Title: RECENT ADV ANCES IN SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES (SPIONs) FOR IN VITRO AND IN VIVO CANCER NANOTHERANOSTICS Author: Ganeshlenin… [615995]

Accepted Manuscript
Title: RECENT ADV ANCES IN SUPERPARAMAGNETIC
IRON OXIDE NANOPARTICLES (SPIONs) FOR IN VITRO
AND IN VIVO CANCER NANOTHERANOSTICS
Author: Ganeshlenin Kandasamy Dipak MaityPII: S0378-5173(15)30325-2
DOI: http://dx.doi.org/doi:10.1016/j.ijpharm.2015.10.058Reference: IJP 15313
To appear in: International Journal of Pharmaceutics
Received date: 19-8-2015
Revised date: 20-10-2015Accepted date: 22-10-2015
Please cite this article as: Kandasamy, G., Maity, D.,RECENT ADV ANCES IN
SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES (SPIONs) FOR IN
VITRO AND IN VIVO CANCER NANOTHERANOSTICS, International Journal of
Pharmaceutics (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.10.058
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RECENT ADVANCES IN SUPERPARAMAGNETIC IRON OXIDE NAN OPARTICLES 1
(SPIONs) FOR IN VITRO AND IN VIVO CANCER NANOTHERANOSTICS 2
3
Ganeshlenin Kandasamy a, Dipak Maity a* [anonimizat] 4
aNanomaterials Lab, Department of Mechanical Enginee ring, Shiv Nadar University, Uttar 5
Pradesh – 201314, India 6
7
Department of Mechanical Engineering, Shiv Nadar Un iversity, Uttar Pradesh – 201314, India. 8
Tel: 91 120 3819100, Extn 216 9
10
11
Abstract 12
Recently superparamagnetic iron oxide nanoparticles (SPIONs) have been extensively used in 13
cancer therapy and diagnosis (theranostics) via mag netic targeting, magnetic resonance imaging, 14
etc. due to their remarkable magnetic properties, c hemical stability, and biocompatibility. 15
However, the magnetic properties of SPIONs are infl uenced by various physicochemical and 16
synthesis parameters. So, this review mainly focuse s on the influence of spin canting effects, 17
introduced by the variations in size, shape, and or ganic/inorganic surface coatings, on the 18
magnetic properties of SPIONs. This review also des cribes the several predominant chemical 19
synthesis procedures and role of the synthesis para meters for monitoring the size, shape, 20
crystallinity and composition of the SPIONs. Moreov er, this review discusses about the latest 21
developments of the inorganic materials and organic polymers for encapsulation of the SPIONs. 22
Finally, the most recent advancements of the SPIONs and their nanopackages in combination 23
with other imaging/therapeutic agents have been com prehensively discussed for their effective 24
usage as in vitro and in vivo theranostic agents in cancer treatments. 25
26
Keywords 27
Magnetic Nanoparticles; Superparamagnetic Iron Oxid e; Synthesis & Surface Engineering; Spin 28
Canting; Cancer Theranostics; Magnetic Resonance Im aging; Hyperthermia 29
30
1. Introduction 31
Cancer is one of the most dreadful diseases among a ll human diseases. According to World 32
Health Organization (WHO) statistics, close to 8.2 million cancer related deaths had happened 33
till 2012 and more than 14 million people are newly diagnosed with cancer (www.who.int/en/). 34
All over the world, cancer related research is ongo ing to control cancer as well as to kill cancer 35
cells completely. Nanotechnology has garnered a gre at deal of attention in medical research and 36
has created a vast impact on the economy (www.nano. gov) in recent days. A commission for 37
regulating the usage of nanotechnology in biologica l applications is expected by approximately 38
43% of people, in a poll recently taken from more t han 18,000 people via social media 39
throughout the world (Sechi et al., 2014). Nanomedi cines are significantly involved in cancer 40
research because of their ability to provide an imp rovised therapeutic and diagnostic 41
(theranostic) approach by overcoming multi-drug res istance of cancer cells and drawbacks of 42
conventional cancer treatments such as poor solubil ity of hydrophobic anti-cancer drugs, 43
biocompatibility, usage of harmful radiations etc. In cancer nanomedicine, different 44
nanoparticles, anticancer drugs and imaging agents are encapsulated and/or embedded within the 45

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biocompatible organic/inorganic shell structures to form a multifunctional system for combined 46
therapy and imaging. 47
Among various nanoparticles, superparamagnetic iron oxide nanoparticles (SPIONs) particularly 48
magnetite (Fe 3O4) and meghamite ( γ-Fe 2O3) nanoparticles are used primarily in cancer 49
theranostic applications such as magnetic resonance imaging (MRI) and magnetic hyperthermia 50
due to their significant magnetic properties and bi odegradability. Yet, toxicity of SPIONs 51
towards normal cells has been pointed out by scient ific communities, when SPIONs are involved 52
in in vivo cancer treatments. SPIONs exhibit superparamagneti c behavior at size below 30 nm at 53
room temperature. Superparamagnetism can be defined as the ability of magnetic nanoparticles 54
to show robust paramagnetic nature with high suscep tibility and saturation magnetization under 55
the influence of a magnetic field and the tendency of losing the same nature completely once the 56
magnetic field is removed, resulting in zero magnet ic remanence and zero coercivity. The 57
surfaces of SPIONs at reduced sizes are so reactive due to the increased surface area-to-volume 58
ratio. So, the surfaces of SPIONs are usually coate d with surfactants/capping agents/polymers to 59
prevent agglomeration in colloidal solution, and to maintain the size and shape of SPIONs. 60
Otherwise, SPIONs tend to aggregate to form bulk st ructures and settle down in colloidal 61
solutions. However, these surface coatings affect t he inherent magnetic properties of SPIONs 62
depending upon the nature, amount/length, compositi on and thickness of the surface coatings. 63
The SPIONs are conjoined with other contrast agents , fluorescence tags/dyes, quantum dots, etc. 64
for effective imaging of cancer cells/tissues throu gh fluorescence imaging, near infra-red (NIR) 65
imaging, computed tomography (CT), ultrasound imagi ng, positron emission tomography (PET), 66
single photon emission computed tomography (SPECT), etc. The SPIONs are also combined 67
with chemotherapeutic drugs (such as anthracyclines , antimetabolites, platinum-based-drugs, 68
taxanes, vinca alkaloids and so on), nucleic acids (deoxyribonucleic acids and ribonucleic acids), 69
unconjugated monoclonal antibodies (for instance, r ituximab, trastuzumab), targeting agents 70
(peptides, proteins, and small biological), photody namic, photothermal and sonodynamic 71
agents/nanoparticles to form combinatorial nanopack ages for effective cancer treatment. 72
However, the magnetic properties of SPIONs are dete riorated by the conjugation of these 73
drugs/antibodies/other nanoparticles with SPIONs. 74
Many review articles have already discussed about v arious synthesis procedures, surface 75
coatings, encapsulations and biomedical application s of the SPIONs. However, there is a serious 76
lack of studies on the magnetic properties of the S PIONs at the fundamental level. Moreover, 77
there is a lack of comprehensive studies on the rec ently developed SPIONs and their in vitro and 78
in vivo applications for cancer theranostics. Therefore, w e have discussed here the effects of 79
physicochemical parameters such as size, shape and surface coatings on the magnetic properties 80
of the SPIONs. Furthermore, we have discussed the m echanism of different predominant 81
chemical synthesis of SPIONs and their encapsulatio n using silica and polymers. Finally, we 82
have discussed the recent progress made in the usag e of SPIONs independently and also in 83
combination with other therapeutic and imaging agen ts for advanced in vitro and in vivo cancer 84
theranostic applications. 85
2. Magnetic Properties of SPIONs 86
The transformation from multi-domain phase to singl e-domain phase in a material begins at the 87
nanometer scale. When the magnetostatic energy equa lizes the domain-wall energy in magnetic 88
nanomaterials, the single-domain phase dominates at specific dimensions. The critical diameter 89
for a spherical Fe 3O4 nanoparticle to possess a single-domain is believed to be 128 nm. At single- 90
domain phase, SPIONs possess one huge magnetic mome nt and exhibit superparamagnetism 91

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above the blocking temperature (T B) (size and shape dependent phenomena), while the t hermal 92
energy overcomes the anisotropy energy of magnetic materials. The relationships for anisotropy 93
energy (E( θ)) and superparamagnetic relaxation time ( τ) (with respect to temperature) are given 94
below: 95
96
(1) 2E( ) KVsin θ θ = 97
(2) ()0 B exp KV/k T τ τ = 98
Where K is the magnetic anisotropy constant, V is t he particle volume, θ is the angle between an 99
easy axis and a magnetization vector, k B is Boltzmann’s constant and T is the temperature. τ0 is 100
in the range of 10 −13 –10 −9 s. Usually, in a colloidal solution, the magnetic moments of suspended 101
SPIONs align themselves along the easy axis during the absence of magnetic field. However, the 102
magnetic moments tend to align themselves in a dire ction parallel to the applied field, when 103
magnetic field is applied, thereby resulting in hig h magnetization values. But, once the field is 104
removed, the moments of SPIONs revert back to their original easy axis positions due to 105
longitudinal and transverse relaxivities of SPIONs. 106
In SPIONs, the magnetic moments appear due to the p resence of unpaired 3d electrons in Fe 3+ 107
and Fe 2+ cations in the cubic fcc lattice, where an electron spin coupling of Fe 2+ and Fe 3+ ions 108
takes place at octahedral sites and an anti-paralle l electron spin coupling of Fe 3+ ions takes place 109
at tetrahedral sites. Exchange interaction/coupling between these two sites across oxygen anions 110
is called as superexchange interactions which are r esponsible for the magnetic behavior of 111
SPIONs. Destruction of superexchange interactions c an also happen at the surface of SPIONs, 112
due to the inclination of the surface atomic spins of magnetic nanoparticles to a particular angle 113
(called canting angle). The inclination effect (or spin canting effect) is due to: (i) the lack of 114
number of atoms necessary for the formation of sing le magnetic moment and/or (ii) less 115
organized spins at the surface as compared to the c ore of SPIONs. Moreover, spin glass like 116
layers (i.e., slow relaxation behavior of surface a toms of SPIONs) may also occur with respect 117
the magnetic frustrations between the Fe 2+ and Fe 3+ atoms located on the surface of SPIONs. 118
Oxygen vacancies, edge roughness, defects in cation ic positions (cationic vacancies), Laplace 119
pressure and changes in surface and/or core chemica l ordering and anisotropies (i.e., local 120
symmetry breaking due to the presence of dead magne tic layer and/or anti-ferromagnetic layer 121
formed by surfactants) (Maity and Agrawal, 2007) of SPIONs could be the other factors that 122
affect the magnetic properties of iron oxide nanopa rticles, besides the surface spin canting 123
effects. Hyperfine spectral lines (obtained from Mo ssbauer spectroscopy), field cooled (FC) and 124
zero field cooled (ZFC) methods (obtained from supe rconducting quantum interference device) 125
are used for determining canted spins, presence of non-magnetic layers (from surface coatings) 126
and spin-glass like behavior. All these magnetic ph enomena change with the modifications in 127
size, shape and surface coating of SPIONs, which ar e discussed in following sections with 128
typical examples. 129
2.1. Effect of Size and Crystallinity 130
The size reduction of SPIONs results in the decreas e of magnetic moments of SPIONs, thereby 131
reducing the saturation magnetization of SPIONs. Ne vertheless, the spin canting effect is 132
increased at size reduced nanoparticles, ascribing to the increase in surface area-to-volume ratio 133
of SPIONs. The inter-relationship between the size, magnetization and spin-canted surface layer 134
can be given by equation (3): 135
(3) 3
s s m M [(r-d)/r] = 136

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Where m s is saturation magnetization of size-reduced nanoparticle, Ms is s aturation 137
magnetization of bulk materials, r is the size of n anoparticle and d is the thickness of disordered 138
surface layer. In a recent investigation, it was es timated that about 93.6% of surface spins in 3- 139
nm sized iron oxide nanoparticles were canted as co mpared to 38.6% of surface spins in 12-nm 140
sized nanoparticles (by assuming 0.9 nm spin cantin g layer thickness). As a result of the 141
increased canting effects in 3 nm sized iron oxide nanoparticles, the magnetization values 142
decreased correspondingly (Kim et al., 2011). Simil arly, in another study, 5 nm sized Fe 3O4 143
nanoparticles exhibited a low magnetization value o f 27 emu/g, attributed to its reduced size 144
(Chen et al., 2011). 145
Aside from surface spin canting, volume canting als o occur in magnetic nanoparticles, attributed 146
to the presence of cationic vacancies, improper cry stallinity and reduced magnetic core volume 147
arising from variations in magnetic nanoparticle pr eparation methods (Serna et al., 2001). In a 148
recent investigation, the internal/volume and surfa ce canting effects were studied on 149
nanoparticles of 5 different sizes (5,8,11,15 and 2 0 nm), where 5 nm iron oxide nanoparticles 150
possessed a surface canting layer thickness of one atomic layer and exhibited a magnetization 151
value of 51 emu/g approximately (Baaziz et al., 201 4). The thickness of canting layer increased 152
to two atomic layer thicknesses for 8, 9, and 11 nm sized SPIONs due to the disordered structure 153
initiated by perturbed oxidation states, thereby br ought both surface and volume canting effect at 154
these sizes (as shown in Fig 1). However, the canti ng layer thickness fell rapidly for large (>11 155
nm) nanoparticles because of the changes of iron at oms in their respective interstitial sites, and 156
the magnetite compositions of iron oxide nanopartic les, thus the larger sized magnetic 157
nanoparticles (such as 15 and 20 nm) exhibited high er magnetization values (71 and 82 emu/g 158
respectively). In another study, 15 nm sized spheri cal Fe 3O4 nanoparticles displayed 159
magnetization value of 53.3 emu/g, ascribed to the reduction in magnetic effective volume (Ge et 160
al., 2009). Therefore, spin canting effect in a nan oparticle is a mixture of both surface and 161
volume spin canting effects. The spin canting effec ts are dependent on temperature and applied 162
magnetic field. Moreover, volume and surface spin c anting effects also occur in magnetic 163
nanoparticles, when the iron oxide nanoparticles ar e being doped with other atoms (gadolinium) 164
(as shown in Fig 2) (Zhou et al., 2013). 165
In addition to the spin canting effects, anti-phase boundaries (APB – a crystal defect occurs on 166
planes while chemical ordering) also affect the mag netic properties of SPIONs at reduced sizes. 167
Wetterskog et al. observed the presence of APBs in single-phase 20 n m sized SPIONs (shaped 168
via oxidation of the core made of rock salt (Fe 1–xO)), resulting in low magnetization values 169
(Wetterskog et al., 2013). However, in another inve stigation, APB and other magnetic disorders 170
(preceding to exchange bias) were found to be absen t even in multi-core highly-crystalline 171
citrate-coated SPIONs due to their shared crystallo graphic orientations (Lartigue et al., 2012). 172
Herein, the multi-core SPIONs retained superparamag netic properties at room temperature even 173
at large sizes (19-30 nm) with magnetization values up to 82 emu/g. Moreover, the multi-core 174
SPIONs remained equally dispersed as compared to si ngle-core SPIONs even after the 175
application of magnetic field. Further information on size controlled magnetic properties can be 176
obtained from the other reviews (Batlle and Labarta , 2002; Obaidat et al., 2015). 177
2.2. Effect of Surface Coatings 178
The organic/inorganic surfactants/capping agents fo rm a protective layer around SPIONs by 179
attaching to the surface atoms of SPIONs via the en d functional groups either through 180
electrostatic interactions or covalent bonding. The thickness of the protective layer can be in the 181
range of 1-5 nm, if small organic molecules are use d as surfactants/capping agents. This 182

