Iron Oxide Nanoparticles and Nano-Composites: An Efficient Tool for Cancer Theranostics

In recent years, functional Iron oxides nanoparticles and nano-composites have gained a special traction in the field of nano-biomedicine, owing to their multifunctional capabilities that includes the inherent magnetic resonance imaging, magnetic bioseparation, cargo delivery and magnetic hyperthermia behavior. Interestingly, there are various forms of iron oxides available, with each form having their own specific characteristics. The different polymorphic forms of iron oxides are obtained through various synthetic routes and are usually surface modified to prevent their oxidation. The chapter shall encompass the synthesis and surface modification of Iron oxides nanoparticles, physicochemical properties, and theranostic application of the magnetic iron oxide nanoparticles in cancer. Also, the future directions of Iron oxide nanoparticles and nano-composites towards the achievement of clinically realizable nanoformulation for cancer theranostic applications were highlighted.


Introduction
Over the past few decades, nanomaterials have gained a special interest and are being investigated widely for various applications, owing to their unique characteristics [1]. Until now, several nanoparticulate systems have been studied to demonstrate their potential to detect, diagnose and treat cancer effectively with high degree of specificity and affinity to target cells, in comparison to the other onco-therapeutic approaches [1][2][3]. Iron oxides are being investigated widely in the field of nanobiomedicine, owing to their greater degree of variability and versatility [4]. The multifunctional capabilities of iron oxide nanoparticles including tumor labelling, magnetic bioseparation, biological entities detection, transfections, invivo cell tracking, tissue repair, clinical diagnosis, targeted drug delivery, magnetic hyperthermia and altered drug pharmacokinetics can be achieved via surface modification and bioconjugation [5,6]. Iron oxides are found in many forms, namely magnetite (Fe 3 O 4 ), hematite (α-Fe 2 O 3 ), maghemite (γ-Fe 2 O 3 ), wustite (FeO), bixbyite type (β-Fe 2 O 3 ), ε-Fe 2 O 3 [6,7], hydroxides, oxide/hydroxides [7] and ζ-Fe 2 O 3 [8]. Each polymorphic form of Iron oxides has their own specific features [4]. Among these various forms, magnetite and maghemite are widely studied and found to be promising due to their proven biocompability [6] and unique magnetic properties [9]. With recent progress in nano-platforms, majority of studies are focused on the development of magnetic systems with long circulation half-life, high specific absorption rate, low Curie temperature [Wei Wu] and shortened transverse relaxation times, i.e., T 2 and T* 2 [9,10].
In general, the magnetic behavior of nanomaterials is greatly influenced by various features, such as size, size distribution, shape, polymorphic form, surface modification and purity [11][12][13]. Iron oxide nanoparticles of various polymorphic forms are synthesized through various approaches, including co-precipitation [14,15] [27][28], bacterial synthesis [29], laser pyrolysis [30], and electrochemical synthesis [31]. For effective practical applications, the nanoparticles and nano-composites must be readily aqueous dispersible, stable and biocompatible with fascinating magnetic properties and interactive surfaces. The particle characteristics of Iron oxides such as size, shape and surface charges serves as a determining factor in achieving biological distribution and elimination [32]. Multiple studies evidence that the particle characteristics play an important role in the toxicity, i.e. smaller particles showed increased toxicity than larger particles [33-35].

Synthesis and surface functionalization of iron oxides
In the last two decades, several synthetic approaches have been followed to synthesize iron oxide nanoparticles with controlled size and morphology, biocompatibility and monodisperse nature [32,36]. The schematic depiction of commonly employed chemical approaches is shown in Figure 1.
