Biological applications of magnetic nanoparticles

View Article Online / Journal Homepage / Table of Contents for this issue Chem Soc Rev Dynamic Article Links Cite this: Chem Soc Rev., 2012, 41, 4306–4334 CRITICAL REVIEW www.rsc.org/csr Biological applications of magnetic nanoparticles Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H Miriam Colombo,wa Susana Carregal-Romero,wb Maria F Casula,c Lucı´ a Gutie´rrez,de Marı´ a P Morales,d Ingrid B Bo¨hm,f Johannes T Heverhagen,f Davide Prosperi*a and 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 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 miniaturization 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 a Dipartimento di Biotecnologie e Bioscienze, Universita` di Milano-Bicocca, Milan, Italy E-mail: davide.prosperi@unimib.it b Department of Physics and WZMW, Philipps Universita¨t Marburg, Marburg, Germany E-mail: wolfgang.parak@physik.uni-marburg.de c Dipartimento di Scienze Chimiche e INSTM, Universita` di Cagliari, Cagliari, Italy d Departamento de Biomateriales y Materiales Bioinspirados, Instituto de 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 Universita¨t Marburg, Marburg, Germany w Both authors contributed equally to this work 4306 Chem Soc Rev., 2012, 41, 4306–4334 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 applications: molecular detection, imaging (including a focus on regenerative medicine), delivering, and heating 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 temperatures (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 This journal is c The Royal Society of Chemistry 2012 Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H View Article Online Fig 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 Due to their small volume, MNPs usually present superparamagnetic 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 electromagnetic 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 paramagnetic 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, superparamagnetism 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 superparamagnetic 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 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 biological applications, biocompatibility reaches great importance, as the accumulation or toxic effects of the MNPs need This journal is c The Royal Society of Chemistry 2012 to be reduced as much as possible Therefore, most of the time a balance between larger magnetic moments and biocompatibility 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 biomedical 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) The MNP coating is also a key element in order to specifically bind other compounds to the MNPs or to prevent agglomeration, 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 spinpinning, 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 magnetisation 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 monodomain 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 (Ne´el 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, Ne´el relaxation prevails over Brown relaxation while for larger sizes and low viscosity media, Brown relaxation is the rotation mechanism.11 Micrometre disc shaped MNPs have been observed to produce mechanical cell damage due to Brown motion.12 The frontier Fig (left) Rotation of the moment within the MNP, overcoming their anisotropy energy barrier that leads to Ne´el 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 Chem Soc Rev., 2012, 41, 4306–4334 4307 View Article Online Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H Fig Frontier MNP size between multidomain and monodomain MNPs depending on their crystalline structure.13 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 Ne´el 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 relaxation 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 4308 Chem Soc Rev., 2012, 41, 4306–4334 MNPs are magnetically saturated at common field strengths used for MRI and their presence produces a marked shortening of T2 along with a less marked reduction of T1 Therefore, 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 applications is that they can be tracked in vivo by magnetic measurements, 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 Quantitative 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 Synthesis of magnetic nanoparticles Tailored design of colloidal MNPs plays a crucial role in determining the effectiveness of functional magnetic nanostructures 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 nonstoichiometric iron oxide nanostructures has been often overlooked due to the difficulty to distinguish magnetite and maghemite at the nanoscale by conventional characterization This journal is c The Royal Society of Chemistry 2012 View Article Online Table Saturation magnetisation Ms and magnetic anisotropy K of selected magnetic ferrites (taken from the studies of Cornell et al., Cullity and Bate,25 Viswanathan,26 and Buschow27) Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H Fe3O4 g-Fe2O3 MgFe2O4 MnFe2O4 CoFe2O4 NiFe2O4 Ms/A m2 kgÀ1 K/J mÀ3 98 82 31 110 94 56 À1.1 Â 104 À4.6 Â 103 À2.5 Â 103 À2.8 Â 103 1.8 Â 105 À7.0 Â 103 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 functionalized 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 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 concerns 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, CoFe2ÀxGdO4 (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, stabilization, 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 stoichiometric 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 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 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 Cheap chemicals Mild reaction conditions Synthesis in H2O Ease surface modification Ease formation of ferrites Ease conversion to g-Fe2O3 Ease scale-up Broad size distribution Low reproducibility Uncontrolled oxidation Improved size control Narrow size distribution Synthesis in H2O Tunable magnetic properties Improved size control Narrow size distribution Ease size tunability Uniform magnetic properties Narrow size distribution High size control High cristallinity