Advanced Drug Delivery Reviews 65 (2013) 732–743

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Advanced Drug Delivery Reviews

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New forms of superparamagnetic nanoparticles forbiomedical applications☆

Chenjie Xu a,b,⁎, Shouheng Sun a,⁎a Department of Chemistry, Brown University, Providence, RI 02912, USAb Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457, Singapore

☆ This review is part of the Advanced Drug Delivery Revnanoparticle platforms”.⁎ Corresponding authors at: Department of Chemis

dence, RI 02912, USA.E-mail addresses: cjxu@ntu.edu.sg (C. Xu), ssun@bro

0169-409X/$ – see front matter © 2012 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.addr.2012.10.008

a b s t r a c t

a r t i c l e i n f o

Article history:Accepted 3 October 2012Available online 2 November 2012

Keywords:Magnetic nanoparticlesSynthesisModificationBiomedical applicationDrug deliveryMolecular imaging

Magnetic nanoparticles (MNPs) based on iron oxide, especially magnetite (Fe3O4), have been explored as sensi-tive probes for magnetic resonance imaging and therapeutic applications. Such application potentials plus theneed to achieve high efficiency and sensitivity have motivated the search for new forms of superparamagneticNPs with additional chemical and physical functionalities. This review summarizes the latest development ofhigh moment MNPs, multifunctional MNPs, and porous hollow MNPs for biosensing, molecular imaging, anddrug delivery applications.

© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7322. Synthesis of MNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 733

2.1. MNPs with high magnetic moment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7332.1.1. Structure controlled ferrite MNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7332.1.2. Metallic MNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 733

2.2. Multifunctional MNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7342.2.1. Molecular functionalization of MNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7342.2.2. Heterogeneous MNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7342.2.3. Core/shell MNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735

2.3. Hollow MNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7363. Modification and functionalization of MNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7374. Biomedical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738

4.1. High magnetic moment MNPs for biosensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7384.2. Molecular imaging with multifunctional MNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738

4.2.1. Tumor imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7384.2.2. Cell tracking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738

4.3. Drug delivery with hollow MNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7395. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 740Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741

iews theme issue on “Inorganic

try, Brown University, Provi-

wn.edu (S. Sun).

l rights reserved.

1. Introduction

Nanomedicine is an emerging field that provides novel approachesto address the ever-increasing challenges in conventional medicine [1].Magnetic nanoparticles (MNPs), due to their comparable sizes to biolog-ical molecules and their unique physical properties, have been exploredextensively for medical applications. Among various functional MNPs

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studied, superparamagnetic nanoparticles (NPs) are especially promisingas contrast enhancement agents for magnetic resonance imaging (MRI)and also as a platform for drug delivery. These MNPs, represented bymagnetite (Fe3O4) NPs, are oftenmade in sizes smaller than 20 nm in di-ameter and their magnetization directions are subject to thermal fluctu-ation at room or biological temperatures. Therefore, without an externalmagnetic field, their overall magnetization value is randomized to zero.Such fluctuation in magnetization direction minimizes the magnetic in-teractions between any two NP in the dispersion, making the dispersionstable in physiological solutions and facilitating NP coupling with biolog-ical agents. Once exposed to an external magnetic field, these MNPs canalign along the field direction, achieving magnetic saturation at a magni-tude that far exceeds that from any of the known biological entities. Thisunique property of MNPs allows not only the detection of the MNP-containing biological samples, but also the manipulation of these biolog-ical samples with an external magnetic field [2,3].

Conventional MNPs like iron oxide NPs have been demonstratedsuccessfully in biomedical applications both in vitro (magnetic separa-tion, magnetic sensing/detection, and magnetic transfection) and invivo (MRI, targeted drug delivery, and tissue engineering). However,limitations exist including low magnetic moment [4], low sensitivityin MRI diagnosis [5], and low cargo capacity [6]. For example, one ofthe commercial iron oxide NP products, Feridex IV, has r2 relaxivity of98.3 mM−1S−1 under normal MRI conditions, which requires ~35 mgiron for imaging liver of an adult with 60 kg of bodyweight [7]. Further-more, due to the wide distribution of both size and magnetic moment,traditional MNPs tend to generate large magnetic field gradients thatcause dephasing and signal loss in and around the MNP-concentratedregions. This makes it difficult to distinguish MNP-targeted area fromother “field perturbers” such as air bubbles and blood clots [8]. Whenacting as drug carriers, traditional MNPs offer only one chemical surfacefor drug loading and targeting-molecular conjugation, lowering the ca-pacity of either targeting agent or drugmolecular (usually less than 10%byweight) aswell as the targeting and therapeutic efficiency [9–11]. Toovercome the limitations of mono-functional traditional MNPs, newforms of MNPs with high magnetic moment, multifunctionality, andhigh drug loading have been actively pursued [4,12–15]. Here, we sum-marize the recent developments in the synthesis of newMNPs for sen-sitive biomedical applications, including spinel structured ferrite NPsand metallic NPs with high magnetic moments, multifunctional MNPsfor controlled coupling of different biological agents, and porous hollowMNPs with increased capacity for drug loading.

2. Synthesis of MNPs

Traditional iron oxide based MNPs are synthesized by co-precipitation of ferrous and ferric ions in alkaline media in the pres-ence of surfactants (e.g. Dextran). In the synthesis, other metal saltscan be added to form ferrite MFe2O4. Despite the versatility of thesynthesis, iron oxide NPs made from this method do not have thedesired control on the morphology and magnetic moment. Monodis-perse iron oxide NPs are now often produced by high-temperature re-ductive decomposition of metal salt or organometallic precursors inorganic solutions [16,17]. In this method, a burst nucleation event firstoccurs when the concentration of metal precursors quickly increasesto the critical saturation pointwithout further formation of nuclei after-wards [18]. The remaining precursors deposit on the pre-formed nuclei,forming NPs with narrow size distribution. The method produces notonly monodisperse ferrite NPs with high magnetic moment, but also aseries of new MNPs with multifuctionalities.

