ISBN No: 8184871597

5
Magnetic Nanoparticles and
Thermosensitive Carriers for Hyperthermia
and Drug Delivery
Pallab Pradhan1, Manish K. Jaiswal2, Manashjit Gogoi1, D. Bahadur2, R. Banerjee1
1 Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai,
India, 2 Department of Metallurgical Engineering and Materials Science, Indian Institute of
Technology Bombay, India

1. INTRODUCTION

Magnetic nanoparticles (MNPs) have attracted lot of attention due to their immense potential
in biomedical applications like magnetic hyperthermia treatment (MHT) of cancers (Jordon et
al., 1999a), magnetic drug targeting (MDT) (Lübbe et al., 2001), magnetic resonance imaging
(MRI) contrast enhancement agents (Sun et al., 2008). For MHT, MNPs in the form of aqueous
colloidal suspension, know as magnetic fluid/ ferrofluid, are subjected to alternating
magnetic field to generate heat, which occurs due to different losses namely hysteresis, Néel
and Brownian relaxation (Cullity, 1972 and Mornet et al., 2004). Thus, cancer cells containing
the magnetic nanoparticles can be heated upto 42-45 °C under alternating magnetic field,
known as ‘Hyperthermia’, which results in the killing of cancer cells (Jordon et al., 1996). A
temperature more than 46 °C and upto 56 °C is also known to be used for cancer treatment
and the process is called ‘Thermoablation’ (Hilger et al., 2001). On the other hand, in MDT,
chemotherapeutic drugs attached to MNPs are physically targeted to the tumors and retained
there by application of an external permanent magnetic field and thus undesirable side
effects due to uncontrolled drug distributions in the healthy tissues are minimised for surface
tumors (Alexiou et al., 2000). When both MDT and MHT approaches are combined,
bioavailability of the chemotherapeutic drug and its therapeutic efficacy can be enhanced. In
this direction, efforts have been made to develop smart drug delivery systems composed of
MNPs encapsulated in thermosensitive lipids or polymers containing chemotherapeutic
drugs (Viroonchatapan et al., 1998 and Liu et al., 2009).
68 NANO TECHNOLOGY

Magnetic nanoparticulates can be targeted to tumors with the help of permanent

magnetic fields using the principle of MDT and the encapsulated drugs can be released
locally by heating the magnetic nanoparticulates upon application of alternating magnetic
field using the principle of MHT. Thus, enhanced therapeutic effects may be achieved due to
combined treatment by MHT and chemotherapy (Fig.1). In this chapter, recent advances in
the development of magnetic nanoparticles and thermosensitive carriers for hyperthermia
and drug delivery applications are reviewed.

Figure 1. Schematic showing the concept of targeted and temperature triggered drug delivery

The figure depicts the use of different thermosensitive magnetic nanocarriers by combined magnetic
drug targeting (MDT) and magnetic hyperthermia treatment (MHT) for thermo-chemotherapy of
cancers

MAGNETIC HYPERTHERMIA TREATMENT (MHT)

Theory of MHT

When magnetic nanoparticles are subjected to an alternating magnetic (AC) field, they
generate heat which may be measured by Specific Absorption Rate (SAR). SAR, is also
denoted as specific loss power (SLP), and is an important parameter to evaluate the heating
 MAGNETIC NANO PARTICLES FOR THERAPY 69

efficiency of magnetic materials. SAR may be defined as the power of heating of a magnetic
material per gram and can be represented as the following equation.

∆T 1
SAR = C × × (1)
∆t mFe

where C is the specific heat capacity of the sample and ∆T/∆t is the initial slope of the time
dependent temperature curve and mFe is the weight fraction of magnetic element (like
iron/ferrite) in the sample (Mornet et al., 2004, Hilger et al., 2002a, Vasseur et al., 2006). It has
been observed that the effect of AC field on SAR depends on particle size. In the range of
superparamagnetic particle size, the SAR follows a square law with the AC field amplitude
(H) (Hergt et al., 2004a and 2004b). The importance of a high value of SAR for heating
applications is obvious. In particular, in cancer treatment by hyperthermia a high value of
SAR will allow attainment of desired temperatures with low particle load.
The heat generation of magnetic materials is due to three different types of losses,
namely hysteresis loss, Néel and Brownian relaxations (Cullity, 1972) and these mechanisms
are shown in Fig. 2. Hysteresis loss occurs mainly in bulk ferro/ferromagnetic (FM)
materials. However, in superparamagnetic (SPM), Néel and Brownian relaxations are
responsible for heat generation. For magnetic hyperthermia, SPM nanoparticles are mostly
used as their heating ability is higher as compared to FM particles in clinically tolerable
magnetic field amplitudes (Cullity, 1972).

Figure 2. Schematic showing mechanism of different types of losses in magnetic particles under AC
magnetic field

Hysteresis Loss

FM particles possess hysteresis properties. When an alternative magnetic field is applied to

these multidomain FM particles, the change in magnetization is mainly due to the rotation of
spontaneous magnetization of elementary domains and to the domain wall movements
separating them which is called "domain wall displacement"(Mornet et al., 2004). In general,
70 NANO TECHNOLOGY

the latter effect is more dominant. Due to this domain wall displacement the magnetization
value of these multidomain magnetic particles changes and follows the path of a hysteresis
loop which results in generation of heat. The amount of heat generated is given by the
frequency multiplied by the area of hysteresis loop and can be expressed as in equation 2:

HFM =µ0 f (2)

Where, HFM, µ0, f, H and dM are heat generated by FM materials, permeability of the free
space, frequency of magnetic field, applied magnetic field and change in magnetization value
respectively. Here the heat generation due to eddy current is neglected since the sizes of the
magnetic particles as well as the range of applied magnetic field for hyperthermia are too low
to produce any eddy current. HFM for the FM can be determined from the M-H loop obtained
from VSM or SQUID magnetometer (Pankhurst et al., 2003).

