Superparamagnetic iron oxide based nanoprobes for imaging and theranostics

Advances in Colloid and Interface Science xxx (2013) xxx–xxx

CIS-01291; No of Pages 19

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Advances in Colloid and Interface Science

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Superparamagnetic iron oxide based nanoprobes for imaging and theranostics

Tina Lam a, Philippe Pouliot b,c, Pramod K. Avti a,b,c, Frédéric Lesage b,c,⁎, Ashok K. Kakkar a,⁎⁎a McGill University, Department of Chemistry, 801 Sherbrooke Street West, Montreal H3A 0B8, Quebec, Canadab École Polytechnique de Montréal, Génie Électrique/Biomédical, C.P. 6079 succ. Centre-ville, Montreal H3C 3A7, Quebec, Canadac Montreal Heart Institute, 5000 Bélanger Street, Montreal H1T 1C8, Quebec, Canada

⁎ Correspondence to: F. Lesage, École Polytechnique d4711x7542.⁎⁎ Correspondence to: A. Kakkar, Department of Chem

E-mail addresses: [email protected] (F. Lesa

0001-8686/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.cis.2013.06.007

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Keywords:Superparamagnetic iron oxide nanoparticles(SPIONs)Magnetic resonance imaging (MRI)Cardiovascular diseaseSynthesisPhysicochemical properties

The need to target, deliver and subsequently evaluate the efficacy of therapeutics in the treatment of a diseasehas provided added impetus in developing novel and highly efficient contrast agents. Superparamagnetic ironoxide nanoparticles (SPIONs) have offered tremendous potential in designing advanced magnetic resonanceimaging (MRI) diagnostic agents, due to their unique physicochemical properties. There has been tremendouseffort devoted in the recent past in developing synthetic methodologies through which their size, hydrody-namic radii, chemical composition and morphologies could be tailored at the nanoscale. This enables one tofine tune their magnetic behavior, and thus their MRI response. While novel synthetic strategies are beingassembled for directing SPIONs to the diseased site as well as imparting them stealth and biocompatibility,it is also essential to evaluate their biological toxicological profiles. This review highlights recent advancesthat have been made in the synthesis of SPIONs, subsequent functionalization with desired entities, and a dis-cussion on their use as MRI contrast agents in cardiovascular research.

© 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 01.1. MRI contrast mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 01.2. Sizes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 01.3. Shapes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2. Synthesis of SPIONs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1. Chemical means of synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.1.1. Co-precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1.2. Hydrothermal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1.3. Microwave . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1.4. Microemulsion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1.5. Ultrasound irradiation/sonochemical/sonolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1.6. Thermal decomposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1.7. Electrodeposition/plating/electrospray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1.8. Sol–gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1.9. Flow injection synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1.10. Green chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.2. Summary of synthetic methodologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03. Characterization techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.1. Shapes, morphology, and coverage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.2. Surface potential and hydrodynamic radii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.3. Magnetic properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

e Montréal, Génie Électrique/Biomédical, C.P. 6079 succ. Centre-ville, Montreal, H3C 3A7, Quebec, Canada. Tel.: +1 514 340

istry, McGill University, 801 Sherbrooke St. West, Montreal H3A 0B8, Quebec, Canada. Tel.: +1 514 398 6912.ge), [email protected] (A.K. Kakkar).

rights reserved.

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4. Conjugation (surface coating) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.1. Passive targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4.1.1. Inorganic shells/components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.1.2. Organic shells/components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.1.3. Drug molecules/fluorescent dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4.2. Active targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05. Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 06. Applications of SPIONs in cardiovascular theranostics using MRI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

6.1. Stroke (neuroinflammation) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 06.2. Atherosclerosis/inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 06.3. Stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 06.4. Cardiac viability/function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 06.5. Magnetic particle imaging for angiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

1. Introduction

Developments in the field of nanotechnology have brought to-gether distinct fields such as chemistry, engineering, biology, andmedicine, and have paved the way to design novel molecularcontrast probes for imaging and theranostics [1–4]. The rationalefor the design of these nanoprobes includes improved detectionof diseased areas, enhanced sensitivity of the pathological sites tothe probe, and biocompatibility. The use of molecular contrastnanoprobes in imaging, which were initially investigated using pos-itron emission tomography (PET) and fluorescence, has seen growthin other modalities including X-ray and computed tomography (CT),magnetic resonance imaging (MRI), and ultrasound. Multimodalityand multifunctionality have also been rendered to these nanoprobeswhich offer the potential to facilitate early detection, accurate diag-nosis and efficient therapeutic intervention. The use of magneticnanoparticles based on iron oxide for MRI is receiving significantattention. In particular, superparamagnetic iron oxide nanoparticles(SPIONs) are emerging as the optimal choice for many MRI applica-tions. SPIONs offer unique properties including quantum effects,large surface area, and easy surface chemistry. Thus, SPIONs mayyield better MRI contrast agents compared to conventional onesbased on transition metal ions. This review aims to highlight ad-vances in the strategies to synthesize SPIONs, and their applicationsas MRI contrast agents.

Magnetic resonance imaging (MRI) is one of the most powerfulnon-invasive imaging tools in the biomedical field [5]. It relies onlarge magnetic fields and radio frequencies, and makes use of relaxa-tion times of water protons present in various organs at differentconcentrations, to produce high resolution soft-tissue anatomical im-ages with good endogenous contrast. Protons align themselves in thedirection of a magnetic field due to their spin. When a radio frequencypulse is applied to the sample, a change occurs in the direction of themagnetization of an ensemble of protons within an imaging volumeelement (voxel). The time taken by the magnetization to return toits original alignment with the magnetic field is called the relaxationtime. It is characterized by three relaxation constants of importance:longitudinal (T1), transverse (T2), and apparent transverse relaxation(T2*). T1 relates to how fast the magnetization parallel to the staticmagnetic field recovers after a perturbation is applied to the system.T2 relates to how rapidly the magnetization in the plane transverseto the static magnetic field loses coherence, and is much shorterthan T1. T2* is the effective relaxation time seen by most MRIsequences, is typically much smaller than T2 and is due to magneticfield inhomogeneity (fluctuations in space and in time). One of themajor disadvantages of MRI is its inherently low sensitivity. Whilethis can be improved by increasing the magnetic field (4.7 to 14 Tand beyond), acquiring data for longer duration, or designing more

Please cite this article as: Lam T, et al, Adv Colloid Interface Sci (2013), ht

sensitive sequences and probes, an important alternative is usingexogenous contrast agents.

One role of exogenous nanoprobes in MRI is to modify the relaxa-tion times of water protons to provide better anatomical contrastregions. Metal oxide-based nanoparticles used as MRI contrast agentsinclude iron oxide, manganese oxide, and gadolinium oxide [6]. Con-trast agents are generally divided into two main categories: T1 andT2 MRI contrast agents which affect the longitudinal and transverserelaxation times of water protons respectively. The classic examplesof T1 contrast agents include gadolinium and manganese oxide,whereas iron oxide nanoparticles form T2 contrast agents. Gadolinium(Gd) based contrast agents, also called T1 or positive contrast agents,produce hyperintense signal in T1-weighted images. However, dueto the toxicity associated with Gd-based contrast agents, the researchfocus has shifted to SPIONs [7]. SPIONs produce hypointense signal inT2- and in T2*-weighted images [5] (Fig. 1).

A major disadvantage with negative contrast agents is that thesedo not distinguish the presence of SPIONs from many other sourcesof signal-degrading effects, such as magnetic field inhomogeneity.Many reviews on the subject of iron oxide nanoparticles have beenwritten, and many different classifications of these particles as pertheir sizes have been employed, which are summarized in Table 1.For example, based on their overall size, Shan et al. classified themas oral SPIONs (300 nm to 3.5 μm), polydisperse SPIONs (50 to150 nm), and ultrasmall SPIONs (b50 nm). The other classificationsnoted in Table 1 are based on the authors' choice, and there is yetno clear and concise agreement between different categories.

1.1. MRI contrast mechanisms

While the basic and widely used MRI mechanism for SPIONs is anegative contrast due to T2 and T2* shortening, a more detailed anal-ysis has shown that they also lead to a shortening of T1, which leads toa positive contrast. However this T1 effect becomes less exploitable athigher field strengths. For SPIONs at high magnetic field, R1 = 1/T1relaxivity falls to zero, while R2 = 1/T2 asymptotes to a positive con-stant [8]. This makes the applicability of SPIONs for specific purposesstrongly dependent on field strength. However, much effort has beenspent in obtaining positive contrast, which falls into two broad mech-anisms, typically producing a contrast depending on both T1 and T2*.One class of mechanisms is dependent on magnetic field perturba-tions or frequency shifts caused by SPIONs [9], the main variantsbeing susceptibility gradient mapping [10] and phase gradient map-ping [11]. In another class, the positive contrast is generated byusing an ultrashort echo time (TE), as short as a few microseconds.This is done by differentiating two images, one of which is acquiredwith this ultrashort TE [12]. The T1 contrast is then recovered with astandard inversion recovery with a sequence that can be an ordinary

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Fig. 1. (a) T2 weighted contrast measured at 7 T of gadolinium oxide and iron oxide nanoparticle samples of increasing concentration; (b) R2 relaxation is high for both compounds,but a greater effect is achieved for the SPIONs than for the Gd oxide nanoparticles.

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gradient echo (GRE). However, this technique has no specificity in theorigin of the positive signal, which can be due to SPIONs as much as tomagnetic field heterogeneities. In an especially promising techniquecalled sweep imaging with Fourier transformation (SWIFT, Fig. 2),the contrast relies upon frequency modulation of the excitationpulse, followed by immediate signal acquisition [13,14].

Finally, the T1 and T2 effects of SPIONs depend on whether theyare free (in the blood stream) or cell-bound. In general, the so-calledstatic dephasing theory helps predict relaxation properties ofSPIONs in cells [15], and it has led to the demonstration of a special-ized T2–T2* contrast mechanism to distinguish free SPIONs fromcell-bound SPIONs in vitro and in vivo [16]. This imaging contrastexploiting the difference between T2 and T2* effects was achievedby combining gradient and spin-echo imaging sequences. The over-all changes in the magnetic properties of SPIONs depend on variousfactors such as the size, shape, composition, crystallinity, concentra-tion within an imaging voxel and data acquisition parameters [17].Therefore, it is very important to understand how different syn-thetic strategies could be applied to obtain SPIONs of desired sizesand shapes. Specifically, T2 reduction capabilities of SPIONs increasewith their size, since a lesser proportion of iron atoms is near thesurface of the nanoparticle, where their magnetic properties are re-duced. T2 reduction capabilities also decrease with polymer chainlength which coats the iron oxide nanoparticles, and is related topolymer coating thickness.