Possible scale-up Tunable magnetic properties High temperature Safety of the reactants Toxic organic solvents High temperatures Phase transfer required Fe3O4, Fe2O3, MFe2O4 Fe3O4, Fe2O3 Low reaction yield Poor cristallinity Surfactants are difficult to remove Fe2O3, Fe3O4, MFe2O4 10–50 nm, 50–100 nm Sphere 1–0.25 mm, 15–31 nm Disc, sphere 2–30 nm, 20–80 nm Cube, sphere, needle 33–35 36, 37 39–43 Fe3O4, Fe2O3, MFe2O4, Cr2O3, MnO, Co3O4, NiO 3–500 nm Cube, sphere, triangle, tetrapode, rod 17, 24, 28, 44–65 = Advantages, = Drawbacks, = Nature of MNPs, = NP diameter, = NP shape and = References This journal is c The Royal Society of Chemistry 2012 Chem Soc Rev., 2012, 41, 4306–4334 4309 Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H View Article Online 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 hydrolytic 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 hydrothermal 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 conditions, 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 parameters, 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 waterin-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 4310 Chem Soc Rev., 2012, 41, 4306–4334 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 intermediates, complex aqueous chemistry equilibria are prevented and alternative growth patterns can be achieved Non-hydrolytic routes primarily rely on the thermal decomposition of organometallic 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 preparation 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 concentration, temperature of decomposition of the precursor and of MNP growth, reaction time, solvent In particular, successful 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 (polydispersity 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 cubeoctahedral, 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 This journal is c The Royal Society of Chemistry 2012 Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H View Article Online 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 nonbiocompatible 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 decomposition techniques are currently being extended to the preparation 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 substituted 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 copresence 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 nanocrystalline 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 nontoxic 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 interactions 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 This journal is c The Royal Society of Chemistry 2012 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 crosslink 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 molecules 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 epichlorohydrin (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 PEGylation 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 Chem Soc Rev., 2012, 41, 4306–4334 4311 Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H View Article Online 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 biofunctionalization 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 purification 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 biochemical 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 adenoviral vector tagged with MNPs This hybrid system composed of MNPs and an adenovirus (Ad) has applications in 4312 Chem Soc Rev., 2012, 41, 4306–4334 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 polyethylenimine (positively charged) and the Ad,104 (ii) biotinylated 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 maleimidemodified adenovirus and thiol-functionalized MnFe2O4 MNPs.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 doxorubicin (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 biodegradable 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 immobilization 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 This journal is c The Royal Society of Chemistry 2012 View Article Online Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H Fig 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 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 oligonucleotides 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 biocomposite 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 applications demanding high control of the particle–biomolecule interactions in a molecular level Table shows some of the biomolecules and the coating agents mentioned in this manuscript together with their features and applications 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 make MNPs 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 of MNPs and their cytotoxic effect,121,122 have provided new insights in understanding 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 This journal is c The Royal Society of Chemistry 2012 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 physicochemical 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 internalized 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 Chem Soc Rev., 2012, 41, 4306–4334 4313 View Article Online Examples of different coating agents and biomolecules used in the fabrication of magnetic biocomposites Table Biomolecule/coating agent Adenoviral vectors Antibodies Aptamers Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H Dextran DNA or RNA Doxorubicin Enzymes or proteins Folic acid Nitrilotriacetic acid Oligonucleotides Paclitaxel Polyethylene glycol Silica Tat peptide Advantages/applications References MRI and gene delivery They favored cytosolic release High variety of antibodies that bind to specific proteins only present in particular cellular structures Recognition processes, cell capturing and immunomagnetic separation Detection of in vivo enzyme activity Tracking of labelled cells Intelligent drug delivery Accumulation in tumors It is possible to create them artificially for specific targeting Smaller than Abs Recognition processes They bind to specific target molecules Stabilizer agent Enhances blood circulation 104–109 93–98, 198, 199, 204, 247, 350, 368–371, 374 Recognition processes Drug delivery Drug for cancer treatment Biomarkers