2012 Wahajuddin and Arora, publisher and licensee Dove Medical Press Ltd. This is an Open Access [615996]

© 2012 Wahajuddin and Arora, publisher and licensee Dove Medical Press Ltd. This is an Open Access
article which permits unrestricted noncommercial use, provided the original work is properly cited.International Journal of Nanomedicine 2012:7 3445–3471International Journal of Nanomedicine
Superparamagnetic iron oxide nanoparticles:
magnetic nanoplatforms as drug carriers
Wahajuddin1,2
Sumit Arora2
1Pharmacokinetics and Metabolism
Division, CSIR-Central Drug
Research Institute, Lucknow,
Uttar Pradesh, 2Department of
Pharmaceutics, National Institute
of Pharmaceutical Education and
Research, Rae Bareli, India
Correspondence: Wahajuddin
Pharmacokinetics and Metabolism
Division, CSIR-Central Drug Research
Institute, Lucknow 226001,
Uttar Pradesh, India, CSIR-CDRI
communication no 8264.
Tel +9152226124 11-18 extension 4377
Fax +91 52 2262 3405
Email [anonimizat].i nAbstract: A targeted drug delivery system is the need of the hour. Guiding magnetic iron oxide
nanoparticles with the help of an external magnetic field to its target is the principle behind the
development of superparamagnetic iron oxide nanoparticles (SPIONs) as novel drug delivery
vehicles. SPIONs are small synthetic γ-Fe2O3 (maghemite) or Fe3O4 (magnetite) particles with
a core ranging between 10 nm and 100 nm in diameter. These magnetic particles are coated
with certain biocompatible polymers, such as dextran or polyethylene glycol, which provide
chemical handles for the conjugation of therapeutic agents and also improve their blood dis –
tribution profile. The current research on SPIONs is opening up wide horizons for their use as
diagnostic agents in magnetic resonance imaging as well as for drug delivery vehicles. Delivery
of anticancer drugs by coupling with functionalized SPIONs to their targeted site is one of the
most pursued areas of research in the development of cancer treatment strategies. SPIONs have
also demonstrated their efficiency as nonviral gene vectors that facilitate the introduction of
plasmids into the nucleus at rates multifold those of routinely available standard technologies.
SPION-induced hyperthermia has also been utilized for localized killing of cancerous cells.
Despite their potential biomedical application, alteration in gene expression profiles, disturbance
in iron homeostasis, oxidative stress, and altered cellular responses are some SPION-related
toxicological aspects which require due consideration. This review provides a comprehensive
understanding of SPIONs with regard to their method of preparation, their utility as drug
delivery vehicles, and some concerns which need to be resolved before they can be moved
from bench top to bedside.
Keywords: superparamagnetic iron oxide nanoparticles, SPIONs, targeted delivery, coating,
functionalization, targeting ligands, toxicity
Introduction
All great things come in small packages, and products of nanoscience are no exception.
Nanoparticles are simply particles in the nanosize range (10−9 m), usually ,100 nm
in size. Due to their small size and surface area characteristics, they exhibit unique
electronic, optical, and magnetic properties that can be exploited for drug delivery.
Also known as nanovectors in the field of drug delivery, they are promising new tools
for controlled release of drugs because they can satisfy the two most important criteria
for successful therapy, ie, spatial placement and temporal delivery.
No drug is free from side effects, and these side effects usually arise from nonspeci –
ficity in drug action. For instance, in the case of tumor therapy, it is the side effects of
cytotoxic drugs, such as bone marrow depression and reduced immunity, which can
be hazardous to the extent that termination of therapy may be required. Modification Dove press
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International Journal of Nanomedicine 2012:7of the surface characteristics of nanoparticles, such as
superparamagnetic iron oxide nanoparticles (SPIONs) with
biocompatible polymers, and controlling their size within the
desirable range can yield powerful targeted delivery vehicles
which can deal with this issue.
Freeman et al were the first to introduce the concept of use
of magnetism in medicine in the 1970s.1 Since then, much
research has been done in this area, leading to the design of
various magnetic particles and vectors. The main objective
today is optimization of the properties of these magnetic
particles to: provide an increase in magnetic nanoparticle con –
centration in blood vessels; reduce early clearance from the
body; minimize nonspecific cell interactions, thus minimiz –
ing side effects; and increase their internalization efficiency
within target cells, thus reducing the total dose required.2,3
SPIONs are small synthetic γ-Fe2O3 (maghemite), Fe3O4
(magnetite) or α-Fe2O3 (hermatite) particles with a core rang –
ing from 10 nm to 100 nm in diameter. In addition, mixed
oxides of iron with transition metal ions such as copper,
cobalt, nickel, and manganese, are known to exhibit super –
paramagnetic properties and also fall into the category of
SPIONs. However, magnetite and maghemite nanoparticles
are the most widely used SPIONs in various biomedical
applications. SPIONs have an organic or inorganic coating,
on or within which a drug is loaded, and they are then guided
by an external magnet to their target tissue. These particles
exhibit the phenomenon of “superparamagnetism”, ie, on
application of an external magnetic field, they become
magnetized up to their saturation magnetization, and on
removal of the magnetic field, they no longer exhibit any
residual magnetic interaction. This property is size-dependent
and generally arises when the size of nanoparticles is as low
as 10–20 nm. At such a small size, these nanoparticles do not
exhibit multiple domains as found in large magnets; on the
other hand, they become a single magnetic domain and act as a
“single super spin” that exhibits high magnetic susceptibility.
Thus, on application of a magnetic field, these nanoparticles
provide a stronger and more rapid magnetic response com –
pared with bulk magnets with negligible remanence (residual
magnetization) and coercivity (the field required to bring the
magnetism to zero).4,5 This superparamagnetism, unique to
nanoparticles, is very important for their use as drug delivery
vehicles because these nanoparticles can literally drag drug
molecules to their target site in the body under the influence
of an applied magnet field. Moreover, once the applied
magnetic field is removed, the magnetic particles retain
no residual magnetism at room temperature and hence are
unlikely to agglomerate (ie, they are easily dispersed), thus evading uptake by phagocytes and increasing their half-life
in the circulation. Moreover, due to a negligible tendency
to agglomerate, SPIONs pose no danger of thrombosis or
blockage of blood capillaries.
The current research on SPIONs is opening up broad
horizons for their use in the biomedical sciences. They
have been used for both diagnostic as well as therapeutic
purposes. In magnetic resonance imaging (MRI), SPIONs
have been used as targeted magnetic resonance contrast
agents, allowing diagnosis of progressive diseases in their
early stages.6 From a drug delivery point of view, targeting
of cancer is the most pursued area, with emphasis on delivery
of chemotherapeutics and radiotherapeutics.7,8 However,
increasing applications of SPIONs have also been found in
the areas of gene delivery, cell death with the help of local
hyperthermia, and delivery of peptides and antibodies to
their site of action.
Toxicity is an important issue which must be dealt with
before SPIONs can be considered for widespread use in drug
delivery. Much research has been carried out to evaluate
the biocompatibility of these magnetic nanoparticles and
their possible adverse interactions with cellular and subcel –
lular structures.9,10 In this review, we discuss their desirable
characteristics, including shape, hydrodynamic volume,
surface charge and colloidal stability, various methods used
for preparation of these nanoparticles, and their role in the
biomedical sciences. Furthermore, limitations of SPIONs
as drug delivery agents and their toxicity are discussed in
detail.
Physicochemical characteristics
essential for drug delivery
Figure 1 shows the most important physicochemical char –
acteristics of SPIONs, which should be taken into account
while designing a successful drug delivery system. Such
properties mostly govern the blood distribution profile of
these nanoparticles.
Shape
The morphology of Fe2O3 nanoparticles has been known to
be affected by several factors, including the reaction condi –
tions and chemicals involved. In the presence of surfactants
with bulky hydrocarbon chain structures, like oleylamine and
adamantane amine, the steric hindrance exerted by surfactants
has been shown to affect the shape of growing crystals of
iron oxide during synthesis.11 The shape of magnetic nano –
particles has not been extensively studied as far as its effect
on biodistribution of SPIONs is concerned. However, a few
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3446Wahajuddin and Arora

International Journal of Nanomedicine 2012:7researchers studying other nanoparticulate delivery systems
have reported that rod-shaped and nonspherical nanoparticles
show a longer blood circulation time compared with spherical
particles.12 For instance, Huang et al studied the effect of par –
ticle shape on the in vivo behavior of mesoporous silica nano –
particles. They found that the shape of the nanoparticles could
affect their biodistribution, clearance, and biocompatibility
in vivo. Short-rod mesoporous silica nanoparticles which are
more or less spherical were found to accumulate in the liver,
whereas long-rod-shaped particles were distributed to the
spleen. Moreover, short-rod mesoporous silica nanoparticles
showed rapid clearance rates via urine and feces compared
with long-rod mesoporous silica nanoparticles.12 Another
reason which may favor a longer blood circulation time for
rod-shaped nanoparticles may be the fact that the phagocytic
activity of macrophages is stimulated to a lesser extent by
rod-shaped particles than by spherical ones.13 However,
spherical magnetite and maghemite particles offer a uniform
surface area for coating and conjugation of targeting ligands
or therapeutic agents. Mahmoudi et al showed that the shape
of SPIONs exerts a direct effect on cell toxicity. Nanobead-
shaped, nanoworm-shaped, and nanosphere-shaped SPIONs
showed greater cellular toxicity compared with nanorods and
colloidal nanocrystal clusters.14
Size
The size of nanoparticles largely determines their half-life
in the circulation.15 For instance, particles with sizes smaller
than 10 nm are mainly removed by renal clearance, whereas
particles larger than 200 nm become concentrated in the
spleen or are taken up by phagocytic cells of the body,
in both instances leading to decreased plasma concentrations. However, particles with a size range of 10–100 nm are
considered to be optimum, with longer circulation times
because they can easily escape the reticuloendothelial system
in the body. They are also able to penetrate through very
small capillaries.16 Furthermore, biomedical applications of
SPIONs, including MRI, hyperthermia, and magnetic cell
separation, depend on the magnetic properties of these par –
ticles, which in turn are largely dependent upon size.15 The
small size of SPIONs is also responsible for the enhanced
permeability and retention effect, which causes concentration
of the particles in target tumor tissue. However, SPIONs with
a particle size smaller than 2 nm are not suitable for medi –
cal use. This is due to the increased potential of particles in
this size range to diffuse through cell membranes, damaging
intracellular organelles and thus exhibiting potentially toxic
effects. Therefore, control of particle size during preparation
of SPIONs is an important concern.
The techniques most commonly employed for measuring
the particle size of SPIONs are transmission electron micro-
scopy, dynamic light scattering, and the Scherrer method using
x-ray diffractograms. Because only a small number of the
particles prepared are of the desired size, it becomes necessary
to carry out fractionation of magnetic fluids. Currently applied
techniques are centrifugation, size exclusion chromatography,
and field flow fractionation.17–19 While the first two techniques
separate particles on the basis of their density or size, the lat –
ter technique utilizes the magnetic properties of SPIONs for
their efficient fractionation.
Surface properties
The surface charge of nanoparticles gives an indication of their
colloidal stability. Nanoparticles having high positive and
negative zeta potential show dispersion stability and as a result
do not agglomerate on storage. Charge also determines the
distribution of these particles in the body and is an important
parameter affecting internalization of nanoparticles in their
target cells. In one study, it has been reported that uncoated
and pullulan-coated SPIONs are internalized into cells by dif –
ferent mechanisms, demonstrating surface-dependent particle
endocytosis behavior.20
SPIONs having a positive charge are better internalized by
human breast cancer cells than are negatively charged particles.
However, intake of these nanoparticles also depends upon cell
type.2 Particles with a hydrophobic surface are easily adsorbed
at the protein surface (opsonization) and are engulfed by circu –
lating macrophages, resulting in their clearance from plasma.21
Therefore, they show a low circulation time. However, particles
that are surface-engineered with hydrophilic polymers like
Shape
Size
Surface
propertyAffects
biodistibution
Effect of celltoxicity
Determinesresidual time in
blood
Effect on magnetic
properties
Indicates colloidal
stability
Determinesinternalization into
the target cellsPhysiochemical
properties of SPIONs
Figure 1 Physicochemical considerations of superparamagnetic iron oxide
nanoparticles for drug delivery.
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3447Superparamagnetic iron oxide nanoparticles