2.1. MNPs with high magnetic moment

2.1.1. Structure controlled ferrite MNPsFerrite MFe2O4 or MO·Fe2O3, with M being the common divalent

transition metal cations (Mg2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+…) is

a class of materials that has spinel structure with oxygen formingcubic close packing and Fe3+ and M2+ occupying the octahedral andtetrahedral interstitial sites. When Fe3+ takes octahedral sites andM2+ in tetrahedral sites, the structure is said to have a normal spinelstructure. The MFe2O4 can also adopt an inverse spinel structure inwhich half of Fe3+ exchange siteswithM2+. In this inverse spinel struc-ture, the spins in two Fe3+ located in tetrahedral and octahedral sitesare anti-ferromagnetically coupled and cancel each other. Therefore,magnetic moments of the inverse spinel structured MFe2O4 are depen-dent on the un-paired d-electrons fromM2+ and their overall values arereduced by anti-ferromagnetic coupling between Fe3+. When M isFe2+, Co2+, or Ni2+, MFe2O4 NPs have an inverse spinel structure [19].As Fe2+ has larger magnetic moment (4 μB) than Co2+ (3 μB) and Ni2+

(2 μB), the mass magnetization of Fe3O4 (101 emu/g) is larger than thatof CoFe2O4 (99 emu/g) andNiFe2O4 (85 emu/g) [20].WhenM isMn2+,however, nanostructured MnFe2O4 can adopt a normal spinel structureinwhich Fe3+ take octahedral sites and there has no anti-ferromagneticcoupling between Fe3+. The structure provides a higher mass mag-netization than the inverse spinel structured MFe2O4 [20]. Further-more, ZnFe2O4 NPs show a ferromagnetically mixed spinel state(Zn1−xFexO4)A[Fe2−xZnO4]B [21]. Addition of ZnFe2O4 into an in-verse spinel structure (e.g. Fe3O4) can significantly increase the netmag-netic moment [22]. This was demonstrated in the non-stoichiometricZnxFe1−xO·Fe2O3 NPs (x=0.14, 0.26, 0.34 and 0.76) that were synthe-sized by thermal decomposition of diethyl zinc and iron acetylacetonate(Fe(acac)3) [23]. When x was controlled from 0 to 0.34, r2 values in-creased systematically, achieving near 3 fold enhancement from 9.5 to34.7 mM−1 s−1 (measured in a 0.55 T field). Recently the synthesiswas further improved by replacing the unstable and pyrophoric diethylzinc with zinc chloride, ZnCl2 and reacting ZnCl2 with Fe(acac)3 inthe presence of oleic acid, oleylamine, and octyl ether to produceZnxFe1−xO·Fe2O3 [24]. When x in ZnxFe1−xO·Fe2O3 NPs was from0 to 0.1, 0.2, 0.3, and 0.4, r2 values reached from 276 to 397, 466,568, and 687 mM−1 s−1 (measured in a 4.5 T field) respectively.The effect of Zn2+ doping into MnFe2O4 NPs was also studied [24].This led to the formation of Zn0.4Mn0.6Fe2O4 NPs with their relaxivityreaching 860 mM−1 s−1, the highest value ever reported. Table 1summarizes some ferrite MNPs produced recently and their magnet-ic data for bio-imaging applications.

2.1.2. Metallic MNPsMNPs based on transition metals of Fe, Co and Ni have much high

magneticmoments than their oxide counterparts [25]. They are normallyprepared by thermal decomposition or reduction of organometallic pre-cursors. For example, FeNPswere synthesized by thermal decompositionof Fe(CO)5 at 180 °C in the presence of oleylamine in octadecene [26], orin the presence of oleylamine and hexadecylammonium chloride inoctadecene [27], or at 170 °C in the presence of polyisobutene in decalinunder N2 [28]. Similarly, Co NPs were prepared by thermal decomposi-tion of Co2(CO)8 [29–32]. Reduction of Fe[N(SiMe3)2]2 with H2 in thepresence of surfactants under 150 °C led to high moment Fe nanocubes[33]. Similarly, high-temperature reduction of [Co(η3-C8H13)(η4-C8H12)]and Ni(cycloocta-1,5-diene)2 gave Co and Ni nanorods respectively[34,35].

Magnetic alloy NPs can be produced by the combination of thermaldecomposition and reduction of metal precursors. The key to the suc-cessful synthesis is to control the reaction condition so that two differentmetals can nucleate and grow into an alloy structure [36–38]. For exam-ple, FeCo NPs were synthesized by reduction of Fe(acac)3 and Co(acac)2in the presence of oleic acid, oleylamine and 1,2-hexadecanediol under agas mixture of 93% Ar+7% H2 at 300 °C or were made by sodium boro-hydride reduction of ferrous and cobalt salts [39,40]. FePt (or FeAu) NPswere synthesized by the thermal decomposition of Fe(CO)5 and the re-duction of Pt(acac)2 (or AuAc3) by 1,2-hexadecanediol in the presenceof oleic acid and oleylamine [41,42]. To stabilize high moment metallicNPs for biological applications, some robust coating strategies have

Table 1Survey of MNPs, their surface coating, magnetic moments and their relaxivities for MRI under a permanent magnetic field B0.

MNPs Core material Core diameter (nm) Surface coating Magnetic moment (emu/g) B0 (T) Relaxivity r2 (mM−1 s−1) Reference

Feridex® Fe3O4, γ-Fe2O3 4.96 Dextran 45 1.5 120 [44]Resovist® Fe3O4 4 Carboxy-dextran 1.5 186 [45]Combidex® Fe3O4 5.85 Dextran 61 1.5 65 [45]Iron oxide Fe3O4 4 DMSA 25 1.5 78 [20]

6 43 1069 80 130

12 101 218Mn–ferrite MnFe2O4 6 DMSA 68 1.5 208

9 98 26512 110 358

Co–ferrite CoFe2O4 12 DMSA 99 1.5 172Ni–ferrite NiFe2O4 12 DMSA 85 1.5 152Zn–iron oxide Zn0Fe1O·Fe2O3 4.6 DSPE-PEG 19.8 0.55 9.5 [23]

