4306 Chem. Soc. Rev., 2012, 41, 4306–4334 This journal is c The Royal Society of Chemistry 2012
Cite this: Chem. Soc. Rev., 2012, 41, 4306–4334
Biological applications of magnetic nanoparticles
Miriam Colombo,wa Susana Carregal-Romero,wb Maria F. Casula,c
Lucıa Gutierrez,de
Marıa P. Morales,dIngrid B. Bohm,
fJohannes T. Heverhagen,
f
Davide Prosperi*aand Wolfgang. J. Parak*
b
Received 8th December 2011
DOI: 10.1039/c2cs15337h
In this review an overview about biological applications of magnetic colloidal nanoparticles will
be given, which comprises their synthesis, characterization, and in vitro and in vivo applications.
The potential future role of magnetic nanoparticles compared to other functional nanoparticles
will be discussed by highlighting the possibility of integration with other nanostructures and with
existing biotechnology as well as by pointing out the specific properties of magnetic colloids.
Current limitations in the fabrication process and issues related with the outcome of the particles
in the body will be also pointed out in order to address the remaining challenges for an extended
application of magnetic nanoparticles in medicine.
1. Introduction
Magnetic materials, based on metals such as iron, cobalt and
nickel or metal oxides, have been involved in different ways in
the development of modern technology. We can find them in
many devices such as motors, generators, sensors, videotapes,
and hard disks. Therefore, the huge interest on the miniaturiza-
tion of these materials can be easily understood. This is in
particular true as on the very small scale magnetic materials, i.e.
magnetic nanoparticles (MNPs), can also display properties
different from the bulk. In most cases, MNPs smaller than the
single domain limit (e.g. around 20 nm for iron oxide) exhibit
superparamagnetism at room temperature. Meaning that the
MNPs that can be ferromagnetic or ferrimagnetic lose their
magnetism below their Curie temperature and that are
composed of a single magnetic domain. The superparamagnetism
has in particular applications in ferrofluids due to the tunable
viscosity, in data analysis and in medicine. All these disciplines
need to be provided with specific kind of MNPs, stable in
different conditions and with different geometries and physical
properties. In this context, nanotechnology allows for controlled
synthesis, functionalization of materials in the nanometre scale
and provides a toolbox that did not exist before. MNPs can
be specifically designed to increase the current data storage
capacity of magnetic hard disks or to be used as biohybrid
materials. Actually, MNPs based on iron oxide are being used
in clinical trials as contrast agents in magnetic resonance
imaging (MRI), and clinical applications in drug delivery and
diagnosis are seriously being considered. Colloidal MNPs can
be designed in countless different ways, but the number of
systems suitable for biological applications is highly diminished.
The purpose of this review is to outline the conceptual
properties of colloidal MNPs and the motivation for their use
in different areas of biologically related research. We have
classified the uses of MNPs into four main concepts of applica-
tions: molecular detection, imaging (including a focus on
regenerative medicine), delivering, and heating.
2. Basic physical properties
The microscopic origin of magnetic properties in matter lies in
the orbital and spin motions of electrons,1 whose spin and
angular momentum are associated with a magnetic moment.
The interaction between the magnetic moments of atoms from
the same material causes magnetic order below a certain
critical temperature. We can classify bulk materials on the
basis of these interactions and their influence on the materials
behaviour in response to magnetic fields at different tempera-
tures (e.g. ferromagnetism, ferrimagnetism, etc.).2 Bulk magnetic
materials are composed of regions, called magnetic domains,
within which there is an alignment of the magnetic moments.
If the volume of the material is reduced, as in the case of
MNPs, a situation in which just one domain is reached occurs
and the magnetic properties are no longer similar to bulk
materials.
aDipartimento di Biotecnologie e Bioscienze,Universita di Milano-Bicocca, Milan, Italy.E-mail: [email protected]
bDepartment of Physics and WZMW, Philipps Universitat Marburg,Marburg, Germany. E-mail: [email protected]
cDipartimento di Scienze Chimiche e INSTM, Universita di Cagliari,Cagliari, Italy
dDepartamento de Biomateriales y Materiales Bioinspirados, Institutode Ciencia de Materiales de Madrid, ICMM-CSIC, Madrid, Spain
e School of Physics M013, University of Western Australia, Crawley,Australia
f Department of Diagnostic Radiology, Philipps Universitat Marburg,Marburg, Germanyw Both authors contributed equally to this work.
Chem Soc Rev Dynamic Article Links
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Due to their small volume, MNPs usually present super-
paramagnetic behaviour, meaning that the thermal energy
may be enough to change spontaneously the magnetisation
within each MNP. In other words, the magnetic moment of
each MNP will be able to rotate randomly (in reference to the
orientation of the MNP) just because of the temperature
influence. For this reason, in the absence of an electro-
magnetic field (Fig. 1) the net magnetic moment of a system
containing MNPs will be zero at high enough temperatures.
However, in the presence of a field, there will be a net statistical
alignment of magnetic moments, analogous to what happens to
paramagnetic materials, except that now the magnetic moment
is not that of a single atom but of the MNPs containing various
atoms which can be up to 104 times larger than for a para-
magnetic material. This property, marked by the lack of
remanent magnetisation after removal of external fields, enables
the MNPs to maintain their colloidal stability and avoid
agglomeration, which is important for biomedical applications.
Many new interesting phenomena have been observed in
MNPs that are not shared with their bulk counterparts.3 In
bulk materials, the main parameters to characterize magnetic
properties such as coercivity (Hc) and susceptibility (w) are:
composition, crystallographic structure, vacancies and defects,
and magnetic anisotropy. In addition, for MNPs on the
nanoscale range, the shape and size also determine their
magnetic behaviour. The magnetic anisotropic energy barrier
from the spin-up state to the spin-down state of the magnet is
proportional to the product of the magnetic anisotropy
constant (K) and the volume of the magnet. Therefore, super-
paramagnetism itself depends on the size of the MNP. In
general, the smaller the size of the MNP, the lower its
transition temperature from ferromagnetic to superpara-
magnetic behaviour will be. Size reduction also reflects in the
enhancement of the relative contribution of surface effects.
For instance, a reduction in size from 12 to 5 nm in maghemite
(g-Fe2O3) can lead to a reduction in saturation magnetisation
of half the theoretic value, mainly due to surface disorder.4
Besides size effects, the MNP shape is known to strongly
influence K and again the magnetic anisotropic barrier and
the whole magnetic properties of the nanomaterial.
In general, it could seem that large magnetic moments are
preferred for most applications, as this would reduce the
amount of MNPs needed. However, when dealing with bio-
logical applications, biocompatibility reaches great impor-
tance, as the accumulation or toxic effects of the MNPs need
to be reduced as much as possible. Therefore, most of the time
a balance between larger magnetic moments and biocompat-
ibility needs to be reached. This is the reason why iron-based
MNPs are often preferred compared to others based on more
toxic transition metals.5 Ideal diameters of MNPs for bio-
medical applications based on ferrites, i.e. mixed metal oxides
with iron(III) oxide as their main component, are between the
superparamagnetic threshold at room temperature (10 nm)
and the critical single-domain size (B70 nm).
TheMNP coating is also a key element in order to specifically
bind other compounds to the MNPs or to prevent agglomera-
tion, as will be explained later, but it will also affect the
magnetic properties of the MNPs due to the high specific
surface area and the large amount of atoms at the surface.
Some authors have observed a reduction in the saturation
magnetization with coating and explain it in terms of the spin-
pinning, i.e. the decrease in the effective magnetic moment due
to a non-collinear spin structure originated from the pinning
of the surface spins and coating ligand at the interface of the
MNP6 or by the formation of a surface ‘‘dead layer’’ resulting
from the chemical reaction between the stabilizing surfactant
and the MNP.7–10 However, it has also been observed that it is
possible to keep the magnetic properties unchanged when
MNPs have been coated with phosphonates7 which are
supposed to reduce the spin canting in the MNP surface. It
can be concluded that differences in the nature of the coupling
agent and in the type of interaction between the ligand and the
MNP surface would explain the differences in the magnetisa-
tion values.
The application of an external alternating magnetic field
(AMF) to MNPs leads to the production of energy, in the
form of heat, if the magnetic field is able to reorient the
magnetic moments of the MNPs.8 Such an effect can be
exploited to use MNPs as mediators in magnetic hyperthermia.
In bigger multidomain MNPs, this reorientation is produced
through the movement of domain walls, while in small mono-
domain MNPs, the reorientation of the magnetic moments can
occur due to (i) the rotation of the moment within the MNP,
overcoming their anisotropy energy barrier (Neel loss), or (ii)
the mechanical rotation of the MNPs that will create frictional
losses with the environment (Brown loss) (Fig. 2). For maghemite
MNPs, below 15 nm, theoretically, Neel relaxation prevails
over Brown relaxation while for larger sizes and low viscosity
media, Brown relaxation is the rotation mechanism.11 Micro-
metre disc shaped MNPs have been observed to produce
mechanical cell damage due to Brown motion.12 The frontier
Fig. 1 Schematic representation of a superparamagnetic particle.
Note that although the moments within each particle are ordered
(red arrows), the net magnetic moment of a system containing MNPs
will be zero in the zero field and at high enough temperatures. In the
presence of a field, there will be a net statistical alignment of magnetic
moments.
Fig. 2 (left) Rotation of the moment within the MNP, overcoming
their anisotropy energy barrier that leads to Neel Loss, (centre)
mechanical rotation of the MNPs, that will create frictional losses
with the environment and lead to Brown losses and (right) movement
of domain walls in multidomain MNPs that leads to hysteresis loss.
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4308 Chem. Soc. Rev., 2012, 41, 4306–4334 This journal is c The Royal Society of Chemistry 2012
size between multidomain and monodomain MNPs depends
on their crystalline structure as well as on their composition,
although in many cases it ranges between 50 and 100 nm13
(Fig. 3). In general, small monodomain MNPs are preferred
for hyperthermia treatments. In the case of Neel relaxation,
the characteristic relaxation time of a MNP system is given by
tN = t0exp[KV/(kT)], where k is the Boltzmann constant, T
the temperature, V the particle volume and K the anisotropy
constant. On the other hand, Brownian relaxation time (tB =
4pZr3/(kT)) depends on the viscosity of the medium (Z) in
addition to temperature. In each situation, the faster relaxa-
tion mechanisms will be the dominant one, thus the effective
relaxation time may be defined as teff = tNtB/(tN + tB).As a consequence of the magnetisation rotation under an
AMF, an increase in temperature with time (DT/Dt) at a given
MNP mass concentration (m) can be measured and used to
determine the specific absorption rate (SAR), defined as SAR =
C(DT/Dt)(1/m), where C is the sample specific heat capacity,
also called specific power loss. This value is widely used to
compare the heating capacity of different MNPs. Local heating
above physiological temperature has been demonstrated to be
a cause of cell death, although the mechanisms are not fully
understood. Then, if we are able to target the MNPs to
tumorous tissue, we will be able to selectively heat it without
damaging the surrounding healthy tissue.
MNPs are also able to create small local magnetic fields,
which cause a shortening of the relaxation times (T1 and T2) of
the surrounding protons. This effect is named proton relaxation
enhancement and leads to a change of the Nuclear Magnetic
Resonance (NMR) signal intensity in its surroundings. MRI
contrast is improved due to the presence of MNPs acting as
contrast enhancing agents. MRI contrast enhancement relies
on the different engulfment of MNPs by different cells.14 It has
been shown that the use of superparamagnetic iron oxide
MNPs improves lesion detection and diagnostic accuracy of
MRI.15 T1 and T2 are called the longitudinal and transverse
proton relaxation times, respectively. The longitudinal relaxation
involves redistributing the populations of the nuclear spin
states in order to reach the thermal equilibrium distribution.
It reflects an exchange of energy, as heat, from the system to its
surrounding. T1 measures the dipolar coupling of the proton
moments to their surrounding; thereby isolated protons would
show negligible rates of T1 relaxation. The transverse relaxation
is related to the decoherence of the magnetisation of the
precessing protons due to magnetic interactions with each
other and with other fluctuating moments in their surroundings.
MNPs are magnetically saturated at common field strengths
used for MRI and their presence produces a marked short-
ening of T2 along with a less marked reduction of T1. There-
fore, most of the published examples of the use of MNP as
contrast agents are based in T2 weighted imaging even though
the impact on T1 is significant and often higher compared to
paramagnetic chelates.16–19
Another advantage of the use of MNP in biological applica-
tions is that they can be tracked in vivo by magnetic measure-
ments, which are very sensitive and offer substantial information.
The tracking is based on the interaction of the MNPs with
different tissues and cells. The labeling of a biological area of
interest is possible mainly due to the conjugation of the MNPs
with a specific cellular surface or due to the engulfment of
MNPs. Recently, magnetisation curves of lyophilized samples
of liver, spleen and kidney enabled quantitative estimation of
the amount of magnetic material in those organs by comparison
of the normalized saturation magnetisation of the organs to
that of the nanoparticles alone.20 Another promising approach
to measure MNP biodistribution is the measurement of the
AC (alternating current) magnetic susceptibility.21 Quantita-
tive analysis of the amount of MNPs that are localised in a
given tissue is based on the treatment of the temperature
dependent out-of-phase (w00) susceptibility profile.22 Dilutions
of the administered MNPs with different concentrations are
required for the quantitative analysis protocol as a collection
of standards. This protocol also allows the quantitative
determination of MNPs of biological origin as those coming
from endogenous ferritin.22
3. Synthesis of magnetic nanoparticles
Tailored design of colloidal MNPs plays a crucial role in
determining the effectiveness of functional magnetic nano-
structures for a given biomedical application. A number of
key requirements related to the intrinsic properties of the
magnetic material, to the occurrence of size and shape effects,
to the nature of its surface, to the stability in water-rich
environment, as well as to its non-toxicity need to be taken
into account. While the MNP size and shape, surface coating
and colloidal stability can be tuned through appropriate
synthetic procedures, the choice of the magnetic material is
rather restricted to iron based magnetic oxides which to date
represent the best compromise among good magnetic properties
(such as saturation magnetisation) on one hand and stability
under oxidizing conditions and limited toxicity on the other.