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protective layer thickness can increase to more tha n 100 nm, if large polymers are used. The 183
surfactants/capping agents usually have different e nd functional groups such as -OH, -COOH, – 184
PO(OH)2, -S(=O)2–OH, catechols (refer Fig 3), etc. which bring stoichiometric modifications on 185
the surface of SPIONs (Yuen et al., 2012). Moreover , the influence on the magnetic properties of 186
SPIONs differs with the type of end functional grou ps that are attached to the surface atoms of 187
SPIONs. For example, Darbandi et al. showed that th e effects of surfactants (polyoxyethylene (5) 188
nonylphenylether with –OH end group)) on the magnet ic properties of SPIONs were negligible 189
since no modifications on the surface structure or type of magnetic order in SPIONs were 190
observed (Darbandi et al., 2012). In contrast, the results of canting angle measurements of 191
SPIONs showed that the average surface spin canting of SPIONs had decreased after coating its 192
surface with polyoxyethylene (5) nonylphenylether, owing to chemical affinity of –OH end 193
groups of surfactants towards the surface atoms of SPIONs. However, this negligence 194
phenomenon was contradicting with another investiga tion (Roca et al., 2009), where the strong 195
affinity of carboxylic end groups of oleic acid tow ards the surface of SPIONs resulted in 196
increased surface spin canting effects in SPIONs by victimizing the octahedral iron sites. 197
Nevertheless, in another research (Guardia et al., 2007), high quality SPIONs (6 – 20 nm) 198
produced using oleic acid showed high magnetization values ascribing to the diminution of 199
surface spin disorder by oleic acid, and high cryst allinity of SPIONs. Whereas, no notable 200
changes in magnetizations of SPIONs were observed e ven after the passivation of oleic acid with 201
other organic and inorganic surfactants (with spin canting layer thickness near to 0.07–0.08 nm) 202
onto the surface of SPIONs (de Montferrand et al., 2014). In a recent investigation, based on the 203
theoretical calculations and experimental data (obt ained using electron magnetic chiral dichroism 204
(EMCD)), Salafranca et al. found that the magnetization of SPIONs was enhance d near to 205
magnetization of bulk nanoparticles due to the sati sfaction of orbitals occupations of surface 206
atoms of SPIONs by the surfactants (oleic acid) thr ough oxygen bonding (Salafranca et al., 207
2012). But in another study, the moments of oleic a cid (1 nm thickness) encasing the magnetite 208
nanoparticles exhibited a canting angle of 90° with respect to the moments of magnetic core, 209
resulting in a conclusion that the moments of oleic acid were of magnetic in origin (Krycka et al., 210
2010a, 2010b). In another investigation, curcumin a ttachment (through citric acid) facilitated the 211
re-ordering of surface atoms of SPIONs, thus render ing more crystallinity to enhance the 212
magnetization of the curcumin loaded SPIONs (60 emu /g) as compare to the bare and only 213
citrate capped SPIONs (45 emu/g) (Kitture et al., 2 012). 214
Surfactants having phosphonic acid (-PO(OH)2) funct ional groups tend (i) to improve thermal 215
stability of nanoparticles and (ii) to show enhance d binding affinity (1.5 times more than 216
carboxylic acid) towards the core of iron oxide nan oparticles due to the presence of an extra 217
oxygen atom in phosphonic acid, thereby forming a s trong Fe–O–P bond. The results of 218
Mohapatra et al. showed that phosphonic acid moieties showed more p reference to bind with the 219
surface atoms of Fe 3O4 nanoparticles as compared to outward facing carboxy lic or amine groups, 220
when bifunctional organophosphorous based surfactan t was used to cover the surface of 221
magnetic nanoparticles (Mohapatra and Pramanik, 200 9). Similarly, poly(amido amine) 222
(PAMAM) showed enhanced binding towards the surface of SPIONs due to the presence of two 223
phosphonic groups at the same end, which provided m ore negative charges for easy formation of 224
bidentate phosphate-iron complexes (Di Marco et al. , 2007). The direct functionalization of 225
phosphonic acid based surfactant onto the surface o f SPIONs may yield higher saturation 226
magnetization as compared to the indirect functiona lization of surfactants/capping agents (i.e., 227
complete replacement of oleic acid/any other coatin g with phosphonic acid/catechol end group 228

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based surfactant on the surface of SPIONs) because of reduced spin canting effects and enhanced 229
binding affinity and superexchange interactions. 230
Gold coated SPIONs found to have more magnetization values than the uncoated ones, since the 231
gold coating helped in the reduction of surface mag netic disorders of SPIONs, and enhanced 232
their crystallinity. In supporting to this fact, a combined (theory and experiment) study was 233
performed based on the interaction energies between gold, iron and oxygen atoms (Yue et al., 234
2012). In experimental section, the gold atoms indu ced recrystallization of the iron oxide 235
surfaces to maintain structural stability. Theoreti cally two cases were examined; (i) gold atoms 236
exhibited less interaction over smooth surfaces of iron oxide; (ii) gold atoms showcased higher 237
interactions towards iron oxide surfaces with pores /defects (enhanced surface activeness). The 238
theoretical investigations reported that Van der wa als forces dictated most of the interactions 239
between metal oxide and noble metal combinations. 240
In contrast to the improvements in magnetic propert ies, a recent investigation showed that at 5K, 241
the magnetization of SPIONs had reduced drastically from 79 emu/g to 7.47 emu/g after their 242
surface was covered with star shaped gold shells, o wing to the diamagnetic property of gold 243
atoms (Quaresma et al., 2014). As similar to the pr evious study, mostly the reduction in 244
magnetizations of SPIONs were notified due to the p resence of non-magnetic 245
surfactants/capping agents (Wortmann et al., 2014). However, the usage of surfactants/capping 246
agents in the synthesis of SPIONs is inevitable to maintain the colloidal stability for extended 247
period of time. 248
2.3. Effect of Shape 249
Barring the size and interface controls, shape is a nother factor to be premeditated in the control 250
of magnetic properties of SPIONs. Generally, SPIONs with spherical morphologies are prepared 251
and characterized since it has lesser complications in assessing their magnetic properties. But 252
some investigations that assessed the magnetic prop erties of SPIONs with diverse are reviewed. 253
The shapes of the SPIONs with definite surface face ts (for instance, (111) facet) are decided by 254
the concentration of reactants, reaction temperatur e/aging time and interactions between surface 255
of SPIONs and surfactants/capping agents (Khurshid et al., 2013), where the facets may have 256
cationic or anionic lattice index plane termination s (Lovely et al., 2006). In a recent study, the 257
quantitative as well as microscopic information abo ut the magnetic moments stationed at the 258
surface and core of the nano-cubical and nano-spher ical SPIONs were reported using polarized 259
small-angle neutron scattering (SANS) (Disch et al. , 2012). Herein, more surface canting was 260
observed for the nano-cubical SPIONs due to larger amount of non-magnetic layer thickness of 261
the surfactants/capping agents on the surface of SP IONs as compared to nano-spherical SPIONs. 262
Similarly, lower magnetization values and high coer civity values were reported for the 263
ellipsoidal (solid and hollow) magnetite nanopartic les than the spherical (solid) nanoparticles 264
which was attributed to the increased surface spin canting effects in ellipsoidal shaped 265
nanoparticles (Choi et al., 2013). 266
On the contrary, SPIONs with quasi-cubic morphology displayed a higher magnetization values 267
(79 emu/g) (Wortmann et al., 2014), which could be due to an increase in the magnetic core 268
volume or low surface to volume ratio in such kind of distinct morphologies. In another excellent 269
study, Octapod shaped SPIONs (refer Fig 4) were pro duced by taking control of the 270
concentration of sodium chloride (NaCl) (Zhao et al ., 2013). These octapod shaped SPIONs 271
displayed sustained magnetizations (~71 emu/g) beca use of reduced spin canting effects and 272
enhanced core radius (i.e., 2.4 times more than sph erical ones). In another investigation, 273
variations in the geometries (facet, twins, precipi tates and spheres) of iron oxide crystals were 274

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introduced to study their effect in magnetic behavi ors of respective crystals (Luigjes et al., 2011). 275
Based on the results of dark and bright field image s of high-resolution TEM, the magnetic nano- 276
crystals (facet, twins, and precipitates) with near -to-perfect crystallinity presented comparative 277
magnetization values (69, 65 and 53 emu/g) in oppos ition to spherical SPIONs (~30 emu/g), 278
attributed to their enhanced coupling of atomic mag netic spins in such distinct shapes. In a study, 279
magnetization values of 61.73, 65.43 and 67.99 emu/ g were attained for SPIONs with cubic, 280
cuboctahedral and octahedral morphologies (formed b y altering the synthesis parameters such as 281
heating rate and growth time) respectively (Bateer et al., 2013). Nanoparticles with mixed shape 282
distributions illustrated the magnetization values near to that of bulk ones (Guardia et al., 2007). 283
In summary, the size, shape, crystallinity and surf ace coatings play a significant role in 284
determining the magnetic properties of SPIONs by in ducing/deducing the spin canting effects. 285
However, the size, shape and crystallinity of SPION s are manipulated using different chemical 286
synthesis procedures. Table 1 summarizes the size, shape, surface coatings and corresponding 287
magnetization values of the iron oxide (Fe 3O4 and Fe 2O3) nanoparticles. 288
3. Synthesis Methods of SPIONs 289
The basic strategies involved in the formation of S PIONs are physical, wet chemical, and 290
microbial methods. Each method has its own advantag es and disadvantages, and impacts over 291
various properties of SPIONs. In this section, the predominant chemical procedures used for 292
synthesizing SPIONs have been reviewed with typical examples. 293
3.1. Co-precipitation Method 294
Co-precipitation method is the widely used techniqu e for synthesizing black and/or brownish 295
SPIONs by precipitating an aqueous solution mixture containing ferric and ferrous salts (in a 2:1 296
stoichiometric ratio) using a base, at room or elev ated temperatures (70 °C – 90 °C), in the absence 297
of oxygen. The co-precipitation process occur throu gh either one of the two topotactic phase 298
transformation pathways (as shown in Fig 5): (i) ak aganeite phase (birth of crystal nuclei) to 299
goethite phase (to form arrow-shaped nanoparticles) or (ii) ferrous hydroxide phase to 300
lepidocrocite phase to finally form SPIONs, dependi ng upon the slow (for example, 1.88 301
mL/min) or quick (at once) addition of base into th e mixture of precursor solution (Thanh et al., 302
2014). The above transformations include hydroxylat ion and condensation (either through 303
olation or oxolation mechanisms) of Fe 3+ and Fe 2+ ions based on the pH of the colloidal solution. 304
Moreover, surfactants/capping agents are used to co ntrol the growth of SPIONs during the 305
synthesis. However, the magnetic properties of SPIO Ns are significantly influenced by synthesis 306
parameters such as reaction timings, base molarity, stirring rate, and the type of base. Recently, 307
Vikram et al. showed that the stoichiometric ratio of Fe 2+ and Fe 3+ and the base addition rate also 308
have control over the regulation of the magnetic pr operties of SPIONs (Vikram et al., 2014). 309
They reported that the change in concentration of t he base yielded nanomaterials with 310
ferromagnetic behavior instead of superparamagnetis m. 311
The main drawback of this co-precipitation method i s the lack of proper crystallinity and broad 312
particle size distribution which leads to low satur ation magnetization value (30-50 emu/g) of the 313
SPIONs as compared to the bulk magnetization value of Fe 3O4 nanoparticles (92 emu/g). 314
Nevertheless, magnetic nanoparticles with uniform s ize of 9 nm were obtained via co- 315
precipitation method using a tetramethylammonium hy droxide for MRI contrast (Cheng et al., 316
2005). 317
318
319
3.2. Thermal Decomposition Method 320

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Highly crystalline and monodisperse SPIONs with div erse sizes and shapes can be synthesized 321
via thermal decomposition method in the presence of surfactants (for example, oleic acid and 322
oleylamine) and organic solvents with high boiling points. Solvent free thermal decomposition of 323
iron precursors can also be utilized for preparing magnetic nanoparticles (Maity et al., 2009). 324
However, the resulting hydrophobic SPIONs tend to s how good dispersibility only in organic 325
solvents (for example, tetrahydrofuran) because of their hydrophobic interactions between 326
surfactants and solvents. Many synthesis parameters such as concentration of surfactants, 327
reaction temperatures, reaction timings, ratio of p recursors to surfactants, solvents and heating 328
rate during reflux govern the physicochemical chara cteristics and magnetic properties of SPIONs 329
(Maity et al., 2008). 330
The hydrophobic SPIONs are converted to water solub le ones by using either ligand exchange 331
method or bilayer surfactant stabilization method t o involve them instantaneously into cancer 332
theranostic applications. For example, Xu et al. replaced the hydrophobic surface coatings 333
formed of oleate and oleylamine molecules with a hy drophilic coating of dopamine attached 334
PEG through ligand exchange method, since dopamine tended to show more affinity to attach 335
with the surface of iron oxide nanoparticles as com pared to the hydrophobic coatings (Xu et al., 336
2008). In the bilayer surfactant stabilization, the bilayers are formed by the insertion of 337
hydrophobic part of an amphiphilic molecule in betw een the long hydrophobic chains of 338
surfactants whose end functional groups, for instan ce carboxyl groups, are attached to the surface 339
atoms of magnetic nanoparticles. Recently, oleic ac id was used to form bilayers on the surface of 340
magnetic nanoparticles after optimizing their conce ntration with respect to the formed 341
nanoparticles (Prakash et al., 2009). But, these co nversion methods are tedious and seriously 342
affect the magnetic properties, colloidal dispensab ility and yield of SPIONs (Liu et al., 2014). To 343
overcome these problems, hydrophilic SPIONs can be directly synthesized by one-pot 344
thermolysis method using polyol based surfactants a nd/or solvents (Maity et al., 2010). 345
3.3. Hydrothermal Method 346
In hydrothermal method, the precursors are dissolve d in an aqueous solution along with 347
surfactants/capping agents, and sealed in a Teflon coated autoclave, where high temperature and 348
high pressure are maintained to synthesize SPIONs w ith definite sizes and shapes. Finally, the 349
temperature of the autoclave is allowed to cool dow n to room temperature and the resultant 350
supernatant solution is washed to remove unused sur factants/capping agents, impurities and 351
unreacted precursors. The parameters such as heatin g temperatures, reaction timings and the ratio 352
of the precursor to surface coatings are manipulate d to obtain biocompatible SPIONs with 353
various sizes, shapes and magnetic properties for M RI contrast and cancer hyperthermia. In a 354
study, ethylene glycol played an important role in synthesizing SPIONs with three different 355
morphologies (polyhedron/rod shaped, porous sphere and flowerlike) with saturation 356
magnetization 66-73.5 emu/g, in which flowerlike na noparticles possessed the lowest 357
magnetization (66 emu/g) ascribed to the shape and size induced magnetic properties reduction 358
(Ramesh et al., 2012). Nonetheless, this method has disadvantages such as producing moderately 359
crystalline SPIONs as compared to magnetic nanopart icles synthesized via thermolysis method 360
(Wang et al., 2013) and consuming more time. 361
3.4. Microemulsion Method 362
Two immiscible phases (oil and water) are used to f orm SPIONs under the presence of 363
stabilizing agents by forming a monolayer at the in terface between the immiscible phases. 364
Water-in-Oil (W/O) and Oil-in-Water (O/W) are the t wo kinds of microemulsions formed to 365
synthesize different kinds of nanoparticles. The hy drophilic and hydrophobic parts of surface 366

