JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 94, No. 6,6OC613. 2002

REVIEW

Functional Magnetic Particles for Medical Application MASASHIGE SHINKAI’

Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo 113-8656, Japan’

Received 31 July 2002/Accepted 9 August 2002

Magnetic particles for medical applications have been developed by many researchers. Since magnetic particles have unique magnetic features not present in other materials, they can be ap- plied to special medical techniques. Separation, immunoassay, magnetic resonance imaging (MRI), drug delivery, and hyperthermia are enhanced by the use of magnetic particles. Magnetite cationic liposomes (MCLs), one of the group of cationic magnetic particles, can be used as carriers to introduce DNA into cells since their positively charged surface associates with the negatively charged DNA. They can also be used as heat mediators for cancer therapy. Magnetic particles conjugated with tumor-specific antibodies have enabled tumor-specific contrast enhancement in MRI. In addition, antibody-conjugated magnetic particles were shown to target renal cell carci- noma cells, and are applicable to the hyperthermic treatment of carcinomas. The use of magnetic particles with their unique features will further improve medical techniques.

[Key words: magnetic particle, magnetic resonance imaging, hyperthermia, drug delivery, magnetic carrier]

Since the mid-1970s, magnetic particles have increas- ingly been used in the area of bioscience and medicine. The unique feature of magnetic particles is their reaction to a remote magnetic force. Magnetic particles are attracted to high magnetic flux density and this feature is used for drug targeting and bioseparation including cell sorting. Hystere- sis loss in the alternative magnetic field is a very important feature of magnetic particles, because it enables effective thermotherapy (hyperthermia). Of course, magnetic parti- cles themselves generate a magnetic field and influence the local area around them. This feature is exploited in mag- netic resonance imaging (MRI). In these applications, mag- netic particles are given various names, e.g., magnetic mi- crospheres, magnetic nanospheres, and ferrofluids among others. Here, I use the general term ‘magnetic particles,’ and summarize their medical applications.

I. MAGNETIC SEPARATION FOR PURIFICATION AND IMMUNOASSAY

Separation is the most documented and currently the most useful application of magnetic particles. Many mag- netic particles have been developed as magnetic carriers in separation processes including purification and immuno- assay (l-4). Specific ligands are bound to magnetic parti- cles in the same way as to other carriers comprising a poly- mer matrix. Separation techniques are extremely important in process engineering including bioprocessing. For exam-

e-mail: shinkai@bio.t.u-tokyo.ac.jp phone: +81-(0)3-5841-7357 fax: +81-(0)3-5841-8657

ple, in the case of fermentation processes, it has been esti- mated that downstream processing represents 40% or more of the added value of a fermentation product (5). In labora- tory use, automatic DNA/RNA purification apparatuses are commercially available. With respect to medical applica- tions, magnetic cell sorting was developed for cellular ther- apy. In the removal of a specific cell population from blood, other dominant blood cells interfere with conventional im- munoaffinity column chromatography (6). In blood purifi- cation, the elimination of specific cytokines such as inter- leukin-I or tumor necrosis factor, the major pathogenic me- diators in septic shock and multi-organ failure, by magnetic particles has been proposed (7).

Magnetic particles are also used in immunoassays (8, 9). Immunoassays are widely used in the field of medical ex- amination (e.g., detection of viruses or assay of various hor- mones) (10, 11). Magnetic separation is a good way to shorten both adsorption and separation steps (12).

Recently, gene transfer using magnetic carriers has been proposed by some researchers (13). In clinical use, somatic cells transformed to produce specific proteins require quick selection from the limited cell cycle. Therefore, quick selec- tion of transformants among a transiently transformed pop- ulation is important. Selection using antibiotic-resistance marker genes is, however, time consuming and recovery of transformants cannot be guaranteed, because the transfor- mation efficiency depends on the cell line used. Therefore, a more simple and quick selection method was desired. For such an application, magnetite cationic liposomes (MCLs) were used as carriers to introduce DNA into cells since their positively charged surface associates with the negatively

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TABLE 1. Effect of magnetic separation on transformant selection

Cell line Specific luciferase activity (RLU/cell)

Total cells before separation Trapped cells Transformant selection index”

Rat glioma cell T-9 4.37 21.1 4.82 Mouse tibroblast NIW3T3 1.74 12.2 6.99 Monkey tibroblast CV-1 0.623 0.813 1.29 Monkey fibroblast COS-1 904 2830 3.13

a Transformant selection index = (activity of trapped cells)/(activity of total cells before separation). In the case of the mouse tibroblast NW3T3 and the monkey fibroblast-like cell line COS-1, high values of the index were obtained as was the

case for T-9 cells. CV-1 cells did not exhibit a high specific luciferase activity. This may be due to the low transfection efficiency by MCLs. since the specific luciferase activity of total cells before separation was also low.