Of many approaches, co-precipitation is widely used for the synthesis of iron oxide due to their process simplicity [37]. At the same time, it is necessary to consider several factors such as precursor salts used, ratio of Fe 2+ /Fe 3+ ions, nature and type of surfactants used, reaction parameters (pH and ionic strength of media, reaction temperature, stirring rate, drop rate of salt/base solution) and chemical processes employed in the co-precipitation technique to achieve the iron oxide nanoparticles with desirable crystal structure and morphology, monodispersity, and attractive magnetic properties [32,38]. Peternele et al. synthesized magnetite and maghemite nanoparticles by precipitating the mixture of chloride salts of Fe 2+ and Fe 3+ (1:2 ratio) using 1.5 M NaOH or 25% Ammonia under vigorous stirring. The sequences of addition of base strongly affect the formation of phase and size of the magnetite nanoparticles. Further, much smaller sized maghemite was obtained by oxidizing prepared magnetite nanoparticles due to the Ostwald ripening effect. The nanoparticles obtained using ammonia displayed uniform size and were monodisperse in nature [37]. Cui et al. synthesized magnetite, maghemite and hematite using a common epoxide precipitation route involving an ethanolic solution of ferrous chloride tetrahydrate and a gelation agent propylene oxide. Initially, the nucleation of magnetite was observed due to the oxidation of Fe(II) precursor. From this step, the magnetite solution can be transformed into Fe 3 O 4 , γ-Fe 2 O 3 and α-Fe 2 O 3 nanoparticles through centrifugation-followed by vacuum drying, sol-to-xerogel formation followed by oxidation at 150°C and, atmosphere controlled evaporation of sol at 150°C, respectively [39,40]. In another study, Cui et al. synthesized nanoparticles of other polymorphic forms such as lepidocrocite and goethite just by regulating the pH of the obtained greenish precipitate comprising an ethanolic solution of ferrous chloride tetrahydrate and a gelation agent propylene oxide using ammonia, followed by air oxidation under room temperature [41].
In several studies, the microemulsion and reverse micellization has been employed for the preparation of iron oxide nanoparticles with controlled morphology, but at the same time the aggregation of nanoparticles are high and requires several washing, and stabilization [32]. For instance, Lee et al. synthesized magnetite nanoparticles by  adding the hydro-ethanolic Fe (II, III) salt precursor solution into sodium  dodecylbenzenesulfonate (SDBS) dispersed xylene solution under vigorous stirring  for 12 h. Hydrazine was added to the solution maintained at 90°C, and the solution was refluxed for 5 h to obtain nanoparticles. The size of the nanoparticles can be tuned by modifying the relative amounts of precursor and the polar solvent-surfactant ratio [42]. In another study, Jung et al. applied the reverse micelle technique to synthesize uniform and monodisperse Fe 3 O 4 nanoparticles for sensing and drug targeting applications. Herein, the precursor and surfactant immobilized in the organic phase were added to the aqueous phase comprising stabilizer and stirred for several hours to form stable reverse micelles of sub 3 nm size. The formation of large irregular and small worm-like nanoparticles was observed in the absence of stabilizer [43].
In order to achieve iron oxide nanoparticles with high-quality monodisperse nature, thermal decomposition approach has been used widely. However, this requires relatively higher temperature and involves complex operations. Hyeon et al. prepared maghemite nanocrystallites with a high degree of crystallinity and monodiperse nature by subjecting Iron pentacarbonyl-oleic acid complex to thermal decomposition. Initially, the complexation was carried out by transferring the Iron pentacarbonyl into a hot mixture containing oleic acid and octyl ether and maintained at 100°C for 1 h. The resulting mixture was cooled and treated with mild oxidant trimethylamine oxide. Following the addition of trimethylamine oxide, the solution was again heated to 130°C for 2 h under an inert (Ar) atmosphere. Further, the temperature was slowly increased to 300°C and refluxed for 1 h to obtain the maghemite nanocrystallites [44]. Later, Lassenberger et al. used a slightly modified thermal decomposition technique to synthesize the monodisperse oleic acid stabilized Iron oxide nanoparticles [45], which does not involve the use of mild oxidants. The particle size of nanocrystallites is directly linked to the concentration of the complexation agent and the heating rate employed in the synthesis process [44,45]. Zhou et al. successfully synthesized various morphologies of monodisperse Fe 3 O 4 nanoparticles by varying the ratio of Iron oleate/sodium oleate, reflux temperature, and heating rate [46]. The biggest problem with the nanoparticles obtained via thermal decomposition route is their limited solubility in aqueous environments. Thus, phase transformation is required to render them water soluble [45].