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coatings play a major role in (i) stabilizing nanop articles, (ii) catering chemical reactions to 367
happen and (iii) controlling physicochemical parame ters. W/O microemulsion method is 368
frequently used to form SPIONs, where the stabilizi ng agents in continuous oil phase initially 369
protect the droplets formed of iron oxide reactants , which then react to form SPIONs. The sizes 370
and shapes are controllable by varying the concentr ations of iron oxide precursor to base, 371
surfactant and/or solvents. Recently, cetyl trimeth ylammonium bromide (CTAB) (Okoli et al., 372
2011) and synperonic 10/6 (Okoli et al., 2012) were used to tailor the size of the SPIONs. 373
Similarly, SPIONs with different sizes (6.5, 4.2 an d 8.7 nm) were synthesized by varying the 374
ratio of concentrations of iron oxide precursor to base (1:1 and 2:1) (Chin and Yaacob, 2007). 375
Nevertheless, the removal of unreacted precursors, base and surfactants is intricate in this 376
microemulsion method. 377
3.5. Sonochemical Method 378
Sound energy such as ultrasound can be used for syn thesizing SPIONs, where the cavitation 379
bubbles produced by such ultrasound transform the r eactants into desired products at ambient 380
temperatures. The size and shapes of SPIONs can be varied by controlling the refluxing time, 381
irradiation time and power. Recently, Dolores et al reported that production of Fe 3+ ions for 382
making iron oxide nanoparticles (using ethylene gly col as surfactant) increased in a linear 383
fashion with the increase in reaction time at a par ticular ultrasonic frequency (581 kHz) as 384
compared to other frequencies (861, and 1141 kHz) ( Dolores et al., 2015). 385
386
3.6. Microwave-assisted synthesis 387
Microwave energy can also be utilized for synthesiz ing SPIONs in a very short period of time at 388
low energy consumption. A uniform heat is provided by microwaves inside the reaction 389
containers from all sides for synthesizing stable a nd high crystalline iron oxide nanoparticles. 390
But these nanoparticles have reduced surface reacti vity (due to the low energy surface crystalline 391
facets), which is assessed based on their interacti on with water and aggregate formation process 392
(Pascu et al., 2012). Interestingly, the low surfac e reactive nanoparticles can enhance the stability 393
of SPIONs (Carenza et al., 2014), which is an advan tage of this method. The magnetic properties 394
of SPIONs can be controlled by manipulating the con centration of surfactants, microwave 395
power, and reaction time. 396
4. Encapsulation of SPIONs 397
Both organic polymers and inorganic materials are e xtensively used for encapsulating the bare 398
and surfactants/capping agents modified SPIONs (whi ch are prepared using different chemical 399
synthesis procedures) for improving the biocompatib ility, increasing the cellular uptake, 400
enhancing the circulation of SPIONs and preventing protein corona adsorption. The encapsulated 401
SPIONs should be in definite sizes to prevent them from clearing through kidneys and from 402
reticulo-endothelial system (RES) (i.e., clearing t hrough liver and spleen). In organic 403
encapsulation, synthetic polymers (simple polymers and amphiphilic polymers such as di- and 404
tri- block copolymers) and natural polymers (protei ns/polypeptides and polysaccharides) are 405
commonly used for the encapsulation of SPIONs for d irect use in cancer therapy and imaging. 406
Moreover, the polymeric systems can also be made st imuli sensitive for pH, redox environment, 407
and/or temperature sensitive to favor the release o f nanoparticles at the cancer site (Sundaresan et 408
al., 2014; Wadajkar et al., 2013). Among inorganic materials, silica is commonly used to 409
encapsulate SPIONs for cancer theranostics. Moreove r, both organic and inorganic materials are 410
used to envisage simultaneous co-encapsulation and delivery of SPIONs with other 411
chemotherapeutic, photothermal drugs and so on for cancer therapy and imaging. Herein, some 412

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recent developments in organic polymers and inorgan ic materials for encapsulation of SPIONs 413
for cancer theranostics have been discussed. 414
4.1. Organic Encapsulation 415
4.1.1. Synthetic Polymer Encapsulation 416
In synthetic polymer encapsulation, new kinds of di – or tri- block copolymers are prepared 417
through different polymerization process for encaps ulating and/or growing SPIONs. Recently, 418
free radical polymerization process was utilized to prepare poly(poly(ethylene glycol) 419
methacrylate-co-dimethyl-(methacryoyloxy)methyl pho sphonic acid) [poly(PEGMA-co- 420
MAPC1)] for encapsulation of two batches of SPIONs, where the sizes of SPIONs increased to 421
25±2 nm and 37±2 nm after polymer encapsulation (To rrisi et al., 2014). In another 422
investigation, polymerization-induced self-assembly (PISA) method was used to prepare poly- 423
(oligoethylene glycol methacrylate)-block-(methacry lic acid)-block-poly(styrene) (POEGMA-b- 424
PMAA-b-PST) triblock copolymer (Karagoz et al., 201 4). The carboxylic acid groups in this 425
copolymer initiated the formation of iron oxide nan oparticles (as shown in Fig 6), where the 426
length of methacrylic acid (MAA) units in the copol ymer helped in tuning the sizes of SPIONs 427
(i.e. 15, 9 and 5 MAA units resulted in 15, 12 and 9 nm SPIONs respectively). In another study, 428
poly(methylmethacrylate-acrylic acid-divinylbenzene ) (P(MMA-AA-DVB)) formed using 429
emulsion polymerization was manipulated to produce janus-kind Fe 3O4 nanoparticles with sizes 430
ranging from 200-250 nm (Ali et al., 2014). Nanopar ticles were grown in a single direction based 431
on the reduced interfacial energy between the polym er and nanoparticle magnetic domains. 432
Another group used a biocompatible shell made of po ly(N-methyl-2-vinyl pyridinium iodide- 433
block-poly(ethylene oxide) diblock copolymer (P2QVP -b-PEO) (M w/M n : 1.13) and ferritin 434
molecule to encapsulate SPIONs, where the cationic and anionic charges of hydrophilic 435
copolymer and ferritin surface respectively were ma nipulated to form string-like magnetic 436
clusters (with hydrodynamic size of 200 nm) (Tähkä et al., 2014). 437
In a recent investigation, polymerization of 2-(2-m ethoxyethoxy)ethyl methacrylate and 2- 438
(dimethylamino)ethyl methacrylate (through atomic t ransfer radical polymerization (ATRP) 439
process) was induced on the bromine modified surfac e of APTES-coated SPIONs to form brush 440
like structures on the surface of SPIONs for use in cell transfection (Liu et al., 2013). Similarly, 441
ATRP method was used to form the polymer, polyamido amine-b-poly(2-(dimethylamino)ethyl 442
methacrylate)-b-poly(poly(ethylene glycol) methyl e ther methacrylate) (PAMAM-b- 443
PDMAEMA-b-PPEGMA), which formed a dendritic −linear−brush like structure on the surface 444
of the SPIONs when combined with PPEGMA (He et al., 2012). Likewise, poly(glycidyl 445
methacrylate-co-poly(ethylene glycol) methyl ether methacrylate) (P(GMA-co-PEGMA)) 446
(formed via ATRP method) was used to encase high cr ystalline SPIONs (Huang et al., 2012). 447
Importantly, the magnetization value of P(GMA-co-PE GMA) coated SPIONs (82 emu/g) 448
sustained near to magnetization value of oleic acid coated SPIONs (88 emu/g) as shown in Fig 7, 449
where the authors claimed that the polymer quenched the magnetic moments of SPIONs through 450
electron exchange between SPIONs and polymer. Simil arly, poly(acrylic acid) (PAA) decorated 451
SPIONs (with hydrodynamic diameter of 39.4 ± 2.0 nm ) showed a magnetization value of 78.1 452
emu/g, which reduced in further encapsulations (Lin et al., 2009). PAA covered SPIONs showed 453
a high magnetization of 103 emu/g initially and red uced to 76 emu/g after subsequent chemical 454
coating/modification (Sun et al., 2012), but the re asons for high magnetizations at the initial 455
stage were not discussed in detail. 456
4.1.2. Natural Polymer Encapsulation 457

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Chitosan (a pH sensitive polysaccharide and a deriv ative of chitin) was cross-linked with 458
SPIONs through tripolyphosphate (TPP) molecules. Th e results showed that the SPIONs were 459
stable in wide range of pH levels and the release o f SPIONs and other drugs (bortezomib) 460
increased at low pH (4.2) due to swelling of chitos an at this pH level (Unsoy et al., 2014a, 461
2014b). Dextran (another natural polysaccharide) ca n be used as-such and/or cross-linked or 462
used as a derivative form for protecting SPIONs fro m agglomeration. Many commercially 463
available SPIONs used for clinical MRI, are coated with dextran only. In a recent study, 464
carboxymethyl dextran was used to coat SPIONs, but the magnetization value of SPIONs was 465
only 35 emu/g due to the lack of crystallinity of S PIONs (Jiang et al., 2014). A recent study 466
reported that hyaluronic acid (HA) can be used in c ombination with dextran coated SPIONs to 467
target CD44 receptor of cancer cells and to increas e the loading efficiency of SPIONs 468
(Unterweger et al., 2014). Similarly, collagen prot eins can be linked with heparin (a sulfur- 469
containing polysaccharide that inhibits blood coagu lation and induces their uptake more by 470
cancer cells) (J. Lee et al., 2012) and starch (L. Li et al., 2013) to deliver more SPIONs for MRI 471
application purposes. 472
Two different kinds of gelatin such as gelatin A an d B (extracted from collagen of pork and beef 473
respectively) were used for encapsulating and deliv ering SPIONs (Gaihre et al., 2009). Their 474
results of bioactivity and cellular uptake studies showed that the efficiency was improved when 475
co-encapsulated SPIONs and chemotherapeutic drugs ( both inside gelatin) were used for cancer 476
therapy. In a very recent study, gelatin cross-link ed SPIONs (prepared using emulsion and co- 477
precipitation methods) showed an enhancement in the degradability of SPIONs as well as the 478
cellular intake in cancerous cells (Tomitaka et al. , 2014). In another work, rhodamine 479
isothiocyanate (RITC) modified gelatin was used as a substrate to seed and grow magnetic 480
nanoparticles to improve the stability so that they can be conjugated with neurotrophic factors to 481
improve peripheral nerve regeneration (Ziv-Polat et al., 2014). 482
Three dimensional alginate (an anionic polysacchari de) based hydrogel network can be 483
established through ionotropic gelation method by a dding cations with alginate. SPIONs with an 484
average size of 13-15 nm were formed by sonicating iron oxide precursors with alginate to 485
explore the potential of SPIONs in photon activated therapies (Choi et al., 2012), which is 486
usually carried out via X-rays in lieu gold nanopar ticles. 487
4.2. Inorganic Encapsulation 488
4.2.1. Silica Encapsulation 489
Silica encapsulation presents substantial columbic repulsion in a colloidal solution to maintain 490
the stability of SPIONs for long time and influence s the magnetic properties of SPIONs by 491
modifying the thickness of surface spin canting lay er of SPIONs. Usually silica shell on the 492
surface of SPIONs is formed through Stöber process, which involves hydrolysis and 493
condensation of silica precursors. The surface of s ilica shell can be used to attach 494
ligands/polymers (such as amine/carboxylate/PEG) to increase the biocompatibility of SPIONs 495
and to cater chemotherapeutic drugs for effective c ancer therapy. 496
Sodipo and Aziz group modified the surface of co-pr ecipitated SPIONs with (3- 497
aminopropyl)triethoxysilane (APTES) and silica in t wo different studies (Sodipo and Abdul 498
Aziz, 2015; Sodipo and Aziz, 2014) using ultrasonic energy, where the magnetization values of 499
77.7 and 20-30 emu/g were reported for APTES and si lica coated magnetic nanoparticles 500
respectively. Similarly, in another study, a magnet ization value of 77 emu/g was obtained for 501
silica coated iron oxide nanoparticles (Islam et al ., 2013). Recently, Yang et al . formed a 502
microemulsion containing cetyltrimethylammonium bro mide (CTAB)/SPIONs (formed through 503

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thermal decomposition) whose surface was modified w ith silica coating using tetraethyl 504
orthosilicate (C. Yang et al., 2014). A core/shell/ shell nanostructure of SPIONs/zinc oxide 505
(ZnO)/silica was developed, where ZnO layer was use d for microwave-triggered drug release 506
(Qiu et al., 2014). The porous surface of silica en hanced the opportunity to cater drugs but 507
degraded the magnetic properties of SPIONs (i.e., r eduction in magnetization value from 85 to 508
64.7 emu/g). In a similar way, the magnetization va lue diminished to more than half of the 509
original value ( i.e., from 79 to 38 emu/g), when t he SPIONs decorated with silica coatings (refer 510
Fig 8) and reduced further to 18 emu/g for subseque nt functionalization of folic acid over silica 511
surface (Wortmann et al., 2014). Polyelectrolytes b ased DNA transfection was successfully 512
carried out using silica-coated magnetic nanopartic les, where DNA was attached to silica surface 513
through layer-by-layer (LBL) assembly method (Dávil a-Ibáñez et al., 2013). The quasi-cubic 514
shaped magnetic nanoparticles were developed with s ilica coating to improvise shelf life and 515
biological stability of the nanoparticles (Campbell et al., 2011). In another investigation, 516
aminosilane coating over the surface of SPIONs impr oved their cellular uptake efficiency in cell 517
lines such as RAW264.7, L929, HepG2, PC-3, U-87 MG, and mouse mesenchymal stem cells 518
(MSCs) (Zhu et al., 2012). 519
Silica can also act as a linkage molecule between S PIONs and other polymer coatings to make 520
tunable chemical structures. Takafuji exploited the reaction between 4-vinylpyridine with 3- 521
mercaptopropyl trimethoxysilane to form a polymer c omplex of poly(4-vinylpyridine) with 522
alkoxysilyl group to enhance DNA transfection effic iency (Takafuji et al., 2014). Another study 523
involved coating of silica (for stability) and meso porous silica (for further functionalization with 524
gold) on top of ellipsoidal shaped SPIONs to organi ze a core-shell structure to adopt 525
chemotherapeutic drugs for effective cancer therapy (Chen et al., 2010). 526
5. Cancer Theranostic Applications of SPIONs 527
5.1. Magnetic Resonance Imaging (MRI) 528
In general, MRI is a biomedical imaging technique u sed to image soft tissues of human body in 529
very thin slices in two dimensional as well as thre e dimensional spaces. The water present in our 530
body plays an important role in obtaining MRI image s. The hydrogen nucleus in water tends to 531
align them in a direction parallel to applied exter nal magnetic field. Then a radiofrequency (RF) 532
signal is applied to change the direction of alignm ent of protons in the hydrogen nucleus, where 533
the frequency of the RF signal must be in resonance with the frequency of the hydrogen nucleus. 534
As the directions of the protons are changed after applying the RF signal, the protons tend to re- 535
align with the applied magnetic field. So while ret urning to its original position, these protons 536
release energy as an RF signal that can be detected by detectors in MRI machine. The re- 537
alignment speed of protons varies for various tissu es in our body, which is helpful in imaging 538
such tissues precisely and the time taken for this re-alignment is called as the relaxation time. T1 539
(longitudinal – spin–lattice relaxation) and T2 (tr ansverse – spin–spin relaxation) are the 540
relaxation times based on the time needed for the c omponents of respective magnetization 541
vectors, in both the cases, to return to their orig inal thermal equilibrium state. The relaxivities (r 1 542
& r2), that changes with the applied magnetic field in longitudinal and transverse directions, are 543
the inverse of the relaxation times at the respecti ve directions (i.e., r1=1/T1; r2=1/T2 ), where the 544
ratio of relaxivities is significant in deciding th e fate of the nanoparticles to be used either as a 545
positive or a negative contrast. Both T1 and T2 rel axations are dependent on the saturation 546
magnetization of nanoparticles and their magnetic i nteractions with the protons of surrounding 547
water molecules. 548