charged DNA (14). MCLs are cationic liposomes contain- ing magnetite particles and have been developed to promote the uptake of magnetic particles by target cells (15). When MCLs associated with plasmid vector were transfected into NIW3T3 cells, the specific activity of luciferase gene-trans- fectants was about 7 times higher than that of the cells be- fore separation by the magnetic field (Table 1). Some re- searchers have reported on the separation of transiently transfected cells using magnetic particles associated with antibodies (16, 17). However, this method requires co-trans- fection of a gene for expression of a surface marker. In our method, gene expression of an additional marker is not required for separation and magnetic separation can be performed immediately after the transformation. This is a major advantage for separation of transformants.

II. DRUG DELIVERY AND TARGETING

The treatment of solid tumors using chemotherapy has been limited by systemic toxicity resulting in sub-optimal dosing and thus limited efficacy. Regional therapy achieved through targeted drug delivery could improve efficacy by increasing the drug concentration at the tumor while limit- ing systemic drug concentrations. Magnetic drug targeting should be safe and effective, i.e., with the least amount of magnetic particles a maximum concentration of drug should be easily administered and transported to the site of choice.

The use of magnetic particles for the delivery of chemo- therapeutic agents has evolved since the 1970s. Widder et al. (18) and Morimoto et al. (19) developed albumin micro- spheres encasing anticancer drugs. The magnetic particles injected into an animal were retained at the magnet site de- pending on the magnetic field strength. It has also been shown that magnetic particles can be retained in other parts of the body depending on the placement of the external magnet (20). Therefore, the magnetic force enhances the uptake of magnetic particles by tumor cells. In a rat subcu- taneous tumor, the uptake ratio using a magnet was 70.0% of the total magnetic particles injected, while in the case where no magnet was used it was only 27.4% (21).

I believe that the geometry of the magnetic field is very important. In the conventional magnetic field geometry, the magnetic force works as the magnetic particles are adsorbed on the surface of the magnet. Therefore, the administration method is limited to an artery close to the tumor or to some other target close to the tumor. A novel magnetic field geo- metry is needed to realize completely targetable magnetic drug delivery.

III. MAGNETIC RESORNANCE IMAGING AND DIAGNOSIS

Magnetic resonance (MR) imaging offers the advantage of high spatial resolution of contrast differences between tissues. Due to the unique function of this imaging modality, there is a need to develop effective contrast agents that will enhance and widen its diagnostic utility. Paramagnetic ion chelates and ferromagnetic or superparamagnetic particles have been investigated as contrast agents and used in clini- cal diagnosis.

During recent years, there has been increasing interest in the use of paramagnetic contrast agents like dextran mag- netite in magnetic resonance imaging (MRI) (22, 23). Com- pared with paramagnetic ions, superparamagnetic iron oxide particles have higher molar relaxivities, and, when used as blood pool and tissue-specific agents, may offer advantages at low concentrations (24,25).

Commercial contrast agents do not have targetability but are useful for enhancing contrast differences of the lymph nodes or liver. We and other researchers have been studying the specific distribution of these agents by active targeting (26,27).

Shinkai et al. have also proposed a direct sensing method of magnetic particles for cancer diagnosis. Accurate sens- ing of brain tumors during brain surgery is important, and MRl, for example, has been used during surgical operations. Shinkai et al. developed a small and highly sensitive mag- netic sensor, the magneto-impedance (MI) sensor for the sensing of tumor tissue incorporating magnetic particles and tried to detect magnetite particles in a rat glioma, a malig- nant brain tumor which was transplanted under the skin of the rat leg (Fig. 1) (28, 29). The magnetite cationic lipo- somes were directly injected into the tumor and magnetized by a static magnetic field generator at 1.8 T. By using the MI sensor, the magnetic field distribution of the MCLs was detected, and its distribution indicated that the MCLs were localized in the tumor.

However, such apparatuses for the above methods are ex- perimental and, unlike MRI, are not used in clinical prac- tice. When these apparatuses are available for clinical use, the diagnosis of metastatic cancer with high sensitivity will be possible.

IV. HYPERTHERMIA

Hyperthermia is one of the promising approaches in can- cer therapy. Various methods are employed in hyperthetmia

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Area of the tumor (top view)

FIG. 1. Schematic illustration of the magnetic field sensing of a subcutaneous tumor in a rat and the magnetic field distribution in the transplanted tumor. The shaded circle on the x-y plane indicates the projection of the tumor. The magnetic particles were directly injected into the tumor transplanted under the skin of the rat leg and magne- tized by a static magnetic field generator at 1.8 T. By using the MI sen- sor, the magnetic field distribution of the MCLs was detected, and the distribution indicated that the MCLs were localized in the tumor.

such as the use of hot water, capacitive heating, and induc- tion heating among others (30-33). However, the inevitable technical problem with hyperthermia is the difficulty of the uniform heating of only the tumor region until the required temperature is reached without damaging normal tissue.