Néel and Brownian Relaxation

Magnetic fluids or ferrofluids i.e. SPM nanoparticles suspended in aqueous media, have been
used for cancer hyperthermia (Jordan et al.,1999a; Jordan et al., 1993; Jordan et al 1999b).
When an alternating magnetic field is applied to the ferrofluid, the magnetic moment vector
of these single domain nanoparticles rotate and align themseves along the field direction
against a thermal energy barrier (kBT). Thus each magnetic moment vector possesses
magnetostatic energy of amount KV, where K is the anisotrpy constant of the material and V
is the particle volume. When the field is removed, thermal agitation takes over and the
magnetic moment vector relaxes, called Neel relaxation, dissipating its magnetic energy in
the form of heat. The relaxation of the nanoparticles corresponds either to physical rotation of
the particles themselves within the fluid or rotation of the atomic moments within each
particle. Rotation of the particles is known as ‘Brownian relaxation’ while rotation of the
atomic moment within the particles are known as ‘Néel relaxation’. Brownian relaxation
time τB depends upon hydrodynamic properties of the fluid whereas Néel relaxation time τN
is determined by magnetic anisotropy energy of the SPM nanoparticles relative to the
thermal energy. Both Brownian and Néel relaxation processes may be present in the
ferrofluid, whereas τN is relevant in fixed SPM nanoparticles where no physical rotation is
possible. Considering small amplitude of magnetic field and minimal interaction between the
SPMs’, the response of the magnetization of a ferrofluid to an AC field can be described in
terms of its complex susceptibility χ = χ’ + iχ” where χ’ and χ” are frequency dependent. The
out of phase χ” component is accountable for heat dissipation in the fluid medium and has
been given by Rosensweig depicted in equation 3(2002)

HSPM = µ0 π f χ” H2 .(3)

Where, HSPM, µ0, f, χ” and H are heat generated due to Néel and Brownian relaxation,
permeability of the medium, frequency of magnetic field, magnetic susceptibility and
strength of applied AC magnetic field respectively.
 MAGNETIC NANO PARTICLES FOR THERAPY 71

Superparamagnetism (SPM)

Properties of magnetic particles are changed significantly as their size goes to nano
dimension. As particle size decreases, the coercivity (i.e. the intensity of the applied magnetic
field required to reduce the magnetization of a material to zero after the magnetization of the
sample has been driven to saturation) initially increases and reaches a maximum and then
decreases to zero. Thus, below a critical size the particles do not show any coercivity and
remanence (i.e. the magnetic induction that remains in a material after removal of the
magnetic field) and are known as superparamagnetic. When the particle size is small enough,
thermal energy exceeds anisotropic forces and spontaneously reverses the magnetization of
the particle from one easy direction to the other in absence of an applied magnetic field. This
can be explained by equation 4 (Cullity, 1972)-

KV ≤ kBT (4)

Where K is the effective magnetic anisotropy energy constant (a function of the

magnetocrystalline, shape and surface anisotropies), V is the volume of the particle and kB is
Boltzman’s constant and T is the absolute temperature. The superparamagnetic particle, thus,
has a critical volume (VSP) below which it shows superparamagnetic behavior. The volume
(VSP) can be estimated from equation 5 (Cullity, 1972) -

VSP = 25 kBT/K (5)

where K, V, kB and T have similar meaning as described above. However, one of the
important features of the superparamagnetic nanoparticles is their ability to become
nonmagnetic upon withdrawal of magnetic field. Thus, their biomedical applications are safe
and useful (Neubergera et al., 2005).

Ferro-/Ferrimagnetism

Ferromagnetism is the phenomenon of certain transition metals like iron, nickel, cobalt and
their composites and few rare earth metals such as gadolinium by which they exhibit a very
strong and permanent magnetization even in the absence of a magnetic field. These materials
are called ferromagnetic materials. Magnetic susceptibilities of these materials are in the
order of 106. Permanent magnetic moments of ferromagnetic materials are very high due to
high atomic moments which results from their parallel and unidirectional atomic magnetic
moments in the absence of magnetic fields. Ferrimagnetic materials too have permanent
magnetization but there exist some oppositely aligned electron couples that reduce the net
magnetization value. The net magnetic moment of ferromagnetic materials is positive which
results in possession of permanent magnetism.
The Curie temperature (Tc) is the temperature above which ferro-/ferri-magnetic
particles lose their magnetic properties and become paramagnetic. Thus, the contribution of
hysteresis loss for heating ability of the ferro/ferri-magnetic particles is impaired near the Tc
of the particle. This property can be exploited for the development of smart magnetic
particles for self controlled hyperthermia by tuning the Tc around hyperthermia temperature
72 NANO TECHNOLOGY

i.e. 42-45°C so that they may act as in vivo temperature controlling switches avoiding
overheating of the adjacent healthy tissues of a tumor (Mornet et al., 2004). Though magnetic
nanoparticles play an important role in magnetic hyperthermia, biocompatibility is a crucial
issue for biological application.