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Paramagnetic gadolinium (Gd(III)) complexes are com monly used as MRI contrast agents for 549
about three decades by combining Gd(III) with kinet ically and thermodynamically stable 550
chelating ligands, where the tissues/cells appear b right (T1 or positive contrast) at the contact 551
region (Khan et al., 2014). Moreover, Gd(III) compl exes should be encapsulated into 552
macromolecules such as protein, liposomes and dendr imers to deliver them into in vivo 553
conditions, since free gadolinium ions are very tox ic to the biological systems. The other 554
disadvantages of these Gd(III) complexes at in vivo scenario include the following: (i) short life 555
span, (ii) poor cellular uptake (iii) limited to bl ood and/or extracellular space resulting only in 556
molecular imaging and (iv) induction of nephrogenic systemic fibrosis (NSF). Since the 557
pharmacokinetics of the synthetic Gd(III) complexes are difficult to study in biological systems, 558
manganese (Mn(II) or Mn 2+ ) based T1 MRI contrasts are introduced to image th e anatomical 559
structure of brain. However, these Mn(II) complexes have lower thermodynamic stability, high 560
toxicity (towards heart and liver), low magnetizati on and less MRI signal sensitivity. 561
Nevertheless, metal (Fe, Ni, Co)/metallic alloy (ir on-cobalt and iron-platinum)/metal doped 562
ferrites (for example, CoFe 2O4, MnFe 2O4) are developed as T2 MRI contrast agents for 563
enhancing the signal sensitivity by improving the s aturation magnetization. In T2 MRI contrast, 564
the relaxivities have a tendency to decay rapidly i n transverse direction (faster than r2 relaxivities 565
of protons of water molecules) to yield T2* relaxat ion (corresponding relaxivity – r2*) due to the 566
coupled factors such as spin–spin relaxation and ma gnetic field inhomogeneities, resulting in 567
negative contrast effects (i.e., darkening effect) at the region of contact (molecular/cellular 568
levels). But, the T2 MRI contrast based metals/meta llic alloys are chemically more reactive, 569
resulting in oxidation of these agents under biolog ical conditions. Moreover, the in vivo 570
biodegradability and cytotoxicities of metals/metal lic alloys/metal doped ferrites should be 571
investigated for extensive period of time before th eir usage in clinical trials. 572
5.1.1. SPIONs as T2 MRI Contrast agents 573
SPIONs have been approved to be used as T2 MRI cont rasts (negative contrast) for liver imaging 574
by food and drug administration (FDA) department, a s shown in Table 2. MRI signal contrast of 575
SPIONs is higher than the contrast of Gd(III) and M n(II) complexes, since the relaxivities of 576
SPIONs are enhanced via high saturation magnetizati on due to the presence of more number of 577
Fe atoms that are responsible for such magnetizatio n in SPIONs (Gossuin et al., 2009). 578
Moreover, SPIONs are chemically stable, biocompatib le and biodegradable in in vivo conditions 579
due to their ability to mix as normal iron ions in the blood as compared to metal/metal alloy 580
based contrast agents. In addition, high signal sen sitivity and quick detectability via electron 581
microscopy made SPIONs as potential candidates for in vivo MRI contrast (Yang et al., 2009). 582
Despite the ban on some commercial SPIONs due to it s toxicity, ferumoxytol (Feraheme), 583
ferumoxides, ferucarbotran, ferumoxtran-10, ferrist ene and ferumoxsil are other SPIONs based 584
MRI contrast agents at current clinical trials (htt ps://www.clinicaltrials.gov/). Nevertheless, 585
many investigations have been performed by global r esearchers to improve the T2 MRI contrast 586
effects of SPIONs for enhanced cellular and molecul ar imaging. Very recently, exceptional 587
transverse relaxivity values of 735.3mM –1s–1and 450.8 mM –1s–1 at 3T MRI were achieved for 588
terephthalic acid (TA) and 2-amino terephthalic aci d (ATA) coated SPIONs respectively (Maity 589
et al., 2012). The authors explained that high crys tallinity and the effective spin transfer between 590
the surface atoms of SPIONs and surrounding molecul es through π- π conjugation of TA/ATA 591
facilitated the actualization of high T2* MRI relax ations with low cytotoxicity towards fibroblast 592
cells (NIH3T3 cell lines). Moreover, the manipulati on of distribution of SPIONs inside the 593
encapsulants yielded a high r2 relaxivity of 582 mM–1s–1 at 9.4 T MRI in their another study 594

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14
(Karagoz et al., 2014). Similarly, PEI (with two di fferent formulations) coated SPIONs exhibited 595
relatively high r2 relaxivity values of 514.7 mM –1s–1and 596.8 mM –1s–1 respectively at 1.5 T (Lin 596
et al., 2014) but the reasons behind this increment were inconclusive. Table 3 summarizes MRI 597
relaxivity values of synthetic iron oxide (Fe 3O4 and Fe 2O3) nanoparticles having different sizes 598
and surface coatings. 599
Many studies have been performed to investigate the MRI contrast efficiency of SPIONs in in 600
vivo scenarios. For example, Saraswathy et al prepared citrate-coated ultra-small SPIONs (C- 601
USPIONs) with particle size and r2 relaxivity of 12 nm and 102 mM –1s–1 respectively. The 602
hepatocellular uptake of C-USPIONs was identified b y a 39% decrease in signal intensity in 603
post-contrast MRI images of rat liver (Saraswathy e t al., 2014a). In another study, dextran-coated 604
SPIONs (DSPIONs with r1: 2.5 mM –1s–1 and r2:140.7 mM –1s–1) were injected into male Wistar 605
rats at a dose of 2.17 mg/ml Fe/kg body weight via tail vein to evaluate the liver fibrosis in these 606
animal models, where the post-contrast T2 weighted images showcased a hypointense liver with 607
a 55% decrease in the average MRI signal intensity, indicating a higher hepatocellular uptake of 608
DSPIONs (Saraswathy et al., 2014b). Similarly, fola te-targeted, poly(ethylene glycol)-poly( ε- 609
caprolactone) (FA-PEG-PCL) coated USPIONs were inje cted into BEL-7402 tumor bearing 610
nude mice via tail vein, where the MRI signal inten sity decreased to 41.2% within 3 hours of 611
injection resulting in clear tumor images. Moreover , the intensity further decreased to 32.4% at 6 612
hours after injection, which showed that the accumu lation of folate receptor based SPIONs at the 613
target tumor site increased as compared to non-targ eted ones (Hong et al., 2012). 614
In one investigation, poly (lactic acid)-d-alpha-to copherol polyethylene glycol 1000 succinate 615
copolymer (PLA-TPGS) coated SPIONs were injected in to MCF-7 induced severe combined 616
immune deficiency (SCID) female mice at a dose of 5 mg Fe/kg body weight (Prashant et al., 617
2010). In vivo MRI images of the liver of SCID mice were evaluate d before and 0.33, 2, 5 and 12 618
hours after the injection of PLA-TPGS coated SPIONs , where the MRI signal intensity at the 619
tumor site decreased after the injection of the SPI ONs indicating their potential diagnostic usage 620
in clinical trials. In another investigation, the c ore size (14 nm) of 1,2-distearoyl-sn-glycero-3- 621
phosphoethanolamine-N-[methoxy(polyethylene glycol) ] (DSPE-PEG) copolymer coated 622
SPIONs were tuned resulting in an increase of T2 re laxivity by more than 200 fold in non- 623
biological conditions (Tong et al., 2010). Moreover , these DSPE-PEG coated SPIONs had more 624
half-life (i.e. 23.2 minutes) in blood circulation of human U87 glioblastoma cells induced mice. 625
Similarly in another study, PEG coated SPIONs (9 nm size) and PEG/polyethylenime (PEI) 626
coated SPIONs (10 nm) showed enhanced MRI contrast effects after injecting them at a dose of 627
10 mg Fe/Kg of body weight of Kunming mice (J. Wang et al., 2015) as shown in Fig 9. In 628
addition, the PEG-SPIONs decreased the MRI signal c ontrast at bulbus olfactorius, frontal 629
cortex, temporal cortex and thalamus portions of mi ce after 24 hours of its injection as compared 630
to PEG-PEI coated SPIONs due to their improved half -life in blood circulation. In another 631
research, CXCR4-peptide-attached-PEG-coated-iron-ox ide nanoparticles were formed via self- 632
assembly by initial modifying the surface of nanopa rticles with bioorthogonal azide and alkyne 633
groups (Gallo et al., 2014). A 25% decrease in MRI signal intensity in U87.CD4.CXCR4 634
(implanted in BALB/c nude mice) based tumor after t he intratumoral injection of peptide 635
modified nanoparticles, whereas a 14% intensity dec rement at the tumor site (within 4 hours) 636
was observed when the nanoparticles were intravenou sly injected. 637
5.1.2. SPIONs as T1/ dual mode (T1-T2) MRI Contrast agents 638
The potential of SPIONs as a T1 MRI contrast (posit ive contrast) agent has been identified 639
recently, where the size of SPIONs should be optimu m (< 5 nm) to achieve good T1 contrast 640

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15
effect (Chan et al., 2014). Moreover, both T1 and T 2 relaxations can be enriched in a single iron 641
oxide nanoparticle by optimizing their size, shape and surface coatings. In a recent investigation, 642
SPIONs with both longitudinal and transverse relaxi vities were obtained by simply manipulating 643
the morphology and exposed facet (111) of SPIONs to enhance both positive and negative 644
contrasts in SPIONs (Zhou et al., 2014). The size-o ptimized magnetic nanoplates (8.8 and 4.8 645
nm) showed r2 values of 311.88±7.47 and 182.2± 7.73 mM -1s-1 and r1 values of 38.11±1.04 and 646
43.18±3.33 mM -1s-1 at 0.5 T as shown in Fig 10. In another investigat ion, dendron modified 647
SPIONs showed better T1 and T2 contrasts (Ghobril e t al., 2013), when compared with 648
commercial SPIONs used for MRI contrast (Basly et a l., 2013, 2011). Likewise, liposomes were 649
used to encapsulate ultra-small SPIONs at increased concentrations to improve dual-mode (T1 650
and T2) MRI contrast efficacy (Prassl et al., 2012) . Similarly, ultra-small SPIONs produced T1 651
contrast effect better than other commercially avai lable SPIONs at specific sizes (Sandiford et 652
al., 2013). Analogously, Jung et al. achieved T1 and T2* MRI contrast concomitantly in in vivo 653
and in vitro conditions by controlling the size of the SPION (~ 7 nm), where r1 relaxivities (13.31 654
mM -1s-1 at 1.43 T and 6.84 mM -1s-1 at 3T) of SPIONs were relatively higher than the 655
conventional ones (gadolinium based) and r2* relaxi vity was maintained at 49.50 mM -1s-1 at 3T 656
(Jung et al., 2014). Similar set of studies on this dual-mode MRI contrast enhancement were 657
lately done (Pourcelle et al., 2015). Recently, a n umerical analysis for finding an optimal 658
aggregation for better relaxivities was also studie d (Vuong et al., 2011). 659
5.2. Magnetic Hyperthermia Therapy 660
Hyperthermia therapy (HTP) which is a heat induced malignant cancer treatment by promoting 661
SPIONs as heat producers, can be performed as eithe r localized therapy or systemic therapy. 662
After localization of SPIONs near the cancer site u sing magnetic targeting, an alternating 663
magnetic field (AMF) is applied for a period of tim e to induce heat of about 42-45 ⁰ C for 664
initiating apoptosis in cancer cells, where this he at can be controlled by manipulating the size, 665
shape, crystallinity, corresponding magnetic proper ties of SPIONs and the applied AMF. 666
Specific absorption rate (SAR) is a parameter to qu alitatively and quantitatively measure the 667
efficiency of SPIONs which take part in converting AMF into heat based on Brownian and Néel 668
relaxations of individual SPIONs. The occurrence of whole body and localized side effects can 669
be minimized during magnetic hyperthermia, if the A MF and frequency are below 5*10 9 Am -1 s -1 670
and 1 MHz respectively. In support to the previous study, Cervodoro et al reported that the 671
relaxations required for inducing heat from SPIONs (5, 7 and 14 nm sized) started to take place 672
at a frequency range i.e., less than 1 MHz and stop ped above this frequency range when tested 673
for wide range of frequencies (up to 30 MHz), there by recently confirming the operational 674
frequencies for heat induction (Cervadoro et al., 2 013). Moreover, magnetic nanoflakes, made of 675
deoxy-chitosan polymer stabilized 20 nm sized nanoc ubes, yielded a comparatively high SAR 676
value of 73.8 ±2.3 W/g Fe for a frequency of 512 kHz than individual nanocubes (Cervadoro et al. , 677
2014). In a similar fashion, multi-core magnetic na noparticles exhibited a high SAR value of 678
almost 2000 W/g (applied field of 29 kA/m and frequ ency of 520 kHz) with an increase in 679
temperature rate of 1.04 °C/s for an iron concentration of 0.087 M (Lartigue et al., 2012). 680
Moreover, doping of SPIONs with other metal atoms ( for instance, manganese) can also be done 681
to improve the hyperthermia activity of magnetic na noparticles. But, copper (5%, 10%, 15% 682
mol/mol) doped iron oxide core (7 nm) resulted in v ery low SAR values, owing to the lower size 683
of ferritin molecules coated magnetic core (Fantech i et al., 2014). Table 4 summarizes the SAR 684
values of the iron oxide nanoparticles with respect to different sizes and surface coatings. 685
Though, optimal SAR values are obtained for magneti c nanoparticles outside the biological 686