1.2. Sizes

In terms of practical applications, SPIONs should have a longenough circulation time to allow for imaging modalities to detecttheir localization, and they should circulate through organ and tissuecapillaries unobstructed, in order to prevent vessel embolism in the

Table 1Classification of SPIONs according to their sizes.

Dimensions Classification Reference

Overall size 300 nm to 3.5 μm Oral SPIONs [233]50 to 150 nm Polydisperse SPIONsb50 nm Ultra-small SPIONs (USPIONs)

Hydrodynamicradii

N40 nm SPIONs [29]b40 nm USPIONs

Overall size N300 Oral SPIONs [103]60 to150 nm Standard SPIONs (SSPIONs)10 to 40 nm USPIONs2.9 to 9 nm Monocrystalline SPIONs (MIOs)


patient [18]. SPIONs with sizes less than 100 nm are desired to pre-vent the uptake by macrophages and from the reticulo-endothelialsystem (RES). In a physiologically healthy non-inflammatory envi-ronment, SPIONs larger than 10 nm are unable to pass through ves-sels, but in the presence of inflammation or increased vasculature,i.e. around a tumor site, enhanced permeability and retention effect(EPR) allows these particles to infiltrate tissues [19]. Furthermore,the saturation magnetization (Ms) is linearly correlated with thesize of the particle [20,21]. Generally, for nanoparticles of iron oxideto be considered superparamagnetic, a size range between 4 and10 nm in diameter is often required [22]. One complexity is that asthe sizes of these nanoparticles decrease, the surface-to-volumeratio effectively increases. This often results in pronounced surfaceeffects (spin-canting, non-collinear spin) which can have adverseeffect on the Ms values [21]. One of the goals in the synthesis ofSPIONs has been to obtain a narrow range of size distributions toachieve high Ms, and increased circulation times in vivo (only incase of repeated imaging over longer durations) [23]. However, thesize of these SPIONs should not be smaller than 5.5 nm whichwould make them rapidly removed via renal clearance [21]. Further-more, for in vivo applications, not only does the inherent size of theSPIONs matter, but the size and type of the coating is very important.The types of surface functionalization and hydrophilicity also influ-ence the T2 relaxation times of the SPIONs, depending on their abilityto facilitate the interaction of water protons with the core of themetal ions [24,25]. Some functional groups, called effective groups,have the ability to allow fast exchange of bound water moleculeswith the core of the SPIONs relative to the proton relaxation rates.These decrease the overall T2 relaxation time, and are key in SPIONsacting as effective and desirable contrast agents [26–28]. The size ofSPIONs and their coating properties will reduce SPION agglomeration,and also help in strengthening their stealth properties since morethan 70% of body is water-rich, and the conjugated coating is thefirst entity to interact with the physiological medium [29].

1.3. Shapes

Although the most widely synthesized SPIONs as MRI probes arespherical in shape, other shapes (rhombic, ellipsoidal, cubic, crystal,flower and cube) with desired magnetic properties have also beenreported [19]. Much effort however has been devoted to synthesizingspherical SPIONs because the control over their synthesis is less com-plicated than for other shapes. This allows a better control over theuniformity of spherical SPIONs [30]. Other less common shapes,such as magnetite nanorods, nanorings, nanocubes and nanoflowers

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Fig. 2. In vivo GRE detection and ex vivo SWIFT detection of SPIO-labeled embryonic stem cells in the heart. Short-axis GRE images and SWIFT Im and Mag images obtained from theheart receiving 2.0 M (a–c) and 0.2 M (d–f) cells. Arrows in (a) and (d) point to locations of the stem cells, while those in (b) and (e) point to regions where negative Im signal(compared to the background) was generated by some of the off-resonance spins.Reprinted with permission from John Wiley and Sons, Magnetic Resonance and Imaging, 2010, 63, 1154–1161 [11].


have also been constructed [19,31,32]. The effect of different shapesof SPIONs on the biological systems remains a debatable issue [33].

2. Synthesis of SPIONs

As reviewed above, the shape and size of SPIONs are of consider-able importance for MRI applications, and these parameters have tobe taken into account in choosing an appropriate methodology fortheir synthesis. SPIONs with desired properties have longer circula-tion times, and these properties influence their cellular internaliza-tion and biodistribution.

2.1. Chemical means of synthesis

The synthesis of spherical SPIONs of varying sizes is predomi-nantly carried out using chemical (about 90% of all synthetic routes),physical (less than 10%), and biological methods (about a few %) [19].This review will focus on chemical means of synthesizing magnetiteSPIONs, and different methods used in their construction are listedin the order of synthetic prevalence. A brief summary of “green” syn-thesis in which either more environmentally friendly solvents orbiomimicry (utilizing bacteria) is also listed in this collection ofsynthetic strategies. It is now evident that in chemical synthesis, thespecific method employed and the reaction conditions are determi-nant factors in controlling the shape and size of the SPIONs.

2.1.1. Co-precipitationThe most widely used synthetic methodology to obtain magnetite

SPIONs is through the co-precipitation method. The latter involvesadding a base to amixture of Fe3+ and Fe2+ salts (in a 2:1 stoichiometricratio) in the presence of a base, leading to the formation of a black colloi-dal suspension of magnetite iron oxide nanoparticles (Eq. (1)) [34].

2Fe3þ aqð Þ þ Fe2þ aqð Þ þ 8OH− aqð Þ→Fe3O4 sð Þ þ 4 H2O lð Þ ð1Þ

This reaction must be performed in an oxygen-free environment toprevent the oxidation of magnetite (Fe3O4) into maghemite (γ-Fe2O3).


The latter packing is less desired since they have a slightly lower Ms

and stability in ambient air [29]. Several factors including temperature,reaction time, strength and concentration of different types of bases,the type of iron precursors used, the speed of stirring, and the pH ofthe solution mixture need to be carefully controlled to ensure that theparticles fall within a certain desired size range [19]. The nanoparticlesare generally isolated from the supernatant solution either by i) physicalmeans through centrifugation or ultracentrifugation, or ii) magneticdecantation ormagnetic filtration. The advantage of this syntheticmeth-od is that it employs cheap and commercially available reagents with-out invoking very stringent reaction conditions, and does producesub-100 nm diameter nanoparticles. However, the polydispersity of thenanoparticles remains a concern [19]. TheMs of magnetite nanoparticlessynthesized via co-precipitation ranges from 30 to 70 Am2 kg−1, whichis lower than the theoretical value of 96.4 Am2 kg−1of bulk Fe3O4. Thisis most likely due to impurities and/or surface effects [35]. The blackcolloidal suspension of SPIONs can subsequently be functionalizedwith various coating agents to confer desired characteristics such aswater-solubility, and to create positively or negatively charged surfaces[36].

The most commonly used mixture of iron salts to synthesizeSPIONs via co-precipitation is FeCl2·4H2O and FeCl3·6H2O in a 1:2mole fraction which is stirred in the presence of a base in an aqueousmedium protected under an inert gas atmosphere. Employing differ-ent kinds of bases, SPIONs varying in core diameter sizes from3.8 nm [37] to 9.8 ± 3.0 nm [38] to 11.5 nm [39] have been synthe-sized. One group used syringes to discharge the iron salts and rapidmixing to form SPIONs that were more crystalline, and also had a nar-row size distribution with mean size core diameter of 4.46 nm [40]. InFig. 3, the TEM image of the SPIONs synthesized via rapid mixingmethod (a) together with the plot of size distribution, and (b) TEMimage of SPIONs synthesized via conventional mixing method areshown. It can be seen that the rapid mixing method gives larger,more crystalline SPIONs with narrower size distribution than conven-tional mixing method. It is also possible to use a different combina-tion of iron salts, namely anhydrous FeCl3 and FeSO4·7H2O again ina 2:1 molar ratio to obtain SPIONs with 9.5 nm core diameter [41].

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Fig. 3. TEM images and plots of size distribution of magnetite nanoparticles prepared via rapid mixing methods (a) and (b), and via conventional mixing methods (c) and (d).Reprinted with permission from American Institute of Physics, Applied Physics Letter, 2011, 99 [40].

Fig. 4. TEM image (a) and HRTEM image (b) of as-prepared SPIONs dispersed in water.Reprinted with permission from ACS Publication, ACS Nano, 5, 6315–6324, Copyright2011, American Chemical Society [42].


2.1.2. HydrothermalAnother common method through which magnetite SPIONs have

been synthesized involves heating various iron salt precursors inaqueous solution at high temperatures (150 °C and higher). The solu-tion mixture is sealed in Teflon-line autoclaves to reach high pressure,and after the reaction is complete, it is cooled to room temperatureand washed with various solvents to remove impurities and non-conjugated stabilizing agents. The SPIONs are generally recoveredas a black powder after drying in vacuo at elevated temperatures(around 60 °C) or through lyophilisation. The choice of iron salt pre-cursor, the reaction time, and the reaction temperatures have to becarefully monitored to obtain SPIONs of varying sizes.

The most widely utilized methods to produce SPIONs via the hy-drothermal route make use of a single type of iron salt. SPIONs rang-ing in size from 5.1 ± 0.5 nm core diameter and capped by vitamin C[42] have been prepared using this method, and it was found that in-creasing the reaction time as well as the reaction temperatures gavebigger SPIONs with greater crystallinity. The TEM image of theseSPIONs capped with vitamin C is shown on Fig. 4(a). The crystallinityof the lattice fringes (Fig. 4b) can be used to determine the crystalpacking of the magnetite particles. The presence of a magnetite crys-tal packing is both supported by the lattice spacing (Fig. 4b) as well asthe electron diffraction pattern (inset of Fig. 4b). Using FeSO4·7H2Oat elevated temperatures (around 250 °C), SPIONs of approximately20 nm in core diameter [43] were obtained. It was noted thatlowering the temperature to 90 °C and keeping the same iron saltprecursor did not affect the core diameter [44]. If the temperature iskept high but the iron salt is changed to tris(acetylacetonato) iron(II)(Fe(acac)3), SPIONs decreased in size from 5 to 12 nm [45] in corediameter. Iron chloride salts are also widely used to obtain highlycrystalline SPIONs ranging from 15 to 31 nm cores by varying thereaction conditions [46]. FeCl3·6H2O has also been used extensivelytogether with different capping agents, namely tartaric acid and


ascorbic acid, to give SPIONs ranging in core size from 10 to 15 nm[47], and less than 10 nm [48] respectively. Cheng et al. [49] preparedhollow nanospheres using FeCl3·6H2O in the presence of polyacryl-amide, sodium citrate and urea at 200 °C. The particles were mono-disperse, water-soluble, and hollow 240 nm nanoshells with a wallthickness of 20 nm.