of process such cancer, apoptosis, inflammatory reactions Protein separation, purification, detection and analysis Accumulation in tumors (drug delivery and hyperthermia) Effective tumor targeting agent Ligand bearing Ni2+-chelating species Protein separation Probes for detecting or separating DNA or RNA Nanoswitches Drug for cancer treatment Protein repellent, nonimmunogenic and nonantigenic Long-term circulation capability in blood vessels Widely used for protein conjugation Stabilizer agent that can be loaded (within the pores) Biological compatibility and optical transparency Useful in the fabrication of multifunctional MNPs Facilitate cellular uptake of MNPs 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 of MNPs 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 4314 Chem Soc Rev., 2012, 41, 4306–4334 99–103 66, 120–123, 143, 149–155, 177–183, 274, 323, 390 104, 117, 349, 381–389 114–116, 306, 330–332 118, 119, 144, 165, 166, 347 322, 372, 373 160–164 167–169, 177 110–113 16, 73, 76–81, 99, 100, 322, 328, 354–363 88–92, 203, 273, 327, 369–372 75 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 internalization 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 degradation and clearance of MNPs from circulation by the endogenous 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 This journal is c The Royal Society of Chemistry 2012 Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H View Article Online Fig 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 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 threemonth 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 Molecular detection with magnetic nanoparticles In the language of nature, biological entities exploit highaffinity 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 applications 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 Inhomogeneous matter such as protein production mixtures are This journal is c The Royal Society of Chemistry 2012 incompatible with the particulate-free conditions required for a correct usage of commercial columns Magnetic separation exploiting MNPs 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 Â 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 significantly 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 approaches 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 Chem Soc Rev., 2012, 41, 4306–4334 4315 View Article Online 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 Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H 6.4 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 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 doublestranded 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 nanoassembly 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 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 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 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 4320 Chem Soc Rev., 2012, 41, 4306–4334 This journal is c The Royal Society of Chemistry 2012 Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H View Article Online 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 standardised 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 fluorescein isothiocyanate, indocyanine green) and different loading methods (Fig 12).245–247 Complex MNPs have been designed to both trigger DCs with antigens and monitor them by MRI 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 protamine sulfate, polylysine in concert with mDCs (Fig 12).233,247–251 MPIOs have a diameter of at least 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 remarkably inefficient labelling (0.59 Æ 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 mlÀ1 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 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 Â 105 cells up to 1.0 Â 106 cells, and 100 cells mmÀ3 at T or 50 cells mmÀ3 at 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 userThis journal is c The Royal Society of Chemistry 2012 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 concentration 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 implement more sophisticated applications (e.g rapamycin inhibition on lymphoid homing and tolerogenic function of nanoparticlelabeled 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 Â 200 Â 200 mm) showing the popliteal lymph nodes from one representative mouse, days after injection with (a) Â 106 MPIO-labelled DCs or (b) Â 106 unlabelled DCs (images taken from the study of Rohani et al.).244 Chem Soc Rev., 2012, 41, 4306–4334 4321 Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H View Article Online 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 visualization 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 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 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) mesenteric lymph node (images taken from the study of Reichardt et al.).247 Copyright 2008 The American Association of Immunologist, Inc 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 compromising the patient’s care.267–269 T2 relaxivity MRI 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 4322 Chem Soc Rev., 2012, 41, 4306–4334 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 minutes.301 Macrophages engulfed these MNPs via phagocytosis Afterwards 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 atherosclerotic plaques were hypointensive on T2 =TÃ2 weighted MRI after active engulfment of MNPs Peak concentrations of iron were found in the liver after hours and in the spleen after 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 carboxydextran 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 macrophages could phagocytose them 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 biological 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 externally 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 This journal is c The Royal Society of Chemistry 2012 Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H View Article Online 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 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 mm accumulate 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 the MNPs.313 Hence, passive nanocarriers This journal is c The Royal Society of Chemistry 2012 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 administered 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 experience 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 polymercoated MNPs The difference is attributed to the higher volume fraction of magnetite in the phospholipid agglomerates.