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16
environment, those values tended to decrease when S PIONs are introduced into in vitro and in 687
vivo conditions because of the dissipation of heat to t he surrounding tissues through blood flow 688
(inside and outside tumor area). 689
However, SPIONs showed good therapeutic results in cancer treatments in in vitro and in vivo 690
scenarios. For example, 14 nm magnetic nanoclusters (with SAR value of 500 Watt/g) killed 691
almost 74% of MCF-7 cancer cells in in vitro conditions, where a therapeutic temperature of 692
45 °C for 1 hour was maintained (Maity et al., 2011) as shown in Fig 11. Similarly, the cell 693
viability of HeLa cells was reduced to 42% as these cells were exposed to a temperature of 43 ⁰ 694
C (for 1000 seconds) which was induced by applying an alternating magnetic field to silica 695
coated iron oxide nanoparticles (Majeed et al., 201 4). In another study, the magnetic 696
nanoparticles reached their in vitro hyperthermia levels (42-45 ⁰ C) in less than 200 seconds at a 697
frequency of 26.48 kA/m, when these nanoparticles w ere incubated with three different cancer 698
cell lines (DA3, MCF-7 and HeLa) (Gkanas, 2013). Mo reover, the induction of apoptosis in 699
cancer cells through magnetic nanoparticles increas es with an increase in the 700
concentration/quantity and the size of these nanopa rticles. For instance, Jadhav et al reported that 701
the induction of apoptosis process in WEHI-164 tumo r cells increased near to 80%, when the 702
quantity of sodium carbonate-stabilized-oleic acid- functionalized magnetic nanoparticles was 703
increased from 0.22 mg to 0.44 mg (Jadhav et al., 2 013). In another study, only 40% of Jurkat 704
cells survived for a low dose (490 µg Fe/ml) of 16 nm magnetic nanoparticles as compared to 705
80% and 90% survival rate for 12 nm and 13 nm nanop articles at 600 µg Fe/ml concentration 706
(Khandhar et al., 2012). In a recent study, polymer (combination of poly(vinyl alcohol) and 707
polyvinylpyrrolidone)-stabilized-iron oxide-graphen e nanocomposite attained a heat of ~42 °C 708
for a concentration of 2.5 mg/mL within 15 minutes of application of AMF at 418 Oe, where 709
⁰40 ± 4% and ⁰76 ± 3% of cell death was observed after 4 and 8 ho urs incubation of 710
nanocomposites with HeLa cells (Swain et al., 2015) . 711
Hayashi et al reported that the exposure of magneti c nanoclusters to AMF intensity of 8 kA/m 712
and frequency of 230 kHz decreased the size of tumo r in Female CB17/Icr-Prkdcscid mice, 713
where the folic acid attached magnetic nanoclusters (with an average SAR value of 248 W/g) 714
were injected intravenously (Hayashi et al., 2013). A rise in the temperature of 6 °C was observed 715
at 20 minutes as compared to the surrounding tissue s. Moreover, the volume of the tumor 716
decreased to one-tenth times of the tumor in contro l mice after 35 days of treatment (as shown in 717
Fig 12), where the life-span of hyperthermia treate d mice extended by 4 weeks. In a similar way, 718
intraperitoneally injected magnetic nanoparticles h elped in the reduction of tumor created via 719
injection of Pan02 cells into C57BL/6 mice, after g etting exposed to 15-20 minutes of AMF, 720
thereby improved the life expectancy rate of mice b y 31% (Basel et al., 2012). In another case, 721
the volume of SCCVII squamous cell carcinoma induced in mice was compara tively reduced 722
through magnetic nanoparticles at specific intraven ous dose and applied field of 38 kA/m at 980 723
kHz (Huang and Hainfeld, 2013). In a similar fashio n, polypyrrole coated Fe 3O4 nanoparticles 724
showed an SAR value of 487 W/g, where the nanoparti cles considerably inhibited the growth of 725
myeloma tumor induced in Female CB17/Icr-Prkdcscid mice but completely when a 726
combination of Fe 3O4 nanoparticles and a chemotherapeutic drug at a quan tity of 5 mg/Kg was 727
used for cancer therapy (Hayashi et al., 2014). Sim ilar in vivo hyperthermia studies using 728
SPIONs are summarized in Table 5. 729
5.3. Magnetic Targeting 730
Magnetic targeting is the targeting of iron oxide n anoparticles to a specific cancer site by 731
applying an external magnetic field. In one study, the transcellular transport of heparin coated 732

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17
magnetic nanoparticles into the cellular membrane o f MDCK strain II cells was made easy with 733
the presence of magnetic field as compared to the a bsence of magnetic field (Min et al., 2010). In 734
another investigation, N-methyl-2-pyrrolidone media ted lipid based iron oxide nanoparticles 735
were combined with green fluorescence protein (GFP) -nucleic acids system, where the nucleic 736
acid transfection efficiency inside HeLa cells was improved with magnetic targeting as compared 737
to the untargeted ones (Jiang et al., 2010). Human mesenchymal stem cells (used in regenerative 738
cell therapies) were incubated and attached with PE G coated nanoparticles through concentrated 739
magnetic field exposure up to a range of time (1 ho ur to 24 hours), where no changes in the cell 740
viability and cell structure were observed after th e internalization of magnetic nanoparticles into 741
those stem cells (Landázuri et al., 2013) as shown in Fig 13. Later, the magnetic nanoparticles 742
loaded stem cells showed enhanced accumulation in b oth in vivo and in vitro situations, after the 743
application of magnetic field. Similarly, the inter nalization of poly (dopamine) coated magnetic 744
nanoparticles increased by 14-15% more in case of H eLa cells and HepG2 cells after exposing 745
them to a magnetic field for about 4 hours (Wu et a l., 2015). 746
In a recent investigation, the in vivo magnetic targeting of PEI-modified magnetic nanopa rticles 747
to intracerebral 9L tumors was achieved using an ex posure to magnetic field of 350 mT for about 748
30 minutes, after the injection of 12 mg Fe/kg of n anoparticles through intra-carotid catheter 749
(Chertok et al., 2010). The results showed a 30 fol d improvement of accumulation of magnetic 750
nanoparticles onto tumors. The cellular uptake and the accumulation of iron oxide nanoparticles 751
at two different 4T1-tumor sites (induced in a mice ) were enhanced through magnetic targeting 752
(Z. Li et al., 2013). In another study, the magneti c targeting helped in improving the intake 753
efficiency of dextran coated SPIONs in VERO and MDC K cell lines as well as in tumor induced 754
BALB/c mice (nearly160% improvement) (Mojica Piscio tti et al., 2014). Likewise, α,β-Poly(N- 755
2-hydroxyethyl)-D,L-aspartamide-co-(N-2-ethylen-iso butirrate)-graft-poly-(butyl methacrylate) 756
(PHEA-IB-p(BMA) copolymer-coated SPIONs were acted as nano-carriers for a 757
chemotherapeutic drug (flutamide), where the retent ion concentration of magnetic flutamide in 758
kidneys of Winstar rats was higher after the applic ation of magnetic field (0.3 T) as compared to 759
non-magnetic flutamide (Licciardi et al., 2013). In addition to magnetic targeting, active 760
targeting of the magnetic nanoparticles can also be performed with the help of surface attached 761
targeting ligands such as folic acid, peptides, apt amers, proteins, antibodies, etc. 762
5.4. Magnetofection 763
Transfections performed using SPIONs are called mag netofection, where different types of 764
negatively charged nucleic acids (DNA and RNA) are conjugated with magnetic nanoparticles 765
through positively charged polymers such as polyeth ylenimine (PEI) or polyaziridine via 766
electrostatic interactions. PEI polymer is widely u sed in encapsulating SPIONs for 767
magnetofection applications since PEI has a tendenc y to induce “proton sponge effect” in order 768
to escape from lysosomal degradation inside the cel l. Moreover, the size and zeta potential of 769
polymer coated SPIONs complexes should be optimized for effective nucleic acids delivery. 770
In an investigation, SPIONs were encapsulated with two kinds of polymers such as (i) a 771
polycationic polymer, i.e., poly(hexamethylene bigu anide) (PHMBG) and (ii) PEI, (Castillo et 772
al., 2012). Then small interfering RNA’s (siRNAs) r esponsible for luciferase gene knockdown 773
were functionalized onto both of polymer encapsulat ed SPIONs, where PEI modified 774
siRNA/SPIONs delivery system showed high magnetofec tion efficiency and reduced toxicity 775
towards CHO-K1 tumor cells as compared with PHMBG m odified siRNA/SPIONs delivery 776
system. Polyacrylic acid (PAA)-PEI modified SPIONs was conjugated with a therapeutic gene 777
for plasmid DNA interleukin 12 (pDNA IL−12 – immune response stimulant against tumors), where 778

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a 12.6 fold increase in transfection was observed i n B16F1 cells after applying magnetic field 779
(Prijic et al., 2012). In another study, the magnet ofection efficiency of SPIONs-PAA-PEI 780
complexes had improved when the functionalization o f PEI over SPIONs-PAA system was done 781
in an alkaline medium (Prosen et al., 2013). Branch ed PEI-SPIONs were conjugated with pDNA 782
(containing gWIZ (an eukaryotic expression vector)- IL-10 (interleukin 10)) for transfecting 783
primary vascular endothelial cells (HUVEC), where t he expression of PAI-1 in HUVEC cells 784
was effectively inhibited after the magnetofection of IL-10-branched PEI-SPIONs (Namgung et 785
al., 2010). 786
An effective silencing of melanoma cell adhesion mo lecule (MCAM) was achieved in both in 787
vitro and in vivo levels through magnetofection, when pDNA (encoding for short hairpin RNA 788
against MCAM) was combined with PEI-SPIONs (Prosen et al., 2014). Similarly, the growth of 789
HepG2 cancer cells was inhibited effectively by tra nsfecting them with human telomerase 790
reverse transcriptase (hTERT) gene silencing siRNA modified disulphide-PEI-SPIONs (SSPEI- 791
SPIONs) (D. Li et al., 2014). In another study, the luminescence expression of pGL3-control 792
plasmid (with Hind III/Xba I firefly luciferase cDN A fragment – introduced into E. Coli strain 793
DH5a) in HEK 293T cells was improved with the incre ase in w/w ratio of pGL3-control plasmid 794
loaded PDMAEMA-iron oxide nanoparticles (Huang et a l., 2013) (as shown in Fig 14). 795
However, a 1000 fold increase in transfection effic iency was observed on the application of 796
magnetic field onto nanoparticles incubated HEK 293 T cells. 797
5.5. SPIONs in Combination with Dopants/Other Imagi ng Agents for Multimodal Imaging 798
Dual mode MRI contrast (T1 and T2 contrasts) can al so be achieved either by doping gadolinium 799
(Gd) atoms into the core of SPIONs or by attaching the Gd-complexes onto the surface of 800
SPIONs for retaining their corresponding relaxiviti es. For example, Xiao et al prepared PEG- 801
coated-Gd-doped-Fe 3O4 nanoparticles (mean diameter of 4.74±0.51 nm) with r1 and r2 values of 802
65.9 and 66.9 mM -1 s -1, where these nanoparticles showed an enhanced in vivo T1-T2 dual MRI 803
contrast effects (performed using 7 T MRI scanner) in C6 glioma-bearing mice after post- 804
injection of nanoparticles at a dose of 5 mg Fe/kg (Xiao et al., 2014). In another recent study, a 805
dual-mode contrast was achieved by forming a hybrid dumbbell-shaped nanostructure, in which 806
one bell consisting iron oxide core (covered with P EG) was connected through platinum cubes 807
with another bell (formed of Gd coated gold core) ( Cheng et al., 2014). The r1 and r2 relaxivities 808
were optimized by controlling the distance between the two bells (i.e., the size of platinum 809
cubes) to prevent the magnetic influences between i ron oxide and gadolinium. 810
Likewise, Fe 3O4/Manganese oxide (MnO) hybrid nanocrystals in the f orm of core/shell, 811
dumbbell and flower-shaped were respectively synthe sized from 5, 11, and 21 Fe 3O4 812
nanoparticles to detect in vivo human hepatocellular carcinoma (HCC) (Im et al., 2 013). The T1 813
and T2 MRI signal intensities were effectively enha nced, since the Fe 3O4 nanoparticles remained 814
in the Kupffer cells of the liver, after the releas e of Mn 2+ ions in the extracellular area (for T1 815
contrast) after opsonization of the hybrid nanocrys tals, thereby the quenching of T2 contrast 816
effect due to Mn 2+ ions was avoided. In another similar study, a brai n-tumor-targeting-agent 817
(chlorotoxin) and a fluorescence agent (Cy5.5) were attached to the dumbbell shaped 818
Fe 3O4/MnO nanoparticles through N-(Trimethoxysilylpropyl ) ethylene diamine triacetic acid, 819
trisodium salt (TETT) and N-Succinimidyl-3-(2-pyrid yldithiol) propionate (SPDP) conjugated 820
with PEG 2000 (Li et al., 2015). The T1–T2 dual modal signal enha ncement was significantly 821
obtained at the glioma-bearing brain of mice, after the intravenous injection of respective doses 822
of 10 and 5.5 mg of Mn and Fe per kg of body weight (with respective relaxivities (r1 and r2) of 823
5.37 and 203.82 mM -1 s -1) as shown in Fig 15. 824

