany c Chemicell, 10777 Berlin, Germany
Abstract ‘‘Magnetic drug targeting,’’ a model of locoregional chemotherapy showed encouraging results in treatment of VX2squamous cell carcinoma in rabbits. In the present study we investigated the biokinetic behavior of Iod[123]-labelled ferrofluids in vivo and showed in vitro that the ferrofluid concentration is dependent on the magnetic field strength. r 2002 Elsevier Science B.V. All rights reserved. Keywords: Cancer therapy; Ferrofluids; Magnetic nanoparticles
1. Introduction Biocompatible ferrofluids are magnetic nanoparticles, that may be used as a delivery system for anticancer agents in locoregional tumor therapy, called ‘‘magnetic drug targeting’’. In previous studies we have shown that magnetic drug targeting is a sufficient and successful treatment modality of VX2 squamous cell carcinoma in rabbits [1]. Through this form of target directed drug application, one attempts to concentrate a pharmacological agent at its site of action in order to minimize unwanted side effects in the organism and to increase its locoregional effectiveness. Magnetic fluids are used in medicine since 1960 for e.g. the magnetically controlled metallic thrombosis of intracranial aneurysms [2]. Ferrofluids are also used as a contrast agent for MRI. In our studies we could demonstrate that after intraarterial application $
Supported by the Margarete Ammon Foundation, Munich, . and Forschungsforderung Medizin-Technik of the Technical University, Munich, Germany. *Corresponding author. Tel.: +49-9131-8533141; Fax: 9131853687. E-mail address: [email protected] (C. Alexiou).
ferrofluids are concentrated in a circumscript tumor region at least for 6 h with an external magnetic field. These results could also be confirmed in histological investigation [1,3]. In the present study we investigate the enrichment of Iod[123]-labelled ferrofluids in vivo and the dependency of magnetic field strength in vitro.
2. Materials and methods 2.1. Ferrofluids The ferrofluids used in the experiments were obtained from Chemicell (Berlin, Germany; German patent application No. 19624426.9) and consisted of a biocompatible colloidal dispersion formed by wet chemical methods from iron oxides and hydroxides to produce special multidomaine particles [4]. These ferrofluids are covered with hydrophilic starch polymers coupled with endstanding functional groups (e.g. phosphate) allowing ionic binding to many cationic therapeutic drugs. The hydrodynamic diameter of the whole magnetic nanoparticle is about 100 nm. Several agents can ionically bound to the functional group phosphate or in the case of Iod in this study as a iodine–starch complex [5].
0304-8853/02/$ - see front matter r 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 8 8 5 3 ( 0 2 ) 0 0 6 0 5 - 4
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C. Alexiou et al. / Journal of Magnetism and Magnetic Materials 252 (2002) 363–366
2.2. Iod[123]-, Iod[125]-ferrofluid particles Iod[123] has been received by CYGNE bv, Eindhoven, The Netherlands and Iod[125] by Amersham Pharmacia Biotech, Buckinghamshire, England. Concentrate suspension of ferrofluids has been incubated with 11 MBq Iod[125], respectively, 64 MBq Iod[123] in a IODOGEN (Pierce, USA) coated vial and separated from Iodid by a Sephadex column and magnetic sedimentation. For in vitro experiments volumina of 200 ml of a highly diluted stock solution has been distributed in 96-well plates and has been incubated on a fitting 96 magnet plate. After 5, 10, 15, 30, 60 and 90 min the upper and the lower phase has been separated: 100 ml each has been filled in countervials and the activities of upper and lower phase has been countered separate in a gammacounter. Three probes have been measured for each time and condition (0, 0.2 and 0.4 T). For in vivo experiments ferrofluids (amount: 1 ml ff; 6 mg Fe3O4/ml ff) have been labelled under sterile conditions and injected in the femoral artery by focusing an permanent neodym magnet (NdFeB) on the tumor and during the detection of activity by a gamma-camera for 100 min.