International Journal of Nanomedicine 2012:7polyethylene glycol (stealth particles) containing, eg, hydroxyl
or amino functional groups, are able to evade engulfment by
the reticuloendothelial cells or circulating macrophages, thus
having better therapeutic efficacy due to increased residence
time in the blood.22 Surface-engineering of magnetic nano –
particles with different functional groups imparts different
surface characteristics, making them suitable for a wide variety
of biomedical and other industrial applications. Coating the
surface of SPIONs with different polymers will be discussed
in the following sections.
As the size of iron oxide particles reduces into the
nanorange, iron ions on the surface of nanoparticles play an
important role in determining the magnetic properties of these
particles. The oxidation state of the iron ion on the surface of
the nanoparticle is sensitive to its surrounding environment,
particularly to surfactant exposure. Analysis of the oxidation
state of iron ions on the surface of nanoparticles can help in
identifying their structure and chemical environment. It has
been reported that the oxidation state of the iron ion can have a
potential effect on the morphology of nanoparticles prepared.
For instance, iron ions in the trivalent state ( +3) favor formation
of spherical nanoparticles, whereas metal ions in the divalent
state (+2) favor formation of nanorods.23 The primary objec –
tive of research at present is the preparation of uniform and
stable SPIONs by controlled synthesis and coating processes.
The synthetic route selected not only determines the physical
features of SPIONs, but also has a profound effect on their
crystallochemical characteristics.24
Core fabrication
Nucleation and crystal growth are the two fundamental
steps in preparation of crystals from solution. Taking into
consideration the classical model of crystallization proposed
by LaMer and Dinegar, monodispersed nanoparticles can
be produced by a single short burst of nucleation, which
occurs when a solution reaches its critical supersaturation
concentration.25 Nuclei thus obtained then grow as a result
of diffusion of solute particles from the solution onto the
surface of the nuclei, until a suitable size is reached. For
achieving monodispersity, care must be taken to ensure
that nucleation does not occur during the crystal growth
phase. Multiple nucleations can also result in formation of
uniformly dispersed nanoparticles. This occurs as a result
of Ostwald ripening, a self-reforming process whereby
small nuclei crystals formed get redissolved and deposit
onto larger nuclei, forming large uniform crystals.26 Aggre –
gation of smaller units may also result in uniform-sized
nanoparticles.27The most commonly used methods for preparation
of uniform iron-based nanoparticles in solution are
coprecipitation and microemulsion.28 Preparation of SPIONs
utilizing the coprecipitation method involves two approaches,
ie, partial oxidation of ferrous hydroxide suspension by
different oxidizing agents such as nitrates, as explored by
Sugimoto et al, and addition of base to an aqueous solution
containing a mixture of ferrous (Fe2+) and ferric (Fe3+) ions
with 1:2 stoichiometry in an oxygen-free environment.29
Massart et al prepared SPIONs utilizing the second approach
and obtained a black precipitate of spherical magnetic nano –
particles in the size range of ,20 nm.30 On the other hand,
particles obtained using the method reported by Sugimoto
et al were larger, ranging from 30 nm to 200 nm. The size
of magnetic nanoparticles prepared by the coprecipitation
method largely depends upon the pH and ionic strength of
the precipitating solution. It has been demonstrated that as
the pH and ionic strength of the medium increases, the size
of the particles decreases.24 These parameters not only affect
the size of the nanoparticles formed, but also determine the
electrostatic potential on the surface of these nanoparticles,
which is indicative of their dispersion stability.
Smaller and more uniform particles can be synthesized
using the microemulsion approach. Water-in-oil (w/o) micro –
emulsions (ie, reverse micelle solutions) are transparent,
isotropic, and thermodynamically stable liquids. In these
systems, the aqueous phase is dispersed as microdroplets in
the continuous oil phase, ie, entrapped within the micellar
assembly of stabilizing surfactants.28 The main advantage
of utilizing this approach is that these microdroplets serve
as nanoreactors, providing a confined space which limits
growth and agglomeration of nanoparticles during their
synthesis, thus regulating their size and surface properties.
Using this method, iron precursors like ferrous chloride and
ferric chloride are precipitated as oxides in the aqueous core.
Iron precursors in the organic phase remain unreactive. By
controlling the size of the microdroplets, particles in the
desired size range can be obtained. A novel nanocomposite
consisting of nanometric cores of silver embedded in a matrix
of γ- Fe2O3 was prepared by sequential reaction of different
mixtures of reverse micelles.31
An emerging method for preparation of uniform nanopar –
ticles is the polyol technique, whereby fine metallic particles
can be made by reduction of their dissolved metallic salts
and direct metal precipitation from solution containing a
polyol. Iron particles around 100 nm prepared by this process
have been reported. High temperature decomposition of iron
precursors in the presence of suitable surfactants results in
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3448Wahajuddin and Arora

International Journal of Nanomedicine 2012:7 synthesis of uniform magnetic nanoparticles with a desirable
size range and surface properties. Sun et al reported synthe –
sis of monodispersed magnetic nanoparticles sized 3 nm to
20 nm by thermal degradation of iron (III) acetylacetonate
in phenyl ether in the presence of alcohol, oleic acid, and
oleylamine at 265 °C.32 Use of dendrimers as templating hosts
for synthesis of magnetic nanoparticles has drawn consider –
able attention. This is because of the fact that by properly
selecting the appropriate dendrimer host, biocompatible
SPIONs suitable for in vivo application can be made via
a single-step process.33 High-energy ultrasound waves can
also be utilized for the synthesis of magnetite and maghemite
nanoparticles. These high-energy sound waves create acous –
tic cavitations, ie, formation, growth, and implosive collapse
of empty cavities, resulting in transient localized hot spots
with a temperature of about 5000 K.34,35 Formation of these
cavities sends out shock waves, leading to particle size reduc –
tion with concomitant formation of magnetite nanoparticles.
However, large-scale synthesis of magnetic nanoparticles
utilizing this approach is not very feasible. Magnetic nano –
particles have also been synthesized by electrochemical
deposition of metal on a cathode, produced by reduction of
metal ions dissolved from the anode.24
Spray and laser pyrolysis are two further emerging
approaches for preparation of uniform magnetic nanopar –
ticles, with great commercial scale-up potential. In spray
pyrolysis, a solution of Fe3+ salt and reducing agents is
sprayed through a series of reactors where aerosol droplets
undergo evaporation of solvents with solute condensation
within the droplets. This is followed by drying and thermoly –
sis of the precipitated product at high temperature, resulting in
microporous solids finally sintering to form dense particles.24
Laser pyrolysis involves heating a flowing mixture of gases
with a continuous wave carbon dioxide laser, initiating and
sustaining a chemical reaction. Homogenous nucleation of
particles results when a critical concentration of nuclei is
reached within the reaction zone above a certain pressure
and laser power. Usually, iron pentacarbonyl is used as a
precursor for synthesis of γ-Fe2O3 nanoparticles by the laser
pyrolysis method.24,36
Magnetotactic bacteria, a group of Gram-negative
prokaryotes, have demonstrated an ability to synthesize fine
iron oxide nanoparticles in the size range of 50–100 nm.
These bacterial magnetic nanoparticles are covered with
phospholipid layers, making them biocompatible and hence
useful for a variety of bioapplications.37,38 Researchers have
also used a variety of natural protein components, such as
ferritin, which serve as nanoshells consisting of a central core within which iron oxide nanoparticles 6–8 nm in size can be
synthesized.39,40 Table 1 summarizes the various methodolo –
gies, along with their advantages and disadvantages, used for
preparation of SPIONs.
Coating
The next step after fabrication of SPION cores is their coating.
Coating with suitable polymers endows some important char –
acteristics to these nanoparticles that are essential for their
use as drug delivery vehicles. Coating of SPIONs is essential
because: it reduces the aggregation tendency of the uncoated
particles, thus improving their dispersibility and colloidal
stability; protects their surface from oxidation; provides
a surface for conjugation of drug molecules and targeting
ligands; increases the blood circulation time by avoiding
clearance by the reticuloendothelial system; makes the par –
ticles biocompatible and minimizes nonspecific interactions,
thus reducing toxicity; and increases their internalization
efficiency by target cells. The presence of amino groups on
the coating shell of amino-polyvinyl alcohol-functionalized
SPIONs increases their uptake by human melanoma cells, as
reported by Petri-Fink et al.41 Table 2 lists the most commonly
used coating materials for SPIONs.
SPIONs can be coated either during their synthesis or
can undergo adsorption after synthesis.21 Both methods have
been reported to produce particles with a uniform coating.
However, coating of SPIONs with nonmagnetic polymers
like polysaccharides leads to a decrease in saturation mag –
netization as compared with uncoated SPIONs. Amstad
et al have reported a decrease in saturation magnetization
of mPEG(550)-gallol-stabilized SPIONs as compared with
bare particles, from 58 emu gFe−1 to 50 emu gFe−1, as mea –
sured by a superconducting quantum interference device.6
A large decrease in saturation magnetization is undesirable
because it will result in failure to attain an effective con –
centration of SPIONs at the target site on application of the
external magnetic field. The SPIONs should be coated in
such a manner that the coating not only imparts favorable
characteristics but also preserves the desirable properties of
uncoated SPIONs.
Drug loading
The primary requirement is that the drug should be loaded
in such a manner that its functionality is not compromised.
Moreover, these drug-loaded nanoparticles should also
release the drug at the appropriate site and at a desired
rate. Drug loading can be achieved either by conjugating
the therapeutic molecules on the surface of SPIONs or by
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3449Superparamagnetic iron oxide nanoparticles

