Biological applications of magnetic nanoparticles

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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This journal is c The Royal Society of Chemistry 2012 Chem. Soc. Rev., 2012, 41, 4306–4334 4307

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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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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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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Biological applications of magnetic nanoparticles

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Transcript of Biological applications of magnetic nanoparticles