In particular, the US Food and Drug Administration (FDA)
and the European Medicines Agency (EMA) have already
approved the medical use of magnetic iron oxides MNPs
which include magnetite (mixed Fe2+ and Fe3+ ions), and
maghemite, which retains the same spinel structure as magnetite,
but is fully oxidized to Fe3+. As bulk materials, both are
ferrimagnetic. As magnetite has a larger magnetisation its use
should be preferred. In contrast, maghemite is usually more
stable in aqueous media, ensuring therefore a more durable
magnetic behaviour.23 Actually, the ease of formation of non-
stoichiometric iron oxide nanostructures has been often over-
looked due to the difficulty to distinguish magnetite and
maghemite at the nanoscale by conventional characterization
Fig. 3 Frontier MNP size between multidomain and monodomain
MNPs depending on their crystalline structure.13
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techniques24 and many of the reported results refer to a
mixture of magnetite and maghemite. In general, MNPs are
termed depending on the final size of the coated and functio-
nalized particle. MNPs smaller than 50 nm are called ultra
small superparamagnetic iron oxide nanoparticles (NPs), and
superparamagnetic iron oxide NPs (SPIONs) when they are
larger than 50 nm.
Although iron oxides have certainly been and still are the
most widely encountered, biomedical applications of magnetic
ferrites are currently being intensely investigated. In particular,
substituted magnetic spinel ferrites of the general formula
MFe2O4 (where M = Zn2+, Mn2+, Co2+, Ni2+, Mg2+. . .)
offer the opportunity to fine-tune the magnetic properties of the
inorganic MNP core as a function of the kind of divalent ion, as
summarized in Table 1. Incorporation of the divalent cation
into the ferrite structure changes not only the saturation
magnetisation but also the magnetic anisotropy of the material.
In particular, iron oxide MNPs containing manganese or
cobalt have shown improved magnetic properties compared to
Fe3O4 or g-Fe2O3 MNPs,28 leading to an enhancement of the
magnetic resonance signal, as it is the case for manganese
ferrites,29 or a more efficient SAR, as it is the case of cobalt
ferrites.30 One consequence is that despite toxicological con-
cerns related to the leakage of toxic ions, substituted ferrites
with high anisotropy such as CoFe2O4 may be suitable for
biomedical use as MNPs of significantly smaller size than iron
oxide MNPs, which are likely to be easily cleared by the body,
could be employed. Inhomogeneities in composition and/or
the geometric cation distribution within the MNPs can be
responsible for important reductions in saturation magnetisation
and alteration in the anisotropy as it has been shown recently for
different ferrite MNPs.31 Recently, substituted magnetic spinel
ferrites have been doped with gadolinium producing a new
magnetic material, CoFe2�xGdO4 (x = 0–25) MNPs, which
has a strong influence of gadolinium doping on the saturation
magnetisation and coercivity due to large lattice distortion and
grain growth of small MNPs.32 Based on the above-mentioned
key features of ferrites, the main protocols for the preparation
of biohybrid MNPs based on spinel iron oxides and substituted
magnetic spinels will be described. Accordingly, the most
widely encountered strategies for MNP protection, stabili-
zation, functionalization and bioconjugation, which will be
reviewed in the following paragraphs, mainly refer to the
surface chemistry properties of ferrite materials.
3.1. Synthesis of Fe3O4 and MFe2O4 nanoparticles and their
magnetic properties
Wet chemistry routes for the preparation of ferrite MNPs can
be conveniently grouped into hydrolytic and non-hydrolytic
approaches, which exhibit distinctive advantages and suffer
from specific drawbacks (Table 2). Magnetic ferrites for
biomedical use are most commonly prepared by hydrolytic
synthesis, with particular reference to coprecipitation techniques.
A widespread approach relies on the Massart method,33 where
magnetite is obtained by alkaline coprecipitation of stoichio-
metric amounts of ferrous and ferric salts (usually chlorides).
The experimental parameters affecting this process, which
involves the formation of intermediate hydroxyl species, such
as temperature, pH, concentration of the cations and nature of
the base, have been studied in order to vary the average MNP
size in the range from 3 to 20 nm. It is noteworthy that the
pH is a critical parameter in affecting both the MNP size
(by increasing the pH the repulsion among primary MNPs is
induced and smaller magnetite MNPs are obtained) and the
Table 1 Saturation magnetisation Ms and magnetic anisotropy K ofselected magnetic ferrites (taken from the studies of Cornell et al.,Cullity and Bate,25 Viswanathan,26 and Buschow27)
Ms/A m2 kg�1 K/J m�3
Fe3O4 98 �1.1 � 104
g-Fe2O3 82 �4.6 � 103
MgFe2O4 31 �2.5 � 103
MnFe2O4 110 �2.8 � 103
CoFe2O4 94 1.8 � 105
NiFe2O4 56 �7.0 � 103
Table 2 Comparison of different general characteristics of MNPs (mainly based in iron oxide) synthesized through different wet chemistry methods
Hydrolytic Non-hydrolytic
Coprecipitation Hydrothermal coprecipitation Reversed micelles coprecipitation
1 Cheap chemicalsMild reaction conditionsSynthesis in H2OEase surface modificationEase formation of ferritesEase conversion to g-Fe2O3
Ease scale-up
Improved size controlNarrow size distributionSynthesis in H2OTunable magnetic properties
Improved size controlNarrow size distributionEase size tunabilityUniform magnetic properties
Narrow size distributionHigh size controlHigh cristallinityPossible scale-upTunable magnetic properties
2 Broad size distributionLow reproducibilityUncontrolled oxidation
High temperatureSafety of the reactants
Low reaction yieldPoor cristallinitySurfactants are difficultto remove
Toxic organic solventsHigh temperaturesPhase transfer required
3 Fe3O4, Fe2O3, MFe2O4 Fe3O4, Fe2O3 Fe2O3, Fe3O4, MFe2O4 Fe3O4, Fe2O3, MFe2O4, Cr2O3,MnO, Co3O4, NiO
4 10–50 nm, 50–100 nm 1–0.25 mm, 15–31 nm 2–30 nm, 20–80 nm 3–500 nm5 Sphere Disc, sphere Cube, sphere, needle Cube, sphere, triangle, tetrapode,
rod6 33–35 36, 37 39–43 17, 24, 28, 44–65
1 = Advantages, 2 = Drawbacks, 3 = Nature of MNPs, 4 = NP diameter, 5 = NP shape and 6 = References.
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stability of the MNP dispersion. In particular, thanks to the
surface electrostatic repulsion stable ionic ferrofluids can be
obtained in a wide range of pH. The coprecipitation approach
offers a wide range of advantages including: the use of cheap
chemicals and mild reaction conditions; the possibility to
perform direct synthesis in water; the ease of scale-up; the
production of highly concentrated ferrofluids thanks to the
high density of surface hydroxyls. Most important, the synthetic
route is extremely flexible when it comes to the modulation of
the core and surface properties. Conversion to maghemite is
easily obtained by chemical oxidation of the magnetite colloids,
and substituted ferrites can be prepared by alkalinization of
aqueous mixtures of a ferric salt and a salt of a divalent metal
under boiling conditions.34 Likewise, thanks to the high
density of reactive sites surface modification can be easily
performed by direct incorporation of additives, which is
particularly useful for large scale production as it is the case
of the carbonate method.35 A general limitation of the hydro-
lytic approach lies in the large number of parameters, which
have to be carefully monitored in order to control the synthetic
outcome, which deals with the complex aqueous chemistry and
rich phase diagram of iron oxide phases.
To improve the magnetic properties of the produced MNPs
the coprecipitation method can be performed under hydro-
thermal conditions.36 Hydrothermal routes have also been
used for hydrolytic procedures starting from iron complexes,
such as the ageing in aqueous acidic/basic solution of iron
polyolates followed by digestion in autoclave at 80–150 1C for
several days.37 In this synthetic approach, the reaction condi-
tions, such as solvent, temperature, and time, usually have
important effects on the synthetic outcome. The MNP size in
crystallization is controlled mainly through the rate processes
of nucleation and particle growth, which compete between
each other. Keeping constant other parameters, these rates
depend on the reaction temperature being the nucleation
process faster than MNP growth at higher temperatures what
ends in a decrease of MNP size. In contrast, prolonging the
reaction time would favor MNP growth.
As a major drawback of hydrolytic routes lies in the limited
control of the MNP size distribution, synthesis in confined
environments such as microemulsions has been proposed. In
particular, reverse micelles have been used to carry out the
classic coprecipitation reaction38 and the hydrolysis of metal–
surfactant complexes39 in water-in-oil emulsions. The para-
meters, which affect the MNP size, are the microstructure and
composition of the microemulsion (both the surfactant most
commonly sodium bis(2-ethylhexyl) sulfosuccinate (AOT),
cetyl trimethylammonium bromide (CTAB), dodecylsulfonate
(DS) and the hydrocarbon which constitutes the continuous
phase—such as hexane, heptane, octane), the temperature,
and the kind of counterion. Reverse micelles have been
successfully used as a means to mediate the formation of iron
oxide MNPs as well as substituted ferrites with improved size
distribution in the 4–12 nm range (typical size of the water-
in-oil droplets of the microemulsion).40,41 On the other hand,
both lower yields and poor crystallinity are obtained compared
to conventional coprecipitation routes and the sensitivity of
the preparation conditions complicates reproducibility and
scale up. Heating the reverse micelles at 90 1C was recently
shown to improve the crystallinity of iron oxide and substituted
ferrites.42 It should be mentioned that the synthesis of Fe3O4
MNPs from oil-in-water emulsion has also been proposed to
improve the yield and reduce drastically the amount of surfactant
and oil compared to the water-in-oil reverse microemulsions.43
More recently, a great effort has been devoted to the
synthesis of high quality magnetic ferrites by non-hydrolytic
routes in order to achieve dependable solution-based procedures
to obtain high quality ferrite MNPs. In particular, by avoiding
the formation of hydroxy and oxyhydroxy species as inter-
mediates, complex aqueous chemistry equilibria are prevented
and alternative growth patterns can be achieved. Non-hydrolytic
routes primarily rely on the thermal decomposition of organo-
metallic precursors into organic media including polyol and
have enabled us to obtain MNPs with well-defined magnetic
properties thanks to their high crystallinity, controlled size and
narrow size distribution. Polyols are especially interesting
media because they are hydrogen-bonded liquids with high
permeability, able to dissolve to some extent ionic inorganic
compounds. Polyols act as high-boiling solvent (reactions can
be carried out at temperatures up to 250 1C), reducing agent
and stabilizer being able to control the MNP growth and
prevent MNP agglomeration.44 Using polyols with different
molecular weights and polarities enabled us to control not
only the MNP size but its stability and solubility.45
Synthetic protocols based on the rapid decomposition of
thermally unstable precursors into hot non-aqueous solutions
of coordinating surfactants were first optimized for the prepara-
tion of monodisperse chalcogenide semiconductor NPs and
then extended to metal46,47 and metal oxide NP colloid synthesis.
The success of this procedure relies on the occurrence of a
controlled fast supersaturated burst nucleation followed by
crystal growth, and the experimental parameters which can be
tuned to control the outcome of the synthesis include: the kind
of organometallic precursor and of surfactant(s), relative ratio
of inorganic precursor and surfactant and overall concen-
tration, temperature of decomposition of the precursor and
of MNP growth, reaction time, solvent. In particular, success-
ful synthesis of iron oxide MNPs has been achieved by either
single molecular precursor systems made out of complexes
which bind iron through oxygen such as iron(III) cupferronate,48
acetylacetonate,49 and oleate complexes50–52 or dual source
systems, where the use of an iron precursor (most commonly
iron pentacarbonyl) and of an oxidizer in appropriate ratios
offers the possibility to produce the desired magnetic phase by
controlled oxidation.24,49,53,54 Nearly monodisperse (poly-
dispersity 5% or less) highly crystalline MNPs with well-defined
morphology can be obtained. Materials prepared by these
routes are contributing to shed light on the properties–
structure relationship of MNPs.17,55–58 Best results are
obtained for pseudo-spherical MNPs with size in the range
4–20 nm, although strategies to extend the size limits59 as well
as to tune the crystal shape from pseudospherical to cube-
octahedral, tetrapod, and hollow sphere by adjusting the
reaction temperature, the oxidizing/reducing conditions, and
the kind of surfactant(s) have been proposed.53,60,61 The most
commonly used surfactants are fatty acids and aliphatic
amines, which effectively mediate crystal nucleation and
growth and in addition enable us to obtain ferrofluids made
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out of optically clear dispersions of MNPs in non-polar media
(such as toluene, hexane, chloroform). A major limitation of
this technique for the preparation of MNPs is that non-
biocompatible precursors and solvents are being used, and a
phase transfer into aqueous media of the nanocrystals is
required prior to their use in biomedicine (see Section 3.2).
On the other hand, based on the improved magnetic properties
of the prepared iron oxide MNPs surfactant-mediated decom-
position techniques are currently being extended to the pre-
paration of substituted ferrites. Although MNPs with good
magnetic properties and with controlled size and ion doping
are being obtained,62–64 tuning of the MNP shape for sub-
stituted ferrites still needs to be largely improved.
A successful route for non-hydrolytic synthesis of MNPs
which was first proposed by Sun and coworkers makes use of
the decomposition of Fe(acac)3 in an ether solution of a
mixture of surfactants (such as oleic acid and oleylamine) in
the presence of a polyol such as 1,2-diol acting as reducing
agent. Although the reaction mechanism has not been fully
elucidated, the reduction of iron(III) to an iron(II) intermediate
which then undergoes decomposition is likely to take place.
Key parameters for optimal synthetic outcome are the copre-
sence of a mixture of surfactants, the control of the reducing
atmosphere, and the heating ramp (typically first up to 200 1C
and then up to 260–300 1C). Size control is best achieved
through a seeded growth mechanism: by using 3–4 nm nano-
crystalline seeds, monodisperse MNPs with size up to 20 nm
have been obtained.28 A major advantage is that this approach
can be extended to the preparation of magnetic ferrites (such
as MnFe2O4 and CoFe2O4) by using relatively cheap and non-
toxic Fe(acac)3 and M(acac)2 or MCl2 as starting precursors.28,65
For these reasons polyol-based routes are regarded as valuable
methods for the large scale production of magnetic ferrites
intended for biomedical use, although to date the synthetic
sophistication attained by more conventional non-hydrolytic
decomposition routes has not been reached yet (in terms of
adaptability to other magnetic materials and shape control).