Zn0.14Fe0.86O·Fe2O3 4.5 26.8 14.5Zn0.26Fe0.74O·Fe2O3 4.5 43.1 22.4Zn0.34Fe0.66O·Fe2O3 4.9 54.1 34.7Zn0.76Fe0.24O·Fe2O3 4.5 30.0 7.4

(ZnxFe1−x)Fe2O4 Zn0Fe1Fe2O4 15 DMSA 114 4.5 276 [24]Zn0.1Fe0.9Fe2O4 15 126 397Zn0.2Fe0.8Fe2O4 15 140 466Zn0.3Fe0.7Fe2O4 15 152 568Zn0.4Fe0.6Fe2O4 15 161 687Zn0.8Fe0.2Fe2O4 15 115 307

(ZnxMn1−x)Fe2O4 Zn0Mn1Fe2O4 15 DMSA 125 4.5 422Zn0.1Mn0.9Fe2O4 15 140 516Zn0.2Mn0.8Fe2O4 15 154 637Zn0.3Fe0.7Fe2O4 15 166 754Zn0.4Fe0.6Fe2O4 15 175 860Zn0.8Fe0.2Fe2O4 15 137 388

FeCo/C Fe12Co88 4 Phospholipid-PEG 162 1.5 185 [43]Fe40Co60 7 215 644

Fe α-Fe 10 PEG 70 1.5 129 [46]Fe Amorphous Fe 15 OAm-PEG 90.6 3 67 [27]Fe α-Fe 15 OAm-PEG 164 3 220

Notes: DSPE-PEG: 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG, PEG MW=5 kD); DMSA: 2,3-dimercaptosuccinic acid;OAm-PEG: oleylamine-α,ω-bis(2-carboxyethyl)poly(ethylene glycol).

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been applied. For example, a layer of graphitic shellwas coated onto FeCoNPs to protect their highmagneticmoment from fast decay [36,43]. Crys-talline Fe3O4 shell was also used to protect metallic Fe NPs as demon-strated in the Fe/Fe3O4 NPs through the controlled oxidation of theas-synthesized Fe NPs with (CH3)3NO [26]. Metallic NPs that have beenstudied as contrast agents for MRI are listed in Table 1.

2.2. Multifunctional MNPs

Iron oxide MNPs are already multifunctional. Due to their responseto external magnetic field, they have been studied as contrast agentfor MRI and as magnetic heating element for magnetic fluid hyperther-mia. However, such MNPs only have one kind of chemical surface. Tocouple these NPs with different biological agents and drug molecules,MNPs with different chemical surfaces are preferred [47]. This can beachieved through molecular functionalization of existing MNPs, designof multi-componentMNPs (heterogeneous MNPs), or co-encapsulationof MNPs with other functional components in a matrix (core/shellMNPs).

2.2.1. Molecular functionalization of MNPsConjugation of other functional molecules onto the surface of

MNPs is an easy approach to achieve multifunctionality. This strate-gy is cost-effective, time-efficient, and easy to adapt for other circum-stances. The conjugation can be realized by conventional couplingchemistry, “click” chemistry, and chelating coordination [48].

Conventional coupling chemistry uses the well-known reactionamong \NH2, \SH, and \COOH to functionalize MNPs. For exam-ple, NH2-coated MNPs can be coupled with functional moleculecontaining \COOH via an amide bond formed through common

EDC/NHS or sulfo-NHS coupling chemistry. Molecule bearing \SH canbe grafted onto the NH2-coated MNPs via a heterobifunctional linkersuch as succinimidyl iodoacetate, N-succinimidyl-3-(2-pyridyldithio)propionate, or succinimidyl-4-(N-meleiminomethyl)cyclohexane-1-carboxylate.

“Click” chemistry is a copper-catalyzed cyclo-addition of azide-alkyneand offers an alternative strategy for quick and robust coupling of NPswith other functional molecules [49]. The reaction can be performed inrelativelymild conditions and is highly specific. A representative exampleis the functionalization of cross-linked iron oxide MNPs (CLIO) with 18F,in which CLIO functionalized with azide moiety were reacted with3-(2-(2-(2-[18F]-Fluoroethoxy)ethoxy)ethoxy)prop-1-yne (18F-PEG3),producing 18F-CLIO with a decay-corrected yield of 58% [50].

Chelating coordination is often used to conjugatemetal ions toMNPs.For example, 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid(DOPA) pre-conjugated on Fe3O4 NPs through amide bond couldbind 64Cu for additional positron emission tomography (PET) imag-ing capability [51]. Dithiocarbamate-bisphosphonate, (dtbp)2, wasalso studied as both surfactant for CLIO and chelating group for 64Cu[52]. Through chelating bond formation between \COO− and Pt, cis-platin could be conjugated onto the surface of Au–Fe3O4 dumbbellMNPs for controlled platin delivery and release [53].

2.2.2. Heterogeneous MNPsHeterogeneousMNPs refer to those containing two ormore different

functional units within one nanostructure, such as Au–Fe3O4, FePt–CdS,and Fe2O3–carbon nanotube MNPs. In such a heterogeneous NP struc-ture, each NP unit exhibits its unique magnetic, optical, or electronicproperties and provides its distinct surface for selective chemical modifi-cation [54–56].

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Au–Fe3O4 dumbbell-like MNPs were prepared through seed-mediated growth in which Au NPs were used as seeds and Fe3O4 MNPswere grown on Au by decomposition of Fe(CO)5 followed by oxidationof Fe [53,57]. These dumbbell-like MNPs preserve the optical propertyof Au NPs (plasmonic absorption at ~530 nm) and magnetic propertyof Fe3O4 MNPs (saturation magnetic moment at 80 emu/g). The synthe-sis has been extended to prepare noble metal–metal oxide dumbbellMNPs with Fe3O4 MNPs grown over the noble metal (Au, Ag, Pt, orAuAg) NPs (Fig. 1A) [58]. In these dumbbell MNPs, the size of noblemetal NPs was controlled in pre-synthesis and the size of Fe3O4 MNPswas tuned through the concentration of Fe(CO)5 during the reaction.