A simple hydrothermal process can be a better alternative for the preparation of monodisperse, dislocation-free, highly crystalline iron oxide nanoparticles, as it does not require high temperature. The use of surfactants in the hydrothermal process could limit the growth of nanoparticle size, while retaining the crystallinity and magnetic properties close to that of the iron oxides prepared without surfactants and bulk iron oxides [6,32]. Ozel [48]. Xu et al. used urea to synthesize template-free rod-like β-FeOOH structures by varying the reaction temperature and time [49]. Also, urea in combination with ammonia was added to ferric chloride solution and autoclaved at 150°C for 6 h to obtain β-Fe 2 O 3 [50]. Various reports highlight that depending on the type of the reducing agent and surfactants used in the hydrothermal process, different iron oxide phases and nanostructures such as spherical, polyhedral, nanocubes, octahedral, truncated cubes, hollow spheres, nanorods etc. [6,22,23,32,47,49,51,52] can be obtained.
Sonochemical technique is a competitive alternative, to prepare ultrafine, monodispersive iron oxide nanoparticles with unusual properties. Usually, iron oxides prepared through this route are amorphous and possess low magnetization with speromagnetic behavior below magnetic transition temperature [6]. Hassanjani-Roshan et al. demonstrated the effect of ultrasonic power and reaction temperature on the particle characteristics such as crystalline/amorphous behavior and particle size of α-Fe 2 O 3 . The particle can be transformed into a highly crystalline form by subjecting them to higher temperature, following the sonochemical synthesis [53]. A cube-like Fe 3 O 4 nanoparticle with different particle sizes ranging from 20 to 58 nm was obtained by varying the molar concentration of the precursor and the reducing agent [54]. According to the LaMer model, the local supersaturation should be higher to achieve the smaller particles. Ludtke-Buzug et al. state that the maximum local supersaturation is higher at lower ultrasound frequency [55].
Recently, green chemistry and biological methods are being used for the preparation of iron oxide nanoparticles owing to their safety, low cost, non-toxic and eco friendliness approach. However, the consistency of synthetic process is highly influenced by the source of the green and biological reducing agents [40]. The microbial-mediated approach demands a lengthy incubation time for the synthesis of iron oxide nanoparticles [56]. Whilst, the plant-mediated environment-friendly approach requires less time and can be used for the synthesis of various iron oxide nanostructures like spherical, needles, cubical, dendrites, cylindrical, and so on [56] with appreciable biological activities. Many studies utilize plant extracts with mild reducing capability along with bases like sodium hydroxide [57,58], sodium carbonate [59], glycine [60] to synthesize stabilized iron oxide nanoparticles, as this can avoid the use of environmentally-toxic stabilizing agents. Few studies reported the successful synthesis of Iron oxide nanostructures using the extracts like grape berry ferment [61], flower extract of Avecinnia marina [62], and leaf extract of Bauhinia tomentosa [27] alone. The synthesis techniques of Iron oxide nanoparticles with their characteristic size are depicted in Table A1.
Avoiding agglomeration while retaining the stability is the most crucial challenge every magnetic material undergoes. In last two decades, considerable efforts have been devoted for the passivation of iron oxide nanoparticle surface using organic and inorganic materials, to avoid agglomeration, and to have improved solubility, stability and biocompatibility. The surface functionalization of iron oxide nanoparticles could be achieved by coating the iron oxide core with shell material (or) by dispersing the iron oxide core over the shell material (or) through chemical interaction of iron oxides with shell material (or) through bioconjugation reactions (or) by coating a shell-core structure with shell material. The shell materials could be organic or inorganic materials with functional properties. The commonly employed functional materials in the passivation of iron oxide surface include organic small molecules (drugs, fatty acids, polyol, dyes, vitamins, cyclodextrins), surfactants (dextran, polyvinyl alcohol), biological molecules (proteins, nucleic acids, antibody, cells, enzymes, microbes), silanes (p-aminophenyltrimethoxysilane, 3-aminopropyltriethyloxysilane), polymers (synthetic and natural origin), silica, metals and non-metals (gold, silver, carbon, etc.), metal oxides and metal sulphides [6]. In recent times, with the advancement in the polymer technology, several stimulus-induced targeted iron oxide-drug nanoconjugates are developed. Inspired by the different pH conditions of tumor environment, several pH-sensitive systems are being explored to minimize the release of chemotherapeutic drugs in the blood and normal tissues [61,[63][64][65][66]. Also, the thermo-responsive systems are being explored simultaneously to have control over the drug release, i.e., the release of drug is initiated only at the hyperthermia temperature [66,[67][68][69][70]. Interestingly, other stimulus-mediated systems such as enzymemediated [71,72], light-mediated [71,73], ultrasound-mediated [71,74] are also being explored.