3.2. Phase transfer and surface functionalization
The application of MNPs in biology requires their stability in
solutions containing high concentrations of proteins and salts,
as well as in cell culture media. Thus, a crucial issue facing the
use of MNPs for biological applications is the stabilization
and functionalization of their surface. As it was mentioned
MNPs can be synthesized following different approaches that
will lead to hydrophobic or hydrophilic cores. Their inter-
actions with the surrounding media depend on the presence/
absence and nature of molecules on the magnetic surface. In
general, NPs tend to flocculate due to van der Waals forces,
but MNPs can in addition be magnetically attracted between
each other and agglomerate. Even for MNPs formed by
coprecipitation in the presence of a stabilizing agent such as
dextran,66 further functionalization is required to increase
stability, to target the surface and to minimize the response
of the reticuloendothelial system. There are several coating
strategies that depend on the chemistry of initial particle
surface and on the final application of the MNPs, but they
all should have in common the achievement of hydrophilic
NPs, stable in broad pH ranges and high ionic strengths, and
with functional groups that make the attachment of biomolecules,
Abs, etc. possible.67 Comparing the strategies followed for the
surface modification and bioconjugation of different inorganic
nanoparticles one cannot conclude that they depend only on
the nature of the inorganic core. Nanoparticles with biological
applications such as gold colloids have different surface
reactivity compared to any other inorganic NP and they
interact in a different way with the different end groups of
the ligand molecules such is the case of the strong affinity
gold–thiol group.68 Besides the inorganic core influence, the
coating strategy will be mainly based on the initial ligand–
nanoparticle interactions present after the synthesis of the
core. Whether it is necessary to exchange the ligand, to cross-
link it, to modify it by adding new functional groups or to add
new coating layers of polymers or inorganic materials depends
strongly on the initial layer(s) of molecules that cover the
magnetic surface. Coating materials for biological applications
include organic molecules (e.g. methylene diphosphonic acid,
citrate), polymers (e.g. dextran, poly(ethylene glycol),
poly(NIPAAM)), surfactants (CTAB) and inorganic mole-
cules and shells (e.g. silica).69–74 MNPs that are already water
soluble can be chemically modified to improve the stability
and add new functional groups. This is the case of dextran
MNPs, the polysaccharide can be crosslinked with epichloro-
hydrin (resulting in for example cross-linked iron oxide MNPs
(CLIO)) to increase the coating density and treated with
ammonia to provide primary amino groups.75
Poly(ethylene glycol) (PEG) has been widely used for the
conjugation with proteins and to extend the MNPs circulation
time.76,77 Moreover, MNPs covered with PEG are considered
to be nonimmunogenic, nonantigenic, and protein resistant.
Therefore, PEG has been one of the most popular polymers
used for MNP coating.78,79 The phase transfer and PEGyla-
tion of hydrophobic MNPs coated with a layer of oleate and
oleylamine have been possible for example, by replacing these
molecules with a modified PEG containing dopamine.73 It is
also possible to coat easily with PEG MNPs that have been
produced through a hydrolytic approach.80 In a recent work,
two new strategies to PEGylate commercially available MNPs
have been developed for MNPs with a diameter above 100 nm.81
There is a huge amount of different protocols to carry out the
polymer coating of the MNPs. In many cases, the initial NP is
hydrophobic and thus, the two possible approaches to render
it hydrophilic are: (i) to exchange the surfactant for another
ligand molecule that on the one end carries a functional group
that is reactive toward the MNP surface and on the other end
a hydrophilic group,69 and (ii) to add a second layer of an
amphiphilic polymer that is solely stabilized around the first
layer by hydrophobic interactions (by intercalation of the
hydrophobic tail of the second polymer in the surfactant
molecules of the MNPs) and further cross-linking of the
complete polymer shell.82,83 The latter method is more universal
and agglomeration during the coating process is almost
avoided though on the other hand, the hydrodynamic radius
is inevitably increased. MNPs can be treated as well as
building blocks and take part of a polymeric microstructure
material such as magnetic polyelectrolyte capsules by keeping
their magnetic properties and increasing their stability.84
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Inorganic materials have been largely used to give stability to
MNPs but mostly to produce multifunctional materials. Gold has
been used for example to produce multifunctional (with optical
and magnetic properties) and biocompatible materials such as
hybrid nanorings, spherical MNPs decorated with Au NPs on the
surface and core–shell structures.85–87 Silica is another favorable
surface coating or host material for the inclusion of MNPs
because of its biological compatibility and optical transparency
which makes possible to introduce or conjugate to the same silica
matrix molecules or particles with optical properties such as
dyes.88–90 Gadolinium is a promising coating agent for MNPs
because it is a popular positive contrast agent for MRI. In
particular, the growth of a silica shell can be exploited to
functionalize MNPs with gadolinium giving rise to materials
combining T1 positive and T2 negative contrasting efficiency.91,92
3.3. Strategies for bioconjugation
There is a wide variety of procedures for the adequate bio-
functionalization of MNPs which will yield to biocomposites
with the appropriate features for biomedical applications. This
paragraph deals with the strategies commonly followed to bind
or encapsulate biomolecules to MNPs. The main issue here is
how to build up such biomagnetic structures by keeping the
activity and properties of their constituents. In this context,
functionalization of MNPs with monoclonal antibodies (mAbs)
represents an interesting example. These Y-shaped proteins
used by the immune system to indentify foreign objects have
their most reactive amine groups in the antigen binding site
(Fab domain). One of the most popular strategies involves the
covalent attachment of the mAb through their most reactive
amine groups, but it can lead to random orientation of the mAb
resulting in a loss of recognition activity.93,94 Several strategies
have been proposed to avoid random orientation. Mainly they
imply the mAb modification through several steps of purifica-
tion or specific immobilizing proteins.95,96 Recently, another
approach has been published that takes advantage of unspecific
reversible interactions between the mAb and the MNP in order
to orient the mAb on the magnetic surface before being
attached covalently in an irreversible way.97,98
Aptamers are nucleic acid ligands that bind to a specific
target molecule. Their synthetic design is a common bio-
chemical practice even though natural aptamers exist. They
consist of DNA, RNA or short peptides. Their specific
aptamer–protein interaction makes them ideal candidates to
produce recognition and specific uptake of aptamer labelled
MNPs by target cells. There are several strategies to attach
aptamers to the magnetic surface. MNPs can be functionalized
with aptamers by ethyl(dimethylaminopropyl) carbodiimide/
N-hydroxysuccinimide (EDC/NHS) chemistry if the MNPs
have been previously coated with a molecule that provides
carboxy groups to the magnetic surface (e.g. PEGylation).99,100
Streptavidin coated MNPs can also be conjugated to aptamers
that have been labelled with biotin101 and MNPs coated with
Au NPs or Au shells can be functionalized with thiolated
aptamers directly by mixing both constituents.102,103
Another example of magnetic biocomposites is the adeno-
viral vector tagged with MNPs. This hybrid system composed
of MNPs and an adenovirus (Ad) has applications in
simultaneous MRI and gene delivery. Such nanostructure can
be fabricated for example by self-assembly of: (i) MNPs coated
with a fluorinated surfactant combined with branched poly-
ethylenimine (positively charged) and the Ad,104 (ii) biotiny-
lated adenovirus and streptavidin conjugated MNPs,105 (iii)
MNPs coated with N-hexanoyl chitosan (positively charged)
and the Ad,106 (iv) organic matrix of MNPs coated with Ad
binding proteins and the adenovirus107 and (v) Au NPs decorated
MNPs with the Ad (Au surface bind to cysteine and methimine
residues of Ad surface proteins).108 It has been also possible to
bind MNPs to an adenovirus by cross-linking of maleimide-
modified adenovirus and thiol-functionalizedMnFe2O4MNPs.109
The main body of an adenovirus has around 90 nm of diameter,
thus the number of MNPs per virus will vary depending on the
MNP size from thousands to tens or less.104,109
MNPs are regarded as nanocarriers that may enhance the
bioactivities of some drugs by delivering them directly into the
area of the body where they have to act. Even if drugs are not
considered as biomolecules, we include their conjugation with
MNPs here, because they require the production of final
biocompatible MNPs. In this paragraph we will focus only
on two different anticancer drugs, paclitaxel (PTX) and doxo-
rubicin (DOX). There are several problems associated with
their use as effective anticancer drugs: (i) low solubility in
aqueous solutions, (ii) low bioavailability for selectively targeting
cancer cells, and (iii) lack of an efficient method for their
detection and tracking. In theory, these drawbacks could be
solved by including the drugs in an appropriate carrier such as
magnetic biocomposites. PTX is a mitotic inhibitor used for
the treatment of breast, ovarian, lung, prostate, melanoma, as
well as other type of solid tumors. It is administered by
injection and it is an irritant, thus it can cause inflammation
of the veins and tissue damage. Therefore, drug loading into a
matrix is especially convenient. Core–shell structures (where
the core is magnetic and the shell is a polymer) and bio-
degradable polymeric matrix containing both the MNPs and
PTX have been proposed as carrier systems.110,111 PTX can be
also bound on the surface of the MNP.112 For instance, Hua
et al. have produced MNPs covalently labelled with PTX by
modification of polyaniline. This hydrophilic polymer modified
with succinic anhydride which forms water-soluble self-doped
poly[aniline-cosodium N-(1-one-butyric acid) aniline] was
used to functionalize the MNPs previous to covalent immobi-
lization of PTX on the surface.113 DOX is an anthracycline
antibiotic, a family of drugs that works by intercalating DNA
which includes among the most effective anticancer treatments.
It has also side effects. DOX is a vesicant that can cause
extensive tissue damage and blistering if it escapes from the
vein. Most of the strategies to encapsulate DOXs are also
based on the formation of a coating layer of amphiphilic
polymer and the loading of this layer with the hydrophobic
drug via hydrophobic interactions or covalent bonds. However,
the composition of the magnetic nanostructure is generally
selected based on the selected release mechanism (e.g. pH
triggered release, enzymatic degradation, and thermic effects).
As an example, pH-triggered DOX-releasing MNPs can be
based on a change of affinity between DOX and the coating
agent of the MNP upon a pH change (e.g. pyrene based
polymers and DOX can bind to each other by p–p interactions
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at neutral pH, but protonation of DOX under intracellular
acidic conditions can cause its sudden release by decreasing
this p–p interaction)114 or in a change of conformation of the
polymer matrix upon pH variation (from swollen to shrunk
state upon pH decreasing) (Fig. 4).115,116
Many interesting applications of bioconjugated MNPs
require the presence of DNA on the surface of the MNP.
DNA is negatively charged and can be coated with small
positively charged MNPs via electrostatic interactions by
keeping its biological activity.117 In another approach, magnetic
silica particles have demonstrated to attach successfully oligo-
nucleotides upon functionalization of their surface with amino
or thiol groups.89 Tat peptide coatings induce intracellular
accumulation of MNPs, they can be attached on the magnetic
surface via disulfide linkage.75 This biomolecule belongs to the
family of cell-penetrating peptides and facilate the cellular
uptake. MNPs can be coated with enzymes but also be used
to detect them.118,119 In summary, there is a large list of
strategies commonly used for the bioconjugation of MNPs that
depends on the specific application of the magnetic biocompo-
site. Parameters such as stability, activity and orientation of the
biomolecules within the magnetic biocomposite are key aspects
in the fabrication of efficient systems useful in biological appli-
cations demanding high control of the particle–biomolecule
interactions in a molecular level. Table 3 shows some of the
biomolecules and the coating agents mentioned in this manu-
script together with their features and applications.
4. Cellular and in vivo toxicity
As it has been mentioned, within the big family of different
MNPs, some dextran-coated formulations have been already
FDA- and EMA-approved as MRI contrast agents. In the
future, the use of new types of MNPs in clinical trials is expected,
but the factors that makeMNPs suitable for medical applications
are not yet well-known. Some recent findings, such as the
intracellular degradability of MNPs120 or the close correlation
between the cellular localization and concentration ofMNPs and
their cytotoxic effect,121,122 have provided new insights in under-
standing the effect of MNPs in vitro. However studies such as the
toxic effects of inhalation exposure to ferric oxide123 or the long
term in vivo biotransformation of MNPs124 are of crucial interest
for the expansion of diagnosis assays and therapies based on
MNPs. Regardless of the intrinsic differences between the various
MNPs, the size-factor itself appears to cause several adverse
effects. As the superparamagnetic MNPs are in the same size of
natural proteins, these MNPs can reach places where larger
MNPs cannot enter. Furthermore, the confinement of MNPs
in subcellular structures such as endosomes can lead to very high
local concentrations which cannot be achieved by free ions. The
shape of the MNP has also been demonstrated to influence the
uptake of MNP by living cells.125 Thus, size, shape and physico-
chemical properties dictated by the coating agent of MNPs
greatly determine the extent of cellular interactions.
4.1. Cytotoxicity end-points
MNPs such as nickel ferrites have shown potential toxicity
affecting cell proliferation and viability.126,127 In contrast,
some of the iron oxide MNPs are biocompatible when coated
with specific surface modifiers.128 In both cases, there is a lack
of information concerning the molecular mechanisms of toxicity.
In this paragraph some of the most reported and discussed
mechanisms which affect cell homeostasis will be discussed
although they are still all a matter of debate.
One of the frequent concerns related with MNP cell uptake
is the generation of reactive oxygen species (ROS). These
species can initially serve as a defense mechanism against
invading foreign species or, alternatively, they can lead to
the induction of apoptosis. Its riskiness is cell type-dependent,
but most of cells have defense mechanisms that buffer a certain
amount of ROS making possible only transient high levels of
these species.129 For MNPs, the induction of ROS is typically
a transient effect that highly depends on the stability of the
coating agent, in its nature (if it produces ROS or not), and in
the concentration of MNPs that have been internalized by
cells.130–133 In the case of nickel ferrite MNPs several studies
have reported the induced toxic response in cells through ROS
generation and recently its dependence on the concentration of
MNPs has been pointed out.134 As transient higher ROS levels
can sometimes be observed without any clear cytotoxic effects,135
the overall impact of elevated ROS levels associated with the
presence of MNPs remains unclear.