Alternatively, noble metal could be grown on the pre-synthesizedMNPs [59–61]. For example, Ag–Fe3O4 MNPs or Ag-hollow Fe3O4

MNPs were made by controlled nucleation of Ag on the pre-formedFe3O4 MNPs or hollow Fe3O4 MNPs [59,62]. In the synthesis, theas-prepared MNPs dispersed in organic solution and AgNO3 dissolvedin water were mixed and agitated by ultrasonication. The sonicationprovided the energy required for the formation of a micro emulsionwith Fe3O4 MNPs assembling at the liquid/liquid interface (Fig. 1B).Fe(II) ions on MNPs acted as a catalytic center for the reduction ofAg+ and nucleation/growth of Ag NPs. The partial exposure of MNPsto the aqueous phase caused the formation of Ag–Fe3O4 MNPs, whichshowed the typical plasmonic absorption of Ag NPs and magnetic be-havior of Fe3O4 MNPs. Slightly different from this two-phase reaction,hollow Fe3O4–Ag MNPs were prepared (Fig. 1C) [60]. The synthesisstarted from Fe MNPs coated with amorphous iron oxide, followed bythe reduction of Ag on the shell by oleylamine. As iron oxide shell wasamorphous, the mechanical stress caused by the lattice mismatch be-tween iron oxide and Ag was minimized, which led to a low interfacialenergy between iron oxide and Ag. Following the Ag deposition, FeMNPs were oxidized to the hollow Fe3O4 MNPs through the Kirkendalleffect [63].

In addition to the noble metal–metal oxide heterogeneous MNPs,semiconductor–metal alloy, semiconductor–metal oxide MNPs, and car-bon nanotube–metal oxide complex have also been reported [47,64,65].

Fig. 1. A) Schematic illustration of the growth of metal-oxides dumbbell MNPs on pre-madeages of a) Au–Fe3O4, b) Ag–Fe3O4, and c) AuAg–Fe3O4 MNPs. Reproduced with permission frMNPs in aqueous phase. Reproduced with permission from reference [62]. C) Schematic illuswith permission from reference [60]. D) Schematic illustration of the growth of FePt–CdS d

For example, FePt–CdS and FePt–CdSe MNPs were fabricated throughtwo-stage reaction (Fig. 1D) [66,67]. First, an amorphous CdS or CdSelayer was grown on the surface of FePt at a low reaction temperature toachieve a core/shell structure. Subsequently, the temperature was raisedto crystallize the CdS/CdSe phase. Because of the difference in phase tran-sition temperatures between FePt and CdS/CdSe, the CdS/CdSe compo-nents melt and induced their dewetting from FePt cores, resulting inthe heterodimeric MNPs. In the case of carbon nanotube–iron oxide(CNT–IO), the CNT nucleated from CO catalytically on iron clusters [68].The resulted CNT–Fe was oxidized in air to CNT–IO.

We shouldnote that thepresence of heterojunctionbetweendifferentcomponentsmodifies thematerial properties at both sides. This is causedbymany factors including surface reconstruction around the junction, lat-tice mismatch-induced crystal strain, and electron interaction/transferacross the interface, which are still under intensive investigation. Insome cases, the original properties are affected negatively. For example,the quantum yield of Fe3O4–CdSe MNPs was only 38% compared withtheir single-core counterparts (CdSe) [69]. In Au–Fe3O4 MNPs, along theincrease of Au core's size (0 to 3 to 8 nm), the r2 relaxivity of the Fe3O4

core deteriorates (121 to 114 to 105 s−1 mM−1) [70]. In another hand,the original properties could be enhanced. For example, the incorporationof Ag core (13.5 nm) onto Fe3O4MNPs (7–10 nm) increased the coerciv-ity of the MNPs from 300 Oe (Fe3O4) to 500 Oe (Ag–Fe3O4), and alsochanged their magnetic properties from superparamagnetic (Fe3O4) toferromagnetic (Ag–Fe3O4) at room temperature [71]. In Ag–CoFe2O4

MNPs, their magneto-optical Faraday rotation was enhanced by nearlyan order of magnitude at 633 nm compared to the monomer (CoFe2O4

MNPs) [61]. Table 2 lists some common heterogeneous MNPs studiedfor biomedical applications.

2.2.3. Core/shell MNPsMultifunctional MNPs can be further prepared by encapsulating

MNPs into a robust matrix to form a core/shell structure. There havebeen a number of examples reporting on the synthesis of core/shellMNPs using gold, silica, zinc oxide [75], polymer [76], or liposomes

noble metal NPs and high-resolution transmission electron microscope (HRTEM) im-om reference [58]. B) Schematic illustration of the growth of Ag-hollow Fe3O4 dumbbelltration of the growth of Ag-hollow Fe3O4 dumbbell MNPs in organic phase. Reproducedumbbell MNPs. Reproduced with permission from reference [66].

736 C. Xu, S. Sun / Advanced Drug Delivery Reviews 65 (2013) 732–743

[77] as the matrix. The robust shell not only protects the magneticcores, but also prevents the direct contact of magnetic core withother sensitive biological agents. Here, we focus on the gold and silicaencapsulation.

Gold (Au) encapsulation is advantageous in term of their stabil-ity, biocompatibility, and convenience for further functionalization[78]. The Au shell could be deposited on Fe3O4 MNPs through grad-ually reducing HAuCl4 on Fe3O4 MNPs by oleylamine [79]. After ini-tial coating, the surface of MNPs was treated with sodium citrateand cetyltrimethylammonium bromide (CTAB) for the dispersion inaqueous solution. Such aqueous-soluble MNPs then served as seedsto grow multiple layers of Au or Ag on the surface. The change ofshell thickness allowed the tuning of plasmonic properties of thecore/shell MNPs to be either red-shifted (to 560 nm with more Aucoating) or blue-shifted (to 501 nm with more Ag coating). Aushell could also be formed over MNPs by simultaneously activatingMNPs and Au NPs in a hot solution [80]. Specifically, Au NPs (2 nm)coated with alkanethiolate were mixed with MNPs protected witholeylamine/oleate. At an elevated temperature (149 °C), alkanethiolatebonding to Auwasweakened and small Au attached toMNPs, forming acore/shell structure with thiolate re-capturing the enlarged Au surface.Compared with the direct deposition of Au shell on MNPs, thisthermally-driven procedure is time-efficient, cost-effective, andeasy to apply to control the shell thickness. More recently, Au shellwas grown on poly-L-histidine coated iron oxide NPs in an aqueousphase [81]. The core/shell structure showed both strong absorptionin near infra-red spectrum and sensitive MRI response, allowing formultimodality imaging.