2.0 g/l Na HCO3, l-Glutamin, Seromedt, Biochrom, Berlin, Germany) and were immediately implanted under sterile conditions into the hind limb of anesthetized recipient rabbits in the supplied area of the femoral artery. The experiments were performed when the tumors have reached a volume of approximately 3500 mm3. For application of the ferrofluids, the animals were anesthetized with an intramuscular injection of ketamine 35 mg/kg body weight (Narketan 10t, Chassot, Bern, Switzerland) and xylazine 5 mg/kg body weight (Xylapant, Chassot, Bern, Switzerland), the femoral artery was cannulized and an indwelling catheter (Venflon, 0.8 mm, Ohmeda Co., Helsingburg, Sweden) was placed after separation of the femoral vein and the saphenous nerve approximately 2 cm distal to the inguinal furrow. The Iod[123]-labelled ferrofluids were administered over a period of 4 min. To prevent thrombosis, prophylaxis consisting of heparin sodium (Heparin-Natrium-25,000 Ratiopharmt, Ratiopharm, Ulm, Germany) was given preoperatively, once postoperatively and twice daily for 5 days postoperatively (200 IU/kg body weight, s.c.)
4. Results 3. Magnetic field A neodym permanent magnet with a magnetic flux density of a maximum of 0.6 T was used to produce an inhomogenous magnetic field for in vivo experiments. The maximum of the magnetic flux density was focused onto the region of the tumor. The magnetic field was focused on the tumor during the ferrofluid injection and for 100 min in total. For in vitro experiments 96 neodym permanent magnets of 0.2 and 0.4 T have been embedded in perspex, fitting the 96-well plates. 3.1. VX2 squamous cell carcinoma The VX2 squamous cell carcinoma was obtained from Deutsches Krebsforschungszentrum (Heidelberg, Germany). The rabbits develop within 2–3 weeks central tumor necroses, locoregional lymph node metastases and hematogenous metastases. 3.2. Animals The experimental animals were female New Zealand White rabbits (2000–2500 g body weight, 12–15 weeks old; Charles River, Sulzfeld, Germany). 3.3. Surgical intervention Fragments of viable VX2 tissue 1 mm in size were taken from the tumor periphery in donor animals. These fragments were placed in a special medium (RPMI 1640,
4.1. Localization of ferrofluids in the tumor tissue after magnetic drug targeting Biodistribution was studied by the use of Iod[123]labelled ferrofluids. The scintigraphically detected Iod[123]-signal after intraarterial (artery supplying the tumor, femoral artery) application in the left femoral artery (supplying the left hind limb of the rabbit, including the tumor) has been shown to be significantly higher in the magnetically focused region compared to the application without external magnetic field (Fig. 1) over the observation period of 100 min. Seventy-seven per cent of the initial activity has been detected over the tumor area by detection of the gammacamera 10 min after the ferrofluid application versus 40% without magnet. Thirty minutes later 40% of the initial activity has been detected versus 14% without magnet and 60 min later still 22% of initial activity has been measured versus 11%. After 10 min one could observe iodine accumulation in the kidney and bladder of the rabbit, which is a clear index for free iodine in the organism, because ferrofluids are not excreted by the kidney, but by the liver, which is accumulating activity. 4.2. In vitro sedimentation study of Iod[125]-labelled ferrofluids To examine dependency of the magnetic field strength, ferrofluids were labelled by Iod[125]. A homo-
C. Alexiou et al. / Journal of Magnetism and Magnetic Materials 252 (2002) 363–366
genous colloidal dispersion has been distributed on 96well plates and has been incubated on 96 magnetic plates. Sedimentation influenced by different magnetic field strength (gravitation, 0.2 and 0.4 T) has been examined over the time (90 min) as shown in Fig. 2. Application of 0.2 T magnetic plates resulted after 5 min in 10%, 10 min in 20.5%, 15 min in 25.6%, 30 min in 43.7%, 60 min in 57.3% and after 90 min in 62.6% of sedimented activity. Application of 0.4 T magnetic plates resulted after 5 min in 28.8%, 10 min in 38.5%, 15 min in
Fig. 1. Enrichment of Iod[123]-labelled nanoparticles in the region of interest (VX2-tumor) after magnetic drug targeting. The images were taken 10 min after FF-application showing stable concentration of ferrofluids within the tumor tissue.