Due to the physical dimensions of MNPs, their intracellular
accumulation can also affect the structure of the cellular
cytoskeleton network.136 The interaction between MNPs and
the cytoskeleton can be direct if the MNPs have been inter-
nalized in the cytoplasm or indirect if the MNPs are localized
in endosomes. MNPs with a wire shape125 or functionalized
with special coating agents can be found in the cytosol, but
most studies report on the typical endosomal localization. In
this context, it is generally accepted that the coating of MNPs
favors different types of cytoskeleton disorganization and the
Fig. 4 Schematic representation of different bioconjugates: (A) oriented Ab bound to a MNP, (B) aptamer coated MNP, (C) adenovirus coated
with B10 nm MNPs and (D) MNP coated with pH or thermo responsive polymer loaded with a hydrophobic drug.
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engulfed MNPs concentration influences the degree of
disorganization.137 As the cytoskeleton is involved in many
intracellular signaling pathways, it remains to be investigated
whether the MNP-induced cytoskeletal disruption leads to
secondary effects such as cell death, diminished proliferation
or other mechanisms.
The complex intracellular signaling pathways can be altered
not only due to cytoskeleton changes but through several
mechanisms, such as: (1) genotoxic effects caused by high
levels of ROS,138 (2) altered protein or gene expression due
to the perinuclear localization of the MNPs which may hinder
the functioning of the transcription and translation machinery,139
(3) altered protein or gene expression levels due to leaching of
free metal ions,131 (4) altered activation status of proteins by
interfering with stimulating factors such as cell-surface receptors140
or (5) altered gene expression levels in response to the cellular
stress that the MNPs induce.141 To date, the effect of MNPs
on protein or gene expression levels has only scarcely been
investigated and more data need to be generated in order to
get a better idea to what extent MNPs can cause alterations to
intracellular signaling pathways.
The biodegradation ofMNPs is the responsible mechanism for
the generation of free ferric iron and further complete dissolution
of the magnetic core. The different dissolution kinetics of MNPs
has been observed to depend on the surface coating.142 Free
ferric iron was found in some cases to induce high levels of
ROS, apoptosis or inflammation and to alter the transferrin
receptor.143 Another possible source of toxicity is the interaction
of MNPs with biological molecules. Due to the charge of MNPs
serum proteins are prone to bind the magnetic surface unless a
protective MNP coating inhibits this process.144,145
Finally, the application of MNPs in hyperthermia or drug
delivery brings new issues to be taken into account. Hyperthermia
requires the application of an AMF that is used to kill tumor
cells,146 but without full control of this technique non-tumoral
cells can be also damaged. Magnetically guided drug delivery or
MRI employs a constant magnetic field gradient, thus no direct
effect on cells are expected. However, the increased internaliza-
tion of MNPs can induce toxic effects by exceeding the local
toxic threshold of the MNPs147 or by affecting the relative
localization of the endosomes inside the cells and therefore
changing their normal intracellular routing and maturation.148
4.2. Potential in vivo toxicity
There are many MNPs manufactured for application as MRI
contrast agents such as Feridex, Resovist, Endorem, Lumirem,
Sirenem, etc.149 but some of them have been currently
removed from the market.150 They are all based in magnetite
composites and most of them are coated with dextran or
carboxy dextran. The number of in vivo studies performed in
humans so far is limited but in continuous growth151–153 and is
expected to bring more information about the potential toxic
effects of MNPs. It is known that in the case of Feridex
intravenous (i.v.) administration may cause severe back, groin,
leg or other pain, or allergic reactions. Ferumoxtran-10 for
example is also inducing side effects such as urticaria or
nausea, all of which are mild and short in duration.154,155 It
is thought that these mild side effects are due to the degrada-
tion and clearance of MNPs from circulation by the endo-
genous iron metabolic pathways. The clearance mechanisms in
humans will be discussed in Section 6.6.
Long term studies in animals have not been yet been
performed for most of the commercially available contrast
agents based on MNPs. Therapeutic iron dextran products
have been associated with the development of sarcomas at the
intramuscular injection sites; the length of treatment or the
length of time after injection until development of tumor is not
known. The MNPs used as contrast agents with patients are
iron oxides associated with dextran. Whether these MNPs
Table 3 Examples of different coating agents and biomolecules used in the fabrication of magnetic biocomposites
Biomolecule/coatingagent Advantages/applications References
Adenoviral vectors MRI and gene delivery. They favored cytosolic release. 104–109Antibodies High variety of antibodies that bind to specific proteins only present in particular
cellular structures. Recognition processes, cell capturing and immunomagneticseparation. Detection of in vivo enzyme activity. Tracking of labelled cells.Intelligent drug delivery. Accumulation in tumors
93–98, 198, 199, 204, 247,350, 368–371, 374
Aptamers It is possible to create them artificially for specific targeting. Smaller than Abs.Recognition processes. They bind to specific target molecules.
99–103
Dextran Stabilizer agent. Enhances blood circulation. 66, 120–123, 143,149–155, 177–183, 274,323, 390
DNA or RNA Recognition processes. Drug delivery 104, 117, 349, 381–389Doxorubicin Drug for cancer treatment. 114–116, 306, 330–332Enzymes or proteins Biomarkers of process such cancer, apoptosis, inflammatory reactions. Protein
separation, purification, detection and analysis. Accumulation in tumors(drug delivery and hyperthermia)
118, 119, 144, 165, 166,347
Folic acid Effective tumor targeting agent. 322, 372, 373Nitrilotriacetic acid Ligand bearing Ni2+-chelating species. Protein separation. 160–164Oligonucleotides Probes for detecting or separating DNA or RNA. Nanoswitches. 167–169, 177Paclitaxel Drug for cancer treatment. 110–113Polyethylene glycol Protein repellent, nonimmunogenic and nonantigenic. Long-term circulation
capability in blood vessels. Widely used for protein conjugation.16, 73, 76–81, 99, 100,322, 328, 354–363
Silica Stabilizer agent that can be loaded (within the pores). Biological compatibility andoptical transparency. Useful in the fabrication of multifunctional MNPs.
88–92, 203, 273, 327,369–372
Tat peptide Facilitate cellular uptake of MNPs. 75
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have a risk of tumorigenesis that is similar to that of iron
dextran is not known. Therefore studies that deal with the long
term influence of MNPs in the organism are highly required.
In this context, Levy et al. have recently studied the long term
in vivo biotransformation of iron oxide MNPs.124 A three-
month magnetic follow-up of MNPs gave evidence of the
degradation and loss of their superparamagnetic properties.
They observed as well the relocation of iron species from liver
to spleen in the organism.
5. Molecular detection with magnetic nanoparticles
In the language of nature, biological entities exploit high-
affinity specific interactions between molecular pairs to achieve
reciprocal recognition and trigger signaling processes. If one
of the biomolecular entities is immobilized on MNPs, the
resulting magnetic nanoconjugates can specifically bind to
the biomolecular counterpart. There are two immediate
consequences of such an effect. The first is that it is possible
to control the localization of selected biological targets by
applying an external magnetic field gradient and, under certain
conditions, isolate them. A second complementary application
exploits the unique superparamagnetic character of these
MNPs to interact with an external magnetic field inducing
dephasing of spin–spin relaxation times (T2) of the surrounding
water protons of the solvent, in which they are immersed. In
this case, the extent of magnetic interference is dependent on the
size of MNP assembly, which is in turn caused by molecular
recognition events. Based on these concepts, several applica-
tions using biofunctionalized MNPs, including protein and
DNA separation, molecular biosensing and pathogen detection
and sequestration, have been explored.
5.1. Protein and DNA separation
Isolation, purification and controlled manipulation of peptides
and proteins represent a need of paramount importance in
biotechnology and in life sciences. Conventional protocols
may involve electrophoresis, ultrafiltration, precipitation and
chromatography.156 Among the available methods, affinity
chromatography is often considered the choice of election in
terms of efficiency and selectivity. However, the use of liquid
chromatography is limited to pre-treated solutions. Inhomo-
geneous matter such as protein production mixtures are
incompatible with the particulate-free conditions required for
a correct usage of commercial columns. Magnetic separation
exploitingMNPs represents an attractive alternative method for
the selective and reliable capture of specific proteins, DNA and
entire cells, as it makes use of cheap materials and does not
necessitate time-consuming sample preparation.157–159 The
basic principle of magnetic separation is very simple (Fig. 5).
MNPs bearing an immobilized affinity tag, or ion-exchange
groups, or hydrophobic ligands, are mixed with the mixture
containing the desired molecules. Samples may be crude cell
lysates, whole blood, plasma, urine, or any biological fluid or
fermentation broth. After a suitable incubation time, in which
the affinity species are allowed to tightly bind to the ligands
anchored to the MNPs, the complexes are isolated by magnetic
decantation and the contaminants washed out. Finally, the
purified target molecules are recovered by displacement from
the MNPs by proper elution procedures.
At present, the most thoroughly investigated affinity tag-based
approach for magnetic separation of proteins makes use of
MNPs functionalized with ligands bearing Ni2+-chelating species,
such as nitrilotriacetic acid (NTA), which allows for the
selective sequestration of (6�His)-tagged proteins with highly
conserved folding down to picomolar concentrations.160,161
His-tagged proteins cover the surface of MNPs selectively and
quickly, reducing nonspecific adsorption of undesired entities,
which represents a major drawback of commercial microbeads.162
As it is likely that the multivalent action of the chelating agent
plays an important role in enhancing the binding selectivity of
His-tagged proteins at low concentration, the choice of the
anchoring strategy as well as the chelating ligand might signifi-
cantly affect the binding capacity and reusability of biofunctional
MNPs without loosing efficiency.163,164 New advances made in
the separation/extraction of biomolecules by MNPs suggest that
this technology could be general and versatile. Similar appro-
aches can be envisaged for alternative affinity tags, which
selectively bind with different biological targets if proper anchors
and ligands are used. For example, it is possible to utilize MNPs
functionalized with specific peptides, including protein A or G,
having strong affinities for the Fc portion of human IgG Abs to
achieve a tight and reversible capture useful for Ab sorting.165,166
Protein separation with MNPs is advantageous compared
with conventional affinity chromatography for several reasons.
The purification process is simple, rapid, cheap and scalable.
Fig. 5 Schematic representation of magnetic separation of DNA or proteins in solution. The grey rods on the surface of the spherical MNP in the
figure represent the immobilized tag which will tightly bind the DNA strand or protein of interest. The separation process follows the steps:
(1) MNPs and the solution with different components are mixed, (2) particular proteins or DNA (green rings) bind to the MNPs and (3) the
magnetic field is applied to trigger magnetic decantation, followed by further washing steps and collecting the molecule of interest.
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Small amounts of materials are necessary for the separation
process thanks to the high surface-to-volume ratio of nano-
sized sequestrants. Moreover, magnetic separation does not
require dedicated equipment, such as centrifuges, filters or
liquid chromatography systems, and no sample concentration
is needed after elution. It is worth mentioning that automated
systems for the separation of proteins or nucleic acids are now
available.157 DNA or RNA can be isolated and concentrated
using selected oligonucleotides grafted on MNPs, which allow
the capture of complementary strands.167 When using labelled
primers in a polymerase chain reaction (PCR), the amplicons
can be isolated, concentrated, and even used in an automated
assay for sequencing. Nucleic acids deriving from bacterial
and mammalian cells have been captured and purified by
magnetic beads using a microfluidic chip in nanolitre volumes
of untreated samples. This method allowed the automated
extraction of amounts of mRNA from a single mammalian
cell.168 One of the problems often encountered by researchers
when using cationic extractors, including amine-functionalized
MNPs, to purify nucleic acids resides in the low release of
captured genetic materials. Indeed, the efficiency is generally
modest using phosphate buffer as a competitor and even much
lower with other ions. Tanaka et al. found that using deoxy-
nucleotide triphosphates in place of phosphate buffer can
increase significantly the desorption efficiency thus improving
the chances of successful PCR analyses.169
5.2. Biosensing with magnetic nanoswitches
The unique optical, electronic, and magnetic properties of
several metal and metal oxide NPs functionalized with affinity
ligands combined with agglomerative phenomena induced by
specific interactions occurring at their surface has led to the
development of highly sensitive NP-based biosensors. In parti-
cular, gold NPs and semiconductor NPs (so-called quantum dots)
have been largely exploited for colorimetric and fluorescence-
based detection of oligonucleotides, proteases, Abs and other
molecular species.170–176 The major disadvantage in biosensing
assays based on optical responses resides in the necessity of
reducing the sample turbidity or background signals from the
biological extracts. A novel class of nanosensors has thus been
developed by exploiting the peculiar magnetic properties of
MNPs. Magnetic relaxation nanoswitches have been first
proposed by the group of Weissleder in a series of seminal
works, which demonstrated the efficacy of this new nano-
biosensor for the accurate and sensitive detection of a broad
range of biological species, including DNA, proteins, patho-
gens, and processes such as enzymatic function.177–182 These
magnetic relaxation switches consisted of 3–5 nm iron oxide
MNPs coated with a 10 nm thick dextran layer that is
stabilized by crosslinking (CLIOs) and functionalized with
amino groups, useful to covalently anchor the affinity ligands.183
Such nanoswitches are able to undergo reversible assembly in
the presence of a specific molecule that is selectively recognized
by the affinity ligands immobilized on the MNPs, resulting in a
change in transverse magnetic relaxivity (R2 = 1/T2) of water
protons adjacent to the floating nanodipole. According to the
outer-sphere diffusion theory, when clusters of MNPs are
sufficiently small, e.g., within a few hundred nanometres, the
effect of assembly is to reduce the average T2 value. In contrast,
when large agglomerates are formed (with size range of several
micrometres), T2 is instead increased compared with that of
individual MNPs dispersed in the same fluid or matrix.184,185
Both strategies have been demonstrated useful depending on
the experimental requirements,186 magnetic relaxation nano-
sensor assays being designed to form reversible nanoassemblies
upon MNP interaction with specific analytes in solution both in
a forward (clustering) or reverse (declustering) setup (Fig. 6).