Magnetic Fluids and their Biocompatibility

Magnetic fluids are the dispersion of small single magnetic domain particles
(superparamagnetic) of nanometer sizes (<100 nm) in organic or inorganic carrier liquids. For
biomedical applications, aqueous carrier liquids are generally used. For the stable dispersion
of magnetic particles, three primary attractive forces i.e. magnetic attractive forces,
gravitational forces and Van der Waals forces have to be balanced by electrostatic and steric
repulsion forces. In electrostatic stabilization, the surface of the particle is made to carry a
charge and the particles repel each other if their proximity goes below a certain distance. The
electrostatic stabilization is a pH sensitive method. Coating of particles by long chain
molecules (polymers or surfactants) prevent aggregation of the particles by steric repulsion.
Biocompatibility of magnetic fluids is an important issue for evaluation of their
suitability for any biomedical application. Biocompatibility of magnetic fluids is determined
by both the core magnetic material and its coatings (Kückelhaus et al., 2004). The coating of
long chain polymer molecules, phospholipids and other surfactants on ferrite particles serves
as a protective layer on the nanoparticles and minimizes the direct exposure of the ferrite
surface to the biological environment. Several surfactant and polymer based coating
materials like citrate, polyaspartic acid, dextran have been developed mainly for Fe3O4 based
magnetic fluids. Lacava et al.(2004) endovenously administered dextran coated Fe3O4
nanoparticles (average core size 9.4 nm) in female Swiss mice. No significant adverse effects
were observed for a period of 6 months suggesting the biocompatibility of the magnetic
nanoparticles.
Sadeghiani et al., (2005) investigated the cytotoxicity and genotoxicity effects of
magnetic fluid containing polyaspartic acid coated iron oxide nanoparticles in female Swiss
mice. The nanoparticles were biocompatible and its biodistribution was similar to that of
citric acid coated nanoparticles but varied from that of dextran and 2,3-
mesodimercaptosuccinic acid coated iron oxide nanoparticles. Petri-Fink et al., (2005) coated
super paramagnetic iron oxide nanoparticles with polyvinyl alcohol (PVA), carboxylate-
functionalized PVA, thiol-functionalized PVA and aminofunctionalized PVA (amino-PVA).
After evaluation of their interactions with human melanoma cells, it was found that amino-
PVA coated nanoparticles are biocompatible and cellular uptake of these nanoparticles
depended on the presence of amine groups and their ratio to the amount of iron oxide.
Biocompatibility of substitute ferrites (such as MnFe2O4 and CoFe2O4) and their heating
ability have also been studied and reported by our group. Pradhan et al., (2007a) synthesised
lauric acid coated Fe3O4, MnFe2O4 and CoFe2O4. Results showed dose dependent cell viability
of all the magnetic fluids and CoFe2O4 was more toxic than other two ferrites. In-vivo
biocomapatibility study for these ferrites was done in 84 Swiss female mice weighing about
20-25g and of age 6-8 weeks. The magnetic fluids were administered through tail vein at
doses of 50, 200 and 400mg/kg body weight. Blood and tissue analysis showed no significant
changes in haematological and biochemical parameters for Fe3O4 and MnFe2O4 containing
 MAGNETIC NANO PARTICLES FOR THERAPY 73

magnetic fluid while CoFe2O4 showed mild hepatotoxic effect. Thus lauric acid coated
superparamagnetic Fe3O4 and MnFe2O4 nanoparticles were found to be more biocompatible
then CoFe2O4nanoparticles.

2. MAGNETIC NANOPARTICLES FOR HYPERTHERMIA

In magnetic hyperthermia, micro/nano magnetic particles in the form of injectable colloidal
dispersions are used. In 1979, Gordon et al. proposed the concept of inducing intracellular
hyperthermia using dextran coated magnetite (Fe3O4) nanoparticles. They showed that AC
magnetic field induced heating occurred in the mammary tumors of Sprague–Dawley rats
following injection of magnetite particles. However, it was not possible to differentiate the
cell killing effects of extracellular and intracellular heating. In 1993, Chan et al. showed that
cellular inactivation due to magnetic hyperthermia under AC magnetic field using magnetic
nanoparticles was equal to the homogenous heating by water bath. No significant difference
in the cell killing effects of magnetic hyperthermia over water bath hyperthermia was
observed. In 1996, Jordon et al. proposed the term ‘magnetic fluid hyperthermia (MFH)’ for
magnetic hyperthermia where magnetic nanoparticles in the form of magnetic fluid were
used. They also found that MFH was equally effective as water bath hyperthermia for a given
time temperature schedule. No additional biological effect was found for MFH over the
water bath hyperthermia. From transmission electron micrographs of cells, they found that
dextran coated magnetic nanoparticles, which were used for the MFH, lost their dextran shell
upon internalization within the cells and thereby formed large aggregate of the particles
resulting in low SAR and decreased cellular heating. The loss of dextran coating of the
magnetic nanoparticles was due to lysosomal degradation of dextran coating of magnetic
nanoparticles. They concluded that the dextran coated magnetic nanoparticles were not
suitable for MFH.
Further in vitro studies of MFH were done by Jordon et al. (1999b) using a newly
synthesized aminosilane coated magnetite nanoparticles instead of dextran coated particles.
Aminosilane coated magnetite particles were positively charged under physiological
conditions. In glioblastoma cell lines, the aminosilane coated magnetite nanoparticle showed
1000 fold higher uptake as compared with dextran coated particles. Moreover, the uptake of
the positively charged aminosilane magnetite nanoparticles was 500-2000 fold higher in
glioblastoma cell lines as compared to that of normal neuronal cell lines. Thus, without a
targeting ligand, aminosilane coated particle could be actively targeted to cancer cells due to
their differential endocytosis. Other than Fe3O4, γ-Fe2O3 (Hergt et al., 2004a) and different
substituted ferrites (M1-xZnxFe2O4, M=Mn, Co) (Giri et al., 2005, Pradhan et al., 2007a, Prasad
et al., 2007a) have also been explored for magnetic hyperthermia applications. Giri et al.
(2005), synthesised different substituted ferrites of Fe1−xMnxFe2O4 (x=0-1) with co-
precipitation method. Size of these particles is 10-12nm. They evaluated the heating ability of
these ferrites and tested for biocompatibility in BHK 21(Syrian Baby Hamster Kidney) cell
lines. It was found out that all these ferrites are biocompatible. Fe0.6Mn0.4Fe2O4 has SAR value
20% more than that of Fe3O4 which argues strongly for its candidature for hyperthermia
application. Prasad et al.(2007a) synthesised ɤ-MnxFe2-xO3 (0≤x≤1.3) by a single step process
and coated these nanoparticles with Acrypol 934 polymer. SAR measurement showed that
this composition took 2190 sec to reach 50oC and its SAR value was 23.6W/g at 37oC. They
74 NANO TECHNOLOGY