International Journal of Nanomedicine 2012:7Table 1 Different routes of synthesis of superparamagnetic iron oxide nanoparticles along with their reaction conditions, advantages, and disadvantages
Method
employedReaction
temperature ( °C)Solvent
usedSize
range
(nm)Morphology Advantages Disadvantages Feasibility of
large-scale
productionPublished
reports
Coprecipitation 20–90 Water 15–200 Spherical or
rhombicSimplest and most efficient chemical method
By adjusting pH and ionic strength, particle
size can easily be controlled
This method can be easily modified for synthesis in
the presence of dextran and other coating materials,
rendering magnetic nanoparticles biocompatible and
hence suitable for in vivo applications
Almost all commercially available
iron-oxide based MRI contrast agents
are fabricated by this routeLarge particle size distribution
Sometimes, aggregation and
poor crystallinity resulting in low
saturation magnetization values
Sometimes, ions get oxidized
before precipitation, affecting the
physical and chemical properties
of SPIONsYes 28,29,
137–140
Microemulsions 20–50 Organic
compound4–12 Spherical or
cubicAdequate, versatile and simple method
Surfactant-stabilized microcavities provide
confinement effect that limit particle nucleation,
growth, and aggregation, thus better controlling
particle size as well as particle shape
Homogenous particle size distributionAdequate crystallinity of SPIONs
is sometimes difficult to achieve
on a large scale due to low
temperature usage
Complicated purification methods
for separation of surfactants
Yield of nanoparticles is low
compared with coprecipitation
method
Large amount of solvents requiredNo 28,137,138,
141
Hydrothermal
synthesis220 Water-
ethanol520 Spherical This approach takes advantage of increased
solubility and reactivity of metal salts and
complexes at elevated temperature and pressure
without bringing the solvent to its critical point
Well control of growth dynamics and
agglomeration of metal nanoparticles
Important synthetic route for production of ferritesHarsh reaction conditions, hence
experimentally demanding
Unwanted oxidation at the
surface of metal nanoparticles
Longer reaction timeYes 137–140
Sol-gel
synthesis200–400 Organic
compound20–200 Spherical Suitable for producing powders of
magnetic materials
Through nonhydrolytic sol-gel route,
monodispersed nanoparticles can be produced
Particles of desired shape and size can be
fabricated, useful for making hybrid nanoparticlesDifficult to obtain monodispersed
nanoparticles through hydrolytic
sol-gel route
Requires toxic reagents and
complicated synthesis steps
Contamination of products with
sol-gel matrix constituents
Broad size distributionNo 140,142
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International Journal of Nanomedicine 2012:7
Electrochemical
deposition70–100 Organic
compound3–8 Spherical Size can easily be controlled by imposed
current density
very narrow particle size distribution ensuring
homogenous distributionHigh cost of production
Difficult to remove surfactant
used in the processNo 28,143
Sonochemical
method25 water 10–30 Spherical or
rod-shapedMonodispersed nanoparticles of a variety of
shapes can be produced
Produces ϒ-Fe203 nanoparticles useful in
biotechnologyParticle size tunability is not
easily achievable
Utilizes organometallic
precursors, can cause in vivo
toxicityNo 137,144,145
Polyol method 120–280 Organic
compound5–40 Spherical Nonagglomerated iron oxide particles with well
defined shape and size can be obtained
High boiling point of polyols can be used as a
solvent as well as a reducing
agentScale up sometimes lead to
nonuniform particle shape and
size, needs modification
Broad particle size distributionYes 146–149
Thermal
decomposition100–320 Organic
compound3–20 Spherical Can be employed to separate nucleation and growth
processes yielding samples with good size control,
narrow size variation of 5%, and good crystallinity
Monodispersed iron oxide particles can easily
be prepared
very narrow size distribution with no follow-up
size selection procedures
Polar solvents like 2-pyrrolidone along with thermal
decomposition allow use of various surface
ligands to achieve biocompatible SPIONs via a
“one-pot” reactionUses complicated and harsh
preparation procedures
Surfactant (eg, oleic acid) is used
in the process, which hinders
subsequent surface modification
Employ organometallic
compounds which can cause in vivo
toxicity, hence replaced by metal
salts
Requires use of very high
decomposition temperature
leading to formation of
iron oxide
on the surface of iron
nanoparticlesYes
137–140,
149–151
Spray pyrolysis 400–700 Organic
compound5–60 Spherical but
aggregated into
larger particlesIt is a simple, rapid and continuous process
Finely dispersed particles of predictable size,
shape and variable composition can be
prepared
This method allows the preparation of air-stable
superparamagnetic α-Fe metallic and FeCo
nanomagnets encapsulated in silica or
aluminumRequires exhaustive control
of the experimental conditions
Cost of production is highYes
28,137,152
(Continued )
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3451Superparamagnetic iron oxide nanoparticles

International Journal of Nanomedicine 2012:7Table 1 (Continued )
Method
employedReaction
temperature ( °C)Solvent
usedSize
range
(nm)Morphology Advantages Disadvantages Feasibility of
large-scale
productionPublished
reports
Laser-induced
pyrolysis1100 Organic
compound5–30 Spherical but
less aggregatedContinuous method of production
Small particle size
Narrow particle size distribution
Nearly absence of aggregation
High production rate, good alternative to
coprecipitation method for the synthesis of
MRI contrast agentsCost of production is high
Requires exhaustive control
of experimental conditionsYes 28,137
Biomimetic
synthesis
(mediated by
bacteria, fungi
and protein)– – 50–100 Spherical,
cluster, cubo-
octahedralNanoparticles synthesized by this route find
variety of bioapplications
Presence of phospholipid bilayer makes particle
biocompatible and suspension of particles
are very stable
Particles can be easily modified
These magnetic particles can be used for
immobilization of a variety of enzymes,
antibodies and oligonucleotidesLacks large-scale synthesis with
well defined shape and size
Lower yield of magnetic
nanoparticlesNo 2,28
Abbreviations: MRI, magnetic resonance imaging; SPIONs, superparamagnetic iron oxide nanoparticles.
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3452Wahajuddin and Arora

International Journal of Nanomedicine 2012:7Table 2 Encapsulating materials and targeting ligands used to functionalize the surface of superparamagnetic iron oxide nanoparticles and their applications
Coating
materialTargeting
strategyTargeting ligand Target Particle size
distributionProperties bestowed by
coating on particleApplications Advantages Published
reports
Poly(dl-lactic
acid)Active Herceptin®
(Her2 antibody)Human breast cancer 50–200 nm Improves hemocompatibility
and produces no hemolysis,
ensure uniform particle size
distributionRadiotherapy, drug targeting
and imagingBiodegradable, can be
conjugated with other
polymers like PEG
resulting in desired
alternation of magnetic
properties78,
153–155
Anti-CD20 monoclonal
antibody (rituximab)CD-20 antigen
(Non-Hodgkin lymphoma)
Passive – –
PEG Active N-terminated A10
RNA aptamer PSMA 10–50 nm Improves dispersity and
blood circulation time,
provides chemical handles
for drug attachment, reduces
toxicity, decreases enzyme
degradationImaging and drug delivery
particularly to tumors
mainly brain and breastEfficient surface coating,
can easily be crosslinked
to reduce the burst
effect of anchored drugs2,16,129,
156,157
Antihuman vCAM-1
antibodiesvCAM-1 as an early
marker of atherosclerosis
Chlorotoxin MMP-2
Folic acid Folate receptor
Methotrexate Folate receptor
Passive – –
Dextran Active Monoclonal
antibody A7Human colorectal
carcinoma10–200 nm Improves biocompatibility,
enhances blood circulation
time, reduces aggregationImaging and drug delivery
Conjugated with urokinase
for targeted thrombolysisEfficient coating due
to high affinity for iron
oxide surface2,158–159
Herceptin Human breast cancer
Passive – –
Chitosan Active ANP ANP receptors 20–100 nm Improves biocompatibility,
imparts hydrophilicity, ease
of functionalization due
to presence of amino and
hydroxyl functional groupsNonviral gene delivery,
drug therapy, hyperthermia,
tissue engineering, targeted
photodynamic therapyLarge natural abundance,
cheap and easy availability2,130,
160,161
(Continued )
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3453Superparamagnetic iron oxide nanoparticles

International Journal of Nanomedicine 2012:7Table 2 (Continued )
Coating
materialTargeting
strategyTargeting ligand Target Particle size
distributionProperties bestowed by
coating on particleApplications Advantages Published
reports
CEA antibody CEA
Magnetic
targeting– –
Silica Active Annexin v Phosphatidylserine 10–300 nm Provides scaffold for
enzyme immobilization,
serves as template for
radical polymerization
with other moleculesControlled drug delivery,
imaging and separation of
biomolecules, apoptosis
imagingLarge surface area, does
not require utilization
of organic solvent, thus
reduced toxicity due to
residual organic solvents2,162
Passive – –
Silane Active Methotrexate Folate receptors 10–200 nm Increases saturation
magnetization, decreases
wear rate of coated
particlesBreast cancer imaging and
drug delivery, assessing
angiogenic profile of tumors
Magnetic separation and
purification of DNAEnhances dispersion in
biological matrix, can
significantly improve
protein immobilization156,
163–167
Arg-Gly-Asp (RGD)
peptideαvβ3 integrin
Passive – –
Glycerol
monooleateActive Herceptin Her2/neu receptors
(breast cancer)100–200 nm Improves biocompatibility,
can be easily functionalized
with other chemical
moietiesSustained release of
encapsulated drug, capable
of delivering high payload
hydrophobic anticancer
drugsDoes not affect magnetic
properties of Fe2O3, high
entrapment efficiency,
can reduce burst release
of drugs168,169
Albumin Active Anti-EGFR antibody EGFR 100–200 nm Improves biocompatibility,
reduces toxicity, stabilizes
preparationDrug targeting and cell
separation, imaging of
esophageal squamous cell
carcinomaDoes not affect cell
proliferation170
Passive – –
Liposomes Active Antitransferrin
receptor single-chain
antibody fragmentTransferrin receptor 20–200 nm Enhances blood circulation
time, increases SPION
specificity and uptake
into tumor cells, prevents
enzymatic degradation of
drugs encapsulatedUse in gene medicine and
drug delivery, hyperthermia,
and imagingSimple and easy surface
modification Increased
payload, does not reduce
superparamagnetic
property of bare SPIONs
on coating21,48,93
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3454Wahajuddin and Arora