Therefore, some researchers have proposed intracellular hyperthermia and developed submicron magnetic particles for hyperthermia (34-36). This idea is based on the prin- ciple that a magnetic particle can generate heat by hystere- sis loss under a high frequency magnetic field, such as 500 kHz (33). Even in the early days of hyperthermia therapy, the use of magnetic particles was proposed for localized hyperthermia treatment, and local temperature elevations were achieved (37). Administered particles usually migrate to reticuloendothelial systems like the macrophages and Kupffer cells in the liver, spleen, and lymph nodes (38, 39). Lymph nodes are sites in which metastatic tumors easily grow. However, the delivery of magnetic particles is passive and its control is very difficult.

In 1979, Gordon et al. first proposed the concept of intra- cellular hyperthermia using dextran magnetite nanoparticles (40). They administered magnetic particles intravenously to Sprague-Dawley rats bearing mammary carcinomas and showed that alternative magnetic-field-induced heating oc- curred in their in vivo experiments. Jordan et al. have pro-

posed magnetic fluid hyperthermia in several more compre- hensive in vitro studies (41).

Regarding their application to hyperthermia, the impor- tant properties of magnetic particles are non-toxicity, bio- compatiblilty, injectability, high-level accumulation in the target tumor and effective absorption of the energy of alter- nating magnetic fields. Chan et ~2. reported on modified dextran magnetite and its hyperthermic effect using several human carcinoma cell lines in vitro (42). The specific ab- sorption rate (SAR), which indicates the heat evolution rate in hyperthermia, of the conventional dextran magnetite is low. Dextran magnetite behaves as a superparamagnetic particles rather than a ferromagnetic one due to its small size, so that its hysteresis loss is very low. Chan et al. con- trolled the oxygen concentration in the preparation of the dextran magnetite and selected particles of around 15 nm. We have reported that particle size is a critical factor in ob- taining a high SAR value (43). In the case of magnetite, the SAR of 35-nm particles is over two times higher than that of 1 0-nm particles.

Hyperthermia using magnetite cationic liposomes Accumulation of magnetic particles in tumor cells can be enhanced by their surface charge. We developed ‘magnetite cationic liposomes’ (MCLs) with improved adsorption and accumulation properties (16). MCLs, which have a positive surface charge, show ten-fold higher affinity for glioma cells than neutrally charged magnetoliposomes. They have a sufficiently high SAR and a general biocompatibility that is comparable to that of dextran magnetite. We previously re- ported the hyperthermic effect of MCLs (16,44,45). MCLs were injected into solid tumors formed subcutaneously in F344 rats and the rats were irradiated three times for 30 min with an alternating magnetic field. The temperature of the tumor was elevated rapidly by magnetic heating and reached over 43°C after 15 min. In contrast, the rectal tem- perature or that in a tumor lacking the MCLs remained be- tween 35-37°C. When magnetic field irradiation was ex- tended beyond 30 min, the tumor temperature at the outside skin continued to increase gradually. Figure 2 shows the time courses of tumor growth of 5 rats in each group. In control animals (indicated by ‘no irradiation’ in Fig. 2), the tumor volume in each rat steadily increased with no evi- dence of regression. In contrast, complete tumor regression was observed in many of the rats subjected to 3 separate magnetic field irradiations. In most cases of complete re- gression, the tumor volume increased up to the 12th day, after which it began to decrease and the tumor finally dis- appeared. No regrowth of tumors was observed following complete regression over a period of 3 months. In the pre- sent study, SO-90% of the injected MCLs accumulated in the tumor tissue, whereas only 20-25% of the neutral mag- netoliposomes were accumulated. This high accumulation of MCLs is considered to be due to the cationic charge on the surface of the liposome used, and a significant hyper- thermic effect was achieved as shown in Fig. 2.