A variant of this synthetic methodology makes use of a mixtureof iron precursors. The synthesis of nearly spherical SPIONs coatedwith PEG-2000 with an average size of 20 to 50 nm core diameterwas carried out via a hybrid co-precipitation-hydrothermal methodusing FeCl3·6H2O and FeSO4·7H2O [50]. Another group reported thesynthesis of SPIONs with good monodispersity via a hydrothermalcontinuous process at 150 °C using a patented reactor and the antiox-idant citrate. With molar ratio of 1:5 citrate to iron, SPIONs of core

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size 17.8 ± 0.4 nm were synthesized, but decreased to 3.8 ± 0.1 nmwith a molar ratio of 1:1 citrate to iron [51].

2.1.3. MicrowaveSynthesis of SPIONs via microwave irradiation is a variation of the

hydrothermal synthetic methodology since high pressures are gener-ated upon heating in a microwave reactor. The SPIONs are recuperat-ed as a powder following washings in various solvents in combinationwith magnetic decantation, centrifugation, or lyophilization. The sizesof the SPIONs synthesized using microwave irradiation have varied alot under different heating temperatures and times. SPIONs of11.5 nm core size and with narrow size distribution were preparedusing 2:1 molar ratio of ferric to ferrous iron precursor at 40 °C and7 minute cycles in an oven (490 MHz) [52]. It was observed thatthe Ms doubled for the samples which were irradiated with micro-waves versus the ones which were not. At a slightly higher tempera-ture (100 ± 5 °C for 10 min) in a conventional microwave (300 W)using FeCl3·6H2O, the core sizes increased to 17.7 ± 6.6 nm [53].SPIONs functionalized with poly-acrylic acid (PAA) in the size rangeof 100 to 400 nm aggregates were synthesized using various amountsof FeCl3·6H2O in a microwave accelerated reaction system, whiledecreasing the heating time to 15 min [54]. Another group managedto synthesize smaller uniform 3 to 5 nm core diameter SPIONs deco-rated with multi-wall carbon nanotubes (MWCNTs) using Fe(acac)3in a Focused Microwave Synthesis system [55].

2.1.4. MicroemulsionThe microemulsion synthetic methodology makes use of a

biphasic heterogeneous solution of water-in-oil in which iron precur-sors are stirred. Water droplets are used as nucleation sites for theformation of SPIONs, often in the presence of surfactant moleculesdispersed in the oil/bulk, essentially forming micelles. The SPIONsprecipitate within the micelles in the presence of a base but not inthe oily phase where the precursors are unreactive. The size of thenanoparticles can be tuned via control of i) stirring speed which ulti-mately affects the size of the core [56], and ii) the temperature of thebulk [19]. This microemulsion methodology usually leads to the for-mation of hydrophilic SPIONs.

The popular choices of iron precursors within this synthetic meth-odology are FeCl2·4H2O and FeCl3·6H2O in a 1:2 ratio, and a variety ofbases. For example, i) monodisperse 40 to 50 nm core–shell SPIONswere prepared using NaOH(aq) as a base [56], ii) 2 to 10 nm coresize by changing the base to ammonia [57], and iii) monodisperse 7to 10 nm cores using NH4OH(aq) [58]. The latter group noted that in-creasing the concentration of iron precursor yielded cubic-shapedSPIONs that had higher Ms, and that increasing the concentrationof NH4OH(aq) decreased the Ms, but did very little to vary the size ofthe SPIONs.

A new water-in-oil microemulsion method was adopted by thegroup of Choi et al. [59], in which foam nest protein was dissolvedin the water phase as a surfactant to get 8 to 10 nm core diameterSPIONs. This is depicted in the TEM images (Fig. 6 top right and topleft) along with the size distribution plots (bottom right and left)for nonconjugated SPIONs and silica-coated SPIONs respectively. Thegroup used a mixture of FeCl2·4H2O and anhydrous FeCl3 in the pres-ence of NaOH. This method does not require a co-surfactant, andpurification therefore can be easily carried out.

2.1.5. Ultrasound irradiation/sonochemical/sonolysisThis synthetic route consists of utilizing ultrasound to physically

create cavitations in a medium that in turn can heat up to about5000 K. This can give rise to the nucleation of seeds and implosionof bubbles. Ultrasonic waves can generate chemical reactions suchas oxidation, reduction and decomposition to produce cavitations ina solution. During sonochemical processes, three different regionscan form, namely i) the inner environment made up of collapsing


bubbles as well as vaporization and pyrolyzation of water into Hand OH radicals via elevated temperature and pressure; ii) the inter-facial liquid region which lies between the cavitation bubbles and thebulk solution, and iii) the bulk solution which remains at room tem-perature. It is believed that sonochemical reactions take place in theinterfacial liquid region [60]. This synthetic methodology allows forthe synthesis of rather monodispersed SPIONs ranging from tens toa few hundreds of nm in diameter.

A typical sonochemical synthesis was reported in 2000 byVijayakumar et al. [60] in which a degassed aqueous solution ofFe(II) acetate was sonochemically irradiated with an ultrasonic hornat 20 kHz giving 10 nm average core sized SPIONs. The amount ofcontaminants present at the end depended on sonication time, withshorter times resulting in fewer impurities.

Polyethylene glycol (PEG) is a very versatile coating agent; not onlydoes it confer water-solubility properties to the conjugated SPIONs,it is also biocompatible and hence provides stealth capabilities tonanoprobes. PEG-encapsulated (Mn = 400) SPIONs were synthesizedusing Fe(CO)5 at 20 kHz [61], while magnetite PEG-coated SPIONs ofabout 10 nm core diameter were obtained using FeSO4·7H2O(aq). Itwas found that the sizes and distributions of the PEG-coated SPIONsvaried according to the feeding conditions of the FeSO4·7H2O solutionsthrough a micro-feeder. Slower feeding rate with lower concentrationsof the iron salt led to smaller and more uniform PEG-coated SPIONs[62].

Another widely used coating agent for SPIONS synthesizedthrough sonochemical means is chitosan. Chitosan (which is thedeacetylated version of chitin) is known to be non-cytotoxic, biocom-patible and biodegradable [63]. Mixtures of iron salts were used tosynthesize chitosan-covered SPIONs by the group of Lee et al. [64] uti-lizing FeCl2·4H2O and FeCl3·6H2O in the presence of ammonia, whichwas sonicated at 20 kHz. It was observed that the average size of theSPIONs decreased upon longer sonication time from 15 to 60 min.They obtained chitosan-SPIONS with narrow size distributions, withthe average being around 78 nm in hydrodynamic diameter. Morerecently, large scale synthesis via sonochemical means at ambienttemperature was rendered possible with the use of the same iron pre-cursor mixture under ultrasonication for 75 min. It led to SPIONs withan average core diameter of 11 nm [65]. The varying sizes are shownin the TEM images in Fig. 6 (right) together with an enlarged view ofthe same TEM image (left).

2.1.6. Thermal decompositionAnother synthetic strategy to obtain monodisperse SPIONs is

through thermal decomposition of an iron precursor. Originally thiswas carried out with iron carbonyl in the late 1970s in a high boilingsolvent such as decalin. Use of many other solvents and improve-ments to the method have followed since then including decanoicacid [64,66,67] as well as sodium oleate in toluene/ethanol/water[68,69]. Many studies have used a surfactant and/or boiling-point el-evating agent such as oleic acid [69]. It can be seen in the TEM imagesof Fig. 7 for oleic-acid coated SPIONs ranging from 7 to 22 nm in coreradii. Other studies have reported that the aging temperature usedduring and prior to reflux yielded differently sized SPIONs [69]. Anearly typical procedure involved placing Fe(CO)5 in a deoxygenatedenvironment in octyl ether with oleic acid which was then heatedto reflux for a given amount of time. It was subsequently oxidizedby introducing air or an oxidant like CH3NO into the reaction flask.It was observed that longer reflux times yielded larger SPIONs. Thesolution was then cooled to room temperature and washed withdifferent solvents. The SPIONs were collected via centrifugation anddispersed in an organic solvent [69]. SPIONs obtained via this proce-dure were hydrophobic and monodisperse, and could be tuned from7 to 25 nm in diameter. Other groups since have used different ironprecursors such as iron oleate [70–72], and asymmetrical iron organ-ometallic sandwich compound, [Fe(η5-C6H3Me4)2] [73], which is an

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Fig. 5. (a) TEM image of bare SPIONs (left) and SPIONs coated with silica (right) along with XRD insets. (b) Corresponding size distribution plots.Reprinted with permission from Springer, Journal of Nanoparticle Research, 2012, 14, 1092 [59].

Fig. 6. TEM images of SPIONs (a) and enlarged image (b).Reprinted with permission from Elsevier, Thin Solid Films, 2011, 519, 8277–8279 [65].


Please cite this article as: Lam T, et al, Adv Colloid Interface Sci (2013), http://dx.doi.org/10.1016/j.cis.2013.06.007


Fig. 7. TEM images of monodisperse SPIONs of varying sizes; (a) 7 nm; (b) 10 nm; (c) 15 nm; (d) 22 nm.Reprinted with permission from Elsevier, Journal of Alloys and Compounds, 2011, 509, 8549–8553 [69].


air-stable compound compared to Fe(acac)3. Still, the use of Fe(acac)3remains very popular. PAA-coated SPIONs of average core size 7.2 nm[74], as well as zwitterionic SPIONs of less than 10 nm in core size[75], have been reported using Fe(acac)3.