Page 39 of 71Accepted Manuscript
19
SPIONs based MRI can be combined with other imaging modalities such as CT/PET/gamma ( γ) 825
imaging/PET/fluorescence imaging/Cherenkov luminesc ence imaging for better diagnosis and 826
treatment of cancers with different resolutions, by coupling SPIONs with radio nuclides, optical 827
imaging enhancers, and luminescence/fluorescence en hancers. The activity of non-phagocytic 828
primary T cells labelled with SPIONs-64 Cu (Copper-64) nano-complex in the tumor cells was 829
studied using a combined imaging of PET-MRI (Bhatna gar et al., 2014). In this analytical study, 830
confocal microscopy images ensured that dimethyl su lfoxide (DMSO) facilitated the entry of 831
nano-complex into the cellular membrane and after t he entry, charged SPIONs-labelled T-cells 832
were used in targeting the B-cell lymphoma model. 64 Cu-attached SPIONs were prepared for 833
MRI/PET hybrid imaging by using dextran as a conjug ation medium (Wong et al., 2012). 834
Human serum albumin (HSA) decorated SPIONs were fun ctionalized with 64 Cu- 1,4,7,10- 835
tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (D OTA) and Cy5.5 dye (1:5:5 ratio) for 836
combined MRI/PET and near-infrared fluorescence ima ging of U87MG tumor induced in a mice 837
(Xie et al., 2010). In similar manner, PEG molecule s decorated 64 Cu DOTA-SPIONs decorated 838
were formed to provide high resolution and high sen sitive PET/MR imaging in in vivo conditions 839
(BALB/c mice) (Glaus et al., 2010). 99m Tc was conjugated with SPIONs to provide an effecti ve 840
MRI/PET imaging in Balb/C mice in addition to MRI a ngiographic imaging. Moreover, PET 841
imaging studies showed that some amount of SPIONs w ere expelled by kidneys and others were 842
re-circulated inside the body (Sandiford et al., 20 13). Similarly, the surface of SPIONs was 843
modified with radionuclides ( 68 Ga or 89 Z) and ligand molecules to provide coupled MRI/PET 844
imaging analysis of in vivo angiogenesis in a single step (Groult et al., 2015 ). 845
Iodide-131 ( 131 I) was conjugated with L-tyrosine modified SPIONs t o amalgamate the SPECT 846
imaging property with the MRI yielding SPIONs (Park et al., 2011). In another study, radio- 847
labelling of Indium-111 ( 111 In) was done on the surface of APTES-SPIONs using t hiolated 848
3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,1 3-triene-3,6,9,-triacetic acid (PCTA) 849
bifunctional chelators for facilitating SPECT imagi ng. MRI/SPECT in vivo imaging results 850
indicated that the radio-labelled SPIONs reached th e tumor site (Zolata et al., 2015). Similar 851
MRI/SPECT imaging study was performed using 111 In modified SPIONs (Misri et al., 2012). 852
Recently, three radionuclides such as 59 Fe, 14 C and 111 In were attached with SPIONs to identify 853
the biodistribution of oleic acid, iron oxide nanop articles and their togetherness at different time 854
intervals, (Freund et al., 2012; H. Wang et al., 20 15), where 111 In labeling was suggested to be 855
used for combined MRI/SPECT imaging. Commercially a vailable SPIONs were conjugated, 856
through bisphosphonates, with 99mTc-dipicolylamine( DPA)-alendronate for combined in vivo 857
MRI/SPECT and PET imaging (De Rosales et al., 2011) (refer Fig 16). In another study, in vivo 858
SPECT/CT imaging of Female BALB/c mice showed that 99mTc-labeled-lactobionic acid- 859
coated SPION selectively accumulated (38.43±6.45% i njected dose/gram) in hepatocytes, where 860
the specific uptake of the SPIONs by liver was conf irmed by the in vivo MRI images (Lee et al., 861
2009). 862
5.6. SPIONs in Combination with Other Therapeutic A gents 863
5.6.1. In combination with Photodynamic therapy 864
In photodynamic therapy (PDT), a photosensitizer/ph otosensitizing agent and a light source are 865
used to kill cancer bearing tissues by initiating p hotonecrotic effects through the production of 866
free radicals/singlet oxygen (highly reactive state of oxygen) in those tissues. Silicon 867
phthalocyanine photosensitizer Pc 4 [HOSiPcOSi(CH3) 2(CH2)3N-(CH3)2] is introduced as a 868
new generation photosensitizer to overcome disadvan tage of Porfimer sodium or Photofrin® (a 869
former FDA approved photosensitizing agent). SPIONs are combined with these photosensitizers 870

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for combined MRI imaging, magnetic targeting, HTP a nd PDT. Recently, a nano-system 871
comprising carboxylic acid functionalized SPIONs, P c 4 photosensitizer and a fibronectin 872
mimetic peptide (Fmp) was developed to target integ rin β1 overexpressed in head and neck 873
squamous cell carcinoma (HNSCC) xenograft tumors. T he magnetic targeting of hybrid SPIONs 874
helped in the enhanced inhibition of tumor growth ( in ) as compared with magnetically non- 875
targeted IO-Pc 4 hybrid nano-system (D. Wang et al. , 2014) as shown in Fig 17. Similarly, a 876
combination of SPIONs and chlorin e6 (Ce6 – a photo sensitizer) showed increased therapeutic 877
effect at in vivo level with low cytotoxicity to normal cells (Z. Li et al., 2013). 878
Carbon fullerene (C60) was mixed with SPIONs and He matoporphyrin monomethyl ether 879
(HMME) (a photodynamic anti-cancer drug) to create hybrid C60-IONP-PEG/HMME drug 880
delivery system, which exhibited good therapeutic e fficacy with a 23-fold improvement in in 881
vitro studies involving magnetic targeting using SPIONs (J. Shi et al., 2013). The surface of 882
gold-coated-Fe 3O4 nanoparticles was modified with thiolated heparin– pheophorbide a (PhA) 883
conjugate to form a Fe3O4/Au/H–PhA nanocomplex (L. Li et al., 2014). This pheophorbide 884
based nanocomplex caused 89.4% cell death in A549 c ells, and considerably reduced the tumor 885
volume (129.22 mm 3) in A549 tumor-bearing mice considerably as compar ed to free PhA (tumor 886
volume – 162.22 mm 3) and saline (tumor volume – 282.47mm 3) treated mice, on exposure to 670 887
nm laser. 888
5.6.2. In combination with Photothermal therapy 889
Normally, photothermal therapy (PTT) does not use a ny free radical oxygen from 890
photosensitizers to cause damage to cancer cells (a s happening in photodynamic therapy). This 891
method manipulates the nanomaterial’s absorbance of light in infrared and longer wavelength 892
ranges to induce damage in the tumor cells by conve rting the absorbed photons into heat. So the 893
materials absorptivity or molar extinction coeffici ent (ability to attenuate light of given 894
wavelength) of nanomaterials is necessarily to be h igh to produce high thermal ablations. 895
Significantly, the near infrared (NIR) therapeutic window absorbance should be more for 896
nanoparticles-to-be-used. The disadvantage (i.e., p hoto bleaching in organic and polymer 897
materials) can be overcome by using inorganic mater ials, for instance, mixing SPIONs with 898
graphene oxide, gold, etc. 899
In a recent study, a correlation between molar exti nction coefficient and self-assembling nature 900
(chainlike/hollow vesicles) of iron oxide nanomater ials was found (Zhang et al., 2014). A two 901
fold improvement in the molar extinction coefficien t of SPIONs was attained when the size- 902
dependent-packaging density of SPIONs was optimized with respect to polypyrrole (a PTT 903
agent), thereby resulted in increased photothermal effect on HeLa cancer cells (Zhang et al., 904
2014). Recently, Prussian blue, another PTT agent ( ordinarily used for staining and clinically 905
approved drug), was grown on the surface of SPIONs to a thickness of 3-6 nm. The blue-colored 906
Prussian blue coated SPIONs system killed more than 80% of HeLa cells at minimal 907
concentration and achieved 87.2% tumor inhibition r ate due to NIR irradiation and magnetic 908
targeting (Fu et al., 2014). Fluorescent carbon, on e dimensional (1D) magnetic NP chains and 909
doxorubicin were combined to form a nano-package fo r improved MRI contrast, fluorescent 910
imaging (using different excitation wavelengths), a nd PTT/chemotherapy at in vitro level (H. 911
Wang et al., 2014). 912
SPIONs coupled with graphene oxide and stabilized b y PEG (GO-IONP-PEG) were injected into 913
randomly selected three mice groups induced with me tastasis in their regional lymph nodes for 914
studying NIR/MR imaging and NIR treatment modality. The combinatorial nano-package 915
showed the possibility of in vitro and in vivo dual-modality mapping and treatment of metastatic 916

Page 41 of 71Accepted Manuscript
21
lymph nodes (S. Wang et al., 2014). Similarly, anot her group formed GO-IONP-PEG 917
nanocomposite loaded with DOX for combined targeted drug delivery, PTT, chemotherapy and 918
MR imaging (Ma et al., 2012). Gold, iron oxide and graphene oxide based nanocomposite 919
system was prepared for enhanced PTT and MR imaging by creating electrostatic interaction 920
through the introduction of PEI after the incorpora tion of SPIONs onto graphene oxide (X. Shi et 921
al., 2013). After the removal of excess PEI, negati vely charged gold nanoseeds were embedded 922
onto PEI, confirmed by increase in NIR absorption b and. Finally, modified PEG was attached to 923
increase the stability and biocompatibility. 4T1 tu mor induced female BALB/c mice was injected 924
with GO-IONP-Au-PEG nanocomposite and subjected to 808 nm laser irradiation for about 5 925
minutes, where a thermal camera noted the change in temperature due to NIR irradiation. It was 926
concluded that reduction in tumor volume in laser i rradiated mice indicated that the formed 927
nanocomposite acted as an effective PTT agent, whic h was not witnessed in control group and 928
not-laser-radiated group. 929
Not only graphene oxide, silica was also used as a shell for octahedral shaped SPIONs to 930
produce stimuli sensitive and targeted DDS for phot on induced heat therapy (W.-P. Li et al., 931
2014). Initially, octahedral SPIONs were truncated and then coated with trisoctahedral gold to 932
improve the photothermal efficiency. Finally, the m esoporous silica shell was formed outside to 933
provide space for oligonucleotide and doxorubicin a ttachment, where these combinations 934
showed enhanced PTT effect in both in vivo and in vitro conditions. Similarly, polydopamine 935
(PDA – another PTT agent) was used to cover the clu stered Fe 3O4 nanoparticles (with r2 value of 936
78.1 mM-1 s-1) for MRI (Zheng et al., 2015). The Fe 3O4@PDA composite nanoparticles 937
showcased a higher killing efficiency in A549 cells , when these nanoparticles-incubated-cells 938
were exposed to NIR irradiation with a power densit y of 6.6 W/cm 2. Moreover, the volume of 939
A549 subcutaneous tumor (induced in Male Balb/c) mi ce was completely inhibited after post 940
injection and subsequent 808 nm-NIR laser irradiati on of the composite nanoparticles for 3 941
minutes. Nevertheless, Chen et al proved that high crystalline SPIONs with proper surface 942
coating acted as a good PTT agent (Chen et al., 201 4) as shown in Fig 18. In their study, the 943
surface temperature of SUM-159 tumor (induced in BA LB/c mice) was increased from 35 °C to 944
60 °C by exposing PEO-b-P γMPS coated SPIONs to 880 nm for 10 minutes. 945
5.6.3. In combination with Sonodynamic therapy 946
Cancer therapies performed using ultrasound is call ed as sonodynamic therapy (SDT) therapy 947
and agents/chemicals used in this method are called SDT agents or sonosensitizers. The 948
photosensitizers such as TiO 2 and HMME can be used as sonosensitizers because of thei r ability 949
to produce hydroxyl/peroxy/alkoxy radicals when exp osed to ultrasound, thereby increasing the 950
chances of killing cancer cells. Magnetic nanoparti cles can be combined with these 951
sonosensitizers to increase the efficiency of cance r therapy. A nanocomposite constituting 952
Fe 3O4–NaYF 4@TiO 2 and doxorubicin was formed to test both sonodynamic and 953
chemotherapeutic efficiency in MCF-7 cells (Shen et al., 2014). The rate of apoptosis in MCF-7 954
cells was found to be increased in the combined the rapy given through these hybrid materials in 955
association with magnetic targeting as compared to individual therapies. 956
PLGA microbubbles co-encapsulating SPIONs and doxor ubicin (mean diameter after 957
encapsulation – 868.0 ± 68.73 nm) performed well in simultaneous chemotherapy and 958
ultrasound/MR imaging against in vivo metastatic lymph nodes (Niu et al., 2013) as shown in Fig 959
19. In a recent investigation, a combination of iro n oxide nanoparticles, SDT, and microwave 960
treatment showed almost 97% reduction of in vivo tumor volume (Gebreel et al., 2014). PVA 961
microbubbles encapsulating SPIONs were created by c onjugating silane coated SPIONs with 962

Page 42 of 71Accepted Manuscript
22
surrounded bubbles through the amino-aldehyde coupl ing in respective surfaces, where the 963
microbubbles encapsulated SPIONs showed enhanced ul trasound/MR imaging (Brismar et al., 964
2012). The air gap formed between magnetic nanoratt les and poly(vinylsilane) with 965
perfluorohexane (PFH) was concealed to make use of the formed nano-system for improvised 966
ultrasound/MR imaging (P. Yang et al., 2014). 967
6. Conclusion and Perspectives 968
Much advancement has been achieved in preparing hig h quality SPIONs as compared to other 969
nanoparticles/nanostructures for cancer theranostic applications. The SPIONs with different 970
sizes, shapes and surface coatings have been engine ered for achieving high crystallinity, very 971
narrow size distribution, and improved magnetic pro perties for their better performance in 972
biomedical applications. However, the usage of SPIO Ns is only limited to in vitro level except 973
few in vivo studies and clinical investigations till date. The intrinsic reasons behind these 974
limitations are related to the diminishing of magne tic properties of SPIONs at the fundamental 975
level. It can be seen from this review that tailori ng of the magnetic properties of SPIONs by 976
reducing the surface and volume canting effects is very tedious. The influence of size dependent 977
spin canting effects on the magnetic properties of SPIONs is comparatively high as compared to 978
the shape dependent canting effects. Moreover, some surfactants such as polyoxyethylene (5) 979
nonylphenylether have less influence over the magne tic properties of SPIONs as compared to 980
other surfactants, but the crystallinity of SPIONs should also be considered. Moreover, it can 981
also be seen that the high temperature synthesis st rategies yielded high crystalline monodisperse 982
SPIONs with significant magnetic properties, where the synthesis parameters played important 983
roles in preparing such high quality SPIONs with di verse sizes and shapes. 984
Previous reviews on various cancer theranostic appl ications, such as hyperthermia, MRI, 985
magnetofection, and magnetic targeting, indicated t hat the improvements in magnetization, 986
relaxivities and SAR values of the SPIONs have led to enhance the theranostic efficacy. 987
However, SPIONs for early diagnosis of cancers and treating them concurrently are still under- 988
developed. Moreover, the formation of SPIONs with m aximum relaxivities and SAR values (at 989
the synthesis level and after organic/inorganic enc apsulations/chemical modifications) for 990
effective in vivo cancer theranostics still remains challenging. Man y developments in the 991
synthesis/encapsulation procedures of SPIONs and co ating them with novel 992
surfactants/encapsulants/targeting agents have been done lately for effective cancer treatment at 993
the in vitro and in vivo scenarios. In this review, the following are signi ficantly outlined: the 994
recent advancements in (i) improving the magnetic p roperties of SPIONs by manipulating the 995
size, shape and surface coatings, (ii) developing n ovel biocompatible polymers to encapsulate 996
SPIONs with and without cancer drugs/targeting agen ts, (iii) applying SPIONs as in vitro and in 997
vivo T1, T2 and dual mode (T1-T2) MRI contrast agents i ndividually (by modifying size and 998
shape of SPIONs) and in combination with other cont rast agents (manganese/gadolinium 999
complexes), (iv) combining SPIONs with radio nuclid es/optical imaging/fluorescence enhancers 1000
for multimodal in vivo cancer imaging, (v) enhancing the cancer therapy u sing recently 1001
developed SPIONs (with different size, shape and su rface coatings) via in vivo magnetic 1002
hyperthermia, magnetofection and magnetic targeting . This review also showed that the efficacy 1003
in cancer theranostics can be further enhanced by f orming SPIONs based nanopackages via 1004
combining SPIONs with other chemotherapeutic drugs, photosensitizers, photothermal agents, 1005
and sonosensitizers. Nevertheless, very limited stu dies on combinatorial cancer theranostic 1006
agents at in vivo level are involved, and the side effects (acute an d chronic) at this level due to 1007
these theranostic agents are still unknown. Thus, m ore elaborate studies are required on co- 1008