By this approach the concentration of analytes, such as glucose
and calcium in solution has been quantitatively determined.187,188
In particular, a combination of CLIO-glucose and concavalin-A
enabled the measurement of glucose concentration across a
semi-permeable membrane over the clinically meaningful
range, suggesting a future potential clinical utilization in an
implantable biosensor. In principle, the same concept could be
applied to simultaneous analysis of multiple metabolites in a
continuous and noninvasive way by MRI in vivo.189
By using functional MNPs endowed with higher inherent
magnetic susceptibilities, several other pathological bio-
markers have been detected with similar experimental setups,
including autoantibodies, toxins and human plasma glyco-
proteins.186,190,191 In a conceptual evolution of this approach,
sets of primary and metastatic cancer cells could be detected,
characterized and distinguished from normal cells using a
library of magnetic glyco-NPs.192
Fig. 6 Schematic drawing showing the principle of superparamagnetic
nanosensors also called ‘‘magnetic relaxation switches’’. The scheme
illustrates the detectable effect on T2 caused by MNP aggregation
sequence in response to the presence of target analyte (blue cross) and
its removal by adding a competing inhibitor (red square).
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Recently, a chip-based diagnostic magnetic resonance
(DMR) system for multiplexed, quantitative and rapid analysis
of unprocessed biological samples has been developed.193 The
source of the signal is the molecular interaction amplification
caused by assemblies of MNPs with enhanced magnetisation.181
The potential of this device has been demonstrated by moni-
toring the presence and amount of proteins in parallel and by
detecting bacteria and analyzing them at a molecular level with
high sensitivity. Miniaturized DMR has several advantages
over the reported conventional bulk experiments: (1) only
microlitre volumes of the sample are required, with a remarkable
increase in detection sensitivity; (2) multiplexed measurements
can be easily performed enabling a rapid screening of analytes
even in opaque biological media provided that they are trans-
parent to magnetic fields; (3) the magnetic field is generated using
a small, portable magnet withmore homogeneous radio-frequency
magnetic fields and less electrical resistance compared with con-
ventional relaxometers; (4) DMRmicrosystem can be produced as
disposable units. All these features suggest a potential of DMR
technology as a friendly handheld diagnostic device.
5.3. Bacteria detection and sequestration by magnetic capture
Magnetic separation, and particularly immunomagnetic
separation using MNPs coated with Abs against surface anti-
gens of cells, has been exploited for separation of eukaryotic
cells.194 Recently, the same approach has been used for the
detection and isolation of bacteria from biological samples
(Fig. 7). Once the microorganism has been selectively captured
and concentrated, the identification can be accomplished by
conventional methods.195–197 In a pioneering study in 1988,
Lund et al. isolated the K88 (F4) fimbrial antigen responsible
for colonization of piglet intestines using magnetic beads
functionalized with a specific mAb and examined the extracts
by fluorescence microscopy.198 Magnetic beads coated with
anti-K88 mAb were also used by Hornes et al. for immuno-
magnetic separation (IMS) of enterotoxigenic E. coli strains
from pigs with diarrhea.199
In general, bacteria at low concentrations are hard to detect
with a conventional analytical method in complex biological
mixtures. However, it is expected that nanotechnology will
improve sensitivity, selectivity and analytical time-efficiency
in clinical diagnosis and environmental monitoring. Gu et al.
developed a vancomycin-conjugated FePt MNP system
(FePt@Van) to capture and detect Gram-positive bacteria at
ultralow concentrations.200 Polyvalent vancomycin tightly binds
to the D-Ala-D-Ala dipeptide, which is a major constituent of the
microbial capsule, enabling magnetic capture and enrichment of
bacteria. The declared detection limit of this method was 4
colony-forming units (cfu) per mL, on the same level of the best
assays based on polymerase chain reaction. FePt@Van MNPs
were also used to isolate and detect Gram-negative bacteria such
as E. coli.201 Combining FePt@Van with fluorescent dyes
allowed for the detection of bacteria in blood samples.202
El-Boubbou et al. developed silica-coated magnetic glyco-NPs
that could detect E. coli strains in 5 minutes, concomitantly
enabling up to 88% removal from the sample exploiting the
bacterial interaction with mammalian cell surface carbohy-
drates.203 MNPs functionalized with a single-domain Ab proved
to be highly efficient and selective in targeting and capturing
Staphylococcus aureus cells in a mixed cell population.204 The
authors suggest that the superior specificity obtained with these
nanocomplexes can be ascribed to the unique targeting selectivity
of multimerized VH Ab small domains. In another study, a
surrogate of Mycobacterium tuberculosis was detected in native
biological samples, such as sputum, in less than 30 min analysis
with a sensitivity of 20 cfu.182 The authors used MNPs with high
inherent susceptibility to target the pathogens in a selective
manner. The detection signal was amplified by concentrating
the specimen in a microfluidic chamber and measured by a mini-
aturized NMR system. More recently, magnetite- and cobalt-
based MNPs functionalized with poly(hexamethylene biguanide)
were demonstrated to be able to bind tightly to lipid A, a glyco-
lipid constituent of endotoxins, and DNA from E. coli, allowing
for the sequestration of bacterial strains and inhibiting the cell
growth and viability at concentration levels below 10 mg ml�1.205
6. Contrast agents for magnetic resonance
In vivo molecular imaging has been identified by the National
Cancer Institute of the United States of America as an extra-
ordinary opportunity for studying diseases non-invasively at the
molecular level.206 The aim is to visualize molecular character-
istics of physiological or pathological processes in living
organisms before they manifest in the form of anatomic changes
without invasive procedures. During the past, worldwide many
working groups showed the feasibility to image molecules in vivo
by different scanning modalities. MRI offers several advantages
such as lack of irradiation, possibility to generate 3D images,
excellent spatial resolution with optimal contrast within soft
tissues, and a very good signal-to-noise ratio. In this paragraph,
we present the current advances in the development of new
generation contrast agents for MRI and their applications.Fig. 7 Schematic drawing showing the principle of cellular detection
and sequestration by tagged MNPs.
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6.1. Molecular imaging with targeted contrast agents
Paramagnetic (e.g. gadolinium (Gd)-, europium (Eu)-, neodynium
(Nd)-, and manganese (Mn)-containing materials) and super-
paramagnetic (iron oxide in the form of maghemite (g-Fe2O3)
and/or magnetite (Fe3O4)) compounds can be used as MRI
contrast materials.14 With respect to molecular imaging, iron
oxide based MNPs have been favored because iron oxide
induces a stronger contrast. Moreover, Gd-contrast materials
may induce severe adverse effects with lethal outcome that have
been observed in patients with compromised renal function and
subsequent deposition in different organs/tissues and release of
Gd3+ which is called nephrogenic systemic fibrosis (NSF).
The first and major prerequisite of targeted contrast agents
is the identification of cells and/or disease and/or function-
specific biomarkers. Ideally, the biomarkers should be solely
and abundantly expressed on the desired cell types. Further-
more, disease-specific biomarkers should be clearly different
from healthy status. Biomarkers for targeted contrast agents
are cell surface receptors (i.e. transferrin receptor, folate
receptor, avb3 integrin, Her2/neu), phospholipids of the outer
leaflet of the cell membrane (i.e. phosphatidylserine (PS)), and
enzymes (See Section 6.3).206–214 TargetedMNPs are composed of
at least two components: (1) the magnetic iron oxide represents the
imaging or sensing component and (2) the attached molecule
represents the targeting or affinity component. MNPs without
targeting component are engulfed by monocytes/macrophages.
Thereby, they can be used to image monocytes/macrophages and
their phagocytic capacity in vivo (Fig. 8).
In most cases the used MNPs are completely artificial
products, but naturally occurring MNPs such as lipoproteins
can be also used.215 Recently, it has been shown that both
loading lipoprotein NPs with signaling substances (e.g.MNPs,
Au-NPs) and functionalisation of the surface are possible,
which transform them into molecular probes for the specific
recognition of molecular targets.215 Several studies demonstrated
the feasibility to specifically image molecules on cells in living
organisms. The major drawback of this approach is the need
of specific mAbs for each molecule, cell type or cell function.
Moreover, the desired molecules are specific species.
6.2. Multimodal magnetic resonance imaging probes
Since different imaging modalities provide complementary
information bi- or multimodal imaging probes have been
designed.83,216 So far, two different classes of multimodal
MRI probes have been synthesized: (1) magneto-optical
probes, and (2) magneto-radioactive probes. Most of the
multimodal MRI studies deal with MNPs conjugated with
organic fluorophores, because they cover several advantages
like high anatomical resolution of MRI, and high sensitivity
provided by the optical component that is comparable with
radioactive tracers but lacks all the disadvantages that are
associated with the latter.121 The optical component can be
detected by a broad range of in vivo (Fig. 9) and in vitro
techniques such as optical scanners (e.g. fluorescence mediated
tomography, fluorescence reflectance tomography, optical
coherence tomography) as well as fluorescence microscopy,
laser scanning (confocal) microscopy, flow cytometry, spectro-
photometry, intravital microscopy, intravascular noninvasive
near-infrared (NIRF) imaging, clinical endoscopy, and detec-
tion during surgery.217
Since near-infrared (NIR) fluorescence (700–1000 nm)
avoids interferences with background fluorescence of mole-
cules of living organisms, and thereby provides an excellent
contrast between desired target and background tissues, NIR
fluorophores should be favoured for imaging living organisms
in real time. Several chemical compounds and materials fulfil
the criteria of NIR fluorophores: (1) organic fluorochromes
(e.g. fluorescent cyanine dyes such as Cy5.5), (2) fluorescent
semiconductor quantum dots (QDs), (3) lanthanides, and (4)
gold NPs.103,212,216,218–223 Organic fluorochromes are widely
available and can be easily coupled to the shell of MNPs to
build up Cy 5.5-MNPs for example. As these compounds
suffer from fast photobleaching, QDs have been introduced
due to their improved photo-stability. QDs are NPs composed
of semiconductors such as CdSe, CdTe, ZnS, or InGaAs212,216,224
having narrow, symmetric emission spectra with long, excited-
state lifetimes220 and possessing high extinction coefficients
combined with a quantum yield comparable to fluorescent
dyes.220 Like MNPs, QDs can be coated with polymeric
materials to allow subsequent functionalization with the above
listed molecules.225 Due to their toxic components nowadays
their use is still limited to in vitro experiments and animal
studies, though QDs from more biocompatible materials are
currently being developed.220,226,227 Au NPs are ideal for
molecular imaging studies because they reduce cellular toxicity,
and have a bright NIR fluorescence emission of 700–900 nm.228–231
Gold MNPs have been used for both in vivo and in vitro
applications including in vivo imaging of small animal models
and protein purification system using novel peptide tags.216,223
Moreover, sensitive biosensing gold MNPs combining
magnetic relaxation switch diagnosis and colorimetric detection
of human a-thrombin have been produced to be specifically
activated by matrix metalloproteinases expressed in tumors and
Fig. 8 Target-specific detection of two different breast cancer types
(SKBR-3 and KB) by anti-Her2/neu-MNPs (A–D). (A) and (B) are
tradional black–white MRI, and (C) and (D) show color maps. (A)
and (C) MRIs show the pre-contrast, and (B) and (D) display the post-
contrast (data taken from Chen et al.).209 Both post-contrast MRI-
images show that SKBR-3 breast cancer cells express the Her2/
neu-receptor, while KB cells do not.
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to be sensitive fluorescent biosensors for detection of DNA
hybridation.103,177,232,233 Lanthanides are both paramagnetic
and fluorescent substances. Europium(III) oxide (Eu2O3)
shows intense red fluorescence after excitation with UV-light.
NPs have been synthesized in Co : Nd : Fe2O3/Eu : Gd2O3
core–shell geometry.219 On the other hand, it is possible to
produce lanthanide-doped MNPs that contain 5 mol% of
Eu exhibiting nearly identical physical superparamagnetic
behaviour than the pure MNPs, and additionally have attrac-
tive optical properties combined with high photostability, a
narrow emission band, and a broad absorption band for long-
term multilabelling studies.221
Although positron emission tomography-magnetic resonance
(PET-MR) hybrid scanners are now increasingly established in
several radiological clinics and scientific units, routine radio-
tracers such as fluordeoxyglucose18 have been used unmodified,
as conjugation to MNPs has to the best of our knowledge not
yet been done. However, recently dual modality tumor imaging
MNPs have been described, namely RGD-conjugated radio-
labelled MNPs.211
6.3. In vivo MRI of enzyme activity
Enzymes are essential molecular players in many physiological
and pathological events, and can be used as biomarkers for
different processes such as apoptosis, atherosclerotic plaques,
inflammatory reactions, cancer and metastasis, to name a few.
Detection of in vivo enzyme activity is achieved either directly by
detection of the active form of an enzyme (e.g. by mAbs directed
against the active enzyme molecule) or indirectly by detection of
enzymatic induced changes of specific substrate molecules.
Currently, enzyme-sensitive MR contrast agents use the latter
principle. They are functionalized or made of mAbs with affinity
to enzyme modified substrates. Upon enzyme activation the
probes are specifically modified, and these induced molecular
changes relate to MR signal changes. The mAb-based approach
has been demonstrated to image cell surface ADP-ribosyltrans-
ferse 2 upon lymphoma cells.232 Protein ADP-ribosylation was
successfully determined with R2 and R�2 relaxometry on a clinical
3T MR scanner after application of both specific anti-ADP-
ribosyl-mAbs and secondary SPIO conjugated mAbs.
Smart MR probes, also called nanosensors, for depiction of
enzyme activity are mainly Gd-complexes. Only some approaches
demonstrate the feasibility of superparamagnetic nanosensors.