Shortly after, many groups adopted the use of water compatiblesolvents such as polyols (ethylene glycol, triethlylene glycol (TREG)[76], tetraethylene glycol (TEG), poly-(ethylene glycol) (PEG) [72],and poly(vinyl alcohol) (PVA)) while others resorted to using2-pyrrolidone. These media can all reach high reflux temperaturesand yield water-soluble SPIONs which are collected after filtrationthrough a 0.2 μm filter [77]. Synthesis of silica-coated [78] andsilane-coated [79] SPIONs via thermal decomposition have also beenexplored.

Recently, water-soluble SPIONs coated with phenol-benzoquinonewith average core size of 19.3 ± 4.4 nm to 9.7 ± 1.5 nm have beenprepared [80] using Fe(acac)3 as the iron salt precursor. It is also pos-sible to obtain water-soluble SPIONs from water-insoluble SPIONscoated with oleic acid/oleylamine synthesized via thermal decompo-sition of Fe(acac)3. This is achieved through ligand exchange withvarious water-soluble sugars [81].

2.1.7. Electrodeposition/plating/electrosprayThe group of Alqudami et al. [82] synthesized SPIONs with core

sizes ranging from 10 to 50 nm in distilled water using very thiniron wires and sheets which were subjected to a potential difference.High current density caused tensile fractures to the wires or sheets,producing nanoparticles. These SPIONs have been proposed to poten-tially have applications as ground water decontaminants. Using asimilar electric explosion of wire (EEW) technique, the group of


Beketov et al. [83] managed to synthesize nanoparticles that behavelike SPIONs in the bulk phase with an average core size of 12 nm.

The electrochemical synthesis of SPIONs can also be achieved inaqueous or non-aqueous electrolytes using either iron or platinum elec-trodes [84] or stainless steel electrodes [85]. The particle size obtaineddepends on the electrolyte, the type of electrodes and current densityused [86,87]. Particle of sizes 3 to 8 nm were prepared using an ironelectrode in an aqueous system, while the size of ca. 20 nm wasobtained using a stainless steel electrode in non-aqueous system.

2.1.8. Sol–gelThe sol–gel synthetic strategy makes use of a gelling agent such

as agarose to form a homogeneous gel in which a metal salt is stirred.In a typical synthesis of this type, the group of Alagiri et al. [88]used agarose polysaccharide which was gelled at high temperature(300 °C). It contained 1% weight iron (III) nitrate nonahydrate,(Fe(NO3)3·9H2O (aq)), and led to the formation of spherical SPIONsof 2 nm average core diameter. More recently, tetraethylorthosilicate(TEOS) has been used to form the gel to which a solution ofFe(NO3)3·9H2O (aq) was added in a 10% weight ratio over a long peri-od of time (10 days at 80 °C). This led to the formation of SPIONs of4 nm in core size [89].

One variant of the sol–gel methodology involves the use of polyolsas solvents rather than water to achieve well defined shapes and con-trolled sizes [90,91]. The process involves the use of metal precursorssuspended in liquid polyol and heated close to the polyol's boilingpoint [92]. This allows the metal precursor to be solubilized in thepolyol, which is then reduced to form metal nuclei through an

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Fig. 8. TEM image(left) and HRTEM image (right) along with the corresponding SelectedArea Electron diffraction pattern (SAED) (left inset) for 15 nm SPIONs.Reprinted with permission from Elsevier, Journal of Alloys and Compounds, 2011,509, 8549–8553 [69].


intermediate complex formation. The latter will then nucleate to formmetal nanoparticles.

2.1.9. Flow injection synthesisFlow injection synthesis (FIS) involves the use of a capillary reac-

tor with continuous or segmented mixing of reagents under laminarflow conditions [93]. The SPIONs obtained from this technique havea narrow size distribution core range of 2 to 7 nm. The advantage ofthis technique is the high reproducibility and little influence of theflow parameters on the material composition and characteristics.

2.1.10. Green chemistryGreen chemistry synthesis of nanoparticles aims to encourage

designs that minimize or eliminate the use and generation of hazard-ous chemicals or reagents. Here we describe two such green methodsfor the synthesis of SPIONs.

2.1.10.1. Microbial synthesis. In this method, fermentation ofβ-Fe-OOH precursor in the presence of Fe(III) reducing bacteria(Thermoanaerobacterethanolicus strain TOR 39 or Shewanellaloihicastrain PV-4) is carried out under anaerobic conditions at 65 °C for3 weeks in the presence of electron donors (e.g. glucose). The parti-cle sizes obtained using this method range between 5 and 90 nm[94–96]. This method is used in the large scale production of SPIONs,as it leads to high yield, good reproducibility and scalability, anduses low temperatures, energy, and production cost.

2.1.10.2. Supercritical fluid method. Supercritical fluids such astrifluoromethane, chlorodifluoromethane, acetone, carbon dioxide,diethyl ether, propane, nitrous oxide and water exist as a single phaseabove their critical temperature and pressure. These are utilized in a con-trolled fashion to obtain the desired geometry of iron oxide nano-particles [97–99]. The use of supercritical carbon dioxide (~8300 kPa,~35 °C) and H2O (22.11 MPa and ~374.3 °C) employed in the synthesisof SPIONs reduces the use of organic solvents [100–102]. The advantageof using these fluids is attributed to the mild conditions (low tempera-ture and pressure), as well as their non-toxic and non-inflammablenature [100–102].

2.2. Summary of synthetic methodologies

There is not one global method for the synthesis of SPIONs whichcan be prescribed for the combination of all the desired chemical andphysical characteristics. The method that is ultimately employedremains much of a personal choice, and depends on obtaining a bal-anced set of properties. Generally, thermal decomposition affordsSPIONs which are spherical, crystalline, and with narrow size distri-butions. A drawback of this strategy is the use of high temperatureswhich are achieved in high boiling solvents. Hydrothermal synthesisoffers similar advantages, but the dispersity of SPIONs in sizes is some-what larger than via the thermal decomposition method. Microwavesynthesis can yield monodisperse SPIONs in a fraction of the timecompared to the classical methods mentioned above, but has similarlimitations as the hydrothermal method. Co-precipitation synthesisis a simpler approach. In general, it is versatile enough in terms ofthe solvents which can be used in the synthesis, and the purificationprocess is straightforward since there is no surfactant moleculeadded to the SPIONs. However it can suffer from low monodispersityin terms of the sizes of SPIONs and the use of large quantity of solventsis often required. Sol–gel synthesis also affords SPIONs with lowdispersity, but the challenge is to extract the SPIONs from the gel. Itmay be really tedious to clean the surface of these SPIONs effectively.Electrospray/deposition technique is another attractive syntheticroute, since SPIONs are rather narrow in size and are devoid of surfac-tants which make their purification simpler.


3. Characterization techniques

After synthesis, it is essential to characterize nonconjugatedSPIONs in terms of their morphology, size, shape, magnetism, etc. Itis equally important to have detailed information such as the extentof coverage once these are conjugated or coated with, for example,surface stabilizers to decrease electrostatic attraction between theSPIONs. The surface coating is often also employed to provide stealthproperties and biocompatibilities, and hence the determination ofthe nature of the coverage is of high importance. We provide belowa brief discussion of different techniques used to elucidate the desiredinformation about the SPIONs.

3.1. Shapes, morphology, and coverage

A plethora of characterization techniques is employed to obtaininformation about size distributions of SPIONs, their shape, morphol-ogy, crystal packing, and magnetic properties [103]. To assert theirshapes, sizes and crystal packing, transmission electron microscopy(TEM) as well as high-resolution TEM (HTEM) [104], which makeuse of an electron source beam to image high electronic densitieson a TEM grid, have been extensively used. Such TEM and HRTEMimages obtained for 15 nm core sized SPIONs are depicted in Fig. 8[69]. HRTEM provides a more crystalline image of the SPIONs thanregular TEM as can be seen when comparing the left from the rightimage of Fig. 8.

X-ray diffraction (XRD) pattern can be used to determine the crys-tal packing in SPIONs, as well as to determine size of the nanoparticleswhich can be calculated using the Scherrer equation [105,106]. InFig. 9, the top XRD pattern corresponds to that of magnetite, whereasthe bottom pattern is that of the maghemite crystal packing [44]. XRDpattern analysis can provide information about the crystal packing,but is not sensitive enough to determine the presence of non-metalsinside the nanoparticles. On the other hand, energy dispersive X-raydiffraction (EDXD or EDX) can help elucidate fine structural detailsby the determination of the elemental components of a given samplethrough software fitting with known diffraction patterns of variouspure samples. For example, in Fig. 5(a), the insets show the EDXpattern of uncoated SPIONs (left) which contains Fe and O, and ofsilica-coated SPIONs (right) which contains Fe, O and Si [59]. Anothermethod used to find out the elemental constituents of a sample isinductive coupled plasma (ICP). This technique makes use of a plasmato decompose samples, and hence obtain various informationdepending on the analyzers and detectors used to decipher thesesignals [107,108].

The use of scanning electron microscopy (SEM) is sometimes alsoemployed, but this requires that the sample be conductive or be madeconductive, often by sputtering a layer of conductive metal on thesurface of the grid [109]. In terms of finding out the nature and the

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Fig. 9. XRD patterns of (a) magnetite SPIONs and (b) maghemite SPIONs.Reprinted with permission from Springer, Metals and Materials International, 2010, 16,225–228 [44].


amount of coverage on coated SPIONs, thermogravimetric analysis(TGA) is commonly employed [110,111]. It makes use of high tem-peratures to decompose the coating around coated SPIONs, andmeasures the weight loss that results at varying temperatures. For ex-ample, increasing size of the hyperbranched macromolecular layer(dendrimeric generations) on SPIONs results in greater weight lossfor a given acquisition temperature as shown in Fig. 10 [112]. This isexpected since increasing the weight of the dendrimers by increasingits generation results in more weight being added onto the surface ofthe SPIONs, hence a larger drop in weight is expected as the temper-ature is raised for increasing generations of dendrimers.