studied the effect of magnetic nanoparticles on HeLa cells under ac magnetic field. It was
observed that the effect on HeLa cells was dose and time dependent which was confirmed by
confocal microscopy images which showed a damage in cytoskeleton structures due to the
effect of MHT.
In vivo efficacy of the MFH has been demonstrated by Jordon et al. in several tumor
models such as murine model of mammary carcinoma and glioma (Jordan et al.,1997, Jordan
et al., 2006), and rat model of prostate carcinoma (Johannsen et al., 2006). Other animal tumor
models such as melanoma, breast tumor have also been reported to be evaluated for efficacy
and feasibility of magnetic hyperthermia in vivo by several researchers (Ito et al.,2003a,
Tanaka et al., 2005, Hilger et al., 2002b). Promising results of Phase I clinical trial of MFH have
recently been published for prostate carcinoma (Johannsen et al., 2005, 2007a and 2007b) and
glioblastoma multiforme (Maier-Hauff et al.,2007 and Landeghem et al., 2009). Phase II
clinical trials are already in progress to evaluate the efficacy of MFH in patients with
glioblastoma multiforme and prostate cancer.

Self Controlled Hyperthermia

Presently iron oxide nanoparticles are extensively used in magnetic hyperthermia. But the
problem with the iron oxide nanoparticles is that their curie temperature is very high
(magnetite and maghemite show Curie temperature of 585 and 645 oC respectively) that
causes overheating of tissues upon continuous application of magnetic field. To avoid
overheating of tissues, one needs to use some temperature probes at the tumour site(s) and
monitor the temperature. Once the temperature reaches 42-43oC, temperature could be
controlled by switching on/off the magnetic field. If the magnetic nanoparticles are designed
with Tc in the range of 42-43oC, upon application of an alternative magnetic field, the
temperature of these nanoparticles will rise up to its Tc only and then the nanoparticles
become paramagnetic and lose their heating ability above Tc. Thus the problems of
overheating of the tissues will be avoided and online monitoring of temperature of the
magnetic hyperthermia treatment site, (e.g. tumor) will not be required. This whole process is
called self controlled hyperthermia.
Ln1-xAxMnO3 (where Ln= rare earth and A= a divalent cation such alkaline earth)
group of materials are rare earth manganates having perovskite structure. Researchers are
exploring the feasibility of using La1-xSrxMnO3 for self controlled hyperthermia, because curie
temperature of this material can be tuned to around 42oC.
Vasseur et al.(2006), synthesised La0.75Sr0.25MnO3 through citrate gel precursor
method. The size of nanoparticles is in the range of 20-180 nm. They claimed, preparation of
curie tuned MNP with a curie temperature of about 45oC which is suitable for self controlled
hyperthermia (Pollert et al., 2007).Shlyakhtin et al. (2007) synthesised La-Ag and La-Na
perovskite smart magnetic materials for self controlled hyperthermia. La1-xAgxMnO3 (Tc=40-
45oC) and La1-yNayMnO3 (Tc=43-53oC) where x=0.22-0.25 and y=0.15-0.17 were prepared by
freeze drying method. The magnetization and curie temperature of these samples were
measured in a vibrating sample magnetometer at H≤7500 Oe and H≤200 Oe respectively.
Specific Absorption rate of the aqueous suspensions were determined at f=440 kHz and
H=350 Oe. The size of these MNPs are in the range of 50-70nm. Since these are multidomain
structures, heating results from the domain wall motion and related hysteresis losses. The
 MAGNETIC NANO PARTICLES FOR THERAPY 75

advantage of these groups of MNPs over La, Sr group of perovskite manganates is that these
are more stable in aqueous medium.
Prasad et al.(2007b) synthesised La0.73Sr0.25MnO3(LSMO) nanoparticles of size 20-
100nm with the help of microwave refluxing technique and the curie temperature of these
material was reported to be 45oC. Acrypol-934 coated magnetic nanoparticles were found to
be biocompatible.
Melnikov et al.(2008) synthesised La1-xAgxMnO3+ nanoparticles with curie
temperature around 43oC. In-vitro evalution showed that these nanoparticles are
biocompatible. It was found that Ag- cations were leached out from La1-xAgxMnO3+ to the
water in the aqueous suspension which makes this suspension biocompatible. It is reported
in literature that the presence of Ag cation in the surrounding is not harmful for cells and
tissues. These Ag+ doped nanoparticles were used to MRI imaging in mouse. The electrical
and magnetic properties of these nanoparticles are found to be comparable with
superparamagnetic iron oxide based nanoparticles (SPIO). Thus it may be concluded that
though this material is not used for hyperthermia application it may be promising candidate
for self controlled hyperthermia as well as MRI imaging.
Saito et al. (2008) reported in vitro and in vivo studies with thermosensitive
ferromagnetic particles with a curie temperature of 43oC. These ferromagnetic particles were
composed of Fe2O3, CuO, ZnO and MgO in the ratio 49:7:30:14 (molar). The average size of
these particles was 100 µm. Since the size of these particles was large, these particles were
directly injected at the tumour site(s). For in vitro studies, the ferromagnetic particles were
placed in 1% agar (dissolved in distilled water) in a 50 ml polypropylene tube. A magnetic
field of 600 A, 188 kHz was then applied to induce heat in the agar phantom. Temperature at
the centre of the agar phantom was measured by a ceramics thermosensor. Temperature of
the agar phantom reached 43oC within 5 min. Before the hyperthermia experiment, each of
the polypropylene tube was incubated at 37oC. For in vivo experiment, tumour bearing mice
were divided into four groups: group I mice were not subjected to alternative magnetic field
(control group), group II mice were subjected to hyperthermia for 30 min, group III mice
were given hyperthermia treatment twice a week for 4 weeks and in group IV mice, PBS was
injected and given hyperthermia twice a week for 4 weeks (this group was control group of
mice for group III). Ferromagnetic particles were injected in the tumour(s) of group II and
group III. Upon application of alternative magnetic field, the temperature of ferromagnetic
particles within the tumour(s) is increased to 43 oC in 7 min. It was observed that growth of
tumour(s) was significantly reduced in group III mice and they lived for a longer period of
time. It was concluded that repeated doses of hyperthermia with Tc tuned ferromagnetic
material may be used for suppressing melanomas.