Fig. 2. Sedimentation of Iod[125]-labelled ferrofluids in dependence of magnetic field strength over the time. No sedimentation has been observed after 90 min with gravitation alone. More ferrofluid-bound activity sedimented by a stronger magnetic field (0.4 T).
365
44.6%, 30 min in 60.3%, 60 min in 71.1% and after 90 min in 75.3% activity. No sedimentation could be detected with gravitation alone. The activity in the lower phase (including FF-pellet) shows to be dependent on magnetic field strength.
5. Discussion The difference between success and failure of chemotherapy depends not only on the drug itself but also on how it is delivered to its target. Because of the relatively non-specific action of chemotherapeutic agents, there is almost always some toxicity to normal tissues. Therefore, it is of great importance to be able to selectively target the antineoplastic agent to the tumor target as precisely as possible, to reduce resulting systemic toxic side effects from generalized systemic distribution and to be able to use a much smaller dose, which would further lead to a reduction of toxicity. At present, i.a. delivery of chemotherapeutic agents is approved and well accepted for treatment of liver metastases [6] and has occasionally been used for other tumor types also (e.g., inoperable head and neck tumors), but it has often necessitated complicated, time-consuming operative procedures, including general anaesthesia [7]. The principle of magnetic drug targeting is to deliver the antineoplastic agent to the tumor region without harming healthy tissue. It has been used as a carrier system in locoregional cancer treatment in tumor bearing rabbits [1]. Enrichment of the intraarterially injected ferrofluids were documented in vivo by MRI and histology after magnetic drug targeting recently. In MRI the magnetic particles strongly reduce the transverse relaxation time (T2) and lower significantly the longitudinal relaxation time (T1). The MRI was made 6 h after treatment with magnetic drug targeting and the concentration of ferrofluids was seen by extinction of signal in the area of the tumor [3]. Biodistribution by Iod[123]-labelled ferrofluids demonstrate the accumulation of ferrofluids in tumor tissue by focusing an external magnetic field of 0.6 T. After 10 min there were approximately 100% more ferrofluids detectable in the tumor region after magnetic drug targeting compared to the ones without magnetic field (Fig. 1). The magnetic flux density is supposed to be an important factor in magnetic drug targeting. We therefore investigated the sedimentation characteristic of Iod[125]-labelled ferrofluids in dependency of magnetic flux density in vitro. In the observation period (90 min) no sedimentation could be seen by gravitation alone. Wells exposed to 0.4 T magnetic plates showed after 10 min approximately 100% more sedimentation compared to the ones exposed to 0.2 T magnetic plates. This
366
C. Alexiou et al. / Journal of Magnetism and Magnetic Materials 252 (2002) 363–366
underscore the importance of the magnetic flux density in this model. Taken together, focused magnetic flux density resulted in a high concentration of ferrofluids and this is dependent on the amount of Tesla applied. Further investigations with magnetic drug targeting have to be done to show, if the increase of magnetic flux density may also increase therapeutic efficacy in vivo.
References [1] C. Alexiou, W. Arnold, R.J. Klein, et al., Cancer Res. 60 (2000) 6641.
[2] J.F. Alksne, A. Fingerhut, R. Rand, Surgery 60 (1966) 212. [3] C. Alexiou, et al., J.Magn. Magn. Mater. 225 (2001) 187. [4] A.S. Lubbe, . C. Bergemann, W. Huhnt, et al., Cancer Res. 56 (1996) 4694. [5] C. Bergemann, D. Muller-Schulte, . J. Oster, L. a" Brassard, A.S. Lubbe, . J. Magn. Magn. Mater. 194 (1999) 45. [6] K.H. Link, M. Kornmann, A. Formenti, et al., Langenbecks Arch. Surg. 384 (1999) 344. [7] J. Scheel, Die intraarterielle chemo-therapie, in: H.H. Naumann, J. Helms, C. Herberhold, E. Kastenbauer (Eds.), Oto-Laryngologie in Klinik und Praxis, Thieme, Stuttgart, 1998, pp. 457–460.