The principle of enzyme sensitive iron oxide nanosensors is a
special design of complex molecules that either agglomerate or
disagglomerate upon enzymatic activity which subsequently
leads to a corresponding switch in the spin–spin relaxation
time (T2) (Fig. 10). They are called magnetic relaxation
switches (MRS, cf. Section 5.2) and they are designed to for
example measure enzyme activity of proteases, methylases,
and restriction endonucleases.177,178,233,234 A peptide substrate
with a protease recognition sequence flanked by two biotin
molecules has been designed that bind to CLIO-avidin (CLIO-A)
MNPs and forms a superparamagnetic nanosensor.234 In the
presence of a specific protease, the peptide linker substrate is
cleaved, and the CLIO-A nanoassembly disagglomerates. By
using this approach, magnetic nanoassemblies responsive to
trypsin, renin, and matrix metalloprotease-2 (MMP2) activity
have been developed and tested.234 Another example of this
technology is a MNP containing a biotinylated caspase-3-
specific peptide substrate that was incubated with a second
MNP to form a caspase-3-sensitive magnetic nanoassembly.178
The used peptide substrate is specifically recognized by caspase-3,
thus serving as an assay for this enzyme. The caspase-3-mediated
Fig. 9 Example of multimodal imaging. C6 cells labelled with a multimodal probe were (A) transplanted into the left and right thighs of nude
mice through intramuscular (i.m.) and subcutaneous (s.c.) injection. Afterwards the animal was scanned by optical bioluminescence (B), positron
emission tomography (PET) (C), MRI (D), and fluorescence (E). Images taken from the study of Hwang et al.217
Fig. 10 Principle of detection of enzyme activity in vivo. The enzymatic
activity produces changes in the stability of the MNPs which induce
changes in the spin–spin relaxation time (T2) and therefore in the
relaxivity R2 (=1/T2) of the protons around the MNPs.
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reaction was associated with a dose-dependent increase in the
T2 relaxation time with kinetics similar to those reported
with fluorogenic substrates. In a similar fashion activity of
restriction endonucleases such as BamHI that cleaves double-
stranded oligonucleotides linked by two MNPs has been
shown to cause a designed nanoassembly to switch to a
dispersed state and produce an increase in T2.177 Two MNPs
(P1 and P2) were designed that hybridized to each other
and form a BamHI recognition site. T2 decreased when P1
and P2 were mixed together since oligonucleotides on these
two NPs hybridize and form a BamHI-sensitive nano-
assembly. Incubation with BamHI resulted in an increase of
T2. Other endonucleases such as EcoRI, HindIII, and DpnI
did not influence T2 when incubated with the BamHI-sensitive
nanosensor.
Although the above described MRS systems are very exciting
particles their introduction for in vivo MR measurements has
not yet been done. The main challenge to image enzyme
activity in vivo is the fact that in most cases enzymes act within
the cytoplasm or within cellular organelles (e.g. mitochondria,
nuclei), and only some types are localized on the surface of
cells (ecto-enzymes) or are secreted into the interstitium. Both
targeting of the desired cell types and delivery of these complex
MNPs into the cytoplasm should be solved in the future to
enable measurements of enzyme activity in vivo as well.
6.4. In vivo tracking of labelled dendritic cells
Dendritic cells (DCs) derive from bone marrow hematopoietic
cells and can be generated in vitro from either autologous
CD34+ progenitors or monocytes.235 DCs present powerful
antigens that play important roles in a huge number of
immune responses including anti-cancer reactions.236 Cancer
vaccines DCs are of special clinical interest because they enhance
the antitumor immune responses due to their capacity to process
and present tumor associated antigen (TAA), and subsequently
to migrate into secondary lymphoid compartments.235,237,238
In therapeutic approaches DCs are loaded with TAA supplied
as whole tumor cell extracts, synthetic peptides, purified whole
TAA protein content or by genetic transfection of TAA
expressing DNA or, mRNA.235 Pulsing of DC based vaccines
with TAA induced their maturation. Afterwards mDCs migrate
to secondary lymphoid tissues and present TAA to specific
T-cell clones (Fig. 11). This event initializes TAA-specific T-cell
responses that might result in tumor cell death.
Although DC-based immunotherapy has been successfully
used in several studies to treat skin, breast, prostate, and
neuronal cancer for example,235,239–241 some patients do not
respond, and only a maximum of 3% of ex vivo generated DCs
reach the lymph nodes.242,243 Still a lot of work needs to
Fig. 11 Principle of preparation steps of DCs/DC-vaccine for MR
tracking studies in vivo. Note that the magnetic labelling procedure can
be either done before DCs are triggered with antigens or afterwards.
Fig. 12 Overview of methods to magnetically label DCs for MR tracking studies in vivo. Upper part (above of the dotted line) shows the different
MNPs that have been used to magnetically label DCs, and the lower part (under the dotted line) shows the different delivery mechanisms to
accumulate MNPs within DCs.
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be done to optimize DC-based vaccination. Both the exact
delivery in vivo and the migration of mDCs to lymph nodes are
critical steps that are necessary for a therapeutic success.
Unfortunately, established methods to ensure DC migration
are invasive.244 Therefore, the possibility to non-invasively
monitor DC-cancer vaccines in real time in vivo would be of
great scientific and clinical impact. Since cellular MRI offers
the possibility to non-invasively visualize in vivo cell delivery
and real-time cell tracking, several groups transformed this
approach for DC-based immunotherapy. Currently, no stan-
dardised labelling protocols exist: DCs have been labelled with
different MNPs (e.g. SPIONs, micro-sized particles of iron
oxide called MPIOs, or multifunctional polymer NPs containing
ovalbumin protein/IgG, MNPs), and fluorophores (e.g. fluores-
cein isothiocyanate, indocyanine green) and different loading
methods (Fig. 12).245–247 ComplexMNPs have been designed to
both trigger DCs with antigens and monitor them byMRI and/
or other imaging modalities. MNP accumulation has been
achieved by simple cell culture when using phagocytosing iDCs
or enhanced by receptor mediated endocytosis via the CD11c- or
Fcg-receptor, addition of transfection agents (TAs) such as prota-
mine sulfate, polylysine in concert with mDCs (Fig. 12).233,247–251
MPIOs have a diameter of at least 1 mm and cover higher
iron contents per particle than conventional MNPs.252 This
fact improved their detection by MRI.244 But on the other
hand MPIOs induced dramatic changes in the phenotype and
morphology of DCs, while ultrasmall SPIONs led to remark-
ably inefficient labelling (0.59 � 0.02 pg Fe per cell) that was
below the detection threshold for cellular MRI.250,253 MNP
DC labelling has been shown to efficiently load DCs without
affecting cellular morphology and functional maturation with
minimal or no effect on viability.233,248–250,254,255 However, a
closer insight demonstrated a dose-per-cell-dependent
decrease of the viability, and an increase of apoptotic cells
especially when higher iron doses of 400 mg ml�1 have been
added to cell cultures.244,251,255 The same was true for the
velocity: magnetic DCs showed efficient migration that was
slightly decreased in parallel to increasing iron doses.251
An iron content of 6 to 78 pg Fe per cell allowed the
depiction of DCs in vivo by clinical and small animal MR
scanners at magnetic field strength ranging from 1.5 T up to
11.7 T.233,244,248–251,254–256 The number of in vivo detectable
magnetically labelled cells ranges from 1.0 � 105 cells up to
1.0� 106 cells, and 100 cells mm�3 at 3 T or 50 cells mm�3 at 7 T
with an iron content of 25 pg Fe per cell, respectively.250,254,256
MR-based DCs tracking in vivo enabled monitoring of the
delivery of the vaccine, trafficking of DCs to lymph nodes and
other lymphoid tissues (Fig. 13 and 14).233,247,255,256
Most DC-tracking studies by MRI in vivo have been
performed in small animals such as mice,233,247,250,255,256 and
to the best of our knowledge only few studies have been
published that present data obtained with patients.254,257 The
reason for this phenomenon is unclear, but there is evidence
for the assumption that the labelling protocols are not
standardized so far, and the migration of the DCs is limited.
For example, iron quantification of magnetically labelled cells
is currently performed by atomic absorption spectrometry
(AAS), a method that is seldom established in clinical units.
Recently, on the basis of absorption spectrophotometry, a user-
friendly and inexpensive method has been described to overcome
these difficulties.258 Other researchers published an optimized
labelling protocol with short incubation time and low concen-
tration of SPIONs.256 Further experimental studies are warranted
to step-by-step improve this cellular treatment regimen with the
assistance of MR-based tracking of DC-vaccines, and/or imple-
ment more sophisticated applications (e.g. rapamycin inhibition
on lymphoid homing and tolerogenic function of nanoparticle-
labeled DCs247 or targeted delivery of nanovaccine MNPs to DCs
in vivo)245 in clinical routine.
6.5. Monitoring stem cell migration
Stem cells transplants are expected to have tremendous
potential for the treatment of many degenerative diseases
because of their capability to perform multiple cell cycle
divisions and of their differentiation efficiency.259,260 Several
clinical trials are ongoing with different types of stem cells.
Mesenchymal stem cells are used for reparation of damaged
tissue, regeneration of bone defects,261,262 spinal cord injury,263
stroke,264 and myocardial infarction,265 while neural stem cells
Fig. 13 (A, B) Example for MR-guided exact DC-vaccination delivery
in vivo. (A) MRI before vaccination; the inguinal lymph node to be
injected is indicated with a black arrow. (B) MRI after injection
showing that the dendritic cells were not accurately delivered into
the inguinal lymph node (black arrow) but in the vicinity, in the
subcutaneous fat (white arrow) (images taken from de Vries et al.).254
(C, D) Migration of DCs is detectable by cellular MRI. Coronal
3D-FIESTA images (200 � 200 � 200 mm) showing the popliteal
lymph nodes from one representative mouse, 2 days after injection
with (a) 1 � 106 MPIO-labelled DCs or (b) 1 � 106 unlabelled DCs
(images taken from the study of Rohani et al.).244
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are investigated for the neural lineages generation of the nervous
system.266 On this basis, one important issue is to identify and
track the stem cells after their injection in the body, to monitor
their motility and to follow the localization and their expansion
thereafter. Among the available in vivo imaging techniques useful
for stem cell monitoring, MRI is particularly promising since
it can provide high spatial resolution images without compro-
mising the patient’s care.267–269 T2 relaxivityMRI contrast agents
based on iron oxides offer a powerful labeling for the in vivo
visualization of the stem cells. As for DCs, MNP sizes for
utilization in stem cell labeling can vary from ultrasmall, within
35 nm diameter,270 to micron-sized.271 To this aim, the MNPs
can be coated by different polymers, including polyethylene
glycol,272 silica,273 dextran,274 and polystyrene,275 to increase
the stability of the suspension and thus avoiding the cell toxicity
caused by the formation of large agglomerates. These chemical–
physical characteristics affect labelling efficiency of MNPs, which
determines the interaction between MNPs and cells.276 The
typical MNP uptake follows an endocytosis pathway that can
be induced by mere incubation of the suspension of MNPs in the
cell medium,277 which, in turn, can be improved by application of
an external magnetic field.278 The addition of adjuvants, such
as transfection agents,130 or MNP functionalisation with Abs
exploiting a ligand-receptor specific interaction,279 could be of
help with some cell types. In alternative, it is possible to induce a
temporary permeability of membrane by electroporation280 or
ultrasound pulses.281
Several MNP based contrast agents were successfully applied
to in preclinical trials.282 The feasibility of MRI tracking after
injection of MNP-labelled stem cells for the treatment of
cardiovascular diseases offers not only a potential regeneration
of heart tissue, but also allows us to follow the long-term
migration cell without impairment of myocardial function and
without altering their cardiac differentiation.283–286
The in vivo cellular imaging after neurotransplantation for
the treatment of acute and chronic central nervous system
diseases, such as demyelination and lysosomal storage disorders,
acute spinal cord injury, Parkinson’s and Huntington’s diseases
and multiple sclerosis, serves several purposes, including
tracking cell migration and integration, postoperative visuali-
zation of stem cells localization, and monitoring graft
conservation.287–296 Although several clinical trials have been
approved by the FDA at the present time,254,297–299 there are a
number of constraints and limitations that remain unsolved:
the stem cells uninterrupted proliferation after transplantation
cause the dilution of the MNPs as labelling agents at the
expense of the long-term tracking and in some cases the cells
divide asymmetrically, leading to an unequal distribution of
the MNPs. Furthermore this kind of labelling prevents the
discrimination between live and dead marked cells.300
6.6. Clearance mechanisms in humans
Clearance mechanisms of MNPs in humans have been studied
with MRI. The MNPs that have been used for this purpose
were ionic ferucarbotran, and non-ionic ferumoxides or AMI-25,
with hydrodynamic diameter of 62 and 150 nm, respectively.
They were rapidly cleared after intravenous injection by
professional macrophages. Their blood half-life was 6 minutes.301
Macrophages engulfed these MNPs via phagocytosis. After-
wards MNPs could be found within lysosomes. This kind of
particle aggregation induced a signal enhancement as explained
above (see Fig. 10). Due to this fact macrophages could be
imaged by MRI. In other words, macrophage-rich organs and
tissues such as liver (Kupffer cells), spleen, bone marrow, and
inflammatory areas with increased macrophages like athero-
sclerotic plaques were hypointensive on T2=T�2 weighted MRI
after active engulfment of MNPs. Peak concentrations of iron
were found in the liver after 2 hours and in the spleen after
4 hours; afterwards MNPs were slowly cleared from these
organs with half-life of 3–4 days.301 In lysosomes MNPs were
enzymatically degraded and free iron were subsequently released
into the metabolic iron pool of the organism. Macrophages
incorporate ferucarbotran into lysosomal vesicles containing
a-glucosidase, which were capable of degrading the carboxy-
dextran shell of the ferucarbotran particles.143 Serum iron and
ferritin levels increased.302 Some of the MNPs remained intact
and were exocytosed by the cells, so that neighbouring macro-
phages could phagocytose them.
7. Magnetic nanoparticles as drug delivery systems
Most pharmacological approaches to cancer therapy are based
on chemotherapeutic substances, which generally exhibit high
cytotoxic activities but poor specificity for the intended bio-
logical target. This practice mostly results in a systemic
distribution of the cytotoxic agents leading to the occurrence
of well documented side effects associated with chemotherapy
caused by the undesired interaction of antitumor drugs with
healthy tissues.303,304 The idea of exploiting magnetic guidance,
making use of an implanted permanent magnet or an exter-
nally applied field, to increase the accumulation of drugs to
diseased sites dates back to the late 1970s. The first preclinical
experiments using magnetic albumin microspheres loaded with
doxorubicin for cancer treatment in rats were reported by
Widder et al.305 Since then, several improved MNP models
Fig. 14 Donor DC traffic to secondary lymphoid organs after local
injection and retention of SPIONs (i.m. injection of lucSPIOCD11c cells
in the right proximal leg 1 h after bone marrow transplant (BMT)
(C57B/63BALB/c)). Trafficking is monitored by bioluminescence
imaging (BLI) on the indicated days. (cLN) Cervical lymph node,
(aLN) axillary lymph node, (iLN) inguinal lymph node, (mLN) mesen-
teric lymph node (images taken from the study of Reichardt et al.).247
Copyright 2008. The American Association of Immunologist, Inc.