Magnetic Liposomes For Hyperthermia Application

Like magnetic nanoparticles, magnetic liposomes have been successfully used for
hyperthermia treatment of cancer. Shinkai et al. (1996) has developed magnetic cationic
liposomes (MCLs) encapsulating magnetite (Fe3O4) based magnetic fluid for hyperthermia
treatment of cancer. The MCLs consisted of N-(a-trimethylammonioacetyl) didodecyl-D-
glutamate chloride (TMAG), dilauroylphosphatidylcholine (DLPC) and
dioleoylphosphatidylethanolamine (DOPE) at 1:2:2 molar ratio. Due to their positive surface
charge, the MCLs showed 10 fold higher affinity for rat glioma cells as compared to neutrally
76 NANO TECHNOLOGY

charged magnetic liposomes. The MCLs had sufficient SAR and biocompatibility comparable
to dextran coated magnetite nanoparticles. In an in vitro experiment, rat glioma cells were
heated to over 43 °C in presence of MCLs by exposing to a high frequency magnetic field (118
kHz frequency and 384 Oe field) and all the cells were found to be killed after 40 minutes of
the hyperthermia treatment.
Hyperthermic effect of MCLs was also evaluated in ex vivo experiments using
implanted cell pellets (Yanase et al., 1997). For this purpose, T9 rat glioma cells were first
incubated with MCLs (at 20 µg/ml concentration) in a petridish for 8 hrs to allow uptake of
the MCLs by the cells. Then, cell pellet was prepared and implanted subcutaneously in the
left femoral region of F344 rats under general anaesthesia. Then the rats were divided into
three groups, each group having five rats. Group I was control with no magnetic field
exposure, group II rats were exposed to magnetic field once for 60 min after implantation of
MCL where as group III rats were exposed to magnetic field three times for 60 min at an
interval of 12hr. The rats were subjected to an AC magnetic field (118 kHz frequency and
384 Oe field) for 60 minutes. Temperature in the cell pellet reached over 43 °C after 20
minutes and was maintained till 60 minutes. After 3 cycles of magnetic hyperthermia, all the
glioma cells were killed in the rats of group III upto 90days.
Further, efficacy of magnetic hyperthermia by MCLs was investigated in vivo (Yanase
et al., 1998). Subcutaneous solid tumors were generated by injecting T9 rat glioma cell
suspension in the left femoral region of rats. After 11th day of the cell injections, tumors with
13-18 mm length were formed. Then, 400 µl of MCLs solution (containing 3 mg magnetite)
was injected in the centre of the solid tumors. After 24 hrs, the tumors were irradiated with
an AC magnetic field (118 kHz frequency and 384 Oe field) for 30 minutes and the
temperature of the tumor was maintained at around 43 °C. The rats were divided into 4
different groups i.e. group-I (no irradiation), group-II (irradiated once), group-III (irradiated
twice) and group-IV (irradiated thrice) consisting 5 animals in each group. Group I did not
show any regression of tumor. But, there was 20%, 60% and 87.5% tumor regression for
group-II, III and IV respectively. Thus, it was observed that repeated irradiations were
necessary for regression of tumor. Histopathology showed homogenous distribution of
MCLs within the tumor following multiple irradiations. In addition to the hyperthermic
effect, MCLs mediated heat treatment induced long lasting antitumor immune response
(Yanase et al., 1998). Complete regression of mammary carcinomas greater than 15 mm was
also achieved by frequent repeated hyperthermia treatments using MCLs in mice models (Ito
et al., 2003b). Apart from the T-9 rat glioma and mouse mammary carcinoma, the efficacy of
MCLs mediated hyperthermia was demonstrated for several types of animal tumors, such as
the B16 mouse melanoma (Suzuki et al., 2003), Os515 hamster osteosarcoma (Matsuoka et al.,
2004), VX-7 squamous cell carcinoma in rabbit tongue (Matsuno et al., 2001), human prostate
carcinoma in nude mice (Kawai et al., 2006).

3. MAGNETIC NANOPARTICLES FOR DRUG DELIVERY APPLICATION (MDT)

Magnetic nanoparticles as a drug delivery system has attracted a lot of attention due to their
potential in targeting chemotherapeutic drugs to tumors by external magnetic fields, which is
known as magnetic drug targeting (MDT) (Lübbe et al., 1996a). In MDT, chemotherapeutic
drugs are retained at the site of their action and therefore drug concentration at the desired
 MAGNETIC NANO PARTICLES FOR THERAPY 77

site is increased. Thus, side effects due to undesirable accumulation of drug in healthy tissues
are minimized. Moreover, combination of targeting and retention of magnetic nanoparticle-
drug complex at the desired site reduces reticuloendothelial system (RES) clearance of drug
and facilitates extravascular uptake (Gonzales et al., 2005). However, for a successful MDT,
several factors need to be considered: (a) size of the particles should be such that they can be
attracted by a magnetic field and can pass through the blood vessels supplied to tumor; (b)
magnetic field should be of sufficient strength to attract the magnetic nanoparticles towards
the desired site like tumor; (c) the magnetic nanoparticle-drug complex should release
sufficient amount of chemotherapeutic drug inside the tumor; and (d) injection method
should be accessible to tumor vasculature and should avoid RES clearance (Alexiou et al.,
2000).
In 1996, the use of MNPs for magnetic field mediated drug delivery was first
reported in an animal model by Lübbe et al. (1996b). They successfully showed tumor
regression in nude mice and rats due to a magnetic field (using a permanent magnet having
0.5-0.8 Tesla field strength) guided targeting of 4'-epidoxorubicin bound to MNPs in
suspension. Further, Lübbe et al. (1996a) conducted first Phase I clinical trials using the 4'-
epidoxorubicin bound ferrofluid administered intravenously in patients with advanced
unsuccessfully treated cancers or sarcomas. The MDT approach was well tolerated by the
patients. MDT approach was also successfully demonstrated using magnetic nanoparticle-
methotrexate drug complexes injected intra-arterially in rabbit VX-2 squamous cell
carcinoma. Magnetically targeted delivery of aerosols to lungs has been reported by Dames et
al.,(2007). They have developed ‘nanomagnetosols’ comprising superparamagnetic iron oxide
which can be targeted to lungs with the help of externally applied magnetic field. They
successfully demonstrated the magnetic field directed delivery of nanomagnetosols of
superparamagnetic nanoparticles-plasmid DNA complex in the targeted lobe of the lung in
mice without any side effects. Thus, magnetic field mediated targeting holds promise in the
treatment of localized lung diseases such as pulmonary infections or cancer.
Magnetic drug targeting requires a very powerful magnet with highly non-uniform
field. In the past, a very heavy and powerful magnet was used for this purpose. Researchers
from Siemens Corporate Technology (CT) designed a very small and powerful magnet for
MDT which produces field gradient according to the treatment requirement. In short,
magnetic drug targeting has become much easier and handy now. The light weight (47kg)
and optimized pole tip of the newly designed magnet will enable precise positioning thereby
allowing safe treatment to even small cancerous lesions.