International Journal of Nanomedicine 2012:7
Gold Magnetic
targeting– – 30–100 nm Improves colloidal stability,
reduces surface toxicity,
provides a strong binding
surface for a self-assembled
monolayer of enzymeMagnetically directed enzyme
prodrug therapy, delivery of
nitroreductasesExhibits strong magnetic
response, uniform
coating
Protects iron oxide core
from oxidation, superior
optical properties171–173
Polyvinyl
alcohol Passive – – 10–50 nm Prevents aggregation of
particles, reduces toxicity,
provides chemical handles
for functionalizationDrug delivery especially
brain, in vivo contrasting
and imagingAmino-PvA exhibit high
uptake efficiency in cells41,131,174
PEI Magnetic
targeting– – 50–100 nm Imparts colloidal stability at
higher salt concentrations,
offers high positive charge
densityNonviral gene delivery,
magnetofectionEfficient and fast delivery
of genetic material,
exhibits proton sponge
effect42,111,112
Erythrocytes Passive/
magnetic
targeting– – 10–100 nm Imparts biocompatibility,
promotes long blood
circulation half-life by
circumventing rapid clearance
by reticuloendothelial
systemDrug targeting, cell
separation, intravascular
imaging and contrastingEasily available,
biocompatible, does not
exert immunogenicity
because of their
autologous nature175
Gelatin Passive -– – 50–100 nm Imparts hydrophilicity and
biocompatibility, ease of
functionalization with drugs
and targeting ligands due to
abundance of amino groupsSeparation of DNA, drug
delivery and targetingNatural polymer,
efficient drug loading130,176
Polyvinyl
pyrrolidonePassive – – 10–25 nm Improves colloidal stability,
enhances blood circulation
timeDrug delivery, imaging,
and contrastingUniform coating,
efficient drug loading177
Polymethyl
methacrylateIn vitro use – – 10–50 nm Facilitates separation
of genetic material and
amplificationmRNA/DNA-purification
Isolation of specific gene
sequences
Immunomagnetic assaysSimple, easy to carry
out, process can easily
be automated78,178
Ethyl cellulose Magnetic
targeting– – 20–50 nm Imparts colloidal stability Sustained release of drugs,
arterial chemoembolizationMaintenance of drug
concentration at desired
site for prolonged period
of time, retains sufficient
magnetic
responsiveness78,179
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3455Superparamagnetic iron oxide nanoparticles

International Journal of Nanomedicine 2012:7Table 2 (Continued )
Coating
materialTargeting
strategyTargeting ligand Target Particle size
distributionProperties bestowed by
coating on particleApplications Advantages Published
reports
Pullulan Passive
targeting– – 40–45 nm Decreased cytotoxicity,
increased cellular uptake,
and reduced aggregationImaging of vascular
compartment, lymph nodes
perfusion imaging, receptor
imaging and target specific
imagingEasily prepared, high
uptake efficiency20
PLGA Magnetic
targeting– – 4–12 μm Ensures homogenous
distribution and reduced
aggregation, increased
biocompatibility with
synovial tissuesIntra-articular treatment of
inflammatory diseases like
arthritis, osteoarthritisAdequate magnetic
retention under the
influence of magnetic
field108, 110,
180
Starch Passive – – 10–20 nm Improves biocompatibility,
reduced aggregationImaging and contrasting
radiotherapyNatural polymer, can
be modified with
other polymers
like PEG92, 181
Abbreviations: ANP, atrial natriuretic peptide; CEA, carcinoembryonic antigen; EGFR, epidermal growth factor receptor; MMP-2, matrix metalloproteinase-2; PEG, polyethylene glycol; PEI, polyethylenimine; PLGA, polylactic-co-glycolic
acid; PSMA, prostate-specific membrane antigen; PVE, polyvinyl alcohol; SPIONs, superparamagnetic iron oxide nanoparticles; VCAM1, vascular cell adhesion molecule 1. coencapsulating drug molecules along with magnetic par –
ticles within the coating material envelope.
A number of approaches have been developed for con –
jugation of therapeutic agents or targeting ligands on the
surface of these nanoparticles. They can be grouped under
two categories, ie, conjugation by means of cleavable cova –
lent linkages and by means of physical interactions. Covalent
linkage strategies involve linkage of the therapeutic agent
or targeting molecules directly with, eg, amino or hydroxyl
functional groups present on the surface of polymer-coated
SPIONs. Alternatively, linker groups such as iodoacetyls,
maleimides, and the bifunctional linker, pyridyl disulfide,
may be used to attach the drug to the particle. This approach
not only leads to enhanced loading capacity, but also results
in more specific linkages, protecting the drug’ s functionality
and hence efficacy. Another advantage of using linkers is that
this method involves milder reactive conditions for attach –
ment, and hence is suitable for drugs like therapeutic peptides
and proteins, which are prone to oxidative degradation.21
Physical interactions such as electrostatic interactions,
hydrophobic/hydrophilic interactions, and affinity interac –
tions can also lead to coupling of drug molecules on the
surfaces of SPIONs. SPIONs coated with polyethylenimine
(PEI), a cationic polymer, interact electrostatically with
negatively charged DNA, demonstrating their applicability
as transfection agents.42 Similarly, dextran-coated SPIONs
functionalized with negatively charged functional groups can
couple with peptide oligomers via electrostatic interactions.43
Because of hydrophobic interactions, lipophilic drugs can
easily be attached to SPIONs covered with hydrophobic
polymers, from where the drug can be released when the
coating degrades. Affinity interactions, such as streptavidin-
biotin interactions, can also be utilized for bioconjugation of
targeting agents or drugs with SPIONs. Unlike electrostatic
and hydrophobic interactions, affinity interactions offer the
most stable noncovalent linkages, which are relatively unaf –
fected by environmental conditions, such as changes in pH
and ionic strength of the medium.
However, drug delivery by conjugating the drugs onto
the surface of SPIONs suffers from some disadvantages.
Low entrapment efficiency of drug molecules is one of the
major drawbacks of delivering the drug by coating the surface
of SPIONs because only a limited amount of drug can be
conjugated in this way. Other disadvantages include highly
stable linkages as a result of covalent bonding between drug
molecules and the surface of SPIONs, leading to failure to
release the drug molecule at the target site. Sometimes, a
catalyst such as copper used during covalent linking of a
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3456Wahajuddin and Arora

International Journal of Nanomedicine 2012:7drug to the SPION surface may cause in vivo toxicity if it is
not purified properly. It has also been shown that it may be
difficult to control the orientation of binding ligands when
attaching them to the surface of magnetic nanoparticles.
This has been observed, particularly in cases where SPIONs
decorated with carboxylic acid groups bind with ligands
with multiple amine groups, often leading to inactivation
of ligands.21
Loading of drug molecules along with iron oxide nano –
particles within the coating material represents another
approach of delivering a drug to the target site. This approach
provides attractive solutions to problems such as low entrap –
ment efficiency and stability. Magnetoliposomes represent
a new class of nanocomposites which not only ensures high
entrapment efficiency but also better stability and magnetic
properties.44 Magnetoliposomes are nanosized, spherical
vesicles consisting of magnetic nanoparticles in a shell
composed of a phospholipid bilayer. These magnetolipo –
somes retain hydrophobic regions that can be used for drug
encapsulation. Loading SPIONs with pharmaceuticals within
the phospholipid envelope offers several advantages. Firstly,
liposomes containing magnetic particles provide simple
and easy surface modifications enabling their targeting to a specific tissue. Secondly, magnetoliposomes containing
high amounts of SPIONs due to increased entrapment effi –
ciency provide optimum magnetic responsiveness and their
nanosize enables exploitation of the enhanced permeability
and retention effect for tumor targeting. In addition, a larger
amount of both hydrophilic as well as hydrophobic drug can
be loaded within the liposomal structure along with SPIONs.
Thirdly, encapsulation of SPIONs within liposomes further
improves the biocompatibility of SPIONs. Fourthly, the
liposomal barrier protects encapsulated pharmaceuticals from
the degradative effects of the surrounding environment.45
SPIONs can be loaded within the liposomes in two ways, ie,
by incubating previously prepared liposomes and SPIONs
under the influence of an external force resulting in localiza –
tion of SPIONs within the hydrophilic core of liposomes,46,47
or by directly precipitating the SPIONs within the hydrophilic
core of liposomes, yielding highly uniform nanoparticles with
sizes around 15 nm.48 However, during coating, agglomerates
of SPIONs sometimes get coated instead of discrete SPION
cores being coated within a phospholipid shell. This results
in poor magnetic and physicochemical properties. Exposing
magnetoliposomes to a strong permanent magnetic field over
the target tissue leads to alignment of magnetic particles
SPIONS
Active targeting Passive targeting Magnetic targeting
Targeted
nanop articleNon-targeted
nanopa rticle
Figure 2 Superparamagnetic iron oxide nanoparticle targeting approaches.2
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3457Superparamagnetic iron oxide nanoparticles