Ohno et ~2. have also improved the method of administra- tion of the magnetic particles. The needle-type magnetite consists of magnetite and carboxymethyl cellulose (Fig. 3) and it is as easy to administer as an injection needle (46). This method was applied to rat brain tumor and high eff-

VOL. 94,2002 MAGNETIC PARTICLES FOR MEDICAL APPLICATION 609

x lo4 6

No irradiation 1

Days after MCL injection

Irradiated twice

x lo4 6

Irradiated once

0 10 20 30 Days after MCL injection

x lo4 h I

Irradiated three times

"0 10 20 30 Days after MCL injection

“0 10 20 30 Days after MCL injection

FIG. 2. The time courses of tumor growth of 5 rats in each group. In non-irradiated animals, the tumor volume in each rat steadily increased with no evidence of regression. In contrast, complete tumor regression was observed in 90% of the rats irradiated by the magnetic field. Regrowth of the tumor over a 3-month period was not observed following complete regression.

cacy was obtained. The time taken to administer two mag- netite needles was only 3 min, although the injection time for the magnetite cationic liposomes (MCLs) was at least 120 min because the backflow of the MCLs had to be pre- vented. The needle-type magnetite is composed of magnetic nanoparticles and carboxymethyl cellulose, and this mate- rial is dispersed in water gradually. Dispersed magnetic par- ticles in tissue are removed eventually by the blood flow. Therefore, it is not necessary for the magnetite needles to be removed following therapeutic treatment. These results sug- gest that administration of the needle-type magnetite is a simple and effective method for mediating therapeutic par- ticulate heating in hyperthermia treatment.

Hyperthermia using antibody-conjugated magnetic

FIG. 3. Photograph of needle-type magnetite. Average diameter is 0.6 mm.

particles Administration of the MCLs and the needle- type magnetite is limited to direct injection into the tumor tissue. If magnetic particles adsorb to only target cells, the particles could be administered via blood vessels. This fea- ture would be of great advantage in terms of the quality of life of patients and would make it possible to diagnose can- cer by MRI as mentioned above. Conjugation of antibodies to magnetic particles is one approach to achieving such an aim. Shinkai et al. have developed magnetic particles conju- gated to the Fab’ fragments of human MN antigen-specific antibody and their hyperthermia effects were demonstrated using a mouse renal cell carcinoma model (47, 48). The Fab’ fragment of the G250 antibody which binds to the MN antigen on many types of human renal cell carcinomas was cross-linked to N-(6maleimidocaproyloxy)-dipalmitoyl phosphatidylethanolamine (EMC-DPPE) in the liposomal membrane. The targetability of the G250-Fab’ fragment- conjugated MLs (G250-FMLs) was investigated using a mouse renal cell carcinoma (mRCC) and MN antigen-pre- senting cells, MN-mRCC. In an in vivo experiment using MN-mRCC-harboring mice, the accumulation was 7.9 mg of the FMLs per gram of carcinoma tissue (the tumor weight was 0.19 g), which corresponded to approximately 50% of the total amount injected. This value was 27 times higher than that of the MLs. After injection of the FMLs, mice were exposed to intracellular hyperthermia using alter- nating magnetic field irradiation. The temperature of the

610 SHINKAI

Carcinoma Liver Blood Heart Lung Spleen Kidney

FIG. 4. Magnetite uptake for carcinomas and various organs. Ani- mals in group I (open columns), the first control group, were trans- planted with MN-mRCC and magnetoliposomes (MLs) were injected. Group II animals (shaded columns), the second control group, were transplanted with mRCC and the G250-FMLs were injected. Groups III animals (closed columns) were transplanted with MN-mRCC and the G250-FMLs were injected. G250-FMLs or MLs (0.4 ml; net mag- netite, 3 mg) containing 0.3 mol% Tween 20 were injected via the heart. The five mice in each group were sacrificed and the carcinoma and organ were removed to assess accumulation of magnetite at 48 h after G250-FML or ML injection.

tumor tissue increased to 43°C and the growth of the carci- noma was significantly arrested for at least 2 weeks. These results indicate that the G250-FMLs could target the renal cell carcinoma cells in vitro and in vivo, and are applicable to the efficient hyperthermia treatment of carcinomass. As shown in Fig. 4, the G250-FMLs could target the renal cell

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carcinoma cells, and are applicable to the hyperthermia of carcinomas (Fig. 5).

Gene therapy using magnetic particles Hyperthermia is often combined with other therapies (49, 50). We have combined TNF-a gene therapy driven by a heat-inducible promoter with hyperthermia produced by the irradiation of MCLs. The gadd (growth arrest and DNA damage) 153 pro- moter, a member of the gadd transcription factor family, was selected as a control promoter of the TNF-a gene (51, 52). The plasmid including the TNF-a gene under the con- trol of the gadd 153 promoter (pGadTNF) was introduced into the cells of a human glioma cell line, U251-SP cells. When the transfected cells were exposed to heat treatment, the TNF-a gene expression was induced. The heated pGadTNF-transfected cells were co-cultured with non- heated pGadTNF-transfected cells as bystander cells. At the 1 : 0 ratio (only heated cells) or the 0 : 1 ratio (only non- heated cells), low concentration was detected (46.2 and 13.7 pg/ml, respectively). In the case of the co-culture at the 1: 1 ratio, total TNF-a gene expression was 172 pg/ml. This value was 3 times higher than the sum of the values of both the heated cells alone and non-heated cells alone. This result means that the TNF-cr expressions of both cells were en- hanced by each other expression. According to this cyclic induction by both heated and non-heated cells, a strong cytotoxic effect on the bystander cells was observed. As a result of this bystander-killing effect, it was concluded that the transfection of the TNF-cx gene under the control of the gadd 153 promoter into tumor cells would be a potent tool for hyperthermia. This system was applied to an in vivo ex- periment using tumor-bearing athymic mice. The heat stress caused by the magnetic particles resulted in a 3-fold in- crease in TNF-a gene expression driven by the gadd 153