Magnetic Liposomes As Drug Delivery Systems

Magnetic liposomes containing chemotherapeutic drug either within the bilayer or in the
inner aqueous chamber have also been successfully targeted to tumors by MDT approach.
Nobuto et al. prepared magnetic liposomes (composed of egg-PC and cholesterol at 1:1 molar
ratio) encapsulating doxorubicin (Nobuto et al., 2004). They showed that intravenous
administration of the doxorubicin containing magnetic liposomes with subsequent
application of magnetic field (0.4 Tesla) for 60 minutes produced 3-4 fold higher doxorubicin
concentration in osteosaromas of hamsters as compared to the magnetic liposomes
containing doxorubicin in the absence of magnetic field. Zhang et al. reported
pharmacokinetic studies on negatively charged paclitaxel containing magnetic liposomes
78 NANO TECHNOLOGY

(Zhang et al., 2005). They showed that paclitaxel containing magnetic liposomes had
extended circulation half life (t 1/2ß 19.37 hrs against 4.11 hrs) as compared with the drug in
cremophor EL/ethanol. Moreover, paclitaxel concentration of lyophilized paclitaxel magnetic
liposomes in EMT-6 breast cancers in mice was higher than that of the lyophilized
conventional liposomes or cremophor EL/ethanol following subcutaneous or intravenous
injection under the influence of external magnetic field. Dandamudi and Campbell have
developed magnetic cationic liposomes for targeting tumor vasculature through MDT
approach (Dandamudi and Campbell, 2007a and 2007b). They demonstrated that under the
influence of an external magnetic field, vascular accumulation and retention of magnetic
cationic liposomes in tumors was enhanced.
Magnetic liposomes have also been emerged as MRI contrast agents due to the T2
shortening effect of magnetic nanoparticles (Martina et al., 2005 and Morais et al., 2005].
Hence, in vivo distribution of magnetic liposomes can be traced by MRI. A feasibility study
on the magnetic targeting of magnetic liposomes to solid tumors coupled with MRI
monitoring was reported in mice (Fortin-Ripoche et al., 2006). In this study, efficient targeting
of sterically stabilized magnetic liposomes (composed of egg phosphatidylcholine (egg-PC)
and 1,2-diacyl-SN-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-
2000(DSPE-PEG2000)] at 95:5 molar ratio and anionic maghemite nanoparticles) to PC3
prostate carcinoma was evaluated swiss nude mice using an external magnet (magnetic field
0.3 Tesla). (Fortin-Ripoche et al., 2006).

Figure 3. Schematic representation showing mechanisms of magnetic hyperthermia triggered drug

release from thermosensitive magnetic liposomes and magnetic nanogel
 MAGNETIC NANO PARTICLES FOR THERAPY 79

Thermosensitive Magnetic Liposomes For Temperature

Triggered Drug Delivery
Chemotherapeutic drugs encapsulated within magnetic liposomes composed of
phospholipids with a gel to liquid transition temperature (Tm) of around 43-45 ºC can be
used for both MHT and site specific temperature triggered drug release (Fig. 3). A better
therapeutic efficacy may be achieved due to synergistic effects of hyperthermia and
chemotherapy (Issels, 2008). Moreover, the magnetic liposomes may be targeted physically to
the specific site by MDT.
Viroonchatapan et al. (1996) developed thermosensitive magnetic liposomes (TMs)
encapsulating dextran coated magnetite for MDT combined with temperature triggered drug
release by MHT. They used DPPC, a phospholipid having Tm of about 42°C, for the
liposomes and demonstrated successfully the feasibility of their use in MDT using an on-line
flow apparatus in vitro. Further, they encapsulated 5-flurouracil, a chemotherapeutic drug,
within the liposomes by reverse phase evaporation method and provided the first evidence
of magnetic hyperthermia mediated drug release within mice B16 melanoma in vivo
(Viroonchatapan et al., 1998). Different magnetic liposomes formulations for MDT and MHT
applications are summarised in Table1.