Silica coating is another popular choice to make MNPs stableand multifunctional. By simply hydrolyzing silica precursors(e.g. tetraethylorthosilicate (TEOS)) under the basic solution, a uniformand thickness-controllable silica shell can be obtained. Silica formedthrough this approach (Sol–gel approach) is usually amorphous andhas strong affinity to MNPs [82–84]. For example, quantum dots (QDs)and iron oxideNPs had been co-encapsulated inside the silica NPs to pre-serve magnetic property of iron oxide NPs and optical property of QDs[85], enabling the magnetic manipulation with real-time fluorescencemicroscope imaging [86]. This silica shell can also act as a carrier for

Table 2Heterogeneous MNPs studied for potential biomedical applications.

Name Functional components Size of components (nm)

Au–Fe3O4 Au 3, 5, 6, 8Fe3O4 12, 18, 20, 25

Ag–Fe3O4 Ag 2–15, 13.5Fe3O4 9, 12, 13

AuAg–Fe3O4 AuAg 6Fe3O4 10

Ag–CoFe2O4 Ag 6CoFe2O4 14

Ag-hollow Fe3O4 Ag 4–8, 4Hollow Fe3O4 5–10, 12

Cu-hollow Fe3O4 Cu 17Hollow Fe3O4 17

TiO2–γ-Fe2O3 TiO2 Length: 50–70; 18Thickness: 5–6

γ-Fe2O3 5.6, 11.3–8.1CdSe–Fe3O4 CdSe 2–4

Fe3O4 4XS (X=Zn, Cd, Hg)–γ-Fe2O3 XS (X=Zn, Cd, Hg) 4.6

γ-Fe2O3 8Carbon nanotube–Fe2O3 Carbon nanotube Diameter: 1

Fe2O3 2–5FePt–Au Au 10

FePt 6FePt–CdS FePt 3

CdS 5FePt–CdSe FePt 3

CdSe 6–8

anticancer drugs (e.g. paclitaxel) and fluorescentmolecules (e.g. fluores-cein isothiocyanate (FITC)) [87]. By simply mixing FITC modifiedaminopropyltriethyoxysilanewith silica precursor (TEOS) during the en-capsulation step, FITC was incorporated onto the surface of MNPs. Theanticancer drugs were inserted into the porous silica matrix throughsoaking MNPs in a concentrated drug solution in dimethylsulfoxide(DMSO).

Thesemodifications as discussed above can allowMNPs to have exot-ic properties such as plasmonic resonance and enhanced chemostability.However, it should be noted that these are achieved at the price of in-creased distance between the superparamagnetic core and biological en-vironment (water molecules mostly), which might subsequently resultin reduced sensitivity in MRI. The issues have been discussedmore thor-oughly in a recent review by Hyeon et al. [88].

2.3. Hollow MNPs

As a potential drug-delivery tool, MNPs offer the possibility ofbeing directed toward a specific target and eventually remaining lo-calized by means of an applied magnetic field. Research on the syn-thesis and modification of MNPs has enabled the further studies onMNP biocompatibility, chemical stability, uniformity, and controllablecirculation in vivo. And emerging reports also provide solid evidencefor the effectiveness of the MNP-based delivery system. However,limited by the high density of the inorganic core and the necessarycoating for MNP stabilization, a drug in the conjugates can only occu-py a very small mass percentage [6]. One solution is to use hollowMNPs that have a magnetic shell and void core. In this case, drugscan be loaded both outside and inside of the MNPs. Considering thebiocompatibility requirements, the ideal candidates are Fe3O4 hollowMNPs (HMNPs) [63,89], Fe hollow nanoframe [90], MnxFe3−xO4 hol-low nanotube [91], Fe3O4/ZnS HMNPs [92], and porous Fe3O4 orFe3O4–SiO2 double layer hollow nanorods [93,94].

Fe3O4 HMNPs were synthesized through controlled oxidation ofcore/shell structured Fe/Fe3O4 MNPs by an oxygen transfer agent(CH3)3NO (Fig. 2A) [63]. Core/shell Fe/Fe3O4 MNPs were obtainedby high-temperature solution-phase decomposition of Fe(CO)5 andair oxidation of the amorphous Fe MNPs at room temperature [26].

Biomedical applications Reference

Optical imaging, MRI, magnetic manipulation, and platin delivery [53,58,70]

Two photon fluorescence imaging, and magnetic manipulation [58,71,72]

[58]

[61]

[60,62]

[60]

Magnetically induced hyperthermia; magnetic induced targeting;and photodynamic therapy

[64,73]

Fluorescent imaging and magnetic manipulation [69]

[65]

MRI and near infra-red mapping [68]

Biological detection, MRI, and optical signal enhancing [74]

[67]