FIG. 5. Photographs of a kidney at 14 days after hyperthermia treatment (scale bar, 5 mm). At 48 h after the injection, the mice were anesthe- tized and subjected to magnetic tield irradiation (118 kHz and 30.6 kA/m [384 Oe]). The mean initial carcinoma weight was 0.19 g. Treatment was repeated three times at 24-h intervals. a,-a,,,, MN-mRCC/G250-FMLs; b,-b,,,, mRCC/G250-FMLs. Carcinomas shown in a,-a,,, were only ob- served on the surface of the kidney and the mean weight gain of carcinomas was only 0.08 g. Since G250-FMLs could not be adsorbed on the car- cinoma surface, the carcinomas were not heated, grew vigorously and their weight gain was 1.38 g over the course of 14 days (b,-b,,,).

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FIG. 6. Rats photographed on the 28th day after the MCL injection. Rat glioma T-9 cells (1 x 10’ cells) were transplanted subcutaneously into the left femoral region of F344 rats. On the 9th day after transplantation into the left side, another aliquot of the T-9 cell suspension (1 x 10’ cells) was transplanted subcutaneously into the right femoral region. The MCLs were directly injected into the tumor on the 1 lth day after the first trans- plantation. The rats were anesthetized and subjected to magnetic field irradiation (118 kHz and 30.6 kA/m [384 Oe]). Treatment was repeated three times at 24-h intervals. (A) A non-irradiated rat; (B) an irradiated rat.

promoter as compared with that of non-heated tumor cells. TNF-a gene expression was also observed in the peripheral area where the hyperthermic effect was not sufficient to cause cell death. The combined treatment of hyperthermia and gene therapy strongly arrested tumor growth in athymic mice over a 30-d period, suggesting great potential for can- cer treatment.

Immunity induction by hyperthermia using magnetic particles Hyperthermia can induce immunity to cancer (53). Yanase et al. demonstrated this using two tumors transplanted to both femurs of a rat. Although only one tumor was subjected to hyperthermia, the other tumor also disappeared completely (Fig. 6). Immunocytochemical as- say revealed that both CDS+ and CD4+ T cells migrated in the tumors after the hyperthermia treatment. These results suggest that our therapeutic magnetic particles are poten- tially effective tools for hyperthermic treatment of tumors, because in addition to the killing of tumor cells by heat, a host immune response is induced. In addition, we have re- vealed that the augmentation of MHC class I antigens on the tumor cell surface and/or the complex of heat shock protein (HSP) and antigen cause the immunity induction (54). Re- cent results have shown the importance of HSPs in immune reactions (55). It has been demonstrated that tumor-derived HSPs, such as HSP70, HSP90 and glucose-regulated pro- tein 96 (gp96), can elicit cancer immunity (56, 57), and in- vestigators have suggested that HSPs chaperone tumor anti- gens. In these proposals, since the HSPs have to be ex- tracted from tumors in the body, surgery is needed. How- ever, if hyperthermia is used, surgery and extraction will be unnecessary.

CONCLUSION

In this review, I have only been able to highlight a limited number of medical applications using magnetic particles. These techniques are based on biochemistry, electronics and magnetics, and physiology. Currently, magnetic techniques are complementary to other methods presently used in med- ical applications and this combination therapy should result in more effective medical treatment. Greater understanding of the properties of magnetic particles will increase their potential for medical application.

ACKNOWLEDGMENTS

I am grateful to Prof. Takeshi Kobayashi, Nagoya University for his guidance and advice.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