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encapsulation and delivery, protein corona formatio n, destruction pathways in tumor cells, 1009
pharmacokinetics/pharmacodynamics of combinatorial theranostic agents and their inter- 1010
interactions to further involve them in clinical st udies. Moreover, the advancements in 1011
contemporary techniques are very much expected to o vercome the constraints in characterization 1012
of the physicochemical properties of the surface en gineered SPIONs and their combination with 1013
other drugs/targeting agents. 1014
In summary, an efficient in vivo cancer treatment can be achieved via multimodal im aging and 1015
therapy by enlarging the therapeutic window via the usage of SPIONs (with high relaxivity and 1016
SAR values) along with other contrast/chemical agen ts/drugs. Although the advancements of 1017
combinatorial SPION based nanopackages are at their preliminary stages, the researchers around 1018
the world are developing new strategies/designs to enhance the performance of these 1019
combinatorial nano-packages in both in vitro and in vivo cancer theranostics. Thus, these multi- 1020
modal SPIONs based nanopackages might enable the co mplete inhibition of cancer in the human 1021
trials in future, if intensive research is performe d by global scientists on these nanopackages. 1022
1023
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1701
1702
Fig. 1 Schematic illustration of change in composit ion of magnetic nanoparticles that evolve 1703
from meghamite for small sizes (typically <8 nm) to a core of rather stoichiometric magnetite 1704
surrounded by an oxidized shell for large sizes (>1 2 nm) via a perturbed oxidized state for 1705
intermediate sizes along with change in surface and volume spin canting. Reproduced with 1706
permission from (Baaziz et al., 2014), © American C hemical Society. 1707
Fig. 2 Schematic illustration of spin phenomena in small-sized (a) iron oxide (IO) and (b) 1708
gadolinium doped iron oxide (GdIO) nanoparticles. T he gadolinium species (Gd 2O3 1709
nanoclusters) in GdIO nanoparticles ( ⁰5 nm in diameter) cause an inner spin-canting effec t, 1710
while the IO nanoparticles ( ⁰5 nm in diameter) contain a spin-canted surface and spin-oriented 1711
core. Reproduced with permission from (Zhou et al., 2013), © American Chemical Society. 1712
1713
Fig. 3 Influence of surface ligands on the overall magnetic moment ( µp) of a SPION. Canted 1714
surface spins are partially realigned upon exchange with strongly-interacting catecholate ligands, 1715
i.e. µp1 <µp2 . Reproduced with permission from (Yuen et al., 201 2), © The Royal Society of 1716
Chemistry. 1717
Fig. 4 I – Controlled synthesis of octapod iron oxi de nanoparticles with different edge lengths. 1718
The octapod iron oxide nanoparticles with edge leng ths of (a) 14, (b) 20, (c) 30 and (d) 36 nm 1719
formed after 1, 1.5, 2 and 2.5 h reaction times in the presence of NaCl, respectively. II – 1720
Schematic cartoon shows the ball models of octapod and spherical iron oxide nanoparticles with 1721
the same geometric volume (the black dotted lines r epresents the magnetic field of the octapod 1722
and spherical iron oxide nanoparticles. The same le ngth of black arrow means the same Ms of 1723
octapod and spherical iron oxide nanoparticles). Wi th the same geometric core volume, the 1724
octapod nanoparticles have much larger effective vo lume (radius, R) than the spherical 1725
nanoparticles (radius, r) with R ~ 2.4r under an ex ternal magnetic field B 0. (b) The smooth M–H 1726
curves of Octapod-30, Octapod-20, Spherical-16 and Spherical-10 measured at 300 K using a 1727
superconducting quantum interference device magneto meter (inset: M–H curves of Octapod-30 1728
and Octapod-20 in low-magnetic field areas). The M s values of Octapod-30, Octapod-20, 1729
Spherical-16 and Spherical-10 are about 71, 51, 67 and 55 emu g -1 respectively. Reproduced with 1730
permission from (Zhao et al., 2013), © Macmillan Pu blishers Limited. 1731 Figure Captions:

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Fig. 5 Formation pathways of magnetite nanoparticle s by co-precipitation method. Main 1732
intermediate phases are shown in yellow areas. Repr oduced with permission from (Thanh et al., 1733
2014), © 2012 American Chemical Society. 1734
Fig. 6 Schematic illustration of possible iron comp lexations and iron oxide formation in poly- 1735
(oligoethylene glycol methacrylate)-block-(methacry lic acid)-block-poly(styrene) (POEGMA-b- 1736
PMAA-b-PST) triblock copolymer aqueous dispersion. Reproduced with permission from 1737
(Karagoz et al., 2014), © 2014 American Chemical So ciety. 1738
Fig. 7 I – Schematic representation of the synthesi s of SPIONs- poly(glycidyl methacrylate-co- 1739
poly(ethylene glycol) methyl ether methacrylate)-fo lic acid (P(GMA-co-PEGMA)-FA). II- TEM 1740
images of (a) pristine oleic acid-stabilized SPIONs dispersed in hexane, and (b) SPIONs- 1741
P(GMA-co-PEGMA)-FA dispersed in DI water. III – Mag netization curves of (a) pristine oleic 1742
acid-stabilized SPIONs and (b) SPIONs-P(GMA-co-PEGM A)-FA at 25 °C. Reproduced with 1743
permission from (Huang et al., 2012), © 2014 Americ an Chemical Society. 1744
Fig. 8 I – Schematic outline of the synthetic pathw ay for folic acid-attached silica-coated iron 1745
oxide (FA@SiO 2@Fe 3O4) nanoparticles, where α-Fe 2O3 quasicubic particles were synthesized by 1746
a facile solvothermal process (i), followed by the silica shell formation with tetraethyl 1747
orthosilicate (TEOS) (ii) and the core reduction to magnetite (iii). The surface of the 1748
SiO 2@Fe 3O4 nanoparticles was activated with amine groups (iv) and vectorized by covalent 1749
attachment of FA as the targeting unit (v). II – Ma gnetization measurements of the nanoparticles 1750
after each step of functionalization at room temper ature (A). FC and ZFC curves of all samples 1751
measured at an applied field of 50 mT (B). Reproduc ed with permission from (Wortmann et al., 1752
2014), © 2011 American Chemical Society. 1753
Fig. 9 T2* MR images of the mouse brains before and after intravenous injection of 1754
poly(ethylene glycol) (PEG)-SPIONs (a1–a7) and PEG/ polyethylenimine (PEI)-SPIONs (b1– 1755
b7), relative T2* value of different brain regions extracted from T2* MR images of mouse brains 1756
before and 24 h after the injection of PEG-SPIONs a nd PEG/PEI-SPIONs (c). The white arrows 1757
highlight the brain blood vessels enhanced by the S PIONs. Reproduced with permission from (J. 1758
Wang et al., 2015), © Elsevier. 1759
Fig. 10 MRI relaxivity and phantom study. (a) Colum ns to show the r1 and r2 values (0.5 T) of 1760
the three nanoplates, respectively. (b) T1 (left) a nd T2 (right) MRI phantom studies (0.5 T) of the 1761
IOP-8.8 (top), IOP-4.8 (middle), and IOP-2.8 (botto m) at different iron concentrations (mM) in 1762
1% agarose. The capability of displaying T1 or T2 c ontrasts is denoted as ON for good contrast 1763
and OFF for poor contrast. (c) T1 nuclear magnetic relaxation suspension (NMRD) profiles of 1764
the three nanoplates as the function of applied mag netic fields, measured by aqueous colloidal 1765
suspensions of each samples. Reproduced with permis sion from (Zhou et al., 2014), © American 1766
Chemical Society. 1767
Fig. 11 (A) Time dependent temperature rise of 1 ml aqueous suspension of MNC-14 1768
nanoclusters with different iron concentrations on exposure to 89 kAm -1 AC field at 240 kHz 1769
frequency. Inset shows field dependent SAR values o f 1ml sample with 0.5 mg ml -1 iron 1770
concentration. (B) Cell viability plot shows the cy totoxic effect on MCF-7 breast cancer cells 1771
treated with magnetic hyperthermia (~ 45 °C), treated with MNC-14 nanoclusters only, and 1772
treated with magnetic field only in comparison with the control cells. (C and D) Optical 1773
microscope images of control MCF-7 breast cancer ce lls treated with magnetic field only and 1774
magnetic hyperthermia using 14 nm sized magnetic na noclusters (MNC-14) (scale bar 100 mm). 1775
Reproduced with permission from (Maity et al., 2011 ), © The Royal Society of Chemistry. 1776

Page 60 of 71Accepted Manuscript
40
Fig. 12 (A) Photograph (left) and thermal image (ri ght) of a mouse 24 h after intravenous 1777
injection of folic acid-modified poly(ethylene glyc ol)-coated SPION nanoclusters FA-PEG- 1778
SPION NCs) under an AC magnetic field with H = 8 kA /m and f = 230 kHz. (B) Tumor-growth 1779
behavior and (C) survival period of mice without tr eatment and treated by intravenous injection 1780
of FA-PEG-SPION NCs, application of an AC magnetic field, 24 h after intravenous injection of 1781
FA-PEG-SPION NCs (n = 5). (D) Photographs of mice 3 5 days after treatment. Reproduced with 1782
permission from (Hayashi et al., 2013), © Ivyspring International Publisher. 1783
Fig. 13 Labeling with SPIOs does not affect the via bility or functionality of human mesenchymal 1784
stem cells (hMSCs). a) hMSCs were incubated with va rious concentrations of SPIOs for 24 h 1785
over a magnet. Their enzymatic activity was assesse d immediately after or 7 days after labeling. 1786
b–h) hMSCs were incubated with 10 µg cm −2 of SPIOs for 24 h over a magnet. b–e) SPIO- 1787
labeled and unlabeled cells were immunophenotyped, and f–h) cultured under conditions to 1788
induce differentiation into adipogenic, osteogenic, and chondrogenic lineages. Cells were stained 1789
with Oil Red O (a marker of adipocytes), Alizarin R ed (a marker of osteocytes), or collagen II 1790
antibody (a marker of chondrocytes). f,g) The prese nce of DiI-labeled SPIOs inside the cells 1791
after the differentiation period was assessed by re d fluorescence emitted by DiI-labeled SPIOs or 1792
h) by Prussian Blue stain. Reproduced with permissi on from (Landázuri et al., 2013), © Wiley- 1793
VCH Verlag GmbH & Co. KGaA, Weinheim. 1794
Fig. 14 (a) Gene expression of poly(2-dimethylamino )ethyl methacrylate-bound iron oxide 1795
nanoparticles with pDNA (IO-PDMAEMA)/pDNA incubated with and without an applied 1796
magnetic force for 20 min and analyzed 48 h post-in cubation in the presence of 10% FBS (n = 3, 1797
*p < 0.05). Confocal microscopy images of IO-PDMAEM A/pDNA, (b) without, and (c) with an 1798
assisted magnet for 20 min; (d) quantification of t he pDNA fluorescence intensity (*p < 0.05). 1799
Reproduced with permission from (Huang et al., 2013 ), © The Royal Society of Chemistry. 1800
Fig. 15 T1- and T2-weighted MR images of glioma-bea ring brain before and after intravenous 1801
injection of iron oxide/manganese oxide conjugated with Cy5.5 and chlorotoxin (CTX) 1802
(Fe 3O4/MnO–Cy5.5–CTX) (A) and Fe3O4/MnO–Cy5.5 NPs (C) wit h a dose of 10 mg Mn per 1803
kg, respectively. Corresponding CNR analysis of T1 (B) and T2 (D) MR images. Reproduced 1804
with permission from (Li et al., 2015), © The Royal Society of Chemistry. 1805
Fig. 16 Dual-modality In Vivo studies. Short-axis v iew (top) and coronal view (bottom) images: 1806
(A) T2*-weighted MR images before injection of 99m Tc-DPA-ale-Endorem, (B) T2*-weighted 1807
MR image 15 min postinjection, and (C) nanoSPECT-CT image of the same animal in a similar 1808
view 45 min postinjection. Contrast in the liver (L ) and spleen (S) changes after injection due to 1809
accumulation of 99m Tc-DPA-ale-Endorem, in agreement with the nanoSPECT -CT image which 1810
shows almost exclusively liver and spleen accumulat ion of radioactivity. MR images were 1811
acquired with a TE of 2 ms. Reproduced with permiss ion from (De Rosales et al., 2011), © 1812
American Chemical Society. 1813
Fig. 17 Inhibition of xenograft tumor formation by Pc 4 PDT delivered by IO nanoparticles. (A- 1814
D) Tumor growth and representative images of tumors on both sides of the mice in the PBS 1815
control, free Silicon phthalocyanine (Pc 4), combin ation of iron oxide and Pc-4 (IO-Pc 4), and 1816
combination of fibronectin mimetic peptide, iron ox ide and Pc-4 (Fmp-IO-Pc4) groups, 1817
respectively. Pc 4 was given at a concentration of 0.4 mg/kg. Laser treatment was performed 48 1818
h after the drug administration. Three out of six m ice from each group are shown as 1819
representatives. Statistical analysis indicated a s ignificant difference in the longitudinal tumor 1820
volume across the 5 groups within the right side (l aser treated), (p < 0.0013). Both IO-Pc 4 and 1821
Fmp-IO-Pc 4 groups had a significantly lower tumor growth volume than the PBS control group 1822

Page 61 of 71Accepted Manuscript
41
(p < 0.022 for IO-Pc 4 and 0.0038 for Fmp-IO-Pc 4). The Pc 4 group had a marginally 1823
significantly lower tumor growth volume than the co ntrol group (p < 0.071). The Pc 4 group had 1824
a significantly higher tumor growth volume than bot h the IO-Pc 4 and Fmp-IO-Pc 4 groups (p < 1825
0.049 for IO-Pc 4 group and 0.040 for Fmp-IO-Pc 4). No tumor growth difference was found 1826
between IO-Pc 4 and Fmp-IOPc 4 groups (p = 0.98). T here was no significant difference in the 1827
longitudinal tumor volume across the 4 groups on th e left side tumor (no laser treatment, p = 1828
0.4987). None of the pairwise comparisons in tumor volume between any two groups with 1829
untreated left tumors was significantly different ( results are omitted). (E) Tumor growth curve 1830
using a lower dose (0.06 mg/kg) and shorter period of time between drug administration and 1831
laser treatment than used in (A-D). Tumors in the F mp-IO-Pc 4 (targeted) group grew 1832
significantly slower than those in the IO-Pc 4 grou p (nontargeted) (p < 0.025). Reproduced with 1833
permission from (D. Wang et al., 2014), © American Chemical Society. 1834
Fig. 18 (a) Representative photos of SUM-159 tumor- bearing mice of both immediate and 3 1835
weeks after laser treatment. Laser wavelength = 885 nm. Power density = 2.5 W cm 2. Irradiation 1836
time = 10 minutes. Arrows point to the tumor sites. H&E staining of tumor tissues from mouse 1837
treated with nanoparticles plus laser irradiation ( b) and control mouse without any treatment (c). 1838
(d) Anti-tumor efficacy of four different groups of mice before and 3 weeks post various 1839
treatments. Four groups (5 mice for each group) are magnetic nanocrystals injected mice with 1840
laser irradiation (G1), nanocrystals injected mice without laser irradiation (G2), laser treated 1841
mice without injection of nanoparticles (G3), and c ontrol mice injected with PBS (G4). Error 1842
bars are based on standard deviations. Reproduced w ith permission from (Chen et al., 2014), © 1843
The Royal Society of Chemistry. 1844
Fig. 19 (a) A microscope image of Prussian blue dye stained tissue. The iron in the 1845
multifunctional polymer microbubbles (MPMBs – poly( lactic-co-glycolic acid) (PLGA) 1846
microbubbles encapsulating Fe 3O4 nanoparticles and chemotherapeutic drug) injected lymph 1847
nodes tissue slices was stained blue; (b) no blue s tain was observed in the tissue slices from 1848
lymph nodes injected with microbubbles that do not contain Fe 3O4. (c) A TEM image of MPMBs 1849
injected lymph nodes. The black mass with high elec tronic density revealed the presence of iron. 1850
It also can be observed that the Fe 3O4 nanoparticles are distributed in the shell of the 1851
microbubbles. (d) There was no detectable high elec tronic density in the microbubbles without 1852
Fe 3O4 injected lymph nodes. Magnification, x 100 (a and b), x 40,000 (c), x 10,000 (d). 1853
Reproduced with permission from (Niu et al., 2013), © Elsevier. 1854
1855
Table 1 Size, shape, surface coatings, magnetizatio n values of iron oxide (Fe 3O4 and Fe 2O3) 1856
nanoparticles 1857
Size (nm) Shape Surface Coatings Magnetization
(emu/g) References
14±4 Spherical Uncoated 74.9 (Ozel and Kockar, 2015 )
9 to 20 Quasi-spherical Uncoated 77 (Klein et al., 2014)