Magnetic drug targeting: biodistribution and dependency on magnetic field strength$ Ch. Alexioua,*, A. Schmidtb, R. Kleinb, P. Hulinb, Ch. Bergemannc, W. Arnoldb b
a Department of Otorhinolaryngology, Head and Neck Surgery, University of Erlangen-Nurnberg, Erlangen, Germany . Department of Otorhinolaryngology, Head and Neck Surgery, Klinikum Rechts der Isar, Technical University of Munich, Isamaninger Strasse 22, 81675 Munich, Germany c Chemicell, 10777 Berlin, Germany
Abstract ‘‘Magnetic drug targeting,’’ a model of locoregional chemotherapy showed encouraging results in treatment of VX2squamous cell carcinoma in rabbits. In the present study we investigated the biokinetic behavior of Iod[123]-labelled ferrofluids in vivo and showed in vitro that the ferrofluid concentration is dependent on the magnetic field strength. r 2002 Elsevier Science B.V. All rights reserved. Keywords: Cancer therapy; Ferrofluids; Magnetic nanoparticles
1. Introduction Biocompatible ferrofluids are magnetic nanoparticles, that may be used as a delivery system for anticancer agents in locoregional tumor therapy, called ‘‘magnetic drug targeting’’. In previous studies we have shown that magnetic drug targeting is a sufficient and successful treatment modality of VX2 squamous cell carcinoma in rabbits [1]. Through this form of target directed drug application, one attempts to concentrate a pharmacological agent at its site of action in order to minimize unwanted side effects in the organism and to increase its locoregional effectiveness. Magnetic fluids are used in medicine since 1960 for e.g. the magnetically controlled metallic thrombosis of intracranial aneurysms [2]. Ferrofluids are also used as a contrast agent for MRI. In our studies we could demonstrate that after intraarterial application $
Supported by the Margarete Ammon Foundation, Munich, . and Forschungsforderung Medizin-Technik of the Technical University, Munich, Germany. *Corresponding author. Tel.: +49-9131-8533141; Fax: 9131853687. E-mail address: [email protected] (C. Alexiou).
ferrofluids are concentrated in a circumscript tumor region at least for 6 h with an external magnetic field. These results could also be confirmed in histological investigation [1,3]. In the present study we investigate the enrichment of Iod[123]-labelled ferrofluids in vivo and the dependency of magnetic field strength in vitro.
2. Materials and methods 2.1. Ferrofluids The ferrofluids used in the experiments were obtained from Chemicell (Berlin, Germany; German patent application No. 19624426.9) and consisted of a biocompatible colloidal dispersion formed by wet chemical methods from iron oxides and hydroxides to produce special multidomaine particles [4]. These ferrofluids are covered with hydrophilic starch polymers coupled with endstanding functional groups (e.g. phosphate) allowing ionic binding to many cationic therapeutic drugs. The hydrodynamic diameter of the whole magnetic nanoparticle is about 100 nm. Several agents can ionically bound to the functional group phosphate or in the case of Iod in this study as a iodine–starch complex [5].
0304-8853/02/$ - see front matter r 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 8 8 5 3 ( 0 2 ) 0 0 6 0 5 - 4
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C. Alexiou et al. / Journal of Magnetism and Magnetic Materials 252 (2002) 363–366
2.2. Iod[123]-, Iod[125]-ferrofluid particles Iod[123] has been received by CYGNE bv, Eindhoven, The Netherlands and Iod[125] by Amersham Pharmacia Biotech, Buckinghamshire, England. Concentrate suspension of ferrofluids has been incubated with 11 MBq Iod[125], respectively, 64 MBq Iod[123] in a IODOGEN (Pierce, USA) coated vial and separated from Iodid by a Sephadex column and magnetic sedimentation. For in vitro experiments volumina of 200 ml of a highly diluted stock solution has been distributed in 96-well plates and has been incubated on a fitting 96 magnet plate. After 5, 10, 15, 30, 60 and 90 min the upper and the lower phase has been separated: 100 ml each has been filled in countervials and the activities of upper and lower phase has been countered separate in a gammacounter. Three probes have been measured for each time and condition (0, 0.2 and 0.4 T). For in vivo experiments ferrofluids (amount: 1 ml ff; 6 mg Fe3O4/ml ff) have been labelled under sterile conditions and injected in the femoral artery by focusing an permanent neodym magnet (NdFeB) on the tumor and during the detection of activity by a gamma-camera for 100 min.