International Journal of Nanomedicine 2012:7within the bilayer, causing magnetoliposomes to aggregate,
fuse, and release the drug only at the target site.
Magnetodendrimers represent another class of nanocom –
posites which are well suited to imaging of cell trafficking
and migration using MRI.49–51 Carboxylated polyamidoamine
dendrimers are commonly used to coat and stabilize iron
oxide nanoparticle suspensions.33 In general, at an elevated
temperature and pH in the presence of dendrimers, oxida –
tion of Fe(II) yields highly stable and soluble SPIONs with
dendrimers.33 Lamanna et al developed dendronized iron
oxide nanoparticles for multimodal imaging. SPIONs coated
with dendrimers having a hydrodynamic size less than
100 nm and displaying either carboxylate or ammonium
groups at the periphery provide a unique opportunity for
labeling with a fluorescent dye. Magnetic resonance and
fluorescence imaging have been demonstrated to be simul –
taneously possible.52
SPION targeting approaches
SPIONs can be properly engineered to reach their target
tissue with minimum nonspecific cellular interactions.
Targeting strategies can be grouped into three classes as
shown in Figure 2. Passive targeting takes advantage of
the innate size of the nanoparticles as well as the unique
characteristics provided by the tumor microenvironment.53
Tumor cells exhibit leaky vasculature due to incomplete
angiogenesis.53 Nanoparticles enter tumor tissue through
these pores, and due to the poor lymphatic drainage system
of cancerous tissues are retained for longer periods of time
than normal cells. This is also known as the enhanced per –
meability and retention effect, discovered by Matsumura
and Maeda.54,55 Thus, particle size in the nanorange itself
provides targeted delivery, without any modification. The
enhanced permeability and retention effect is considered
to be the “gold standard” for developing new anticancer
agents.56
Active targeting involves targeting ligands which are
coupled at the surface of magnetic nanoparticles to interact
with receptors that are overexpressed at their target sites.
Such particles accumulate in larger quantities in target cells
due to “homing” of these ligands onto the receptors and
subsequent ease of internalization. Various active targeting
agents engineered and attached to the SPION surface not
only ensure specific target binding but also minimize the dose
required and nonspecific cellular interactions. Commonly
used targeting ligands are shown in Table 2.
Magnetic focusing uses external magnets to create a
suitable magnetic field gradient over the targeted area and ensure significant accumulation of drug-loaded SPIONs.57
The strength of the applied magnetic field can be altered to
modulate release of the drug in the desired fashion, resulting in
maximum therapeutic benefits. A permanent NdFeB magnet is
generally used for magnetic targeting of SPIONs. Mondelak
et al increased the permeability of dextran-encapsulated
SPIONs over an artificial three-layered membrane on appli –
cation of an external magnetic field of 0.410 Tesla.58 Kumar
et al synthesized magnetic nanoparticles coupled with plasmid
DNA-expressing enhanced green fluorescent protein and
coated with chitosan. The particles were successfully directed
to the heart and kidney via an external magnetic field when
injected into the tail vein of mice. The results demonstrated
that application of an external magnetic field is sufficient to
target a drug to its specific site of action, excluding the need
to develop functionalized nanoparticles.59 In another study,
Lamkowsky et al demonstrated time-dependent, temperature-
dependent, and concentration-dependent accumulation of
dimercaptosuccinate-coated iron nanoparticles in cultured
brain astrocytes. The accumulation of iron oxide nanopar –
ticles was proportionally increased with an increase in the
applied magnetic field strength, increasing the cell-specific
iron content from an initial 10 nmol/mg of protein within
4 hours of incubation at 37 °C to up to 12,000 nmol/mg of
protein. The results suggested that application of an external
magnetic field enhanced both binding of iron nanoparticles
to cell membranes as well as their internalization in cultured
astrocytes, demonstrating the utility of SPIONs as potential
diagnostic and drug delivery vehicles for imaging and treat –
ment of neurodegenerative diseases.60 Prijic et al studied
the potential increase in cellular internalization of SPIONs
by exposure of different cells to an external magnetic field
generated by different permanent magnets. They found that
exposure to neodymium-iron-boron magnets significantly
increased the cellular uptake of SPIONs, predominantly into
malignant cells. Accumulation of SPIONs within malignant
cells was found to be dependent on duration of exposure to
the external magnetic field. 61
Absorption, distribution, metabolism,
and excretion of SPIONs
For a chemical to be called a drug, it ought to have optimal
pharmacokinetic properties as well as be formulated into an
appropriate delivery system with desirable pharmacokinetic
parameters. If these magnetic nanoparticles are intended to
translate from benchtop research to clinical settings, detailed
investigation of their pharmacokinetics, ie, their absorp –
tion and internalization mechanisms in different cells, their
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3458Wahajuddin and Arora

International Journal of Nanomedicine 2012:7biodistribution, and metabolic fate, as well as their elimina –
tion from biological systems, is of prime importance.
Internalization and biodistribution
in different cells
The literature reports a wide variety of processes for the
internalization of SPIONs by different cells. Given intrave –
nously, these magnetic particles are predominantly taken up
by phagocytes in the reticuloendothelial systems of the liver,
spleen, and lymph tissue.62,63 In a study conducted by Gu et al,
biocompatible, dimercaptosuccinic acid-coated SPIONs were
internalized into macrophage-like RAW264.7 cells utiliz –
ing multiple endocytic uptake pathways, such as clathrin-
dependent endocytosis, caveolae-dependent endocytosis,
and macropinocytosis or phagocytosis. Marked inhibition of
SPION internalization was observed when RAW264.7 cells
were pretreated with chlorpromazine (inhibits clathrin-
dependent endocytosis by promoting loss of clathrin and
the AP2 adapter complex from the cell surface), multilayer
β-cyclodextrin (inhibits caveolae-dependent endocytosis
by depleting membrane cholesterol), and amiloride (inhib –
its macropinocytosis by blocking activity of the Na+/H+
exchanger in the plasma membrane). These results con –
firmed that several endocytic uptake mechanisms worked
cooperatively for uptake of SPIONs by macrophage-like
RAW264.7 cells.64 However, in another study by Y ang et al,
ferucarbotran, a clinically approved SPIO-coated carboxy –
dextran with a mean diameter range of 45–60 nm, was found
to be internalized in macrophage-like RAW264.7 cells via
clathrin-dependent endocytosis.65 It seems that there is a cor –
relation between SPION factors (size, surface charge, and
surface coating) and endocytotic pathways, that needs to be
explored in more detail.
SPIONs administered intravenously have to cross the
vascular endothelium, ie, the primary barrier, in order to
reach their target site. Hanini et al evaluated the interac –
tion between human endothelial cells and γ-Fe2O3 nano –
particles using confocal microscopy. Human umbilical
vein endothelial cells were incubated with FITC-labeled
γ-Fe2O3 nanoparticles at a concentration of 10 μg/mL. The
γ-Fe2O3 nanoparticles were efficiently internalized by the
human umbilical vein endothelial cells, with the major –
ity of fluorescence localized within the cytoplasm. These
investigators demonstrated that γ-Fe2O3 nanoparticles could
penetrate cells without protein-coated vesicles, suggesting
a micropinocytotic process.66
Wang et al evaluated the pharmacokinetic parameters and
tissue distribution of magnetic Fe3O4 nanoparticles prepared by a chemical coprecipitation method in imprinting control
region mice. Using atomic absorption spectrophotometry,
they found a wide distribution of SPIONs in various target
organs and tissues, including the heart, liver, spleen, lungs,
kidney, brain, stomach, small intestine, and bone marrow.
However, the concentration of SPIONs was highest in the
liver and spleen, thus demonstrating more pronounced cura –
tive effects of SPIONs in these organs.67
Particle size, surface coating, and surface charge are
major determinants of the biodistribution, pharmacokinet –
ics, and possible toxicity of SPIONs.15 Tissue distribution
is mainly affected by particle size. SPIONs with a particle
size smaller than 50 nm evade opsonization, thus increasing
their circulation time and hence are gradually taken up by
macrophages in the reticuloendothelial systems of the liver,
lymph tissue, spleen, and bone marrow, whereas magnetic
particles smaller than 50 nm are rapidly cleared from the
bloodstream by sinusoidal Kupffer cells in the liver.63 In
addition to particle size, the coating material used on iron
oxide particles also determines the rate of hepatic clearance.
In general, SPIONs covered with coating materials which
hinder access of water to the iron oxide core show slower
degradation and hence an increased half-life in blood.68
Surface charge, in addition to particle size and the coating
material, affects the uptake of SPIONs by different cells. For
instance, positively charged particles adhere nonspecifically
to cells because the majority of the cell membranes have a
net negative charge, whereas strong negative charges on
the surface of magnetic particles facilitate their uptake by
the liver.68 Schlorf et al compared SPIONs with different
core materials (magnetite, maghemite), different coatings
(none, dextran, carboxydextran, and polystyrene) and dif –
ferent hydrodynamic diameters (20–850 nm) with regard
to their internalization mechanisms, release of internal –
ized particles, toxicity, and ability to generate contrast in
MRI. For this study, they utilized U118 glioma cells and
human umbilical vein endothelial cells exhibiting different
phagocytic activity. Noncoated, carboxydextran-coated, and
polystyrene-coated nanoparticles with a varying size range
(10–850 nm) showed very nonspecific phagocytic uptake by
tumor cells and endothelial cells. The coating and surface
charge of the particles were demonstrated to exert a much
larger influence on their nonspecific uptake than their size.
Dextran-coated particles on the other hand were found to
have completely different uptake behavior. The reason for
this different uptake behavior was attributed to the dextran
coating, making these nanoparticles suitable for specific
labeling of molecular targets.69
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3459Superparamagnetic iron oxide nanoparticles