Page 62 of 71Accepted Manuscript
42
7 to 17 Citric acid 70
6 to 16
Maleic acid 74
24 Mixed shapes Uncoated 89
21 Hexamine 59
25 Poly(ethylene
glycol) 60
14 Spherical
Polyvinylpyrrolidone 62 (Kumar et al., 2014)
18 Spherical Oleic acid 67 (Mojica Pisciotti et al.,
2014)
10 63
14 Clusters Tri(ethylene glycol)
and Triethanolamine
75 (Maity et al., 2011)
22 Octahedral Uncoated 80
100 Trisoctahedral Gold 20 (W.-P. Li et al., 2014)
10 Uncoated 70
150 Spherical Poly(N-
isopropylacrylamide-
acrylamide-chitosan) 68 (Sundaresan et al.,
2014)
7 – Uncoated 61
8 – Gold 63 (León-Félix et al., 2014)

Page 63 of 71Accepted Manuscript
43
11 Uncoated 60
8 L-arginine 52
7 Spherical
L-lysine coating 42 (Ebrahiminezhad et al.,
2012)
13.1 ± 0.3 Mannose 59.1
9.7 ± 0.99 Maltose 37.4
3.8 ± 0.21 Lactose 22.02
12.4 ± 0.3 Mixed shapes
Galactose 58.08 (Demir et al., 2014)
14 ± 4 Uncoated 74.9
74 ± 9 Spherical
Uncoated 93.5 (Ozel and Kockar, 2015)
1858
Table 2 MRI relaxivity values of commercial iron ox ide (Fe 3O4 and Fe 2O3) nanoparticles having 1859
different sizes and surface coatings 1860
Name/Company Magnetic
Core
Size
(nm) Total
Hydrodynamic
Size (nm) Surface coatings r1
Relaxation
(mM -1S-1) r2
Relaxation
(mM -1
Ferumoxides, AMI-25
Endorem/Feridex Guerbet,
Advanced Magnetics – 120–180 Dextran 10.1 120
Ferumoxtran-10, AMI-227,
BMS-180549
Sinerem/Combridex, Guerbet,
Advanced Magnetics – 15–30 Dextran 9.9 65
Ferumoxytol Code 7228,
Advanced Magnetics – 30 Carboxylmethyl
dextran 15 89

Page 64 of 71Accepted Manuscript
44
Ferucarbotran SHU-555A,
Resovist Schering – 60 Carboxydextran 9.7 189
SHU-555C Supravist Schering – 21 Carboxydextran 10. 7 38
VSOP-C184 Ferropharm – 7 Citrate 14 33.4
AMI-121 Lumirem and
Gastromark 300 – Silica 3.2 72
AMI-25 Endorem and Feridex 5.6 – Dextran 23.9 98.3
SHU-55A Resovist 4.2 – Carbo-Dextran 25.4 151
AMI-227 Sinerem and Combidex 4-6 – Dextran 21.6 44.1
NC1001 50 Clariscan 5-7 – Carbohydrate-PEG 20 35
SHU-55C Supravist 3-5 – Carbo-Dextran 7.3 57
Resovist 4.6 60 Carboxydextran 10.9 190
ferumoxytol (Combidex) 6.4 30 carboxymethyl-dextran 15 89

Page 65 of 71Accepted Manuscript
45
Sinerem 4-6 15-30 Dextran 9.9 65
1861
1862
Table 3 MRI relaxivity values of synthetic iron oxi de (Fe 3O4 and Fe 2O3) nanoparticles having 1863
different sizes and surface coatings 1864
Name Magnetic
Core Size
(nm) Total
Hydrody
namic
Size (nm) Surface
coatings r1
Relaxatio
n (mM –
1S-1) r2
Relaxatio
n (mM –
1S-1) Magnetic
field (T) Referenc
es
SPIO-14 13.8 28.6 – 385
SPIO-5 4.8 14.8 DSPE-
mPEG
1000 – 130 0.47 (Tong et
al., 2010)
10.96 ±
1.9 12.5 ± 1.3 DSPE-
PEG-500 12.7 ± 3.7 317 ±
58.8
13.63 ±
1.3 10.35 ±
2.6 DSPE-
PEG-750 12.6 ± 2.4 360 ± 40
13.23 ±
1.1 12.0 ± 0.8 DSPE-
PEG-
1000 25.2 ± 4.9 194 ± 57
14.6 ±
0.34 16.4 ± 3.1 DSPE-
PEG-
2000 24.4 ±
5.88 147 ± 34 MIONs
16.2 ± 1.3 21.6 ± 3.6 DSPE-
PEG-
5000 21.5 ±
3.22 173 ± 30 0.47 (LaConte
et al.,
2007)
USPIO-
PEG 24 30.4 62.2
USPIO-
PEG-
RGD 17 31.9 73.9
USPIO-
PEG-
RGDp 7.7-7.9
34 PEG
30.1 106.5 0.47/1.41 (Pourcell
e et al.,
2015)
Fe3O4 – – Uncoated – 100.4 0.5 (Shen et
al., 2012)

Page 66 of 71Accepted Manuscript
46
Fe3O4@
APTS 6.5±1.3 247.8 ±
18.6 APTS 83.8
Iron
Oxide 7.4 80-170 poly(ethy
lene
oxide-b-
D,L-
lactide) 2.4-3.4 90-229 1.4 (Balasub
ramaniam
et al.,
2014)
SPIOs 11 107.5 PEI–b–
PCL–b–
PEG – 256 1.41 (Pöselt et
al., 2012)
BSA-
SPION 8 18 Bovine
serum
albumin 11.6 154.2 1.41 (X. Wang
et al.,
2014)
D-
SPIONs 12 50 Dextran – 140.7 1.5 (Saraswa
thy et al.,
2014b)
SPIONs 10 100 Chitosan 1.56±0.19 369 ± 3 1.5 (Szpak et
al., 2013)
WFION 22 – – – 761 3 (N. Lee et
al., 2012)
PEG/PEI-
SPIONs
(200 ░°C) 6.8 – 3.11 58.8
PEG/PEI-
SPIONs
(260 ░°C) 10 21.8±1.5 PEG/PEI
1.65 142.99 7 (J. Wang
et al.,
2015)
SPIONs 5 30 Ascorbic
acid 0.95 22 9.4 (Sreeja et
al., 2015)
Where DSPE-mPEG – distearoyl-sn-glycero-3-phosphoet hanolamine-n-[methoxy 1865
(polyethyleneglycol)], APTS – 3-aminopropyltrimetho xysilane, PEI-b-PCL-b-PEG – 1866
poly(ethylene imine) – block – poly- ( ε-caprolactone) – block – poly(ethylene glycol), and 1867
PEG/PEI – poly(ethylene glycol)/poly(ethylene imine ). 1868
1869
Table 4 SAR values of iron oxide (Fe 3O4 and Fe 2O3) nanoparticles having different sizes and 1870
surface coatings 1871
Core Size
(nm) Surface coatings Magnetic
Field
Intensity Magnetic Field
Frequency (kHz) SAR
(W/g) References

Page 67 of 71Accepted Manuscript
47
5-13 Dextran 58 kA/m 292 83.5 (Kruse et al., 2014 )
8 10
11 40
13 APTES 7.5 kA/m 522.3
55 (de la Presa et al., 2012)
24 Citrate 21.5 700 1992 (Hugounenq et al., 2012)
11 Oleic acid 47.7 kA/m 194 33.4 (Shah et al., 2015)
9.5 29
9.6 DMSA 30 kA/m 100
35.1 (Song et al., 2012)
10 Methylmethacrylate
and glycine
modified 0.251,
0.335 and
0.419 kOe 265 58.5,
131.7
and
204.0 (Barick et al., 2014)
155.2±0.17 PLA-TPGS/TPGS-
COOH 43 kA/m 240 146 (Mi et al., 2012)
19±3 Decanoic acid 29 kA/m 520 2452 (Guardia et al ., 2012)
21.8 Uncoated 79.32
15.1 Chitosan 26.7 kA/m 26.5
118.85 (Shete et al., 2014)
7 164
9 DPPC and DSPC 27 kA/m 300 kHz to 1.1 MHz
438 (Béalle et al., 2012)
5 180
10 130
12.5 200
14 Pluronics 127 24.5 kA/m 400
447 (Gonzales- Weimuller et al., 2009)
PEG 320
18
Dextran 13 kA/m 256
400 (Mojica Pisciotti et al., 2014)

Page 68 of 71Accepted Manuscript
48
Where APTES – (3-Aminopropyl)triethoxylsilane, DMSA – 2,3-Dimercaptosuccinic acid, PLA- 1872
TPGS – Poly (lactide)-D-a-tocopheryl polyethylene g lycol 1000 succinate copolymer, DPPC – 1873
1,2-dipalmitoylsn-glycero-3-phosphocholine, PEG – P oly(ethylene glycol) and DSPC – 1,2- 1874
distearoyl-sn-glycero-3-phosphocholine. 1875
1876
Table 5 Comparison of different animal models used to evaluate cancer hyperthermia therapy 1877
using magnetic nanoparticles 1878
Surfac
e
Coatin
gs SAR Fe 3O4
weight
admini
strated Cell
line
and
Anim
al Tu
mor
size Injec
tion
site Maxi
mum
Ther
apeut
ic
Tem
perat
ure
(in
°C) Time
to
reach
Thera
peutic
Tempe
rature
(in
minute
s) Appli
cation
time
of
magn
etic
field
for
hyper
therm
ia (in
minut
es) Hypert
hermia
Assess
ment
method Refer
ence
TMAG,
DLPC,
DOPe,
1:2:2
cationic 175
W/g 2 mg DMB
A-
induc
ed
mam
mary
cance
r in
Sprag
ue-
Dawl
ey
rats 10
mm Direc
t
inject
ion to
tumor 45 – 30 Immun
ological
reaction (Moto
yama
et al.,
2008)
TMAG,
DLPC,
DOPe,
1:2:2
cationic – – EL4
T-
lymp
homa
and
C57B
L/6
mice 6
mm Subc
utane
ous
space 45 5 30 Tumor
suppres
sion (Tana
ka et
al.,
2005)
TMAG,
DLPC,
DOPe,
1:2:2
cationic – 20 mg-
magnet
ite/ml MM4
6
mam
mary
carci 15
mm Subc
utane
ous
inject
ion 45 5 30 Tumor
volume,
Heat
Shock
Protein (Ito et
al.,
2003)

Page 69 of 71Accepted Manuscript
49
noma
and
C3W
HeN
mice 70 ,
Immun
ohistoc
hemistr
y
(CD3,
CD4,
CD8,
and
NK)
Carbox
ydextra
n – 1.8
mol/L 39
Aminos
ilane – 2.0
mol/L RG-2
and
Fishe
r rat
F-344 3-4
mm Thala
mus
regio
n
43-47 10 40 Prussia
n blue/
Immun
ohistoc
hemistr
y (Jorda
n et
al.,
2006)
Dextran 286
W/g 3
mg/150
µL C6
and
Fishe
r rat
F-344 5-10
mm Subc
utane
ous
(anter
ior
breg
ma
regio
n) – – 20 Hemato
xylin-
eosin (Rabi
as et
al.,
2010)
Uncoat
ed 864.1
±16.6
J
g−1 m−
1 1 ml,
80 mg
of
Fe 3O4
dissolv
ed in
10 ml
glyceri
n Ehrli
ch
carci
noma
and
Swiss
albin
o
mice 78.2
±3.5
mm 3 subcu
taneo
us
inocu
lation 47±1 15±2.2 40 Histolo
gical
examin
ation
and
Apopto
sis
percent
age
from 30
days of
the
starting
of
therapy (Elshe
rbini
et al.,
2011)

Page 70 of 71Accepted Manuscript
50
0.226
mg Fe
per 100
mm 3 of
tumor
volume BxPC
-3
and
Athy
mic
mice 658 ±
53
W/g
0.535
mg Fe
per 100
mm 3 of
tumor
volume MDA
-MB-
231
and
Athy
mic
mice
0.24
mg Fe
per 100
mm 3 of
tumor
volume MDA
-MB-
231
and
Athy
mic
mice DMSA
900 ±
22
W/g
0.087
mg Fe
per 100
mm 3 of
tumor
volume BxPC
-3
and
Athy
mic
mice 80 –
500
mm 3 Subc
utane
ous
impla
ntatio
n 43 24 60 Immun
ohistoc
hemical
KI-67
Stainin
g (Koss
atz et
al.,
2014)
TCPP 64 ±
2 W/g 3 times
injectio
n of
120 µL
with 1
mg
Fe/mL B16-
F10
mela-
noma
cells
and
C57/
BL6
mice 50
mm 3 subcu
taneo
us
trans
plant
ation
into
rear
limb 10 Histolo
gical
Analysi
s (Baliv
ada et
al.,
2010)
Poly(st
yrene-
co-
acrylic
acid) – 0.3 –
0.4mg Sarco
ma
180
cells
and
Swiss
mice 6 ×
6
mm subcu
taneo
us
trans
plant
ation 45–
49 – 30 – (Nguy
en et
al.,
2012)
Anioni
c
Surfact
ant 535
W/kg 0.5 ml Tu21
2 cell
line
and
Nude 1
cm 3 – 40 5 20 Histopa
thology (Zhao
et al.,
2012)

Page 71 of 71Accepted Manuscript
51
Nude
(NCr)
mice
TMAG,
DLPC,
DOPe,
1:2:2
cationic – 3
mg/0.4
ml osteo
sarco
ma
and
Syria
n
femal
e
hamst
ers – Subc
utane
ous
inject
ion 42 10 30 Tumor
volume (Mat
suok
a et
al.,
200
4)
Where TMAG – N-( α-trimethylammonioacetyl)-didodecyl-D-glutamate chlo ride, DLPC – 1879
dilauroylphosphatidylcholine, DOPe – dioleoylphosph atidyl-ethanolamine, DMBA – 7,12- 1880
Dimethylbenz(a)anthracene, DMSA – 2,3-dimercaptosuc cinic acid, and TCPP – 4- 1881
tetracarboxyphenyl porphyrins 1882
1883
1884
1885