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 Chem Soc Rev., 2012, 41, 4306–4334 4323 Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H View Article Online 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 chemotherapeutics 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 dispersion 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 radionucleotides.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 PEG molecules 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 4324 Chem Soc Rev., 2012, 41, 4306–4334 The ‘‘stealth’’ character of PEGylated MNPs confers them a long-term circulation capability in the blood vessels circumventing 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 correspondence 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 vasculature 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 receptormediated endocytosis, resulting in higher intracellular drug concentration and increased cytotoxicity.360,364,365 The exploitation 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 containing 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 hyperthermic 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 This journal is c The Royal Society of Chemistry 2012 Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H View Article Online 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 multifunctional 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 doublestranded 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 for MRI-guided therapeutic targeting GBM, after convectionenhanced delivery (CED),374 allowing for the effective intratumoral 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 nanomedicine 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 associated 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 This journal is c The Royal Society of Chemistry 2012 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 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 application 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) hyperthermic 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) thermoablation 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 chemotherapy, 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 Chem Soc Rev., 2012, 41, 4306–4334 4325 Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H View Article Online 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 MNPbased hyperthermia treatment of cancer animal models and in view of a comprehensive knowledge of the molecular mechanisms, this therapy is now being established in clinical routine leading to an industrial development.419 Hyperthermia treatment has been approved by the FDA for use alone or in combination with radiation therapy in the palliative management of certain solid surface and subsurface malignant tumors (i.e., melanoma, squamous- or basal-cell carcinoma, adenocarcinoma, 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 4326 Chem Soc Rev., 2012, 41, 4306–4334 Sirtex Medical’s targeted hyperthermia research program treats the majority of liver cancer patients that not have localized tumors with small magnetic micro-spheres (ThermoSpheres) 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 1C These MNPs have been already injected in patients with prostate cancer demonstrating stable intra-tumoral deposition of the MNP 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 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 hyperthermia 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, since MNPs 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 This journal is c The Royal Society of Chemistry 2012 Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H View Article Online 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 degradation, 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 MNPspecific Specifically the metabolic degradation could be influenced 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 proliferationdependent MNP dilution can be influenced by using nonproliferating 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 localisation 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, proliferation, 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 This journal is c The Royal Society of Chemistry 2012 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 nonradiative 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 acceptable 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 nanoparticle-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 AMF CLIO DC DOX EMA EPR FA FDA mAb MNP NP antibody alternating magnetic field cross-linked iron oxide magnetic nanoparticles dendritic cells doxorubicin European Medicines Agency enhanced permeation and retention folic acid US Food and Drug Administration monochlonal antibody magnetic nanoparticle nanoparticle Chem Soc Rev., 2012, 41, 4306–4334 4327 View Article Online PTX PEG MR MRI SPION paclitaxel polyethylene glycol magnetic resonance MR imaging superparamagnetic iron oxide NP Downloaded by Universidad de Vigo on 05 January 2013 Published on 05 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CS15337H 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 Andalucı´ a for a fellowship Parts of this work have been supported by the European Commission (project Nandiatream to WJP) 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) References D H Martin, Magnetism in solids, The M.I.T press, Cambridge, Massachussets, 1967 D Jiles, Introduction to magnetism and magnetic materials, Chapman & Hall, 1991 Y.-W Jun, J.-W Seo and J Cheon, Acc Chem Res., 2008, 41, 179–189 M P Morales, S Veintemillas-Verdaguer, M I Montero, C J Serna, A Roig, L I Casas, B Martinez and F Sandiumenge, Chem Mater., 1999, 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