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have been developed, particularly for cancer therapy. However,
despite very promising results in preclinical investigations, the
first clinical trials have shown poor effective response and thus no
magnetic nanocarriers have been clinically approved yet.306,307
Besides magnetic force delivery, two alternative ‘‘physiological’’
routes can be followed by MNPs, which are common to all
kinds of nanoparticulates. The passive targeting route takes
advantage of the biological function of the reticuloendothelial
system (RES), a cell family of the immune system comprising
circulating monocytes, bone marrow progenitors and tissue
macrophages, which is deputed to the first clearance activity in
mammalian organisms.308 Once unprotected MNPs are immersed
in the blood stream, an array of plasma proteins called opsonins,
including immunoglobulins, complement proteins, fibronectin
and other species, recognize them as an invading agent and
immediately adsorb on their surface. The parameters affecting
the extent of opsonization are essentially related to the physical
properties of the MNP surface, including size, shape, charge
and state of agglomeration. Large objects are rapidly cleared
and highly charged NPs have a tendency to attract opsonins.309
Subsequently, MNPs coated by these plasma proteins are
rapidly endocytosed by the RES cells, resulting in their
removal from circulation and accumulation in organs with
high phagocytic activity, such as liver and spleen. Size is a key
parameter in NP clearance, MNPs smaller than 4 mm accu-
mulate in the liver (70–90%) and spleen (3–10%) quickly. NPs
larger than 250 nm are usually filtered to the spleen; NPs in the
range 10–100 nm are mainly phagocytosed through liver
cells,310 while NPs below 10–15 nm can be cleared by a renal
route.311 Therefore the optimal particle size for drug delivery
treatments ranges between 10 to 100 nm, as these will have the
longest blood circulation time (Fig. 15). It has been suggested
that the particle shape can also play a role. Anisotropic MNPs
with high aspect ratio have demonstrated enhanced blood
circulation compared to spherical MNPs in vivo.312 In the
absence of MNP protecting shells, MNP distribution in the
above-mentioned organs is accomplished within a few minutes,
depending on the size of theMNPs.313 Hence, passive nanocarriers
can be used to deliver drugs for the treatment of hepatic diseases,
such as liver metastases,314 and to favor the internalization of
antibiotics by phagocytic cells of the RES for the treatment of
intracellular infections.315
Magnetically assisted targeting of MNPs will have the
advantage of increasing the local concentration of the admi-
nistered drug, while the overall dose is reduced (Fig. 15).
Controlled transport is crucial for delivery but it is challenging
because of the small MNP size. On one hand, long circulation
time after the MNP injection is desirable to give the MNPs
more chances to be held by the magnetic field close to the
target area. On the other hand, in this application the minimum
diameter for successful MNP capture by a magnet is a limiting
factor. A single MNP in a magnetic field gradient will experi-
ence a force that depends on the magnetic moment and on the
field gradient around it. This force is proportional to the
volume of the MNP and, therefore, decreasing the size by a
factor of 10 decreases the magnetic force by 1000.316,317 For
example, individual Fe3O4 MNPs with a core diameter less
than 20 nm cannot be captured permanently by a HGMS
(high-gradient magnetic separation) column. The minimum
agglomerate size for permanent capture was calculated to be
40 nm for phospholipid-coated MNPs and 70 nm for polymer-
coated MNPs. The difference is attributed to the higher
volume fraction of magnetite in the phospholipid agglomer-
ates.318 The movement of MNPs inside a matrix or fluid
depends directly on a multitude of factors such as the external
magnetic field gradient, the temperature and the viscosity of
the medium, the fluid flow, the interaction between MNPs and
fluid components, and the size and shape of the MNPs. The
dynamics of MNP transport in vivo through a vein or artery to
an area of interest are far from being fully understood, but
there are nowadays several studies in this direction.316,319
Firstly, to hold the MNPs in the area that one wants to target,
field gradients are required, as MNPs will experience no force
in a homogeneous field. For this reason, rare-earth magnets
are generally used. The field gradient has to be high enough to
overcome the blood flow strength that keeps moving the
Fig. 15 Large MNPs (>200 nm) will be easily detected by the immune system and removed from the blood and delivered to the liver and the
spleen.320 Very small MNPs (o5.5 nm) can be excreted through the kidneys.321 The optimal MNP size for drug delivery treatments ranges between
10 to 100 nm, as these will have the longest blood circulation time. Different magnetic biocomposites can be transported to reach the tumor area
inside the body thanks to the applied magnetic field.
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MNPs in the vessels, and for that purpose, the closer to the
magnet surface, the better. The tissue between the target and the
magnet source will also accumulate the MNPs, therefore,
external magnets can be used for targets close to the body surface.
However, internal magnets will be needed for deeper targets.
In contrast with the passive delivery route, active targeting
has the advantage of improving the accumulation of chemo-
therapeutics at the tumor site, but requires multiple synthetic
steps to tailor the chemical properties of MNPs in order to
achieve a suitably bioengineered magnetic nanocarrier. In
principle, it is always necessary to stabilize the MNP disper-
sion in the aqueous environment. Thus, coating the MNPs
with a polymer shell, including organic (PEG,73,322 dextran,323
chitosan,324 polyethyleneimine,325 and phospholipids)326 or
inorganic (silica),327 is usually the first step. Whatever the
stabilizer, the next requirement is to reduce significantly the
possible interactions with opsonins and with the RES, which is
usually accomplished by conjugating the MNPs with an
appropriate protein-repellent molecular species, such as
PEG. The resulting ‘‘stealth’’ MNPs are able to circulate in
the blood for a long period of time without being cleared.328
The final step consists in functionalizing such long-circulating
MNPs with targeting ligands having high selectivity for specific
cancer cell receptors.329 The full-armed magnetic drug delivery
nanosystem is obtained by loading a cytotoxic cargo at some
stage of the above synthetic steps. A wide variety of antitumor
agents has been loaded inside or external to the polymer
coating, either by physical adsorption or by covalent conjugation
(cf. Section 3.3). These include chemotherapeutics (DOX,330–332
danorubicin,333 tamoxifen,334 cisplatin and gemcitabine,335 PTX,336
mitoxantrone,337 cefradine,338 ammonium glycyrrhizinate,339
fludarabine,340 pingyangmycin,341 nonsteroidal anti-inflammatory
pharmaceutics,342 amethopterin,343 mitomycin,344 diclofenac
sodium,345 and adriamycin),346 enzymes,347 toxins,348 genes,349
folic acid (FA),322 Abs,350 growth factors,351 and radionucleo-
tides.352,353 In the three next paragraphs, we summarize some
recent achievements using MNPs for targeted drug delivery
based on these concepts.
7.1. Long-circulating nanoparticles exploiting the
‘‘enhanced permeation and retention’’ effect
In order to avoid rapid clearance from the body by RES while
concomitantly retaining high surface area and activity, the surface
of MNPs needs to be protected. Among the various solutions
investigated so far, PEG has demonstrated to confer the best
performances to the organic/inorganic nanohybrids in terms of
stability, solubility, biocompatibility and capability to shield the
surface charge.78 PEGylation strategies may involve direct MNP
synthesis using PEG precursors or graft copolymers as solvent/
complexant,354,355 or, alternatively, surface conjugation with PEG
molecules modified with suitable anchoring ligands endowed with
high affinity for iron oxide. The most used are siloxanes,322,356
phosphates,16 and catechol derivatives.357 PEGmolecules have also
been tailored to enhance their tumor localization and to promote
the controlled release of therapeutic agents.358 The solubility
increases as a function of PEG molecular weight from 500 to
5000 Da. However, the improved solubility results in a decrease of
magnetic susceptibility and in an increase in hydrodynamic size.
The ‘‘stealth’’ character of PEGylated MNPs confers them a
long-term circulation capability in the blood vessels circum-
venting the possible immune response, opsonin interaction
and clearance by the RES.358 To achieve the best bioinvisibility
properties, the molecular weight of PEG should be in the range
of 1500–5000 Da. As a result, MNPs can flow throughout the
blood for a time long enough to allow them to passively
penetrate through the fenestrations, which are typically in
the range of 200–600 nm, of leaky vasculature in correspon-
dence to the tumor tissue.359 The selectivity of targeting is
essentially due to the absence of such fenestration in healthy
tissues. The diseased vascular condition that favors this
passive selective delivery process is usually termed ‘‘enhanced
permeation and retention’’ (EPR) effect and is associated to a
defective vascular architecture, impaired lymphatic drainage
and extensive angiogenesis. It is worth noting that the release
of drugs from passively diffusing PEGylated nanocarriers
through peripheral tumor tissue by exploiting the EPR effect
has produced some clinically relevant results. However, there
have been contradicting data concerning the real effectiveness
of introducing targeting molecules in these nanocomplexes.360
The diffusion process mediated by the EPR effect is dependent
on the biophysical properties of the MNPs. Therefore, the
chemical and physical characteristics of engineered MNPs
should be carefully optimized, even in the absence of specific
targeting ligands.361 Recently, PEGylated iron oxide MNPs have
been used to associate selective transport of DOX in vivo with
simultaneous MRI tumor localization, demonstrating sustained
drug release and dose-dependent antiproliferative effects in vitro.362
Moreover, clever strategies for ‘‘intelligent’’ drug release have been
attempted by using PEG-containing stimuli-responsive block
copolymers for the coating of MNPs.363
7.2. Targeted delivery of cytotoxic agents
In general, when isolated MNPs extravasate out of the vascu-
lature at the tumor site, they usually exhibit poor retention
unless their surface has been functionalized with specific cell
targeting molecules, which, in turn, can trigger receptor-
mediated endocytosis, resulting in higher intracellular drug
concentration and increased cytotoxicity.360,364,365 The exploi-
tation of the unique multifaceted properties of MNPs has led
to the development of a new concept of ‘‘nanotheranostics’’,
which refers to the simultaneous capability of MNPs to serve
both as diagnostic and as therapeutic agents in the purpose of
treatment of cancer and inflammatory diseases.366 MNPs
functionalized with cytosine–guanine (CG) rich duplex con-
taining prostate-specific membrane antigen showed selective
drug delivery efficacy in a LNCaP xenograft mouse model.367
In another study, an anti-HER2 Ab-conjugated, pH-sensitive
MNP system has been developed for the intelligent release of
DOX inside HER2-overexpressing breast cancer cells.368
Multifunctional MNP clusters encapsulated in an amphiphilic
block copolymer or in a silica@gold nanoshell functionalized
with suitable mAbs were used for MRI-guided Ab therapy or
NIR illumination-based gold nanoshell-triggered hyper-
thermic treatment of different tumors, respectively.369,370 A
new bioengineered iron oxide MNP, presenting the anti-HER2
Ab in an optimal orientation to maximize the binding with
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HER2 receptors in breast cancer cells, proved to be highly
efficient in providing MRI and fluorescence images of the
tumor mass and in strongly reducing HER2 expression in
tumor tissue in vivo, which could be promising for neoadjuvant
therapy of breast cancer.371 FA is also largely utilized as an
effective tumor targeting agent conjugated to composite multi-
functional MNPs. FA-functionalized mesoporous silica
MNPs containing iron oxide NPs and DOX allowed for
simultaneous imaging and improved antitumor drug delivery
in MCF7 and HeLa cells,372 while FA-modified MNPs bearing
b-cyclodextrin encapsulating drug molecules, could release the
payload by applying a controlled high-frequency magnetic
field.373 In a conceptually similar approach, Ruiz-Hernandez
et al. exploited the local temperature enhancement produced
by the heat generated by application of an AMF on double-
stranded DNA fragments capping the pores of mesoporous
silica MNPs, thus enabling the free release of chemotherapeutic
cargo.89 Furthermore, iron oxide MNPs conjugated with an
Ab selective for EGFR receptor deletion mutant (EGFRvIII)
present on human glioblastoma multiforme (GBM) cells were
used forMRI-guided therapeutic targetingGBM, after convection-
enhanced delivery (CED),374 allowing for the effective intra-
tumoral and peritumoral distribution of MNPs in the brain.375
The importance of this proof-of-concept experiment is that it
demonstrates a significant dispersion of the MNPs over days
after the infusion, which may lead to the therapeutic effect
against the primary mass and to the concomitant targeting of
residual peripheral metastases.
7.3. Magnetic field-assisted drug transport and
magnetofection for gene therapy
Magnetic targeting has been recently introduced in nano-
medicine as an innovative approach for the targeted delivery.376
The basic principle of this technique is that MNPs loaded with
the drug of interest are guided to a specific body tissue or
organ by application of an external magnetic field gradient,
achieving a high drug concentration in correspondence to the
diseased area.377
This method is mainly used successfully in cancer treatments.