Magnetite Liposome composition + Purpose of Drug Stages of References

use Encapsulated evaluations

*Egg-PC/cholesterol Doxorubicin In vivo Nobuto et al., 2004

*DMPC/DMTAP/CHOL/DMPE-PEG5000 - In vitro and in Dandamudi et al.,
vivo 2007 a & b
a*Egg-PC/ DSPE-PEG2000 - In vitro and in Martina et al., 2005
vivo Fortin-Ripoche et al.
2006
*HSPC/cholesterol/diacetylphosphate/α- Paclitaxel In vitro and in vivo Zhang et al., 2005
tochopherol
*DPPC (temperature sensitive) 5- In vitro Viroonchatapan et
fluorouracil al., 1996 & 1998
*DPPC/Cholesterol Methotrexate In vitro and in Zhu et al, 2009
vivo
biodistribution
$TMAG/DLPC/DOPE - In vitro and in Shinkai et al., 1996
vivo
$Egg-PC/Cholesterol/ DSPE-PEG2000 - In vitro Pradhan et al., 2007b

Table 1: Different magnetic liposome formulations for MDT and MHT applications
* evaluated for MDT, $ evaluated for MHT. e evaluated as an MRI contrast agent

Thermosensitive Polymers In Magnetic Drug Delivery

The applications of polymer based drug delivery systems have been of considerable interest
in recent years. pH and temperature responsive polymers are being used in triggered release
80 NANO TECHNOLOGY

of drugs locally in tumours. These materials are termed as “smart” materials because of their
ability to receive, transmit or process a stimulus and respond by producing a useful effect. In
case of hyperthermia where local heating is required to destroy cancerous cells at
temperatures around 42-45oC, thermo-responsive polymeric drug carriers play a crucial role
as they change their physical state from one phase to other upon heating. In vitro studies of
some of the known natural polymers like gelatin (Hu et al., 2007), hydroxypropyl cellulose
(HPC) (Gil and Hudson, 2004) and that of biocompatible synthetics e.g. poly(vinylalcohol)
(PVA) (Liu et at 2006), poly(N-isopropylacrylamide) (PNIPAAm) (Gil and Hudson, 2004),
poly(N-isopropylmethacrylamide) (PNIPMAAm) (Duracher et al., 1999) have been
successfully done in their hydrogel form. All these “intelligent” temperature-stimuli
materials exhibit a transition from one physical state to other above a particular temperature,
a property which is fervently sought in temperature triggered release of drug. Table - 2 lists
some thermosensitive polymers commonly used as temperature triggered drug delivery
systems.
PNIPAAm is one of the polymers which remains hydrophilic below its lower critical
solution temperature (LCST) i.e. approximately at 32oC, and precipitates above it. The
polymer particles soak water molecules easily because of hydrophilic amide groups which
make a stable colloidal solution and expel them out due to exposure of the hydrophobic
isopropyl group above LCST. Consequently, a reduction in size occurs which renders its
applicability in temperature triggered drug release at the specific site when intravenously
injected (Liua et al.,2009). These thermosensitive polymers need temperature to get collapsed
above their LCST in order to release drug. In this context, magnetic hyperthermia is an
excellent alternative where magnetic nanoparticles encapsulated in the thermosensistive
polymer can be heated under an AC magnetic field.
PNIPAAm based multiresponsive magnetic carriers have been synthesized and used
as temperature triggered drug delivery systems by Deng et al. (2005). They developed core-
shell particles with a core of fluorescein isothiocyanate (FITC)-labelled magnetic silica and
the shell made of crosslinked poly(N-isopropylacrylamide) which were loaded with
doxorubicin. Futher, magnetic field assisted targeting of these microspheres was successfully
demonstrated in rabbits. (Bisht et al., 2008). One of the key challenges in the current decade
has been to modulate the LCST of PNIPAAm based nanocarriers at around 42-43oC, a desired
temperature for hyperthermia. The LCST of the PNIPAAm, which is about 32oC, can be
shifted upward by hydrophilic modification. A recent effort has been made by Yuan et al.
(2008) in this direction. They functionalized iron oxide nanoparticles surface to conjugate
doxorubicin via acid labile hydrazone bond and then encapsulated the magnetic
nanoparticle-drug complex within a modified thermosensitive smart polymer matrix,
chitosan-g-poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) having LCST of about
38 oC which was higher than the LCST of the PNIPAAm i.e. 32oC. Further, they demonstrated
that the temperature triggered drug release was higher above the LCST as compared to the
temperature below the LCST of the polymer composite. Zhang et.al.(2008) synthesized
copolymers of N-isopropylacrylamide and acrylamide in order to modulate LCST to 42oC.
 MAGNETIC NANO PARTICLES FOR THERAPY 81

Table 2: List of Thermoresponsive Polymers & Their LCST Values

Thermoresponsive polymers Transition temperature (oC)

Gelatin ~40oC
polymers
Hydroxypropylcellulose 45-55oC
Natural

Methylcellulose ~80oC

Poly(N-isopropylacrylamide), PNIPAAm 32-34oC

Synthetic polymers

Poly(N-isopropylmethacrylamide), PNIPMAAm 38-44oC

Poly(N,N-diethylacrylamide) 32–34°C
Poly(methylvinylether) ~37oC
Poly(vinylalcohol), PVA ~125oC
Poly(vinylpyrrolidone), PVP ~160oC
Poly(methacrylic acid), PMAc ~75oC
Poly(N-vinylcaprolactam) 30-50oC
PNIPAAm-co-AAm 33-35oC
Co-polymers

CS-g-PNIPAAm-co-DMAAm ~38oC
PEO-b-PPO 20-85oC

P(NIPAAm-ASS)-18wt% γ-Fe2O3 ~ 40oC

modified

P(NIPAAm-ASS)-38wt% γ-Fe2O3 Above 50oC

MNPs *

In another approach, Rubio-Retama et.al. (2007) reported a positive shift in LCST of

PNIPAAm when they covered its gel particles (size ~ 400 nm) with γ-ferrite nanoparticles
particles. The presence of the magnetic nanoparticles caused an increase in the LCST of the
polymer matrix with a proportionate increase in amount of the nanoparticles.
Apart from thermosensitivity of PNIPAAm, it also has its application in pH sensitive
drug delivery systems. Recently Bertrand et. al. (2009) designed pH-sensitive poly(NIPAAm)
based copolymers having prolonged blood circulation for endosomal drug delivery. They
demonstrated that terminally-alkylated copolymers of NIPAAm and methacrylic acid (MAA)
insert in liposomal bilayers and destabilize at pH~5, thereby triggering the release of
encapsulated drug.