International Journal of Nanomedicine 2012:7Elimination from biological systems
An ideal drug delivery system, after delivering its payload
to the target site, should be completely eliminated from the
biological system with minimal harmful effects. Because
SPIONs are increasingly being investigated for a variety of
biomedical applications, ie, targeted drug delivery, tumor
magnetic hyperthermia therapy, and contrast enhancement
of MRI, detailed elucidation of their metabolic pathways
and clearance mechanisms should be carried out, so as to
rule out any potential toxicity associated with their intended
use. Given that elimination is a broader term, which includes
metabolism as well as excretion, the following section will
provide a better understanding of the metabolic as well as
clearance pathways of SPIONs in biological systems.
Metabolism
The uptake, distribution, metabolism, and excretion of dietary
iron is highly regulated in biological systems. Specialized
mechanisms which govern the body’ s iron regulation pro –
cesses are also thought to be involved in handling iron oxide
nanoparticles. Few reports are available in the literature elu –
cidating their biotransformation in biological systems. Once
internalized, lyzosome-mediated degradation is generally
considered to be the main intracellular metabolic mecha –
nism involved in the degradation of SPIONs.70,71 In order to
evaluate the degradation rate of SPIONs internalized within
acidic intracellular components, Arbab et al utilized an in
vitro lyzosomal environment containing appropriate and/or
specific chelates, such as citrates and phosphates, at an acidic
pH. They demonstrated that sodium citrate at pH 4.5 rapidly
dissolved ferumoxide, a dextran-coated SPION. In the cel –
lular environment, they found that some of the ferumoxide-
transfection agent complex containing endosomes fused with
the lyzosomes, resulting in their rapid dissolution at low
pH, exposing the iron core to chelates, thus liberating the
soluble free Fe(III) into the cytoplasm via divalent cationic
transport.70 In another study, Gu et al proposed three possible
mechanisms for the metabolic fate of SPIONs internalized
in RAW264.7 cells, and supported them with the data gen –
erated. Firstly, during mitotic division of cells, intracellular
SPIONs get distributed to the daughter cells. This mechanism
is considered to be the major route of elimination of SPIONs
from macrophages. Secondly, internalized iron oxide nano –
particles get degraded at low lyzosomal pH, releasing free
Fe(III) within the intracellular medium. The iron released is
stored in the body reserves with the help of iron-regulating
proteins, ie, ferritin and hemosiderin. Thirdly, some of the
internalized SPIONs may potentially be exocytosed out of RAW264.7 cells, although direct evidence for this proposed
mechanism is lacking.64 Levy et al conducted a long-term
(3-month) in vivo biotransformation study of 8 nm SPIONs
coated with hydrophilic glucose derivatives following intrave –
nous administration in mice. Using ferromagnetic resonance
and superconducting quantum interference device measure –
ments, they supported the view of continuous biotransfor –
mation of injected magnetic nanoparticles into iron storage
species within intracellular lyzosomes.72
Excretion
The most desirable pathway for SPIONs to be excreted
from biological systems is via the kidney, because this route
involves minimal intracellular catabolism, reducing the prob –
ability of generating reactive oxygen species and hence asso –
ciated toxicity. Renal excretion represents the safest route of
elimination for SPIONs. However, the shape, hydrodynamic
size, surface coating, and surface charge of SPIONs play a
major role in regulating their renal clearance. It is notewor –
thy that the coating chemistry in particular has a profound
effect on renal clearance of SPIONs. For instance, 55% of
the intravenous dose of SPIONs coated with Pluronics®/oleic
acid with a hydrodynamic diameter of 193 nm was shown
to accumulate in the rat liver.73 On the other hand, SPIONs
with dextran as the coating material have been reported to
undergo 18%–22% elimination via the urine and feces over a
period of 7 weeks in different animal species.74 Some studies
even report more rapid clearance of dextran-coated SPIONs
(nearly 25%) in 19 days via the urine and feces.75 This dif –
ference in clearance rate can be attributed to differential
protein corona compositions which in fact are governed by
the surface chemistry of these magnetic nanoparticles. For
magnetic nanoparticles with a diameter less than 40 nm, the
coating material has much more significance than any other
physicochemical parameter in determining their biodistribu –
tion as well as blood half-life.68
Cell type, in addition to physicochemical parameters,
also plays an important role in determining the cellular
uptake, intracellular metabolism, and toxicity associated
with SPIONs. Mahmoudi et al have shown that at the same
iron concentrations (above 2.25 mM), SPIONs with differ –
ent surface coatings are toxic to human brain cells while
completely compatible with human kidney cells.76,77
Utility as drug delivery vehicles
SPIONs have emerged as an attractive alternative for
targeted delivery of drugs because of their unique magnetic
characteristics. Their role as contrast agents in MRI has
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3460Wahajuddin and Arora

International Journal of Nanomedicine 2012:7Table 3 Role of superparamagnetic iron oxide nanoparticles in different areas of magnetic resonance imaging
Focus area Important remarks References
Molecular imaging • Allows sensitive and specific monitoring of key molecular targets
• Allows monitoring of host responses associated with early events in carcinogenesis
• Imaging of cell trafficking/migration, apoptosis detection and imaging of enzyme activities182–188
Cardiovascular disease imaging • Uptake by macrophages utilized to visualize atheromatous plaques prone arterial sites
• Useful in evaluating the risk of acute ischemic events62,189–192
Cancer imaging • Utilized in clinical imaging of liver and spleen tumors and metastases through
RES-mediated uptake
• Effective in identification of lymph node metastases, useful in treatment of prostate,
breast, and colon cancer
• Prolonged delineation of brain tumor boundaries and quantify tumor volumes
• Useful in imaging CNS tumor neovasculature and assessing therapeutic
response to antiangiogenic chemotherapeutic agents62, 82,191–197
Autoimmune disorders imaging • Used to visualize macrophage infiltration in brain, assess blood-brain barrier
damage and neurological impairment in chronic relapsing experimental autoimmune
encephalomyelitis, rodent model of human multiple sclerosis
• Used to monitor distinct pattern of macrophage migration in acute disseminated
encephalomyelitis82,192,198,199
Central nervous system disease imaging • Serve as useful tool for noninvasive anatomic and temporal tracking of stem
cells in CNS trauma and stroke
• Used to monitor disease progression in epilepsy
• Allows MRI visualization of neuroinflammation in vivo
• Allows monitoring of leukocyte trafficking in the brain82, 200–204
Abbreviations: CNS, central nervous system; MRI, magnetic resonance imaging; RES, reticuloendothelial system.
already been established, particularly for tumor imaging
and cardiovascular disease. Moreover, these magnetic
nanoparticles are increasingly being investigated for their
potential application in the delivery of chemotherapeutic
drugs, radionuclides, and anti-inflammatory agents, as well
as in gene delivery.21,28,78 Magnetic fluid hyperthermia is
another approach where SPIONs can be utilized for local –
ized production of heat, resulting in destruction of cancer
cells.79 SPIONs are also being used in magnetic separation
techniques, particularly for immunomagnetic cell separation
and purification.78,80
The only clinical use for which commercial SPION-based
products are available is as MRI contrast agents for the diag –
nosis of human disease. MRI is one of the most powerful non –
invasive imaging techniques used in clinical medicine today.81
SPIONs have been used as contrast agents for at least 20 years.
Based on their size, SPIONs have been grouped into three cat –
egories, ie, standard SPIONs (50–180 nm), ultrasmall SPIONs
(10–50 nm), and very small SPIONs ( ,10 nm).62 Commonly
used MRI contrast agents fall into the ultrasmall SPION cat –
egory and, for the sake of consistency, we will refer to SPIONs
in general. SPIONs have emerged as an attractive alternative to
gadolinium-based MRI contrast agents, particularly in patients
with renal dysfunction, who are at risk of nephrogenic systemic
fibrosis. Table 3 lists some of the focus areas of MRI imaging
using SPIONs as contrast agents.Given that the major focus of this review is on highlight –
ing the drug delivery applications of SPIONs, their diagnostic
applications are not discussed in detail. Readers who are
interested in a more detailed discussion of the role of SPIONs
as MRI contrast agents are advised to go through some of the
excellent reviews mentioned in the references.21,82–89
Role in cancer therapy
Chemotherapeutic agents are known to exert a plethora of
side effects due to their lack of target specificity. SPIONs
are capable of ferrying anticancer drugs into malignant cells
while sparing healthy cells. This also leads to reduction of
the dose required because the drug is delivered directly to
target cells. Various anticancer drugs, including paclitaxel,
methotrexate, mitoxantrone, and doxorubicin, have been con –
jugated with magnetic nanoparticles to increase their target
specificity.90,91 Coating of SPIONs with polymers like amino-
polyvinyl alcohol and pullulan have resulted in increased
interaction of these nanoparticles with human cancer cells,
as well as suppression of cytotoxicity in healthy cells.20,41
Polyethylene glycol-modified, cross-linked, and starch-
coated iron oxide nanoparticles have been found to enhance
magnetic tumor targeting.92 Because surface modification
or coating can sometimes significantly reduce the super –
paramagnetic properties of SPIONs, a novel approach of
delivering drug-loaded SPIONs by encapsulating them within
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3461Superparamagnetic iron oxide nanoparticles

International Journal of Nanomedicine 2012:7cationic liposomes has been proposed by Y ang et al.93 These
nanocomposites were surface-engineered with antitransferrin
receptor single-chain antibody fragments, which delivered
the complex to transferrin receptors overexpressed on tumor
cells. This nanoimmunoliposome platform technology could
specifically and efficiently deliver SPIONs into tumor cells
after systemic administration.
SPIONs can also be utilized to overcome multidrug
resistance characterized by overexpression of ATP binding
cassette transporters that actively pump a large number of
hydrophobic chemotherapeutic drugs out of cancer cells.
In one experiment, doxorubicin was covalently bound to
PEI via pH-sensitive hydrazone linkage and conjugated to
an iron oxide nanoparticle coated with amine-terminated
polyethylene glycol. The doxorubicin-loaded nanoparticles
were taken up by both wild-type and drug-resistant cells,
and were retained for longer in the drug-resistant cells due
to lack of drug efflux.94
The development of multifunctional magnetic nanopar –
ticle formulations further boosts the potential use of SPIONs
in medicine. These formulations not only serve the purpose
of being drug delivery vectors but also have applications in
MRI imaging,95 targeted thermosensitive chemotherapy,96
magnetically targeted photodynamic therapy,97 and fluo –
rescent/luminescence imaging.98 Y allapu et al developed
water-dispersible SPIONs for hyperthermia, MRI, and drug
delivery applications. SPIONs were prepared by precipita –
tion of iron salts in the presence of ammonia and coated
with β-cyclodextrin and a Pluronics polymer. Multilayer
β-cyclodextrin and Pluronics polymer-coated magnetic nano –
particles were found to have good stability, enhanced cellular
uptake, and sustained release of encapsulated curcumin, an
anticancer drug. This formulation showed improved MRI
characteristics with enhanced therapeutic anticancer activ –
ity. Due to its good dispersibility, this formulation also has
excellent heating effects on application of an alternating
magnetic field, making it suitable for localized treatment
of cancer using hyperthermia. Further, the formulation was
found to be hemocompatible, making it an excellent approach
for drug delivery.99 In another study, Y ang et al prepared
multifunctional wormlike vesicles simultaneously loaded
with SPIONs and doxorubicin for targeted cancer therapy and
ultrasensitive MRI. Stable wormlike polymer vesicles were
formed by heterofunctional triblock polymer R (methoxy
or FA)-PEG114-PLAx-PEG46-acrylate via a double-emulsion
method. The outer surface consisted of long hydrophilic
polyethylene glycol chains bearing methoxy/folate groups,
thereby providing active tumor targeting, while the inner surface was formed by short hydrophilic polyethylene glycol
chains bearing the acrylate group which were cross-linked
via free radical polymerization to provide enhanced in vivo
stability. SPIONs were encapsulated in the aqueous inner
core with a loading efficiency of 48.0 wt% while doxorubicin,
the hydrophobic drug, was loaded into the hydrophobic mem –
branes of the wormlike vesicles, with a loading efficiency of
9%. The multifunctional wormlike vesicles showed enhanced
cytotoxicity in a HeLa cell line as a result of greater cellular
uptake due to folate receptor-mediated endocytosis. More –
over, due to relatively high SPION loading levels as well as
clustering of SPIONs in the aqueous core, these wormlike
polymeric vesicles demonstrated a much higher r2 relaxivity
value than Feridex®, a commercially available MRI contrast
agent.100 Targeted cancer theragnostics seem possible with
the development of such multifunctional nanoformulations,
thereby paving the way for personalized medicine.
In another approach towards killing malignant cells,
radionuclides, particularly β emitters, were coupled with
SPIONs to induce DNA damage to free radicals, resulting
in apoptosis of target cells.101 However, incorporation of
radionuclides on the SPION surface poses unique prob –
lems because of the decaying nature of radiotherapeutics.
The challenge here lies in designing an efficient SPION-
radiotherapeutic complex, which not only achieves a high
local concentration in malignant cells, but also minimizes
interaction between the radioactive agents and healthy cells
in the body. The development of this technique could greatly
reduce exposure to radiation and its associated side effects
in external radiotherapy covering the whole body.
A drug-free approach to combating cancer cells involves
localized heating of these cells from the inside. Cells begin
to show signs of apoptosis when their inside temperature is
raised in the range of 41 °C–47°C. Necrosis results when the
temperature reaches approximately 50 °C. Targeted hyper –
thermia involves administration of magnetic nanoparticles,
which are taken up by cells through endocytotic pathways
and become concentrated in intracellular endosomal vesicles.
When subjected to a high frequency AC magnetic field, these
particles become heated as a consequence of Neel or Brown –
ian relaxation losses in single-domain particles, resulting in
localized killing of these cells.102 The main challenge lies
in selective delivery of these nanoparticles to tumor cells
and optimization of their heating properties for production
of sufficient magnetic hyperthermia. At least a ten-fold
increase in heating power has been reported, with an increase
in particle size of maghemite nanoparticles to 12–14 nm.103
Gonzalez-Fernandez et al showed that Fe2O3 nanoparticles
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3462Wahajuddin and Arora

