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
606
VOL. 94,2002 MAGNETIC PARTICLES FOR MEDICAL APPLICATION 607
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
J. BIOSCI. BIOENG.,
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- nmparticles.
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 43C after 15 min. In contrast, the rectal tem-
perature or that in a tumor lacking the MCLs remained be-
tween 35-37C. 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 43C 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
J.BIoscr. BIOENG.,
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,,,).
VOL. 94,2002 MAGNETIC PARTICLES FOR MEDICAL APPLICATION 611
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.
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