REFFERENCES

Dunnill, P. and Lilly, M. D.: Purification of enzymes using magnetic bioafflnity materials. Biotechnol. Bioeng., 16, 987- 990 (1974). Mosbach, K. and Anderson, L.: Magnetic ferrofluids for prepareation of magnetic polymers and their application in affinity chromatography. Nature, 270,259-261 (1977). Kondo, A., Kamura, II., and Higashitani, K.: Development and application of thermosensitive magnetic immunomicro- spheres for antibody purification. Appl. Microbial. Biotech., 41,99-105 (1994). Kondo, A. and Fukuda, H.: Preparation of therrno-sensitive magnetic hydrogel microspheres and application to enzyme immobilization. J. Ferment. Bioeng., 84, 337-341 (1997). Stechell, C. H.: Magnetic separations in biotechnology - a review. J. Chem. Technol. Biotechnol., 35B, 175-182 (1985). Barclay, A. N., Brown, M. L., Beyers, A. D., Davis, S. J., and Somoza, C., Williams, A. F.: The leukocytes antigen facts book. Academic Press, San Diego, CA (1993). Weber, C. and Falkenhagen, D.: Specific blood purification by means of antibody-conjugated magnetic microspheres, p. 371-378. In Hlfeli, U., Shutt, W., Teller, J., and Zborowski, M. (ed.), Scientific and clinical application of magnetic car- riers. Plenum Press, New York (1997). Guesdon, J. L., Thiery, R., and Avrameas, S.: Magnetic enzyme immunoassay for measuring human IgE. J. Allergy Clin. Immunol., 6,23-27 (1978). Druet, E., Mahieu, P., Foidart, J. M., and Druet, P.: Mag- netic solid-phase enzyme immunoassay for the detection of anti-glomerular basement membrane antibodies. J. Immnol. Methods, 48, 149-157 (1982). Pazzagli, M., Kohen, F., Sufi, S., Masironi, B., and Cekan, S. Z.: Immunometric assay for lutropin (hLH) based on the use of universal reagents for enzymatic labeling and magnetic separation and monitored by enhanced chemiluminescence. J. Immunol. Methods, 114,62-68 (1988). Vonk, G. P. and Schram, J. L.: Dual-enzyme cascade-mag- netic separation immunoassay for respiratory syncytial virus. J. Immunol. Methods, 137, 133-139 (1991). Shinkai, M., Wang, J., Kamihira, M., Iwata, M., Honda, H., and Kobayashi, T.: Rapid enzyme-linked immunosor- bent assay with functional magnetite particles. J. Ferment. Bioeng., 73, 166-168 (1992). Bergemann, C., Miiller-Schulte, D., Oster, J., B Brassard,

612 SHINKAI J. BIOSCI. BIOENG..

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

L., and Liibbe, A. S.: Magnetic ion-exchange nano- and mi- croparticles for medical, biochemical and molecular biologi- cal applications. J. Magn. Magn. Mater., 194,45-52 (1999). Nagatani, N., Shinkai, M., Honda, II., and Kobayashi, T.: Development of a new transformation method using magne- tite cationic liposomes and magnetic selection of transformed cells. Biotechnol. Lett., 22, 999-1002 (2000). Shinkai, M., Yanase, M., Honda, II., Wakabayashi, T., Yoshida, J., and Kobayashi, T.: Intracellular hyperthermia for cancer using magnetite cationic liposome: in vitro study. Jpn. .I. Cancer Res., 87, 1179-1183 (1996). Schneider, S. and Rusconi, S.: Magnetic selection of tran- siently transfected cells. Biotechniques, 21, 876-880 (1996). Padmanabhan, R., Corsica, C. D., Howard, T. H., Holter, W., Fordis, C. M., Willingham, M., and Howard, B. H.: Purification of transiently transfected cells by magnetic affrn- ity cell sorting. Anal. Biochem., 170,341-348 (1988). Widder, K. J., Senyei, A. E., and Scarpelli, D. G.: Magnetic microspheres: a model system for site specific drug delivery in vivo. Proc. Sot. Exp. Biol. Med., 58, 141-146 (1978). Morimoto, Y., Okumura, M., Sugibayashi, K., and Kato, Y.: Preparation and magnetic guidance of magnetic albumin microspheres for site specific drug delivery in vivo. J. Pharm. Dyn., 4,624-631 (1981). Widder, K. J., Morris, R. M., Poore, G., Howard, D. P., Jr., and Senyei, A. E.: Tumor remission in Yoshida sarcoma- bearing rats by selective targeting of magnetic albumin mi- crospheres containing doxorubicin. Proc. Natl. Acad. Sci. USA, 78,579-581 (1981). Suzuki, M., Shinkai, M., Yanase, M., Ito, A., Honda, H., and Kobayashi, T.: Enhancement of uptake of magnetolipo- somes by magnetic force and hyperthermic effect on tumor. Jun. J. Hvnerthermic Oncol.. 15.79-87 (1999). Magin, k L., Wright, S. M., Niesman,‘M. R, Ghan, H. C., and Swartz, H. M.: Liposome delivery of NMR contrast agents for improved tissue imaging. Magn. Reson. Med., 3, 440-447 (1986). Gillis, P. and Koenig, S.H.: Transverse relaxation of sol- vent protons induced by magnetized spheres: application to ferritin, erythrocytes, and magnetite. Magn. Reson. Med., 5, 323-345 (1987). Saini, S., Stark, D. D., Hahn, P. F., Wittenberg, J., Brady, T. J., and Ferrucci, J. T.: Ferrite particles: a superparamag- netic MR contrast agent for the reticuloendothelial system. Radiology, 162,211-216 (1987). Stark, D. D., Weissleder, R, Eliiondo, G., Hahn, P. F., Saini, S., Todd, L. E., Wittenberg, J., and Ferrucci, J. T.: Superparamagnetic iron oxide: clinical application as a con- trast agent for MR imaging of the liver. Radiology, 168,297- 301 (1988). Josephson, L., Groman, E. V., Menz, E., Lewis, J. M., and Bengele, H.: A functionalized superparamagnetic iron oxide colloid as a receptor directed MR contrast agent. Magn. Re- son. Imaging, 8, 637-646 (1990). Suzuki, M., Honda, H., Kobayashi, T., Wakabayashi, T., Yoshida, J., and Takahashi, M.: Development of a target- directed magnetic resonance contrast agent using monoclonal antibody-conjugated magnetic particles. Brain Tumor Pathol., 13,127-132 (1996). Bushida, K., Mohri, K., Katoh, T., and Kobayashi, A.: Amorphous wire MI micro magnetic sensor for gradient field detection. IEEE Trans. Mag., 32,49444946 (1996). Shinkai, M., Ohshima, A., Yanase, M., Uchiyama, T., Mohri, K., Wakabayashi, T., Yoshida, J., Honda, H., and Kobayashi, T.: Development of novel magnetic sensing for brain lesion using functional magnetic particles. Kagaku Kogaku Ronbunnshu, 24, 174-l 78 (1998). (in Japanese) Cavaliere, R., Ciocatto, E. C., Giovanella, B.C., Heidel- burger, C., Jonson, R. O., Margottini, M., Mondovi, B.,