International Journal of Nanomedicine 2012:7around 30 nm in size exhibit maximum heating efficiency.
Coating tends to decrease heating efficiency, hence should
be kept to a minimum.104
Magnetoliposomes have also emerged as an effective
tool for producing localized hyperthermia together with
delivery of encapsulated drugs to the target site, resulting
in the destruction of cancerous cells. In order to achieve
targeted delivery of methotrexate to skeletal muscle tissue,
Zhua et al prepared thermosensitive magnetoliposomes
capable of liberating more than 80% of the encapsulated
methotrexate within 30 minutes when the environmental
temperature was increased from 37°C to 41 °C. The prepared
liposomes enabled increased accumulation of methotrexate
in musculoskeletal tissue when exposed to an external con –
stant magnetic field and heated to 41 °C. These thermosen –
sitive magnetoliposomes showed good magnetic targeting,
with rapid release of the encapsulated drug in response to
hyperthermia.105 Zhang et al prepared lyophilized negatively
charged magnetic liposomes containing paclitaxel for target –
ing breast carcinoma via parenteral administration. Pharma –
cokinetic studies showed that these magnetic liposomes were
selectively taken up by tumor tissue because of their tendency
to migrate exclusively to the tumor mass under the guidance
of an external magnetic field. The antitumor efficacy of the
prepared formulation was found to be better than that of a
commercially available paclitaxel formulation containing
Cremophor® EL and ethanol. In addition, the half-life of
paclitaxel was reported to be increased to 19.37 hours for the
magnetoliposomes versus 4.11 hours for the commercially
available formulation. Paclitaxel-loaded magnetic liposomes
offered targeted delivery of a chemotherapeutic drug to its
site of action with significant anticancer activity and fewer
side effects.106
Apart from therapy, the concept of “magnetic cells”
(magnetic nanoparticles when endocytosed in cells) is pro –
viding new approaches for cellular manipulation in response
to different applied magnetic fields.107 These manipulations
can be used to deform internal cellular membranes or to alter
the cell architecture.
Treatment of arthritis
Corticosteroids are the mainstay of therapy for patients
with arthritis. Intra-articular injections of corticosteroids
provide symptomatic treatment but have severe limitations,
including development of crystal-induced arthritis as well
as increasing the risk of articular infection, particularly on
repeated injection. Development of SPION-corticosteroid
conjugates could be a potential solution to these problems. Moreover, with the application of an external magnetic
field, these magnetic conjugates could be retained for a
longer time at their desired site of action, by delaying their
uptake by macrophages or lymphatic drainage.108 Polyvinyl
alcohol-SPIONs and their fluorescently functionalized
analogs, ie, amino-polyvinyl alcohol-Cy2.5 SPIONs, were
injected intra-articularly or periarticularly in the stifle and
carpometaphalangeal joints of sheep and targeted using a
permanent NdFeB magnet.109 The results confirmed the
biocompatibility of these nanoparticles, which were found
to remain within the synovial membrane for a period of
5 days, demonstrating their potential applicability in pro –
longing the action of medication applied intra-articularly.
SPIONs and dexamethasone acetate coencapsulated into
polylactic-co-glycolic acid microparticles can also serve as
a targeted delivery system for the treatment of joint-related
pathologies. These microparticles have been found to be
biocompatible with synoviocytes.108,110
Gene therapy
The biotechnology industry aims at providing gene-based
therapy to treat otherwise incurable diseases. Expression
of desired genes (sense technology) and silencing of
expression of troubling genes (siRNA technology) are the
two basic approaches of gene therapy. The main concern
arises in successful delivery of these gene-based drugs to
their site of action, while maintaining their integrity and
stability. The conventional approach involves delivery of
the desired genes with the help of viral vectors. However,
this approach raises a lot of safety concerns. Alternative
strategies utilize cationic delivery systems which can form
complexes electrostatically with negative charged DNA.111
SPIONs coated with cationic polymers have been found
to increase the magnetofection efficiency of therapeutic
genes. PEI-coated SPIONs interact with DNA to form
cation-anion complexes which are taken up into cells by
endocytosis. Via the proton sponge effect, DNA-PEI-SPION
complexes evade the endosomal barrier and deliver the gene
of interest to the nucleus. Use of permanent magnets and
a pulsating magnetic field enables enhanced transfection
efficiency due to reduced free diffusion of these particles.
DNA-PEI-SPION complexes also show reduced toxicity
compared with PEI-DNA complexes.42 Namgung et al
designed thermally crosslinked superparamagnetic nano –
particles containing polyethylene glycol, covalently bonded
to branched PEI and complexed with DNA of interest. This
hybrid conjugate demonstrated high transfection efficiency
in vascular endothelial cells; successfully inhibiting the
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3463Superparamagnetic iron oxide nanoparticles

International Journal of Nanomedicine 2012:7expression of plasminogen activator inhibitor-1 involved
in various vascular disorders, including vascular inflam –
mation and atherosclerosis.112 Hwang et al immobilized an
adenoassociated virus delivery system on heparin-coated
SPIONs. The magnetically guided adenoassociated virus
delivery system was found to infect target cells efficiently
and rapidly, opening up new avenues for use of SPIONs in
a variety of gene therapy applications.113
Stem cell therapy
Stem cell therapy involves transplantation of stem cells to
an injured tissue site, where these cells grow, differentiate,
and take over the function of the injured tissue. However,
this therapy involves extensive diagnostic and surgical
intervention. SPIONs have emerged as an attractive tool
for the labeling and delivery of stem cells to desired sites.
Stem cells labeled with SPIONs can easily be tracked using
MRI. This provides a useful noninvasive method for track –
ing the destination of transplanted cells in vivo.114,115 The
increasing use of SPIONs in the labeling of stem cells is due
primarily to the following advantages associated with their
use: SPIONs show good contrast enhancement in MRI as
a result of their high magnetic susceptibility; they are bio –
compatible, as demonstrated by the SPION-based contrast
agents approved by the US Food and Drug Administration
and currently on the market; and the ample opportunity for
modification of their surface architecture, thus allowing
labeling of different types of stem cells. Stem cells can
be labeled with SPIONs by several methods. First, they can
be labeled by prolonged incubation with SPIONs, resulting
in passive accumulation of the contrast agent within the cell.
However, this method does not ensure efficient labeling,
because uptake is limited by the phagocytic behavior of
the cells being labeled.116 Second, coupling of transfection
agents like Superfect®, poly-L-lysine, protamine, and Lipo –
fectamine® with SPIONs can strongly increase the efficiency
of stem cell labeling.117–119 Third, a magnetic field can be used
to increase membrane permeability transiently, resulting
in accumulation of SPIONs within the cells.120 However,
stem cells can also be labeled by conjugating SPIONs to
their cellular surface.
MRI can help to determine the behavior of SPION-
labeled stem cells in vivo, particularly their migration and
potential transformation into desired specialized cells within
a targeted structure. Gathering of such information will help
to bridge our serious lack of understanding of the behavior
of stem cells following transplantation. This will pave the
way for clinical use of cell therapy. SPIONs with differ -ent surface architectures have been employed for efficient
labeling of stem cells. For instance, Horak et al prepared
mannose-modified iron oxide particles using a coprecipita –
tion method for labeling of stem cells. Intracellular uptake
of mannose-modified SPIONs into rat bone marrow stromal
cells was confirmed using optical and transmission electron
microscopy. Due to high relaxivity, which is responsible for
the contrast in MRI and easy internalization into cells, these
mannose-modified iron oxide nanoparticles could serve as
an important tool for diagnostic and therapeutic applications
in cell-based therapy.121 In a similar study, Babic et al evalu –
ated the potential of poly-L-lysine at molecular weights of
146–579,000 as a coating to increase intracellular uptake
of SPIONs in rat bone marrow mesenchymal cells. Their
results showed that poly-L-lysine of high molecular weight
(38,100) was a highly effective surface modifier for achiev –
ing optimal internalization by rat bone marrow stromal cells.
Nanoparticles were found to localize within the endosomal
and lyzosomal compartments, and cellular uptake was found
to be dependent on the poly-L-lysine concentration used to
modify the iron oxide nanoparticles. Such nanoparticles
could serve as a promising tool for noninvasive tracking of
transplanted cells in vivo.122
Apart from being used to label stem cells, SPIONs can
also be used as potential carriers to deliver stem cells to
their target site. Y ang et al biofunctionalized SPIONs with
two kinds of ligands, one specific for stem cells (cluster
designation 34) and another for infarcted myocardium. The
findings showed that SPIONs functionalized in this way can
be guided effectively by an external magnetic field to deliver
stem cells to infarcted myocardium.123
SPIONs can be multifunctionalized using fluorescent and
isotope labeling, thus combining optical and nuclear imaging
with MRI. Such an approach holds great promise in validating
cellular behavior in vivo. In an interesting study conducted by
Lewin et al, SPIONs 5 nm in size were surface-coated with
cross-linked aminated dextran, leading to an overall increase
in size to 45 nm. These iron oxide nanoparticles were then
conjugated with a fluorescent-labeled internalization ligand,
ie, fluorescein isothiocyanate-derivatized HIV -TAT peptide.
In order to impart nuclear characteristics, the dextran coating
was reacted with a chelator, diethylenetriamine penta-acetic
acid, and labeling of the nanoparticles with the 111In isotope.
These triple-labeled (magnetic, fluorescent and nuclear) SPI –
ONs were found to be taken up efficiently by hematopoietic
stem and neural progenitor cells. The modified SPIONs were
found to have no effect on the viability and differentiation
capacity of hematopoietic stem cells.124
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3464Wahajuddin and Arora