5. CLINICAL TRIALS OF MHT

Johannsen et al.(2005) reported the first clinical trial of magnetic fluid hyperthermia. The aim
of this study was to evaluate the feasibility of using magnetic hyperthermia for treating
locally recurrent prostate cancer with minimum invasion. In this study, aminosilan coated
superparamagnetic nanoparticles were transperineally injected into the prostate of a 67 year
old patient. Then magnetic field with a frequency of 100kHz and a variable field strength of
0-18kA/m was applied to increase the temperature of the magnetic nanoparticles. The
82 NANO TECHNOLOGY

magnetic field was applied for 1hr once in a week for six weeks. Temperature at different
points of the prostate was calculated, based on distribution of magnetic nanoparticles inside
the prostate. Maximum and minimum prostatic temperature during the 1st dose of
hyperthermia, were 48.5 and 40oC where as for 6th dose of hyperthermia the respective
temperatures were 42.5 and 39.4oC. These temperatures were determined by invasive
thermometric process. During the hyperthermia, the patient felt pain several times due to
sub-optimal positioning of cooling system, otherwise the treatment was tolerable. The
strength of magnetic field used was in the range of 4-5kA/m for the hyperthermia treatment.
Presence of nanoparticles were detected within the prostate even after 6 week. It was
concluded from this study that magnetic fluid hyperthermia may be used for treatment of
cancer and desired hyperthermia temperature may be achieved by varying the strength of
the magnetic field.
Johannsen et al.(2007a) carried out a phase I trial to study the feasibility of using
superparamagnetic iron oxide nanoparticles in hyperthermia. This study was carried out on
10 patients with recurrent prostate cancer. After administering the magnetic nanoparticles,
the patients received six doses of thermotherapy each of 1hr duration for six weeks. The
frequency of alternative and the field strength of magnetic field were 100kHz and 2.5-
18kA/m respectively. It was observed that a magnetic field of 4-5kA/m was sufficient to
achieve the temperature of the nanoparticles to hyperthermic temperature. Johannsen et
al.(2007b) also condicted a phase I trial of thermotherapy using superparamagnetic
nanoparticles in patients with locally recurrent prostate cancer. The trial was done on 10
patients of age group 62-79 year. The main objective of this study was to investigate the
feasibility of attaining hyperthermia temperature and evaluate the patients’ morbidity and
quality of life during and post treatment period. The nanoparticle dispersion was injected
transperineally into the prostates of the patients. The thermotherapy was given for 60min
with a magnetic field of 100 kHz frequency and variable field strength of 2.5-18kA/m.
Thermotherapy was given six times to each patient with one week interval between two
consecutive therapy sessions. In order to evaluate the patients’ morbidity and quality of life,
post treatment checkups were done at three and six month after the thermotherapy. It was
observed that maximum temperature upto 55oC was attained with application of 25-30%
strength of available magnetic field. Nanoparticles were detected in the prostate even after
one year of the thermotherapy and no systemic toxicity was observed due to presence of
nanoparticles.
Maier-Hauff et al. (2007), conducted a phase I trial of intracranial thermotherapy
using magnetic nanoparticles in combination with radiotherapy on 14 adult patients bearing
glioblastoma multiforme. The estimated life expectancy of these patients was more than three
months. The aim of this study was to evaluate the feasibility of using magnetic nanoparticles
for hyperthermia treatment of glioblastoma multiforme bearing patients. After
administering the magnetic nanoparticles into the tumour region, an alternating magnetic
field was applied to increase the temperature of the tumour region. The amount of magnetic
nanoparticles and their spatial distribution within the tumour were calculated based on MRI
data. Doses of radiation were also applied along with hyperthermia treatment. Temperature
at different points of tumour was determined by optical fibre probe also. It was observed
during this study that the treatment procedure was within the tolerable limit of the patients
and no side effects were observed due to the treatment procedure. It was concluded that
 MAGNETIC NANO PARTICLES FOR THERAPY 83

magnetic nanoparticles may be used for hyperthermia treatment of glioblastoma multiforme

patient with tolerable sufferings.
Recently, Landeghem et al.(2009), reported the neuropathological findings of three
patients from an ongoing phase II clinical trial against glioblastoma multiforme. Estimated
life expectancy of these patients was more than three months. In this study, aminosilane
coated superparamagnetic iron oxide nanoparticles were administered into the tumour
region. Then thermotherapy was applied with a magnetic field of 100kHz frequency and a
variable strength of 2.5-18kA/m. Temperature at different points of the tumour was
measured with optical fibre probe thermometer. This study reported practically no adverse
effects related to MFH treatment however, the magnetic nanoparticles were preferentially
taken up by the macrophages.

6. CONCLUSIONS AND FUTURE PERSPECTIVES

From the above discussion, it is evident that magnetic nanoparticles are promising for
magnetic hyperthermia and drug targeting. Although a seminal work on these fields have
been done on the development and characterizations of the materials over a period of last
two decades, few studies have reached the stage of clinical trials. For magnetic hyperthermia,
local injection of magnetic nanoparticles in the tumor is done as the amount of magnetic
nanoparticles required within the tumor is few milligrams to achieve the temperature of
about 43°C locally, which can hardly be achieved by intravenous administration even after
targeting the nanoparticles both biologically (i.e. by monoclonal antibody or targeting ligand
such as folate) and physically by permanent magnetic field. One possible solution would be
to develop magnetic nanoparticles with very high SAR values so that less than a milligram of
nanomaterial would be sufficient for magnetic hyperthermia locally in the tumor. Also, more
research is needed to develop a Tc tuned smart magnetic particle complexed with
chemotherapeutic agent having high heating ability. The targeting of the magnetic
nanoparticles on intravenous injection has been limited and need further exploration. Once
the magnetic nanoparticles are localized in the tumor, AC magnetic fields may be applied to
raise the temperature of the tumor for hyperthermia treatment, which may give better
therapeutic efficacy due to combined or synergistic effect of chemotherapeutic drug and
hyperthermia. However, an integrated effort is required in this interdisciplinary field prior to
successfully using multifunctional magnetic nanoparticles combining hyperthermia and drug
delivery.

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