2.0 g/l Na HCO3, l-Glutamin, Seromedt, Biochrom, Berlin, Germany) and were immediately implanted under sterile conditions into the hind limb of anesthetized recipient rabbits in the supplied area of the femoral artery. The experiments were performed when the tumors have reached a volume of approximately 3500 mm3. For application of the ferrofluids, the animals were anesthetized with an intramuscular injection of ketamine 35 mg/kg body weight (Narketan 10t, Chassot, Bern, Switzerland) and xylazine 5 mg/kg body weight (Xylapant, Chassot, Bern, Switzerland), the femoral artery was cannulized and an indwelling catheter (Venflon, 0.8 mm, Ohmeda Co., Helsingburg, Sweden) was placed after separation of the femoral vein and the saphenous nerve approximately 2 cm distal to the inguinal furrow. The Iod[123]-labelled ferrofluids were administered over a period of 4 min. To prevent thrombosis, prophylaxis consisting of heparin sodium (Heparin-Natrium-25,000 Ratiopharmt, Ratiopharm, Ulm, Germany) was given preoperatively, once postoperatively and twice daily for 5 days postoperatively (200 IU/kg body weight, s.c.)
4. Results 3. Magnetic field A neodym permanent magnet with a magnetic flux density of a maximum of 0.6 T was used to produce an inhomogenous magnetic field for in vivo experiments. The maximum of the magnetic flux density was focused onto the region of the tumor. The magnetic field was focused on the tumor during the ferrofluid injection and for 100 min in total. For in vitro experiments 96 neodym permanent magnets of 0.2 and 0.4 T have been embedded in perspex, fitting the 96-well plates. 3.1. VX2 squamous cell carcinoma The VX2 squamous cell carcinoma was obtained from Deutsches Krebsforschungszentrum (Heidelberg, Germany). The rabbits develop within 2–3 weeks central tumor necroses, locoregional lymph node metastases and hematogenous metastases. 3.2. Animals The experimental animals were female New Zealand White rabbits (2000–2500 g body weight, 12–15 weeks old; Charles River, Sulzfeld, Germany). 3.3. Surgical intervention Fragments of viable VX2 tissue 1 mm in size were taken from the tumor periphery in donor animals. These fragments were placed in a special medium (RPMI 1640,
4.1. Localization of ferrofluids in the tumor tissue after magnetic drug targeting Biodistribution was studied by the use of Iod[123]labelled ferrofluids. The scintigraphically detected Iod[123]-signal after intraarterial (artery supplying the tumor, femoral artery) application in the left femoral artery (supplying the left hind limb of the rabbit, including the tumor) has been shown to be significantly higher in the magnetically focused region compared to the application without external magnetic field (Fig. 1) over the observation period of 100 min. Seventy-seven per cent of the initial activity has been detected over the tumor area by detection of the gammacamera 10 min after the ferrofluid application versus 40% without magnet. Thirty minutes later 40% of the initial activity has been detected versus 14% without magnet and 60 min later still 22% of initial activity has been measured versus 11%. After 10 min one could observe iodine accumulation in the kidney and bladder of the rabbit, which is a clear index for free iodine in the organism, because ferrofluids are not excreted by the kidney, but by the liver, which is accumulating activity. 4.2. In vitro sedimentation study of Iod[125]-labelled ferrofluids To examine dependency of the magnetic field strength, ferrofluids were labelled by Iod[125]. A homo-
C. Alexiou et al. / Journal of Magnetism and Magnetic Materials 252 (2002) 363–366
genous colloidal dispersion has been distributed on 96well plates and has been incubated on 96 magnetic plates. Sedimentation influenced by different magnetic field strength (gravitation, 0.2 and 0.4 T) has been examined over the time (90 min) as shown in Fig. 2. Application of 0.2 T magnetic plates resulted after 5 min in 10%, 10 min in 20.5%, 15 min in 25.6%, 30 min in 43.7%, 60 min in 57.3% and after 90 min in 62.6% of sedimented activity. Application of 0.4 T magnetic plates resulted after 5 min in 28.8%, 10 min in 38.5%, 15 min in
Fig. 1. Enrichment of Iod[123]-labelled nanoparticles in the region of interest (VX2-tumor) after magnetic drug targeting. The images were taken 10 min after FF-application showing stable concentration of ferrofluids within the tumor tissue.