International Journal of Nanomedicine 2012:7Pitfalls of using SPIONs as drug
delivery vehicles
Creating a suitable magnetic field gradient
A major consideration in successful implementation of
SPIONs in therapy is development of a suitable magnetic field
gradient for effective localization of SPIONs in target tissue,
and their efficient internalization within the desired cells.
Blood flow in the arteries and veins offers resistance to the
development of a magnetic field gradient. The magnetic forces
applied should be strong enough to overcome this resistance
for effective concentration of magnetic nanoparticles at their
desired site.2
Blood vessels and organs deep within the body cannot
be targeted by external magnets because of the lack of an
effective magnetic field gradient. As the distance between
the magnet and targeted area increases, the strength of the
magnetic field at the desired site decreases. Only those areas
which are close to the surface can be properly targeted. To
overcome this limitation, researchers have been using super –
conducting magnets, like SmBaCuO and YBaCuO, which can
exert strong magnetic fields. These magnetic fields have been
found to penetrate the skin surface to a depth of 20 mm.125
Further, neodymium-iron-boron has been used along with
SPIONs to enhance magnetic drug targeting.58,59 Magnetic
stents or implants, which can create strong local magnetic
fields, have also been used to increase the concentration of
drug-loaded magnetic nanoparticles at the desired site.126–128
However, this approach has limited application because of
the surgical procedure involved.
Burst effect
Another limitation of the use of SPIONs for drug delivery is
burst release of drugs grafted onto the SPION surface. Ide –
ally, SPIONs should deliver their entire payload to the site
of action. However, lack of proper surface-engineering leads
to premature release of a significant proportion of the drug
load, which can result in toxicity. Crosslinkable polymers
can reduce burst release of drugs to a large extent. SPIONs
with a rigid crosslinked polyethylene glycol fumarate coating
were found to reduce the burst effect rate by 21% compared
with non-crosslinked tamoxifen nanoparticles.129 Mahmoudi
et al205 utilized starch to test for improvement in the burst
effect of encapsulated theophylline from SPIONs containing
superparamagnetic polyurethane microspheres. They found a
significant decrease in the burst effect of the drug. This may
have been due to entanglement of high molecular weight
starch chains with the polyurethane polymer, forming a bar –
rier to diffusion of the drug into the surrounding fluid.Low bioavailability
Intravenous administration is the route of choice for
SPIONs. Commercially available SPION-based MRI con –
trast agents are generally given by the intravenous route.
Although intravenous administration of these iron oxide
nanoparticles ensures 100% systemic bioavailability, their
specific uptake by target cells is an issue of concern because
of nonspecific uptake by the reticuloendothelial system
and nonspecific binding to plasma proteins. However,
with proper surface-engineering and attachment of suit –
able targeting ligands, along with magnetic focusing, it is
possible to enhance the uptake of drug-loaded SPIONs by
target tissue.2,16,21,130
Interaction with the blood-brain barrier
The blood-brain barrier is relatively impermeable to a
wide variety of drugs due to the presence of tight endothe –
lial junctions. Uncoated SPIONs have demonstrated low
brain-targeting efficacy due to their inability to cross the
blood-brain barrier. Coating of SPIONs with functionalized
polyvinyl alcohol, especially amino-polyvinyl alcohol,
enhances their uptake by isolated brain-derived endothe –
lial and microglial cells. These SPIONs were also found
to be biocompatible, producing no inflammatory response
in brain-derived cell cultures.131 Coating of SPIONs with
appropriate polymers combined with application of a strong
external magnetic field could lead to development of a
potential vector for imaging and treatment of neurodegen –
erative diseases.
T oxicity considerations
For every new drug delivery system that shows promise,
considerable studies of the pharmacokinetic and toxicity
profile must be carried out. These particles are known to
be cleared from the body via endogenous metabolic iron
pathways. Liu et al evaluated single-dose and repeat-dose
toxicity of SPIONs given by subcutaneous injection in mice.
Their results confirmed a low hazard potential for SPIONs,
with no treatment-related deaths or transient clinical signs.
Histopathological evaluation revealed no iron-positive pig –
ments in macrophages from major organs like the liver,
lungs, spleen, brain, kidney, and heart.132 Similarly, Gupta
et al reported SPIONs modified with polyethylene glycol
to be nontoxic to human dermal fibroblasts when using the
MTT assay.16
However, a few reports have suggested some toxico –
logical effects for SPIONs. Iron overload at the target site
is considered to be one of the causes of SPION-associated
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3465Superparamagnetic iron oxide nanoparticles

International Journal of Nanomedicine 2012:7toxicity. After digestion of the coating, high levels of bare
Fe2O3 nanoparticles at the target site can cause an imbalance
in homeostasis. This can alter cellular responses, leading to
DNA damage, an inflammatory response, oxidative stress,
genetic changes, disruption of cytoskeletal organization of
cells, and cytotoxicity. SPIONs, on internalization within
cells, can be digested by lysozymes, resulting in release of
free iron. These free iron ions (Fe2+) can react with hydrogen
peroxide or oxygen in the mitochondria to form very reactive
free radicals which can cause genotoxicity.20,133,134 SPIONs
have also been found to enhance the G2/M phase of the cell
cycle.135 Further, SPIONs can alter immune function in a
dose-dependent manner, as described by Chen et al in an
imprinting control region mouse model.10
In a study conducted by Mahmoudi et al, the main reason
found for SPION-induced toxicity was formation of gas
vesicles as a result of local alteration in ionic equilibrium
and protein function. Mahmoudi et al developed a modified
method for assessing cytotoxicity associated with SPIONs to
establish a better correlation between in vitro and in vivo stud –
ies. Surface-saturated SPIONs were obtained by preincubation
with cell medium for 24 hours. This was done to mimic in vivo
conditions, in which many proteins get adsorbed onto SPIONs
when they are introduced into the circulation. In contrast with
bare SPIONs, preincubated SPIONs induced less cytotoxicity
and gas vesicle formation due to fewer adsorption sites avail –
able for interaction with proteins and other biomolecules.136
Conclusion and future horizons
As the pharmaceutical field has progressed, a large number
of novel drug delivery systems have emerged, and most of
them, including SPIONS, are in the nascent developmental
stages. However, various factors make SPIONs one of the
most promising drug delivery vehicles. They are one of the
easiest systems to produce and have shown the best toxicity
profile so far. Guidance by external magnets enables a third
targeting mechanism, giving them an edge when all three
mechanisms work in unison. They have shown potential
applications in a wide variety of biomedical fields, both
diagnostic and therapeutic. Carcinomas near the body sur –
face, like squamous cell carcinoma, malignant melanoma,
Kaposi’ s sarcoma, and breast carcinoma, are likely to benefit
the most from SPION-based therapy because magnetic
targeting works best in these regions.
In spite of the above advantages and decades of intensive
research, there is still no SPION-based drug delivery product
on the market. There are some serious limitations which have
to be overcome before a commercially successful product can be launched. Methodologies related to SPION preparation
have been improved, but a lot of work still needs to be done on
their characterization. With the discovery of supermagnets, it
has become possible to create a suitable magnetic field gradi –
ent deep inside the skin surface, which can lead to increased
targeting capability of SPIONs. However, such a magnetic
field can only be used in the hospital setting.
Development of transdermal patches containing magnetic
circuitry using supermagnets capable of generating highly
penetrating localized magnetic fields could result in enhanced
and efficient accumulation of drugs carrying SPIONs at
desired sites. This would be particularly beneficial for ambu –
latory patients and could also increase patient compliance
with treatment. Additionally, the complex dosing regimen
according to the individual needs of the patient can be carried
out by modulation of the magnetic field strength used.
Another aspect in need of monitoring is the burst release
effect of SPIONs. Use of crosslinking polymers has demonstrated
promising results in controlling the drug release rate. An alterna –
tive approach may be incorporation of SPIONs within a polymer
matrix that allows retention of their magnetic properties, thus
enabling them to be guided by an external magnetic field. The
matrix would regulate the release of drug as desired.
Detailed investigation of the pharmacokinetics and
biodistribution of SPIONs in vivo should be carried out
if these nanoparticles are intended to be translated from
benchtop research to clinical practice. Elucidation of the
disposition pathways for SPIONs will provide new insights
which will help in further improving the desirable charac –
teristics of SPIONs for targeted drug delivery. Toxicological
considerations concerning use of SPIONs as drug delivery
vehicles warrant further investigation. Interplay of the wide
variety of factors involved in this delivery system could
lead to development of SPION-associated toxicities. These
factors need to be assessed and characterized properly for
development of SPIONs with optimum properties for drug
delivery. Tremendous research in this direction is addressing
these issues, and the positive results so far allow us to hope
that the day is not far off when SPIONs will available for
treatment rather than just research.
Acknowledgments
The authors are grateful to the Director of the Central Drug
Research Institute, Lucknow, India, Dr TK Chakraborty, for
his constant encouragement and support. We are also grateful
to Dr DK Majumdar, whose valuable guidance and constant
encouragement helped us to complete this review, and to
Jasleen Kaur Khurana for editorial assistance.
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3466Wahajuddin and Arora

International Journal of Nanomedicine 2012:7Disclosure
The authors report no conflicts of interest in this work.
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