3.2. Surface potential and hydrodynamic radii

The hydrodynamic radii of nanoparticles can be measured usinglight scattering techniques (LS), such as photon correlation spectros-copy (PCS) or dynamic light scattering spectrophotometry (DLS)(also called quasi-elastic light scattering) [113,114], and a variantcalled electrophoretic light scattering (ELS) [115] which can also cal-culate the surface potential. DLS plots showing the percent number ofparticles of a given mean size, in a solution of SPIONs before and aftercoverage with an enzyme, are shown in Fig. 11 [113]. As expected, thebare SPIONs (left) have a smaller mean diameter than the SPIONs

Fig. 10. TGA curves of SPIONs covered with increasing generation of dendrimers fromG0 to G3, represented on the graph as F020 to F320.Reprinted with permission from Springer, Journal of Superconductivity and Novel magnetism,2012, 25, 1541–1549 [112].


coated with urease enzyme. Hydrodynamic radii measure not onlythe iron oxide as an inorganic entity, but also any organic or poly-meric coating that can be used to stabilize the SPIONs. This can leadto a much larger radial size determination than reported via TEMor XRD measurements. Fig. 12(b) shows the TEM image of SPIONscapped with citrate via a hydrothermal synthesis [51], and the sizedistribution is smaller than that obtained from DLS for the same sam-ple. This difference in size measurement can also be attributed to theswelling of the organic shell in the presence of solvent. The surfacecharge of the nanocarriers has to be determined mainly for biologicalapplications, and zeta-potential (ZP) is used as the most commontechnique to assess it. In some cases, the measurements of particlesranging from 3 to 1000 nm can be carried out using a scanning mobil-ity particle sizer (SMPS). It has also been reported that the size spec-trum could be converted to a mass spectrum due to the strongcorrelation between mobility size of particles and their respectivemolecular weights [116].

The nature of the surface of coated SPIONs and its properties canalso be investigated using various techniques such as solid stateNMR, differential scanning calorimetry (DSC), X-ray photoelectronspectroscopy (XPS), atomic and chemical force microscopy (AFMand CFM), infrared (IR) and Fourier transform infrared (FTIR) spec-troscopy [117].

3.3. Magnetic properties

Superparamagnetism in SPIONs originates from the paramagneticiron centers (Fe(II) and Fe(III)) and is characterized by the presenceof a large magnetic moment (M) in the presence of an externalapplied magnetic field (Bo). No remnant magnetic moment (Mr) isobserved when the applied field is zero. This is what differentiatesSPIONs from purely ferromagnetic nanoparticles containing paramag-netic metal centers, since the latter still present Mr in the absence ofBo [22]. Essentially this translates into the absence of spontaneousagglomeration of SPIONs due to attractive van der Waals forces (inorder to minimize the interfacial surface or energy) [19] in theabsence of Bo, but the presence of magnetization in the presence ofBo. One of the advantages of iron oxide nanoparticles in the sizerange 6 to 15 nm (often then referred to as SPIONs) is that theyshow accelerated spin–spin relaxation (T2 shortening) resultingfrom the dephasing of the magnetic moments due to magnetic-fieldgradients. The T2 shortening is due to the interaction between the di-polar outer-sphere of the water proton spins and the magnetic mo-ment of nanoparticles. Therefore, to be efficient T2 contrast agents,SPIONs should possess large magnetization which could be controlledby their intrinsic material properties (nanoparticle composition andcrystal structure), and extrinsic factors (size, shape and surface prop-erty) [118–121]. The quantitative measure of T2 contrast ability isrelative to the effectiveness with which these SPIONs accelerate therelaxation process of water protons, also known as the relaxivity.The latter relates to changes in the relaxation rate (inverse of relaxa-tion time) per unit concentration of MRI contrast agents. The termsrelaxivity and relaxation rates allow the magnetic characterizationof different SPIONs in regard to their contrast enhancement in MRI.Relaxivity (r1 and r2) measures the ability of magnetic particles to in-fluence either longitudinal relaxation time (T1) or transverse relaxa-tion time (T2) or both, of the surrounding water proton spins.

From the first reports of SPIONs with predominantly T2 effect, sev-eral synthetic and optimization procedures have been used to addresstheir physicochemical properties, biodistribution, pharmacokineticsas well as water proton relaxation properties. Some of the commoncharacterization techniques that are employed in understanding themagnetic properties of the SPIONs include vibrating sample magne-tometry (VSM) [122], super quantum interference device (SQUID)magnetometry [123], and Mössbauer magnetometry [124]. One de-fining characteristic of SPIONs is the presence of a narrow hysteresis

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Fig. 11. DLS plot showing the number (in %) of particles with a given mean size for magnetite SPIONs before (left) and after (right) conjugation with urease enzyme.Reprinted with permission from Elsevier, Journal of Molecular Catalysis B: Enzymatic, 2011, 69, 95–102 [113].


loop upon acquisition of the magnetization M over varying appliedmagnetic field H as depicted in Fig. 13 [125]. Zero-field cooled (ZFC)and field-cooled (FC) acquisitions are often made, and refer to themodes of acquisition in which the sample is either cooled to a certaintemperature when not applying any magnetic field (ZFC) or cooledwhile applying a magnetic field (FC) [126,127].

Additional factors that influence the relaxivity properties ofSPIONs are the degree of aggregation, magnetic field strength andmagnetization saturation [76,128–131]. The overall effectiveness ofSPIONs as MRI contrast agents can be tuned by using different syn-thetic strategies to control the particulate core size, while increasingthe magnetic field strength (4 to 14 T) to increase the probe sensi-tivity. Since SPIONs show superparamagnetic behavior, the loss oftheir net magnetization in the absence of an external magnetic fieldlimits their self-aggregation tendency, and this helps in obtaining agood biological response. To be effectively used for MRI applications,other important factors such as the particle size, charge and surfacecoating need to be accounted for in order to determine the overallstability, biodistribution and metabolism of SPIONs [132].

4. Conjugation (surface coating)

As the cores of SPIONs are made exclusively of inorganic material,it is often required to functionalize or conjugate various coatingagents on the surface to confer desired properties. There are two dif-ferent targeting methodologies that are employed when choosing thecoating for SPIONs, either passive targeting or active targeting. Thefirst allows the conjugated SPIONs to circulate freely in the body,and image their uptake and sequestering at given time intervals, asset by the operator of the imaging technique of interest (i.e. MRI).Passive targeting can confer certain properties to SPIONs such asdecreasing their agglomeration, and would prevent the formation

Fig. 12. (a) TEM images of SPIONs covered with citrate and (bReprinted with permission from the Royal Society of Chemistr


of macromolecular clusters which can decrease the magnetizationvalues of the bulk [19]. Furthermore, the stealth nature of these con-jugated SPIONs can help evade early uptake by macrophages to effec-tively enhance circulation time in vivo. One could also make thembiocompatible through careful choice of the coating agent. Activetargeting requires a careful choice of the predetermined target loca-tion for the accumulation of SPIONs. This methodology relies onusing a targeting moiety on the surface of SPIONs to communicatewith a specific location in vivo through a biological recognition signal,and hence allow the targeted SPIONs to dock onto these locationsrather than move freely in the body.

4.1. Passive targeting

Passive targeting involves the use of SPIONs which are functional-ized or stabilized with a surface coating. Currently, clinical trials in-volving SPIONs as contrast agents for various pathologies aredominantly making use of passive SPIONs [5]. The surface coating isintended primarily for stealth, biocompatibility, longer circulationtimes, decreased aggregation, and decrease rate of cell-type specificuptake [5]. The hydrodynamic radius and surface charge conferredby the coatings are hence carefully chosen to invoke desired proper-ties. The nature of the coating will also have repercussions on theclearance and cytotoxicity of the SPIONs in vivo. Various kinds of coat-ings on SPIONs have been reported, and these surface modificationshave been either done during the synthesis of the SPIONs via methodsmentioned earlier, or post-synthesis [19]. Two general classes of sur-face coatings in passive targeting include i) inorganic shells (Au,/Ag[133], Gd, Zn [134,135], Ni [136,137], Ni silicate [136], quantum dotshell [138] and graphene [139], as well as silica), and ii) organic shellscomposed of surfactants, dendrimers, polymers and combinations ofpolymers as summarized in Table 2.

) size distribution plot.y, Chemical Communications, 2011, 47, 11706–11708 [51].

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Fig. 13. Magnetic hysteresis loop acquired at 300 K showing Ms of 60.5 emu/g, and theabsence of remanence and coercivity indicate the superparamagnetic nature of thesynthesized SPIONs.Reprinted with permission from Elsevier, Journal of Magnetism and Magnetic Material,2010, 322, 1828–33 [125].


4.1.1. Inorganic shells/componentsSilica is used extensively to coat SPIONs since it is known to be

bio-stable and has low toxicity. The silanol surface groups on silicacan react with a plethora of functional groups, making silica a

Table 2Various organic shells, surfactants, dendrimers, polymers and combinations of polymer coa

Coating Diameter (technique used)

Dextran 6.5 ± 1.2 nm (TEM), 39 ± 8 nm50 nm (DLS), 50–100 nm (TEM10 nm (TEM)

Chitosan 10 nm (HRTEM), 21.2 nm (DLS

10.3 nm (XRD), 8.1 nm (TEM)10–20 nm (SEM)

Alginate 193.8 nm (DLS)5–10 nm (TEM), 193.8–483.2 n

Poly(vinyl alcohol) (PVA) 2 mm20–30 nm (TEM), 3.8 nm (XRD56.4 ± 4.1 nm (PCS)21.6 ± 2.5 nm (PCS)9 nm (TEM)

Mannan 37.3 ± 5.6 nm (DLS)46.2 ± 1.9 nm (DLS)9–10 nm (TEM), 52 nm (DLS)

PAMAM dendrimer 8.2 nm (XRD), 10 nm (TEM)10 nm (TEM), 13.5–28.6 nm (DMonodisperse 10 nm (DLS)10–15 nm(TEM), 250–300 nm (Monodisperse 10 nm (TEM), 1310.5 ± 0.5 nm (XRD), 9 ± 0.18.4 nm (SMPS)84.0 ± 1.4 nm (HRTEM)

Other dendrimer 9.5 ± 0.6 nm (TEM), 9.5 ± 1.011.5 nm (TEM, XRD)5 nm (TEM), 16 nm (DLS)7.6 ± 0.7 nm (TEM)5.4 ± 1.2 nm (TEM)

Dodecylbenzenesulfonic acid sodium salt (NaDS) 30 to 50 nm, Polydisperse

Poly(vinyl pyrrolidone) (PVP) 14 nm (TEM)8–10 nm (TEM), 20–30 nm (DL

Poly(ethylene glycol) (PEG) 9, 19, 31 nm (TEM) narrow dist7 nm (XRD), 4.8 ± 2 nm (TEM)10.4 ± 0.8 nm (SEM), 61.7 ± 1

Poly(oligo(ethylene glycol)methacrylate-co-tert-butyl methacrylate)

10.1 to 24.3 nm (DLS)

Poly(lactic-co-glycolic acid) (PLGA) 5 ± 1 nm (HRTEM)Poly(acrylic acid) (PAA) 8 ± 1 nm (HRTEM), 6.4 nm (X


versatile surface coat [133]. Different synthetic methodologies havebeen developed to conjugate silica on the surface of SPIONs viathermal decomposition at different calcination temperatures [140].When the calcination temperature was increased from 350 °C to750 °C the sizes increased from 7.1 ± 0.7 nm to 13.9 ± 1.4 nm. Amore popular synthetic route in recent years makes use of a water-in-oil microemulsion technique. In the presence of varying amountsof tetraethoxysilane (TEOS), varying thicknesses of silica-coatedSPIONs are obtained, ranging from 20 to 30 nm via reverse micro-emulsions and free radical polymerization [141], sub-50 nm [142], 30and 100 nm [143], to between 50 and 60 nm hydrodynamic diameter[144]. Silica-coated SPIONs can also be prepared via the sol–gelmethod-ology as observed recently by the group of Zhang et al. [138]. Theymadeuse of a modified Ströber method in which the SPIONs are first dis-persed as a ferrofluid in the presence of sodium citrate, and sonicatedin an ammonia solution with varying amounts of TEOS. The group ofTadic et al. [89] has prepared non-agglomerated 4 nm size sphericalsilica-coated SPIONs using TEOS.