Moricca, B. G., and RossCFanelli, A.: Selective heat sensi- tivity of cancer cells. Cancer, 20, 1351-1381 (1967).

31. Ikeda, N., Hayashida, O., Kameda, H., Do, H., and Matsuda, T.: Experimental study on thermal damage to dog normal brain. Int. J. Hyperthermia, 10,553-561 (1994).

32. Lin, J. C. and Wang, Y. J.: Interstitial microwave antennas or thermal therapy. Int. J. Hyperthermia, 3,37-47 (1987).

33. Stauffer, P.R, Cetas, T. C., Fletcher, A.M., DeYoung, D. W., Dewhirst, M. W., Oleson, J. R, and Roemer, R. B.: Observation on the use of ferromagnetic implants for induc- ing hyperthermia. IEEE Trans. Biomed. Eng., BME-31, 76- 90 (1984).

34. Takemori, S., Tazawa, K., Nagae, H., Yamashita, I., Kato, H., Kasagi, T., Maeda, M., Honda, T., and Fujimaki, M.: A study of DDS in hyperthermia: inductive heating with use of dextran magnetite (DM). Drug Deliv. Syst., 6, 465470 (1991).

35. Jordan, A., Wust, P., Flhling, H., John, W., Hinz, A., and Felix, R.: Inductive heating of ferromagnetic particles and magnetic fluids - physical evaluation of their potential for hyperthermia. Int. J. Hyperthermia, 9, 5 168 (1993).

36. Mitsumori, M., Hiraoka, M., Shibata, T., Okuno, Y., Masunaga, S., Konishi, M., Okajima, K., Nagata, Y., Nishimura, Y., Abe, M., and other 4 authers: Development of intraarterial hyperthermia using a dextran-magnetite com- plex. Int. J. Hyperthermia, 10,785-793 (1994).

37. Gilchrist, R K. and David V. C.: Selective inductive heating of lymph nodes. Annal. Surgery, 146,596606 (1957).

38. Weinstock, S. B. and Brain, J. D.: Comparison of particle clearance and macrophage phagosomal motion in liver and lungs ofrats. J. Appl. Phys., 65, 1811-1820 (1988).

39. Lonnemark, M., Hemmingsson, A., Bach-Gansmo, T., Hagberg, H., Magnusson, A., Guudersen, H.G., and Nyman, R.: Superparamagnetic particles and oral contrast medium in MR imaging of malignant lymphoma. Acta Ra- diol., 32,232-238 (1991).

40. Gordon, R. T., Hines, J. R, and Gordon, D.: Intracellular hyperthemia - a biophysical approach to cancer via intracel- lular temperature and biophysical alterations. Med. Hypothe- sis, 5, 83-102 (1979).