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During the controlled oxidation process, Fe diffuses outward at afaster rate than oxygen from (CH3)3NO does inward, which pro-duces the Fe3O4 at the metal–oxide interface rather than the interi-or of the core (Kirkendall effect). With different mechanism (acidetching vs. Kirkendall effect), Fe3O4 NPs can evolve from solidMNPs to uniform HMNPs under the continuous heating in the pres-ence of trioctylphosphine oxide (TOPO) and alkylphosphonic acid(Fig. 2B) [89]. In the heating process, alkylphosphonic acid coordi-nated to the metal cations on the surface of MNPs and formediron–phosphonate complexes. The Fe hollow nanoframes were pro-duced from the thermal decomposition of Fe(II)–stearate complexin the presence of sodium oleate and oleic acid (Fig. 2C) [90]. Re-cently, corrosion-aided Ostwald Ripening was used to preparetwo-component HMNPs, Fe3O4/ZnS HMNPs (Fig. 2D) [92]. In thissynthesis, FeS NPs were prepared first and then dispersed in theaqueous mixture containing Zn(acac)2, poly(vinylpyrrolidone), am-monium nitrate, and glycol. After reacting at 150 °C for 10 h, thesuperparamagnetic fluorescent Fe3O4/ZnS HMNPs were obtained. Po-rous Fe3O4 hollow nanorods were prepared a wrap–bake–peel processfrom akagenite (beta-FeOOH) nanorods (Fig. 2E) [93]. In this synthesis,β-FeOOHnanorodswere prepared as a template to deposit a layer of sil-ica precursor, followed by calcinations in air and reduction under thehydrogen/argon flow to transform the inner iron oxide to Fe3O4. The sil-ica removal provided porous hollow Fe3O4 nanorods [93].Without silicaremoval, it became Fe3O4–SiO2 double layer hollow nanorods [94].

Fig. 2. Transmission electron microscope (TEM) image of A) 13 nm Fe3O4 HMNPs. Reproducepermission from reference [90]. C) Cubic α-Fe2O3. Reproduced with permission from refE) Wrap–bake–peel process to obtain nanocapsules from akagenite. Reproduced with perm

3. Modification and functionalization of MNPs

For MNPs, the main forces affecting their stability in solution areattractive van der Waals forces, repulsive double layer, and stericinteractions [95]. Such forces have been extensively discussed inthe literature [96,97] and are out of scope of this review. There arevarious kinds of materials that can act as dispersants for MNPs.The commonly chosen ones include 2,3-dimercaptosuccinic acids,catechol derivatives, dendrimers, polysaccharides, cellulose, chito-san, PEG, and PVP (more details could be found in a recent reviewby Reimhult et al. [98]). They can be grafted onto the surface ofMNPs through either ligand addition or ligand exchange [2]. Ligandaddition does not need to remove the original protecting ligandsand usually generates core/shell or layer-by-layer structure [99].MNPs modified in this strategy usually have better dispersibilityand chemical stability, as demonstrated in FePt NP stabilizationand dispersion by using surfactant addition with DSPE-PEG as dis-persants [100]. The second method, surfactant exchange replacesthe original surfactants with the dispersants (e.g. catechol deriva-tives [101]) that have stronger affinity to the surface of iron oxideMNPs. This has been shown in the functionalization of Fe MNPs[26], Fe3O4 HMNPs [13], and Au–Fe3O4 MNPs [70] with a catecholderivative, dopamine. The strong coordination bond between Feand catechol enables the replacement of the original surfactants,producing stable MNP dispersions in water.

d with permission from reference [63]. B) 21 nm sized Fe nanoframe. Reproduced witherence [89]. D) Fe3O4/ZnS HMNPs. Reproduced with permission from reference [92].ission from reference [93].

738 C. Xu, S. Sun / Advanced Drug Delivery Reviews 65 (2013) 732–743

After modification, hydrodynamic diameter and surface charge(zeta potential) of the MNPs need to be characterized because thesize and charge of MNPs can impact their uptake by cells and theirdistribution in organs [102,103]. Previous studies have indicatedthat when MNPs with a hydrodynamic diameter larger than 200 nmare delivered into the circulation through systematic administration,they are easily sequestered by the reticuloendothelial system (RES)of the spleen and liver. But if the size is less than 10 nm, they are sub-ject to rapid renal clearance [104]. Therefore, to ensure maximal NPcirculation, the optimum hydrodynamic diameter is believed to bebetween 10 and 200 nm. Villanueva et al. showed that the chargeand nature of surface functionalizing molecules on MNPs affectedtheir response to cancer cells [105]. In four different charged carbohy-drates including dextran (neutral), aminodextran (positive), heparin(negative), and dimercaptosuccinic acid or DMSA (negative), cells hadeffective uptake of aminodextran-MNPs, minimal uptake of neutral-charged dextran coated MNPs, low uptake of DMSA-coated MNPs, andconcentration dependent uptake of heparin coated MNPs. In addition,the surface charge also influenced the location ofMNPs after cellular in-ternalization. Schweiger et al. found that negatively chargedMNPswerefirstly in endosomes and lately in lysosomeswhereas positively chargedMNPswere exclusively inside lysosomes [106]. As early as 1996, Choulyet al. have demonstrated that surface charge influenced the capacity ofMNPs to be opsonized right after injection and so changed their phago-cytosis. They found that the negatively charged MNPs enhanced liveruptake greater than neutral MNPs [107].

4. Biomedical applications

4.1. High magnetic moment MNPs for biosensing

Rapid and sensitive measurement of clinically relevant biomarkers,pathogens, and cells in biological samples (e.g. blood or urine) is pricelessfor early detection/screening of disease like cancer, and for real-timemonitoring of personal responses to treatments [108]. Diagnostic mag-netic resonance (DMR) based on MNPs has recently received consider-able attention because of its low magnetic background from biologicalsamples, which enables the sensitive identification of small percentageof biomarkers from the ocean of background entities [109]. One impor-tant parameter in these assays is signal-to-noise ratio or sensitivity,which relates directly to magnetic properties of MNPs. MNPs with highmagnetic moment are ideal candidates to meet this need.