4.1.2. Organic shells/componentsMany small organic molecules containing carboxylic acid groups

have been used to successfully stabilize and cap SPIONs via chelationof the acid group [29]. These can be found in more details in thereview by Qiao et al. [29]. Table 2 provides an overview of varioustypes of organicmolecules, polymers and other hierarchical structuresthat have been employed as conjugating agents on the surface ofSPIONs post-synthesis. The desired application of these functionalized

tings for SPIONs.

Application References

(DLS) MRI contrast agent [53]) Macrophage uptake study [234]

Bioassay agent [235]) Immunoassay, drug delivery agent,

MRI contrast agent[63]

Bacteriacidal agents [236]Tumor embolization agent [237]MRI contrast agent [238]

m (DLS) MRI contrast agent [239]Drug delivery agents [240]

) Cytotoxicity studies [37]Drug delivery agents [154]MRI theranostics [148]Drug delivery agents [241]Drug delivery agents [242]MRI contrast agent [243]MRI theranostics [154]Drug delivery agents [149]

LS) MRI theranostics [145]Drug delivery agents [152]

DLS) Magnetofection (gene delivery) [181]nm (DLS) Drug delivery agents [146]

nm (TEM) Immobilization of invertase [244]MRI contrast agent [245]Targeted tumor imaging [193]

nm (XRD) MRI theranostics [246]Multimodal imaging [39]MRI contrast agent [194]MRI contrast agent [247]Self-assembly [248]Microwave-induced thermoacoustictomography (TAT)

[249]

MRI contrast agent [250]S) MRI contrast agent [251]ribution Magnetic hyperthermia agent [252], 15.7 ± 2 nm (DLS) MRI theranostics [77].5 nm (DLS) MRI contrast agent [72]

MRI contrast agent [253]

MRI contrast agent [254]RD) MRI contrast agent [74]

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SPIONs is sometimes also stated by the authors, and the reader isdirected to these references for detail.

4.1.3. Drug molecules/fluorescent dyesMany drugmolecules have been used to coat SPIONs, and one of the

most widely used is doxorubicin (DOX), an anticancer agent for thetreatment of different types of cancers, carcinomas and sarcomas.DOX in itself has adverse cardiac toxicity. Systematic administration athigh concentrations in the body is required due to its low targetingability, and it also exhibits multidrug resistance effect (MDR). To com-bine imaging through MRI with therapy, SPIONs covered with DOX[145–152] or with dendrimers incorporating DOX [145,146,149,152]have been extensively studied.

Another drug, camptothecin (CPT), is also widely used as a potentanti-tumor agent in vitro, but its use in vivo remains sparse due to itslow water-solubility and cytotoxic effects at conventional dosage[153]. In previous work done by the group of Zhu et al. [153], CPTwas loaded onto magnetite SPIONs which were covered with chitosanand other polysaccharides. They found that CPT loaded magnetiteSPIONs had better in vitro cancer cell inhibition activity than thedrug alone. This was attributed to the amphiphilic properties ofthe polysaccharides which confer increased solubility in aqueousmedia. In the same year, the group of Cengelli et al. [154] made useof esterases to release CPT from the surface of USPIONs coveredwith poly(vinyl alcohol) (PVA) and polyvinylamine. They observeda decrease in proliferation of human melanoma cells in the presenceof CPT-USPIONs, and even more uptake by the cells in the presenceof an applied static magnetic field. More recently, the group of Zhuet al. [155] has carried out studies with CPT-loaded SPIONs coveredwith poly(acrylic acid) (PAA), which has amphiphilic properties.

Other groups have resorted to making use of various dyes conju-gated on the surface of SPIONs often containing biocompatible surfacecoatings in order to provide dual imaging capabilities for theirnanocarriers. One widely used class of dye is the Cy5.5 fluorescentdyes whose absorption and emission profiles are found slightlybelow 700 nm in the near-IR (NIR) region. This absorption/emissionwindow is of importance due to the presence of decreased tissueabsorption in the NIR around 700 to 900 nm, conferring good con-trast capabilities around those wavelengths. Cy5.5 dyes are alsomore photostable than other common dyes such as fluorescein, andare known to be lipophilic [156]. Silica coating to cover the USPIONswith Cy5.5 fluorescent dye has been carried out, and it has been dem-onstrated that such systems had no cytotoxicity even after highincubation concentration, and in vivo, these showed clearance 24 hpost-injection [156]. Many other groups have lately been investigat-ing this dual imaging capability using Cy5.5 anchored onto SPIONsfor various applications, including cancer and tumor imaging contrastagents [63,157–163], effects of amphetamine on neuroglia in thebrain [164], nanoparticle clearance [165], stem cell research [166],enzyme targeting [167], and as pulmonary delivery agents [168].

Another dual approach using MRI and optical detection has beenused by combining SPIONs with another dye, rhodamine, in whichthe relaxation properties at very high magnetic fields were studied[8]. However this work has not been followed up with in vivo studies.

4.2. Active targeting

Active targeting involves utilizing a moiety to direct the functional-ized SPIONs towards a site of pathological interest. Various targetingmoieties have been employed, most of which make use of proteins,peptides, aptamers (functional peptides or oligonucleotides that bindto a variety of chemical and biological molecules with high affinityand selectivity), oligomers and drugs for cardiovascular imaging.The use of SPIONs to target specific sites using MRI is an emergingarea and particularly interesting, as it helps in monitoring cellular andmolecular processes beyond the detection mechanism [169–171].


Targeting specific cell surface helps not only to detect but also charac-terize a disease non-invasively [172]. SPIONs have been linked to avariety of targeting moieties and used as a platform to target recep-tors [173], enzymes [172], integrins [174] and specific cells [167].The targeted SPIONS can also act as smart probes by imaging differentcellular processes [175]. In conventional testing, the accumulationof macrophage at the atherosclerotic site is difficult to understand,and this is a key step during the development of atherosclerosis.Recently, macrophage-targeted SPIONs have provided evidence ofnot only their accumulation at the plaque site, but also to help mon-itor changes in inflammatory burden [175]. The macrophage accumu-lation also plays an important role in the initiation, progression anddisruption of plaque, and subsequently leads to thrombus formation[176]. Therefore, detection of thrombin helps in identifying the plaquevulnerability, and design therapeutic interventions. Earlier, Lu andhis team designed SPIONs-aptamer contrast agents for the detectionof thrombin with sensitivity as low as 10 nM [177]. Macrophagesare also involved in the expression of a variety of proteases includingmatrix metalloproteinases (MMP) leading to plaque vulnerability.These proteases play a very important role in the degradation of avariety of proteins including the extracellular matrix which leads tothrombus formation post plaque vulnerability [178,179]. Synthesis ofmagnetic nanoparticles that can detect these enzyme actions helpsus understand this plaque vulnerability. One such strategy was devel-oped by Bhatia and colleagues [180] in which proteolytic activation,such as MMPs, leads to the degradation of the MMP recognition pep-tide sequence, allowing the aggregation of neutravidin and biotin-functionalized SPIONs. These aggregates show high magnetic suscep-tibility and reduce the T2 relaxation times by approximately 50%of the initial value [180]. Though the strategies developed by thisgroup were directed towards cancer therapy, these targeted magneticnanolabels could also provide information leading to an understand-ing of plaque vulnerability, and therefore help in the developmentof therapeutic strategies.

Proteins can also be used to target specific biological recognitionsites which get expressed or over-expressed in the presence of apathological disease. The group of Lui et al. [181] synthesized SPIONscovered with functionalized PAMAM dendrimers, and performed anMTT assay to evaluate the cytotoxicity of such magnetoplexes againstkidney fibroblast cells COS-7, human embryonic kidney cells contain-ing SV40 large T antigen (293 T) and HeLa cells. Their results showedthat 80–90% of all three cell lines remained viable after 48 h ofincubation.

Gene therapy is a promising therapeutic approach to treat hered-itary (i.e. cystic fibrosis) and acquired diseases (i.e. cancer), but theefficiency of the delivery of genes to desired locations in the bodywith minimal side effects is essential for treatment. Vectors for genedelivery can be viral or non-viral, and of the latter cationic polymers(i.e. polyethyleneimine (PEI), poly(L-lysine) (PLL), chitosan, cationiclipids, and dendrimers) have low immunogenicity and can be syn-thetically tailored to accommodate many other functionalities/payloads. PEI has been shown to have good transfection efficiency,but is not biodegradable. Chitosan on the other hand is biodegradableand biocompatible, and is positively charged, making it a good candi-date for anchoring negatively charged DNA to form stable complexeswhich will prevent premature degradation of the DNA payload. Thedisadvantage of chitosan remains its low transfection efficiency (aswell as other factors such as size, molecular weight, and percent ofdeacetylation). The group of Wang et al. [182] synthesized SPIONscovered with both PEI and chitosan that could deliver both nucleicacid-based therapeutic agents and be used as contrast agent forMRI. They have shown these to be biocompatible (WST assay), anda single injection of these nanocarriers having carrier reporter plas-mids, was able to express genes for at least a week. Other groupshave also made use of chitosan-covered SPIONs for gene therapy,showing good biocompatibility and low immunogenicity [183,184].