41. Jordan, A., Wust, P., Scholz, R., Tesch, B., Filhling, H., Mitrovics, T., Vogl, T., Cervos-Navarro, J., and Felix, R: Cellular uptake of magnetic fluid particles and their effects on human adenocarcinoma cells exposed to AC magnetic fields in vitro. Int. J. Hyperthermia, 12, 705-722 (1996).

42. Chan, D. C. F., Kirpotin, D. B., and Bunn, P. A.: Synthesis and evaluation of colloidal magnetic iron-oxides for the site- specific radiofrequency-induced hyperthermia of cancer. J. Magn. Magn. Mater., 122,374-378 (1993).

43. Shinkai, M., Matsui, M., and Kobayashi, T.: Heat proper- ties of magnetoliposomes for local hyperthermia. Jpn. J. Hy- perthermic Oncol., 10, 168 (1994).

44. Yanase, M., Shinkai, M., Honda, H., Wakabayashi, T., Yoshida, J., and Kobayashi, T.: Intracellular hyperthermia for cancer using magnetite cationic liposomes: ex vivo study. Jnn. J. Cancer Res.. 88.630-632 (1997).

45. Yanase, M., Shin’&; M., Ho&la, H., Wakabayashi, T., Yoshida, J., and Kobayashi, T.: Intracellular hyperthermia for cancer using magnetite cationic liposomes: an in vivo study. Jpn. J. Cancer Res., 89,463-469 (1998).

46. Ohno, T., Wakabayashi, T., Takemura, A., Yoshida, J., Ito, A., Shinkai, M., Honda, H., and Kobayashi, T.: Effective solitary hypertbennia treatment of malignant glioma using stick type CMC-magnetite. In vivo study. J. Neurooncol., 56, 233-239 (2002).

47. Le, B., Shinkai, M., Kitade, T., Honda, H., Yoshida, J., Wakabayashi, T., and Kobayashi, T.: Preparation of tumor- specific magnetoliposomes and their application for hyper- thermia. J. Chem. Eng. Jpn., 34,66-72 (2001).

VOL. 94.2002 MAGNETIC PARTICLES FOR MEDICAL APPLICATION 613

48. Shinhai, M., Le, B., Honda, H., Yoshihawa, K., Shimizu, K., Saga, S., Wahabayashi, T., Yoshida, J., and Kobayashi, T.: Targeting hyperthermia for renal cell carcinoma using hu- man MN antigen-specific magnetoliposomes. Jpn. J. Cancer Res., 92, 1138-l 145 (2001).

49. Shinhai, M., Suzuki, M., Yokoi, N., Yanase, M., Shimizu, W., Honda, H., and Kobayashi, T.: Development of anti- cancer drugs-encapsulated magnetoliposome and its combi- nation effect of hyperthermia and chemotherapy. Jpn. J. Hy- perthermic Oncol., 14, 15-22 (1998).

50. Yanase, M., Shinhai, M., Suzuki, M., Honda, H., and Kobayashi, T.: Study on thermochemotherapy using mag- netic cationic liposomes and anti-cancer drug. Kagaku Kou- gaku Ronbunshu, 24, 179-l 83 (1998). (in Japanese)

51. Ito, A., Shinhai, M., Bouhon, I. A., Honda, H., and Kobayashi, T.: Bystander-killing effect and cyclic induction of TNF-a gene under heat-inducible promoter gadd 153. J. Biosci. Bioeng., 90,437441 (2000).

52. Ito, A., Shinhai, M., Honda, II., and Kobayashi, T.: Heat- inducible TNF-a gene therapy combined with hyperthermia

using magnetic nanoparticles as a novel tumor-targeted ther- apy. Cancer Gene Ther., 8,649-654 (2001).

53. Yanase, M., Shinhai, M., Honda, H., Wahabayashi, T., Yoshida, J., and Kobayashi, T.: Antitumor immunity induc- tion by intracellular hypertbermia using magnetite cationic liposomes. Jpn. J. Cancer Res., 89, 775-782 (1998).

54. Ho, A., Shinhai, M., Honda, H., Wahabayashi, T., Yoshida, J., and Kobayashi, T.: Augmentation of MHC class I antigen presentation via heat shock protein expression by hyperther- mia. Cancer Immmunol. Immunother., 50, 515-522 (2001).

55. Menoret, A. and Chandawarhar, R: Heat-shock protein- based anticancer immunotherapy: an idea whose time has come. Sem. Immunol., 25,654-660 (1998).

56. Tamura, Y., Peng, P., Liu, K., Daou, M., and Srivastava, P. K.: Immunotherapy of tumors with autologous tumor-de- rived heat shock protein preparations. Science, 278, 117-120 (1997).

57. Udono, H. and Srivastava, P. K.: Heat shock protein 70-a.+ sociated peptides elicit specific cancer immunity. J. Exp. Med., 178,1391-1396 (1993).