The magnetic field-assisted transport of cytotoxic agents asso-
ciated with MNPs to tumor cells enhances the therapeutic
efficacy of tumor treatment allowing for the reduction of
administered dosage and minimization of side effects.81,113,378
Recently, in a very interesting approach MNPs have been also
used to boost the oncolytic adenovirus potency. MNPs were
associated to specific virus to improve their uptake by cancer
cells by applying a magnetic force.104 Moreover, a significant
enhancement of the natural immune response to tumor cells
was achieved using MNPs for magnetically guided in vivo
delivery of interferon gamma for cancer immunotherapy.379
The feasibility of the magnetic field-assisted targeting
approach and its therapeutic potential in vitro as well as
in vivo is studied and applied also in other therapeutics
contexts. For instance, Chorny et al. used a PTX-loaded
MNP formulation for the treatment of stent restenosis.111 In
other interesting studies, MRI-guided magnetic delivery of
multifunctional MNPs to the brain enabled crossing the blood
brain barrier reducing the systemic toxicity.380 In the last few
years, because of the importance of nucleic acid delivery to
cells to make them produce a desired protein or to shut down
the expression of endogenous genes,104,381 magnetofection is
rapidly evolving as a novel and efficient gene delivery technique
based on a magnetic force exerted upon gene vectors linked to,
or encapsulated inside, MNPs to direct the genes to the target
cells in vitro, as well as to a target tissue or organ in vivo.382–388
This research area opens new possibilities because through the
development of coupled siRNA- and microRNA-MNPs it is
possible to localize and efficiently deliver genes inside the cells
with a direct cell function interference and tremendous research,
diagnostic and therapeutic applications.389
8. Magnetic nanoparticles as heat mediators for
hyperthermia
8.1. Principles and preclinical investigations
The concept of hyperthermia dates back to more than 4000
years ago when heating was already mentioned as a potential
treatment for some diseases in the advanced cultures of the old
Egypt. Nowadays, hyperthermia has received renewed attention
due to the recent advances, which suggest a potential applica-
tion in cancer therapy. In particular, the use of MNPs as
heat mediators looks promising in the development of novel
thermotherapy treatments, especially in combination with
conventional cancer therapies, including surgery, radio and
chemotherapy.31,328 The pioneering work of Gordon et al. in
1979 paved the way for the intracellular application using
dextran MNPs and a high-frequency magnetic field.390
The hyperthermia procedure, based on heat generation
within cancer cells, takes advantage of the higher sensitivity
of the tumor cells to temperature compared with normal
tissues.391,392 The heating is obtained through the Brown
losses of the MNPs induced by an AMF, to which MNPs
are subjected.393 Depending on the extent of local heat production
two kinds of heating treatments have been defined: (1) hyper-
thermic effect refers to cell apoptosis triggered by controlled
heating in the range 41–46 1C, high enough to modify several
structural and enzymatic functions of cell proteins; (2) thermo-
ablation event occurs as a consequence of cell carbonization as
temperature is raised above 46–48 1C (usually up to 56 1C).394
The thermotherapy efficiency has been successfully applied
to different cancer types, including breast,395–397 brain,398
prostate cancers146,399,400 and melanoma.401,402 The efficiency
of magnetic heating essentially depends on the size and
magnetic susceptibility of the MNPs.403 To reduce systemic
and side effects on the normal tissue, the generated heating has
to be confined to the tumor area and a temperature control is
required. Many efforts have been spent to reach these critical
objectives. Mild hyperthermia in combination with other
traditional cancer treatments, like radiotherapy and/or chemo-
therapy, has provided a substantial therapeutic improvement.
Several studies have shown a reduction in tumor size when a
combination with other therapies is applied.404–406
Exploiting the passive migration to the tumor region
achieved by the EPR effect, magnetite cationic liposomes have
been envisaged as a promising tool for several types of tumors
because of their high accumulation favored by the positive
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charge of the nanocomplex.407–409 The surface modification
and functionalization of the MNPs with biological ligands like
proteins or Abs for active targeting allowed the accumulation
in correspondence to the tumor in a good percentage of the
total intravenously injected amount,410,411 and the binding
with organic fluorophores or fluorescent NPs (QDs), exhibited
simultaneous cancer diagnosis and treatment.412 In order to
minimize the toxicity induced in the body by the chemically
synthesized MNPs and minimize the administered amount of
inorganic material, concomitantly improving the response to
applied AMF, Alphandery et al. developed an innovative
bioproduction approach to the preparation of colloidal
mediators by using extracted chains of magnetosomes, which
exhibited a specific absorption rate remarkably higher than the
chemically synthesized MNPs.413
8.2. Heat shock-induced antitumor immunity
The therapeutic outcome of hyperthermia treatment is not
only due to the direct effect of cell heating but also to the
activation of an immune response which results in a reduction
both of the primary tumor mass and also the metastatic
lesions.400 Heat treatment itself enhances the antitumor effect
through the stimulation of the innate immune response.
A temperature of approximately 42 1C is enough to activate
natural killer cells, which are potent tumor-lytic agents when
activated. Kubes et al. have shown that a high number of
activated monocytes with increased cytotoxic effector function
is recruited into B16-F10 melanoma-bearing mice after mild
local microwave hyperthermia.402,414 The mechanism for the
recognition of tumor cell antigens by the host immune system
involves the release of the content of dying tumor cells,
including heat shock proteins (HSPs). HSPs are responsible
for the activation of neighboring monocytes to produce
proinflammatory cytokines and recruit antigen-presenting
cells.415,416 This stimulation of innate immune system triggered
by hyperthermia continued for an extended period of time
and the treated animals completely rejected new tumor cell
invasion as a metastasis model.417,418
8.3. Clinical trials in humans
As a consequence of the robust results achieved with MNP-
based hyperthermia treatment of cancer animal models and in
view of a comprehensive knowledge of the molecular mecha-
nisms, this therapy is now being established in clinical routine
leading to an industrial development.419 Hyperthermia treat-
ment has been approved by the FDA for use alone or in
combination with radiation therapy in the palliative manage-
ment of certain solid surface and subsurface malignant tumors
(i.e., melanoma, squamous- or basal-cell carcinoma, adeno-
carcinoma, or sarcoma) that are progressive or recurrent
despite conventional therapy. Clinical studies using combined
hyperthermia and radiation therapy have shown that 83.7% of
patients had some tumor mass decrease, of which 37.4% had a
complete tumor regression while 24.5% exhibited a >50%
tumor reduction. There are at least three operating companies
that develop techniques that generate heat by MNPs exposed
to an AMF.
Sirtex Medical’s targeted hyperthermia research program
treats the majority of liver cancer patients that do not have
localized tumors with small magnetic micro-spheres (Thermo-
Spheres). Targeted hyperthermia therapy, used in combination
with targeted radiotherapy using SIR-Spheres microspheres,
should improve even further the efficacy of the SIR-Spheres
microspheres.420 SIR-Spheres microspheres contain resin-based
microparticles impregnated with yttrium-90, a radio isotope
commonly used to treat patients with liver cancer. Aspen
MediSys is developing MNPs, which act as cellular ablation
devices that operate at a size scale typical for drug delivery
vehicles.421 MagForce developed marketable products
(NanoTherms, NanoPlans and NanoActivatort) for the
local treatment of solid tumors (glioblastoma multiforme,
prostate cancer and pancreatic cancer). The principle of the
method is the direct introduction of MNPs into a tumor and
their subsequent heating in an AMF. The water soluble MNPs
are extremely small (approximately 15 nm in diameter), and
contain an iron oxide core with an aminosilane coating. The
MNPs are activated by an AMF, which changes its polarity
100 000 times per second. Thus heat is produced, raising the
temperature of the cancer cells in the order of 5 1C. These
MNPs have been already injected in patients with prostate cancer
demonstrating stable intra-tumoral deposition of theMNP in the
prostatic tissue for at least six weeks, which allows for a series
of thermal therapy treatment without further injections.146,422
Magnetic hyperthermia for bone tumors reduction has also been
studied showing a good clinical outcome.423
9. Remaining challenges
To translate the preclinical settings into clinical applications
for most of the magnetic biocomposites that have been mentioned
along this manuscript a lot of questions should be cleared by
intense basic scientific work. It will be only possible to answer
most of the open questions by adding up the efforts of
interdisciplinary research groups. In this section some of the
general challenges for an extended biological application of
MNPs will be mentioned and discussed.
Firstly, the magnetic properties of the MNPs should be
improved to enhance the magnetic resonance signal in MRI
and to maximize the specific loss power increasing the efficiency
of magnetic thermal induction. Probably it will be necessary to
extend the use of ferrites such as CoFe2O4 and MnFe2O4 or
the new fabrication of nanostructures like core–shell systems
as it was recently demonstrated for improvements in hyper-
thermia applications.31 Toxicological studies of new magnetic
biocomposites will have to be carried out. In the future, more
general and robust bioconjugate chemistries for connecting
biomolecules to particles will be also necessary. The scaling-up
of the fabrication of most of the mentioned composites is still
not possible. Another challenge in the development of coatings
involving active biomolecules for MNPs is to limit the overall
size of particles to below 100 nm, sinceMNPs larger than 100 nm
are rapidly cleared by the liver and spleen.310 Applications such
as drug delivery or hyperthermia will be favored with the
development of new and improved magnetic biocomposites.
Regarding the application of magnetic nanoswitches, the
current studies might lead to the development of implantable
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sensors offering long-term stability when placed in the body.
However, the main drawback is the imaging enzyme activity
in vivo which involves previous cytoplasmatic delivery of
MNPs in the cells of interest (see Section 6.3).
The fate of MNPs in magnetically labelled cells after their
transplantation in an organism is also not fully understood
and requires further study. It is well established that the
MNP-induced signal hypointensity has a maximum (e.g. after
24 hours), and afterwards continuously declines but the reason
is not completely clear. Different possible mechanisms like
proliferation-dependent MNP dilution, metabolic degrada-
tion, exocytosis and/or cell death followed by an uptake of
free MNPs by invading macrophages, and transport to other
organs/tissues in the body of the organism are currently being
discussed. Possibly, not a single but an interplay of these
mechanisms may cause the MR signal intensity decrease. In
addition, this could be cell-type dependent and/or MNP-
specific. Specifically the metabolic degradation could be influ-
enced by a MNP-design with a biodegradable cover that
allows a slow cleavage (retard formulation) in lysosomes
and/or cytoplasmic localisation of the MNPs. The proliferation-
dependent MNP dilution can be influenced by using non-
proliferating cells or slow proliferating ones. Exocytosis of
MNPs has been rarely investigated after magnetically cell
labelling. This process could be cell-dependent as well as
labelling-dependent. Cells that actively take up MNPs by
endocytosis store the foreign material in lysosomes. In this
subcellular compartment MNPs may either undergo metabolic
degradation or may leave the cell via exocytosis. To omit the
latter process cells can be magnetically labelled by physical
methods such as electroporation or magnetofection. This
guarantees cytoplasmic rather than lysosomal MNP localisa-
tion. On the other hand the bombardment of cells with MNPs
leads to a much higher percentage of preparation-dependent
cell death. This means that a greater number of cells is
necessary to finally ensure of having enough labelled cells that
are viable. Instead of physical labelling it is also possible to
modify MNPs by transmembrane localisation peptides (e.g.
HIV tat peptide). Although this method is associated with
a small percentage of preparation-related cell death, all
additional materials used in the synthesis of MNPs must be
FDA-approved. The same is true for transfection agents used
to enhance MNP cellular incorporation.
Robust protocols are necessary to effectively label cells with
MNPs. This includes that neither the Fe-concentration used nor
the total in vitro labelling procedure/preparation steps should
markedly influence cellular functions like viability, prolifera-
tion, differentiation, migration, and chemotaxis for example.
Moreover, this also implicates that neither free MNPs nor
labelled cells might induce harmful reactions in the organism.
The latter point is in preclinical settings largely neglected.
Besides data concerning the biodistribution it is important
to know what happened with the particles in long-term
observations. Especially when non-degradable materials such
as mesoporous silica are used it is necessary to investigate their
fate and their influence on organs/tissues after different time
points. A lot of work is necessary until all of these questions
are fully answered that is a pre-requisite to implement the
MNP-technology into clinical applications.
10. Outlook
Colloidal MNPs possess a broad spectrum of interesting
properties that make them useful for biological applications.
MNPs based on superparamagnetic iron oxide offer the
privileged status of being accepted for clinical purposes. Until
now, they have been used in humans for MRI diagnosis but in
a near future they are expected to be also used for therapeutic
issues and thus becoming theranostic agents. MNPs can be
easily synthesized, they can be made colloidally stable, they are
inexpensive, and they can be conjugated with biological
molecules in a straightforward way. The lack of interference
from complex diamagnetic biological matrix, the use of non-
radiative and non-toxic detection techniques, and the possibility
of analyte magnetic separation and collection are among the
peculiar advantages of the use of MNPs for diagnosis. Due to
their magnetic properties, they are especially interesting in
drug delivery because apart of the possibility of tagging their
surface, that almost all kind of NPs have, they can be driven
inside the organism by the application of an external magnetic
field gradient to the target area of the body where the therapy
has specifically to act. Gene therapy, anticancer treatments
and tissue regeneration are between the most challenging
clinical applications of MNPs. Regardless of the good tolerance
that some MNPs have shown, the long-term outcome of the
MNPs in the body will still need to be determined if their use in
medicine wants to be extended. In depth analysis of the
potential risks associated with the intensive use of inorganic
MNPs cannot be further delayed, including the factors related
with epigenetic phenomena and long-term cardiovascular
effects. Thanks to their broad utilization for research purposes
and to their potential in clinical practice, MNPs represent an
ideal model to attempt to set up a comprehensive and accep-
table nanotechnology platform for the accurate classification
of such risks, for the identification of general protocols for the
evaluation of nanomaterial safety toward human health and
environmental protection, and for the certification of nano-
particle-based drugs and contrast agents for extensive medical
application. Nowadays, it is universally recognized that a
disciplinary point of view is largely insufficient to face a similar
challenge. However, with the joint efforts of chemists, physicists,
biologists, pharmacologists, radiologists and clinical doctors,
soon the mirage of exploiting molecular nanoclinics to assist
conventional diagnosis and therapy will become reality.
Abbrevations
Ab antibody
AMF alternating magnetic field
CLIO cross-linked iron oxide magnetic nanoparticles
DC dendritic cells
DOX doxorubicin
EMA European Medicines Agency
EPR enhanced permeation and retention
FA folic acid
FDA US Food and Drug Administration
mAb monochlonal antibody
MNP magnetic nanoparticle
NP nanoparticle
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4328 Chem. Soc. Rev., 2012, 41, 4306–4334 This journal is c The Royal Society of Chemistry 2012
PTX paclitaxel
PEG polyethylene glycol
MR magnetic resonance
MRI MR imaging
SPION superparamagnetic iron oxide NP
Acknowledgements
We acknowledge Dr Miguel Spuch Calvar for providing us
some of the schemes of the figures within the text. SCR is
grateful to the Junta Andalucıa for a fellowship. Parts of this
work have been supported by the European Commission
(project Nandiatream toWJP). This work was partly supported
by ‘‘Assessorato alla Sanita’’, Regione Lombardia, and Sacco
Hospital (NanoMeDia Project) and ‘‘Fondazione Romeo ed
Enrica Invernizzi’’ (grants to DP).
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