Fig. 2. Sedimentation of Iod[125]-labelled ferrofluids in dependence of magnetic field strength over the time. No sedimentation has been observed after 90 min with gravitation alone. More ferrofluid-bound activity sedimented by a stronger magnetic field (0.4 T).
365
44.6%, 30 min in 60.3%, 60 min in 71.1% and after 90 min in 75.3% activity. No sedimentation could be detected with gravitation alone. The activity in the lower phase (including FF-pellet) shows to be dependent on magnetic field strength.
5. Discussion The difference between success and failure of chemotherapy depends not only on the drug itself but also on how it is delivered to its target. Because of the relatively non-specific action of chemotherapeutic agents, there is almost always some toxicity to normal tissues. Therefore, it is of great importance to be able to selectively target the antineoplastic agent to the tumor target as precisely as possible, to reduce resulting systemic toxic side effects from generalized systemic distribution and to be able to use a much smaller dose, which would further lead to a reduction of toxicity. At present, i.a. delivery of chemotherapeutic agents is approved and well accepted for treatment of liver metastases [6] and has occasionally been used for other tumor types also (e.g., inoperable head and neck tumors), but it has often necessitated complicated, time-consuming operative procedures, including general anaesthesia [7]. The principle of magnetic drug targeting is to deliver the antineoplastic agent to the tumor region without harming healthy tissue. It has been used as a carrier system in locoregional cancer treatment in tumor bearing rabbits [1]. Enrichment of the intraarterially injected ferrofluids were documented in vivo by MRI and histology after magnetic drug targeting recently. In MRI the magnetic particles strongly reduce the transverse relaxation time (T2) and lower significantly the longitudinal relaxation time (T1). The MRI was made 6 h after treatment with magnetic drug targeting and the concentration of ferrofluids was seen by extinction of signal in the area of the tumor [3]. Biodistribution by Iod[123]-labelled ferrofluids demonstrate the accumulation of ferrofluids in tumor tissue by focusing an external magnetic field of 0.6 T. After 10 min there were approximately 100% more ferrofluids detectable in the tumor region after magnetic drug targeting compared to the ones without magnetic field (Fig. 1). The magnetic flux density is supposed to be an important factor in magnetic drug targeting. We therefore investigated the sedimentation characteristic of Iod[125]-labelled ferrofluids in dependency of magnetic flux density in vitro. In the observation period (90 min) no sedimentation could be seen by gravitation alone. Wells exposed to 0.4 T magnetic plates showed after 10 min approximately 100% more sedimentation compared to the ones exposed to 0.2 T magnetic plates. This
366
C. Alexiou et al. / Journal of Magnetism and Magnetic Materials 252 (2002) 363–366
underscore the importance of the magnetic flux density in this model. Taken together, focused magnetic flux density resulted in a high concentration of ferrofluids and this is dependent on the amount of Tesla applied. Further investigations with magnetic drug targeting have to be done to show, if the increase of magnetic flux density may also increase therapeutic efficacy in vivo.
References [1] C. Alexiou, W. Arnold, R.J. Klein, et al., Cancer Res. 60 (2000) 6641.
[2] J.F. Alksne, A. Fingerhut, R. Rand, Surgery 60 (1966) 212. [3] C. Alexiou, et al., J.Magn. Magn. Mater. 225 (2001) 187. [4] A.S. Lubbe, . C. Bergemann, W. Huhnt, et al., Cancer Res. 56 (1996) 4694. [5] C. Bergemann, D. Muller-Schulte, . J. Oster, L. a" Brassard, A.S. Lubbe, . J. Magn. Magn. Mater. 194 (1999) 45. [6] K.H. Link, M. Kornmann, A. Formenti, et al., Langenbecks Arch. Surg. 384 (1999) 344. [7] J. Scheel, Die intraarterielle chemo-therapie, in: H.H. Naumann, J. Helms, C. Herberhold, E. Kastenbauer (Eds.), Oto-Laryngologie in Klinik und Praxis, Thieme, Stuttgart, 1998, pp. 457–460.