DMRmeasures the transverse relaxation rate (R2) of water moleculesin biological samples in which target molecules or cells of interest are la-beledwithMNPs [110]. There are two forms of DMRassays depending onthe size of the targets [108]. For molecule targets smaller than that ofMNPs, molecular targets are used as cross-linking agents to assembleMNPs into clusters, thus effecting a corresponding decrease in T2. Alterna-tively, enzymatic cleavage or competitive binding of molecular targetsdisassembles pre-formed clusters to cause an increase in T2, which iscalled reverse switching. For large biological structures like cancer cellsor bacteria, targeted MNPs tag the surface markers to impact their mag-netic moment. The change of 1/T2 is proportional to the number ofcells/bacteriaMNPs bind, and also indicative of the abundance of relevantsurface biomarkers. Lee and Weissleder et al. utilized MnFe2O4 MNPs tolabel cancer cells from fine-needle-aspirates (FNA) to allow the quantifi-cation and profiling of cancer cellswithDMR-2 systemdeveloped in theirgroup [111]. Thanks for the high magnetic moment of MnFe2O4 MNPs,they could detect as less as 2 HER2/neu positive cancer cells with only1 μL sample and 15 min frames (Fig. 3A and B). While with traditionaliron oxide MNPs, the sensitivity is 1000 cells with the requirement of10 μL sample.With the same DMR device, the same group demonstratedthat another type of MNPs with high magnetic moment, Fe MNPs couldbe conjugated with antibodies for tuberculosis detection [112]. In theassay, the biological sampleswere incubatedwith FeMNPs and then sep-arated from the unbound Fe MNPs before DMR diagnosis. With these

high magnetic moment MNPs, as few as 20 CFUs (colony-formingunits) could be detected in 1 mL of sputum sample in less than 30 min(Fig. 3C and D).

4.2. Molecular imaging with multifunctional MNPs

Molecular imaging is a biomedical research discipline dealing withvisualization, characterization, and quantification of biological pro-cesses at the cellular and sub-cellular levels. The images producedthrough molecular imaging (usually non-invasive) would help reflectcellular and molecular pathways and mechanism of disease in thesubjects. Existing imaging techniques include MRI, PET/SPECT, opticalimaging (fluorescence, bioluminescence, and optical coherence), CT,ultrasound etc., and each of them has their own advantages and dis-advantages. For example, MNP based MRI is advantageous for thehigher spatial resolution (10–100 μm), but suffers from the lowersensitivity, the high background coming from “field disturber”, andlong acquisition time/high cost [113,114]. On the other side, PET aresuper sensitive (10−11–10−12 M) with low background, and opticalimaging is time/cost efficient which facilitates rapid testing of biolog-ic hypotheses and proofs-of-principle in living experimental models.Therefore, using multifunctional MNPs that combine the advantagesof two or more imaging modalities is becoming an attractive strategyin molecular imaging. Here we will describe some representative ex-amples in tumor imaging and cell tracking.

4.2.1. Tumor imagingTo diagnose tumor malignancy early and accurately in clinics, it is

necessary to apply two or more imaging modalities [115]. The emer-gence of multifunctional MNPs fulfills this need and would minimizethe cost in clinical practices.

Au–Fe3O4 dumbbell MNPs (Fig. 4A) have been studied for both MRIand optical imaging of EGFR-positive breast cancer cells (Fig. 4B and C).AuNPs are optically active and have been used in a variety of optical im-aging based on light-scattering, two-photo luminescence and surface-enhanced Raman scattering [116]. Au NPs also present enhanced lightreflection between 500 nm and 800 nm [70]. The combination of Auand Fe3O4 in the dumbbellMNPs allows the integration of reflection im-aging and MRI, which could potentially be used as dual contrast agentsfor MRI diagnosis before surgery and metastasis mapping during sur-gery. Recently, Chen et al. reported a triple functional imaging probefor PET/NIFR/MRI [117]. They modified Fe3O4 MNPs with human serumalbumin (HSA) and functionalized HAS–Fe3O4-NPs with 64Cu-DOTA andCy5.5 (Fig. 4D). In this case, they obtained a novel reporter that had ahigh spatial resolution (MRI), good signal-to-noise ratio (PET), and con-venience for both in vivo and ex vivo analysis (near infrared fluorescence(NIRF)). In a subcutaneous U87MG xenograft mouse model, Chen dem-onstrated the triple-imaging-capabilities of this contrast agent throughexamining the preferred accumulation in tumor after 18 h circulationtime (Fig. 4E–G).

4.2.2. Cell trackingAnother exciting application of multifunctional MNPs is to qualita-

tively and quantitatively monitor transplanted cells in the exogenouscell therapy, which utilizes transplanted cells, in particular stem andprogenitor cells, to replace or regenerate damaged or diseased tissue[118]. The understanding of cell distribution and engraftment track-ing in cell therapy will facilitate prediction of treatment efficacy, re-veal optimal transplantation conditions including cell dose, deliveryroute, and timing of injections, and ultimately improve patient treat-ment [119,120].

Stelter et al. showed that an aminosilane coatedMNPs could be func-tionalized with the fluorescent dye (fluorescein) and the positron-emitting radioisotope (gallium-68) [121]. Hepatogenic HuH7 cells la-beled with these MNPs were intravenously administered and could befollowed through the sensitive γ-ray measurements. Their results

Fig. 3. A) TEM and HRTEM (insert) images of 16 nmMnFe2O4 MNPs. B) Human breast cancer cells (BT474) were labeled with anti-Her2 CLIO and MnFe2O4 MNPs. The change in R2(R2=1/T2) varied linearly with cell counts, and the detection sensitivity was 10× better using the more magnetic MnFe2O4 MNPs. Reproduced with permission from reference[111]. C) Fe MNPs or Cannonballs (CBs) that have an iron core (11 nm) passivated with a thin ferrite shell (2.5 nm). D) Comparison of detection sensitivity. First, a microfluidicchip without a membrane filter was used to determine the intrinsic mass-detection limits. The bacteria were targeted either with CB–BCG (MNPs conjugated with monocolonalantibody of bacillus Calmette–Guerin, a surrogate for tuberculosis) or CLIO–BCG. With CB–BCG, they achieved a mass-detection limit of approximate 6 CFU (1 μL detection volume),much lower than that of approximate 100 CFU for CLIO–BCG. When CB–BCG-targeted samples (100 μL) were filtered, the concentration limit was further reduced to approximate60 CFU/mL. Reproduced with permission from reference [112].