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The use of functionalized SPIONs for active targeting, both in vitroand in vivo, in the field of diagnostic and clinical MRI, is thus emerging.Further developments in the surface chemistry and bioconjugationstrategies that enable linking of biologically active molecules like pep-tides, antibodies, siRNA and drugs with precise orientation and stoichi-ometry would be of considerable future interest.

5. Toxicity

For a variety of imaging applications both in vitro and in vivo,a good SPION candidate should be functionalized with suitable enti-ties, biocompatible, stable under physiological conditions, have sub-nanomolar detection limits, show high contrast, persist long enough(a few hours) in the blood stream for imaging as a blood pool agentand have low toxicity [185,186]. Uncoated SPIONs tend to agglomer-ate and are rapidly taken up by macrophages (phagocytosis), a prop-erty that can be exploited for other purposes, such as imaging of theatherosclerotic plaque [187,188].

One of the quick ways to understand the qualitative or quantita-tive screening of toxicity of SPIONs is by performing in vitro assays(cell viability/cytotoxicity, inflammatory stress, oxidative stress,genotoxicity). However, the choice of assays is still a topic of debate.To obtain adequate and reliable information, authors have arguedthat there is an urgent need to gather reliable and reproducible datafrom various groups in terms of cell types being used, the cell culturegrowth conditions, and SPION treatment strategies [189]. Not onlythe optimization parameters but also the types of SPIONs having dif-ferent coatings and conjugations add even more complexity to the bigpicture. The widely performed toxicity assays include the use of MTS,MTT, and XTT assays for cell viability analysis, bromodeoxyuridinestaining for cell proliferation, annexin-V/propidium iodide stainingfor apoptosis/necrosis analysis, etc. (Fig. 14). The large variation inthe toxicity results obtained could be due to protein adsorption onthe surface of SPIONs that skew the assay analysis [19,189].

There has been intriguing information about the toxicity of SPIONsin vitro. A few studies revealed that dextran and citrate-coated

Fig. 14. TEM image of HepG2 human liver cell line showing various toxicity screeningassays using SPIONs and their representative mechanism of action sites. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) — mitochondrial activity;propidium iodide (PI) staining — DNA staining for cell death; bromodeoxyuridine(BrdU) staining— thymidine incorporation for DNA replication assays; lactate dehydroge-nase (LDH) — membrane integrity assessment.Reprintedwith permission from Chemical Reviews, 2012, 112, 2323–2338. Copyright 2012.American Chemical Society [189].


SPIONs showed in vitro toxicity mediated by the generation of reac-tive free radicals, resulting in oxidative stress and protein oxidation[189]. The choice of coating seems to have a direct effect on the invitro assays [190,191]. On the other hand, SPIONs covered with chito-san showed no cytotoxicity [63]. It is also well known that the pres-ence of iron further enhanced the free radical mediated damage.Recently, Stroh et al. confirmed this by the administration of an ironchelator that reduced ROS levels and oxidative stress mediated invitro toxicity [192]. However, it is difficult to compare the toxicityprofile of SPIONs from various studies as the cellular toxicity dependsnot only on the SPIONs' overall size, charge, concentrations, andsolvent used for dispersing SPIONs, but also on the cell types usedand culture conditions.

A few studies have shown that there is little to no toxicity fromSPIONs. For example, SPIONs covered with chitosan were synthesizedand tested on human osteosarcoma cells (MG-63). MTT assay showedno acute adverse effect on these cell lines for concentrations up to0.2 mg/ml with 83% cell viability after 48 h [63].

Other groups have made use of biocompatible and biodegradablelarge organic architectures, such as dendrons and dendrimers,which have been used to stabilize SPIONs and to provide anchoringsites for drug molecules or peptides. G5-PAMAM stabilized SPIONsfunctionalized with folic acid displayed no cytotoxicity in the con-centration range of 0–80 μg mL−1 via XTT assay on KB cells. Despitehaving a negatively charged surface, these nanocarriers could stillpenetrate negatively charged cell membranes [193]. Chang et al.[146] prepared pH-responsive DOX-loaded PAMAM-SPIONs inwhich 65–70% DOX was released after 24 h at pH 5.0 and 37 °C. Inlater studies, they showed no cytotoxicity of SPIONs which were con-jugated with G3-PAMAM-PEG dendrimers containing DOX tested onMCF7 (breast cancer cell line that over-expresses folate receptors)via MTT assay (85%–92% cells remained viable after 4 hour incuba-tion) due most likely to the presence of PEG on the surface of thesenanocarriers. Thermoresponsive DOX-conjugated SPIONs were alsoinvestigated for their in vitro toxicity on HeLa cell lines (human cervixcarcinoma cell line) via MTT assay [152]. It was shown thatnon cross-linked nanocarriers had better drug release profile thancross-linked ones, but that both are cytotoxic at high concentrations(evaluated up to 1.75 μg/mL).

SPIONs coated with phosphonate-type dendron containing PEGwere evaluated in vitro, and showed high cell viability (up to 94%)of human tumoural cell lines (U87) in a trypan blue test. The sameSPIONs were also tested in vivo and showed no adverse effect after in-travenous injection in rats even 3 months post injection [39]. SPIONsstabilized with polyester dendrons containing guanidine peripheries,led to significantly enhanced uptake of these nanocarriers into mouseglioma cells (GL261), in comparison to dendrons with hydroxyl oramine peripheries, and the former showed no signs of nanoparticletoxicity in the cells over 24 h via MTT assay [194].

Many different factors need to be consideredwhen evaluating in vivotoxicity of SPIONs: not only can the sizes of the SPIONs affect circulationtime prior to uptake by variousmacrophages, especially the Küpffer cellsin the liver, but their respective coatings also have to be taken into ac-count. One study suggests that larger size SPIONs that were chargedshowed higher macrophage uptake than those that were smaller andelectrically neutral [103]. Often enough, there are differences in toxicityin vivo based on the type of cells that are targeted. The group ofMahmoudi et al. reported that coated SPIONs were toxic on humanbrain cells at high concentrations (above 2.25 mM), but showed littleto no effect on kidney cells at 2.25 mM [195]. The group of Song et al.has reported that SPIONs coated with poly(vinylpyrrolidone) (PVP),trisodium citrate (TSC) and maleic anhydride (MAH) present very lowtoxicity to the liver, spleen, kidney, and bone marrow tissues of normalwhite rabbits after 2 or 3 days post-administration [38]. Recently, it hasbeen suggested that SPIONs coated with biocompatible material couldbe used for in vivo applications, due to the fact that biodegradable iron

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can be metabolized through various endogenous salvage pathways, andthat the quantities of iron administered during diagnostic imaging aresignificantly less than that stored in the human body. However, the au-thors were warned against repeated administration of the imagingagent which could cause potential iron overload [196]. Some groupshave reported that SPIONs have no severe toxicity in vivo [189], and itwas rationalized by the observations that after the coatings weremetab-olized and/or cleaved by various macrophages in lysosomal compart-ments and endothelial cells, the leftover iron can then be incorporatedinto the hemes of hemoglobin,making it non-cytotoxic in vivo. The latterwas shown to even reverse iron-deficiency in anemia patients [103].

The general consensus in terms of cytotoxicity on cells cultivated invitro seems to point towards a strong heterogeneity between SPIONscontaining various surface coatings. It not only depends on the assaytype being used to determine cell viability or other such toxicologicalbiomarkers, but also the cell types used. These results underline the im-portance of testing specific compounds individually. It is clear thoughthat in clinical dosages for imaging, the amount of SPIONs injected toobtain good contrast was not sufficiently high to confer iron toxicityto the patients or animals. In addition, the nature of the coating usedin these contrast agents can confer better biocompatibility.

6. Applications of SPIONs in cardiovascular theranostics using MRI

Cardiovascular applications represent a major opportunity forSPIONs, and are the most widely studied contrast agents in cardiovas-cular MRI. This is due to a number of advantages that SPIONs haveover other nanoparticles [197] including higher relaxivities, and adetailed understanding of their chemistry to optimize their proper-ties and conjugate appropriate coatings. Some applications ofSPIONs have already been reviewed in [19,22,198,199] as well as in[200–204]. Only the most recent work and progress with these tech-nologies will be discussed here.

6.1. Stroke (neuroinflammation)

In stroke applications, contrastmainly depends on the accumulationof SPIONs in macrophages (especially for USPIONs). By quantifying thenumber of nanoparticles in macrophages, inflammatory activity in thebrain may be derived. However, using the number of nanoparticle as abiomarkerwould require further investigation. For example, a study in-vestigating accumulation of iron in the brain, followingmiddle cerebralartery occlusion inmice [205], showed a correlation between R2 = 1/T2and iron accumulation. However, there was no significant differenceupon treatment. Challenges arise to control the confounding effect offree SPIONs which have not been phagocytized by macrophages andof tissueswith enhanced permeability thatmay also see SPION accumu-lation without accrued inflammation. So-called multi-parametric MRI,using simultaneous quantification of T1, T2 and T2*, may be able to alle-viate these effects. Recent observations show a reduction of T1 and T2but increased T2* values when the particles are internalized [206]. Astudy showcased the importance of these issues in a separate applica-tion [207] and addressed whether SPIONs are captured by the desiredtarget or by some other tissues. This in turn led to significant effortsput into characterizing the relaxivity properties of SPIONs internalizedin cells at different field strengths [8].

While passive accumulation in macrophages remains moreactively studied, active targeting mechanisms have also been contin-uously developed (see Section 4.2 above). Older contributions includeneutrophil-tagged nanoparticles to investigate abscess [208], inwhich the authors extracted human neutrophils. The latter werethen incubated with magnetite to be re-injected in rabbits with arti-ficial abscesses. At 48 h, dark contrast on T2-weighted MRI imageswas observed showing a potential for this active targeting mecha-nism. More recent approaches that do not require the extraction ofhost cells include the development of a P-selectin binding peptide


iron nanoparticles [209]. Following ischemia and reperfusion, endo-thelial activation was shown to be selectively imaged with this parti-cle compared to non-specific agents or between brain regions withand without infarct. Larger sized, so-called microparticles of ironoxide (MPIO), have also been conjugated to monoclonal antibodiesagainst VCAM-1, and validated in a model of acute brain inflam-mation. The signal created by these targeted particles showed areduction in contrast. These particles were also used to target athero-sclerosis more recently in an ApoE mouse model of the disease [198].