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revealed the predominant localization of the labeled cells in the lungs2 h after injection and the even distribution throughout the animals'body 48 h later. Nahrendorf et al. designed a PET-MRI-fluorescencetrimodality contrast agent through chelating 64Cu onto the dextranatedand DTPA-modifiedmagnetofluorescentMNPs [122]. This platformwasused to image the macrophages and to identify the atherosclerotic le-sions in an Apolipoprotein E deficiency (apoE−/−) mouse model. Thecombination of PET, MRI and fluorescence combined the individual ad-vantages of each imaging modality. After in vivo distribution of MNPs,all imaged apoE−/− mice showed a robust PET signal in the aortic rootand arch, which showed significant differences between accumulateddose in excised aortas and carotids in apoE−/− versus wild-type mice.

4.3. Drug delivery with hollow MNPs

Targeted drug delivery is a Holy Grail in chemotherapy. In an idealtargeted drug delivery, the drug carrier selectively delivers drug mole-cules to the diseased site without a concurrent increase in their intensi-ty in healthy tissues. In the late 1970s, Widder and Senyi proposed theidea of using MNPs to carry drugs to specific sites such as solid tumor,where high-field magnets were positioned [123,124]. Following theirearly studies, the efficacy of this approachwas demonstrated in numer-ous small animal studies and even resulted in a small number of clinicaltrials [125]. However, despite these efforts and achievements, this tech-nique has yet to develop into a workable clinical application. One of thereasons is the low payload capacity of existing MNPs [6] because pay-load (i.e. drugs) can only be attached on the surface or embedded inthe double-layer coating around MNPs. To address this issue, one ofthe solutions is to utilize hollow MNPs discussed in Section 2.3, in

which drugs could be loaded both inside their hollow core and on thesurface.

Our group investigated this hypothesis through utilizing Fe3O4

HMNPs to deliver cisplatin (one of traditional chemotherapeutics)to HER2/neu positive cancer cells [13]. We noticed the shell ofFe3O4 HMNPs (Figs. 2A and 5A) was polycrystalline and its crystal-linity could be further improved by prolonged heating in solutioncontaining oleic acid. With the crystal domain growing larger inthe shell structure, the crystal boundaries in the polycrystallinestructure opened up, resulting in the porous shell with 3 nm poresize (Fig. 5B). The open pores facilitated the diffusion of cisplatininto the cavity of Fe3O4 HMNPs during the ligand exchange process(Fig. 5C). More specifically, we carried out the loading by mixingoleylamine/oleate-coated Fe3O4 HMNPs with cisplatin and replac-ing surfactant (i.e. dopamine-PEG) in chloroform/DMF followed bysolvent evaporation to maximize cisplatin loading. Through thismethod, we could improve the cisplatin percentage on the final con-jugate from 4.82% of Fe3O4 MNPs to 24.8% of Fe3O4 HMNPs.

This highpayload capacity of hollowMNPshas also been confirmedbyother groups.With porousMn3O4HMNPs, Lee et al. improved the loadingamount of a hydrophobic anticancer agent (doxorubicin), where theymixed the water-dispersible porous Mn3O4 HMNPs with doxorubicin inCH3OH/CH3Cl and evaporated the organic solvent [126]. They foundthat the amount of doxorubicin incorporated into the porous Mn3O4

HMNPs was 3.5 times higher than that on the solid Mn3O4 MNPs underthe same NP concentration. The final doxorubicin percentage in Mn3O4

HMNPs was approximately 14% while that in Mn3O4 MNPs was approxi-mately 4%. Shi et al. tested the hollow core, magnetic, and mesoporousdouble-shell MNPs (Fe3O4/SiO2 in Fig. 2E) as carriers for water-insolubleanticancer drugs (docetaxel or camptothecin) [94]. The drugs were

Fig. 4. A) TEM image of 8–20 nm Au–Fe3O4 dumbbell MNPs. Scale bar is 20 nm. B) T2-weighted MRI images of i) 20-nm Fe3O4, ii) 3–20-nm Au–Fe3O4, iii) 8–20-nm Au–Fe3O4 MNPs,and iv) A 431 cells labeled with 8–20-nm Au–Fe3O4 MNPs. Reproduced with permission from reference [70]. C) Reflection images of the A431 cells labeled with 8–20-nm Au–Fe3O4

MNPs. D) Schematic illustration of the multi-functional HSA–Fe3O4-NPs. E) Representative in vivo NIRF images of mouse injected with HSA–Fe3O4-NPs. Images were acquired 1 h,4 h and 18 h post injection. F) In vivo PET imaging results of mouse injected with HSA–Fe3O4-NPs. Images were acquired 1 h, 4 h and 18 h post injection. G) MRI images acquiredbefore and 18 h post injection. Reproduced with permission from reference [117].

740 C. Xu, S. Sun / Advanced Drug Delivery Reviews 65 (2013) 732–743

loaded through soaking the MNPs in a concentrated drug/DMSO solu-tion. After purification, the drugs represented 15–14% (mass percentage)of the final products. In comparison, for solid silica encapsulated Fe3O4

MNPs, the drug percentage was only 1–3% [87].

Fig. 5. HRTEM images of A) Fe3O4 HMNP and B) Fe3O4 porous HMNP. C) Schematic illustratioand functionalization with Herceptin.Reproduced with permission from reference [13].

5. Conclusion

In this paper, we have summarized recent efforts in designing newplatforms of MNPs to address the problems met in the biomedical

n of simultaneous surfactant exchange and cisplatin loading into a Fe3O4 porous HMNP

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application of traditionalMNPs. To improve themagneticmoment, othermetal ions are chosen to dope into the spinel structure of ferrite. Highmagnetic moment metallic MNPs are further synthesized and stabilized.To improve the sensitivity and accuracy ofMRI, other NP components re-sponsible for different imaging modalities are also combined into oneMNP unit. To improve therapeutic efficacy, hollow MNPs are designedso that more drug can be loaded both inside and outside the NPs.These multifunctional MNPs have shown great advantages over the tra-ditional MNPs in disease diagnosis, cancer imaging, cell tracking anddrug delivery. Once their biodistribution and metabolism are better un-derstood, these new forms of MNPs will serve as sensitive probes andplatforms for highly efficient diagnostic and therapeutic applications.

Acknowledgments

This work was supported in part by Nanyang Technological Uni-versity Start-Up Grant to XCJ and Brown Imaging Fund to SS.

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