6.2. Atherosclerosis/inflammation

Applications to atherosclerosis have been reviewed by Mulderet al. [210] and then later by McAteer et al. [198]. USPIONs accumu-late in plaques over several days [211] due to macrophage activitywas also observed [212,213]. This was confirmed by histological andelectron microscope studies. In mice, a recent study investigatedpeptide-linked USPIONs with affinity for VCAM-1 and apoptosis, aspreviously identified by phage display [214]. In both cases, thesetargeted particles displayed co-localization with the plaque, buteach were shown to report on distinct biological processes.

Passive accumulation of USPIONs in inflamed atheroscleroticplaques is an application that has been translated to humans to eval-uate inflammation but also to select patients at high risk (reviewed in[215]). Recently, in a pilot study in humans, the group of Richards etal. [216] has shown that USPION uptake is correlated with the growthof abdominal aortic aneurysms (AAAs). The usefulness of this diag-nostic tool is potentially quite significant clinically, since this condi-tion is common in men (5% of men over 65 years old in the USA),and acute rupture of AAAs has a high mortality rate of 65% to 85%.

Passive mechanisms were also combined with new approaches toinflammation and the investigation of protease activity using activatableMRI particles in vitro [217]. A ‘stealth’ particle with low relaxivitywas created by stabilization due to steric properties of iron oxide withmPEG. The authors used a protease degradable peptide specific forMMP-9, which is expressed in atherosclerosis, to link the mPEG to theiron oxide cores. Upon specific cleavage by MMPs, iron oxide nano-particles accumulate in particle aggregates, thereby increasing therelaxivity of the sample.

6.3. Stem cells

Research on stem cells is one emerging area of application ofSPIONs [218], due to their small size and innocuous nature. A typicalapplication is to label stem cells with SPIONs and to track them withMRI [16]. Cell tagging methods include simply bathing cells in aSPIONs solution, transfecting, and modifying the SPIONs surface forbinding with specific biomolecules [219].

An ambitious application for SPIONs is to use them to label and trackcell populations. Typical workflow will perform initial non-specificlabeling of cells outside the tissue of interest to re-inject marked cellsin circulation. Due to the high contrast generated by SPIONs and themoderate concentration in cells used, living cell activity can be followedin vivo over long periods (days). These studies then enable the investi-gation of the specific role of stem cells in disease and develop specifictreatment strategies.

SPIONs have been used to understand the cardiac pulmonaryvalve remodeling process [220]. This is an important avenue for treat-ment of congenital heart malformations (Fig. 15). SPIONs were usedto label two types of vascular cells found in the pulmonary artery.Cells were then tracked to assess their migration in agar gel. However,much work remains to be done for their proposed application toengineered-tissue implantation in animals and in humans. Endotheli-al progenitor cells (EPCs) derived from blood in rabbits were labeledwith SPIONs and re-injected in animals following balloon injury of theright common carotid artery [221]. Using MRI, EPCs were co-detected

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Fig. 16. ANX-SPIO MRI signal is reversed by apoptosis treatment. Mice were given DOX(25 mg/kg i.p.) and at the same time implanted with osmotic pumps to deliver A6,10 ng/kg/d, or A6 diluent (CON). LW 1/4 lateral wall (of LV). Cardiac MRI 2 d afterDOX, 6 h after ANX-SPIO injection, in long axis (upper) and short axis (lower). Insetshows magnified septum, where T2* signal loss is much less with A6 treatment.Reprintedwith permission from JohnWiley and Sons, Magnetic Resonance inMedicine,2011, 66, 1152–1162 [230].

Fig. 15. Map derived from SPION-labeled and unlabeled vascular smooth muscle cells(VSMCs) in 4% agar gel samples after 13 days of incubation. Brighter contrast wasexhibited by the SPION-labeled cell-encapsulated gel sample.Reprinted with permission from John Wiley and Sons, NMR in Biomedicine, 2012, 25,410–417 [220].


with the injury area in early weeks, following injury displaying a po-tential for following progenitor cells homing after systemic injections.Direct injection in the myocardium was also investigated in vivo inpigs [222]. More recently in rats, mesenchymal stem cells (MSCs)were tagged with SPIONs, and then injected in the hearts of animalsthat were previously cryo-injured [223]. The signal decrease in MRIcould then be observed for up to 10 weeks. Furthermore, animalsinjected with the marked cells had their heart function improve asassessed by left ventricular ejection fraction. The latter confirmedthe preserved function of MSCs following uptake of the SPIONs.

Toxicity and uptake by cells remain active areas of research, andrecently SPIONs coated with unfractionated heparin [224] havebeen investigated. These particles showed increased uptake whencompared to dextran-coated SPIONs, and they were also shown topreserve the differentiation and proliferation potential of humanmesenchymal stem cells. Images in mice were found to have identifi-able contrast from SPIONs for a period as long as a month.

While these studies used MRI contrast to monitor the targetedcells of interest, a recent study has shown the limitations of theseapproaches as a proxy for cell activity [225]. SPION-labeled stemcells were injected in rat hearts after myocardial infarction and anenhancement of function was observed. However, after 8 weeks, thestem cells had disappeared while the effect of SPIONs was still ob-servable with MRI. This was due to the absorption of SPIONs by mac-rophages, not by the stem cells. MRI allowed tracking stem cellslabeled by SPIONs and grafted in the myocardium of rats and mice,but it could not monitor cell survival. A solution to this issue was pro-posed recently in which reporter genes (such as luciferase) were usedto label stem cells. It then required a dual imaging approach combin-ing MRI with bioluminescence imaging (BLI) [226]. This approachwas applied to myocardial infarct remodeling in rats, with stem cellgrafting. In 7 out of 9 hearts two months after grafting, BLI signalincreased, while in the remaining 2 hearts, it disappeared. This corre-lated with MRI and histological findings of successful grafting in the 7out of 9 hearts.

6.4. Cardiac viability/function

At a clinical level, imaging with SPIONs has not shown diagnosticbenefits for patients with acute myocardial infarction compared togadolinium [227]. In a small cohort, the late gadolinium enhanced(LGE) peri-infarct zone was shown to increase, while SPIONs contrastwas left unchanged in a significant portion of patients. The contrastalways decreased over an area that was smaller than that of the LGE


zone. This is in agreement with earlier studies using USPIONs inpigs [228], in which no change in signal intensity was observed inthe infarct zone. Meanwhile an accumulation of the particles wasseen in the liver as expected. However, a separate study in rats andat high field (7 T) indicated that Gd contrast may overestimate thesize of the infarct lesion when compared to histology [229]. UsingSPIONs as a vascular agent, a viable peri-infarct region that remainedperfused was identified.

In a nice example of active targeting in the myocardium, Dash andhis colleagues [230] studied cardiac cell apoptosis (Fig. 16). Apoptosisplays an important role in myocardial diseases. At the initial stage,apoptosis can be reversible and thus a target for therapeutic inter-vention. The authors combined SPIONs with exogenous annexin-V(ANX) which is a protein that binds to membrane phosphatidylserineexpressed in early apoptosis. Injection in mice hearts in vivo allowedthe quantification of apoptosis and its later disappearance after treat-ment by a suitable drug was observed.

6.5. Magnetic particle imaging for angiography

Although not an MRI application, magnetic particle imaging (MPI)is a new tomographic imaging application of SPIONs worth mention-ing for angiography [231]. MPI achieves images of SPION distributionwith sub-mm spatial resolution andmillisecond time resolution. It re-quires different optimizations of SPIONs properties compared to MRI,as their relaxation needs to be tuned to the excitation frequency[232].

7. Conclusions

In recent years, there has been a considerable effort devoted to thedevelopment of synthetic strategies to assemble SPIONs for applica-tions in MRI, an active area of interest for both biomedical and phar-maceutical industries. Various synthetic methodologies now existwhich can help to achieve tunable sizes of SPIONs, however surfacefunctionalizations and toxicity have been issues which have limited

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their use in clinical settings. It is now clear that SPIONs could beeasily uptaken and exocytosed by cells, and effectively metabolized.Bioretention of SPIONs is considered safe, and active targeting byfunctionalized SPIONs has been the most popular choice as advancedMRI contrast agents. The use of SPIONs in the field of cardiovasculartheranostics is increasing, and has shown great promise. However,applications of SPIONs in cellular imaging, stem cells and macrophagemigration are still in a premature stage relative to their use as clinicalcontrast agents. In this review, an effort was made to address andbring together some aspects, if not all, of i) synthetic strategies to ob-tain SPIONs of desired size, ii) surface functionalization, iii) the eval-uation of their magnetic properties, and iv) in vitro and in vivoapplications in cardiovascular research. One strategy to increase theefficacy of SPIONs is by either optimizing the magnetization satura-tion values or by optimizing the in vivo compartmentalization withincells. In vivo compartmentalization influences the cells to retain highconcentration of endocytosed SPIONs in endosomes/lysosomes. Theendocytosed SPIONs generate large field inhomogenities which great-ly enhance R2/R2* effects. This phenomenon has been extensivelyused in cell tracking techniques. The other strategy is to use largersize of SPIONs, as the larger the size of SPIONs, the larger is the mag-netic saturation. However, the increased size might not be favorablefor pharmacokinetics and biodistribution, so the goal in this case isto make use of small-sized SPIONs with high magnetic saturation.To address these issues, various synthetic challenges have to be over-come to generate efficient and specific SPIONs. Currently, there are nosystematic studies which provide information about the influence ofparticle size and their magnetic properties, imaging voxel and dataacquisition parameters, and number of passive or active targetingmoieties affecting their properties. Therefore, there is an urgentneed to establish a structure–property relationship between synthet-ic strategies to obtain desired size of SPIONs (core and hydrodynamicsize), nature of the surface coatings (passive and active targeting moi-eties) and their magnetic properties.

Acknowledgments

Wewould like to thank theNatural Sciences and EngineeringCouncilof Canada (NSERC), Fonds de Recherche Nature et Technologie Québec(FQRNT), the Canadian Institute of Health Research (CIHR), and theCenter for Self-Assembled Chemical Structures (CSACS) for financialsupport.

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Superparamagnetic iron oxide based nanoprobes for imaging and theranostics

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