Crystallographic properties and polymorphism
Iron (III) oxides exhibit distinct physical properties due to its polymorphic forms that has same molecular formula but with different crystallographic structures. Iron (III) oxides occur in various polymorphic forms such as α-Fe 2 O 3 , β-Fe 2 O 3 , γ-Fe 2 O 3 , ε-Fe 2 O 3 , and ζ-Fe 2 O 3 . The polymorphic forms are characterized by crystal structure as: All polymorphic form of Iron (III) oxides can be converted into other phases in response to temperature and pressure. The transformations of phases are interdependent on the precursors, pH value, ionic strength, intrinsic properties of nanomaterials such as crystal structure, particle characteristics, matrix characteristics in which particles are incorporated, reaction parameters and the nature of the treatment applied (e.g. thermal/pressure/both) [6,16,75]. However, under ambient conditions, a thermodynamically stable α-Fe 2 O 3 phase is usually formed directly from β-Fe 2 O 3 , ε-Fe 2 O 3 , and γ-Fe 2 O 3 . Temperature treatment plays a major role in the formation of α-Fe 2 O 3 phase from the other phases such as β-Fe 2 O 3 ($773 K) [76], γ-Fe 2 O 3 ($973 K) [77] and ε-Fe 2 O 3 (>1473 K) [78] phases. In some cases, like hollow β-Fe 2 O 3 , the phase can be transformed directly into γ-Fe 2 O 3 , and depending on the interparticle interactions and size of the nanoparticles, γ-Fe 2 O 3 may exhibit both direct and indirect transforma- Recently, a stable ζ-Fe 2 O 3 was formed by exposing β-Fe 2 O 3 above 30 GPa pressure [75,76]. The other predominantly investigated polymorphic form includes the face-centric cubic Fe 3 O 4 (FeO.Fe 2 O 3 , II and III oxidation states) with tetrahedral and octahedral sites. Like other Iron oxides, Iron (II, III) oxides also tend to undergo phase transformation to other Iron (III) oxide polymorphs under ambient conditions, due to their poor stability in oxygen environment [6]. It is well accepted that the crystalline phase stability of nanoparticles are much dependent on the surface stress, surface strain and surface energy. Recently, much focus has been kept on Cubic Wustite (FeO, space group Fm3m), which under ambient conditions remains in metastable state and undergo oxidation to other polymorphic form of iron oxides [6,[79][80]. Like iron oxides, iron (III) oxyhydroxides also exists in various forms namely an orthorhombic goethite (α-FeOOH, space group Pnma), monoclinic akaganeite (β-FeOOH, space group I2/m), orthorhombic lepidocrocite (γ-FeOOH, space group Cmcm) and hexagonal feroxyhyte (δ-FeOOH) [81]. Ferrihydrite are another polymorphic form of iron (II) oxides that are relatively abundant in the natural systems and can be readily transformed into the goethite phase when there is a rapid oxidation process [82]. Among various iron (III) oxide polymorphs, ε-Fe 2 O 3 particles are observed only in the nanosize and they are size dependent [83]. An amorphous form of Fe 2 O 3 was observed in the particles with less than 5 nm in diameter, wherein the oxygen octahedra are randomly oriented around the Fe (III) ions. However, it is difficult to distinguish the ultrafine particles of amorphous iron oxides and other polymorphs experimentally [84].

Magnetic properties
Magnetic properties of the nanomaterials play a key role in MRI contrasting ability, magnetically-induced heating, externally-targeted drug delivery and bio-sensing applications. The γ-Fe 2 O 3 polymorph exhibits ferrimagnetic and superparamagnetic behavior with a curie temperature of 928 K [16,76], whilst, the ferromagnetic ε-Fe 2 O 3 possess highest coercivity [76,84] with a curie temperature close to 500 K [85]. Interestingly, the magnetic order of ε-Fe 2 O 3 nanoparticles does not get vanished even at 500 K and this different ferromagnetic state persists up to 850 K [86]. A weak ferromagnetic behavior was reported in α-Fe 2 O 3 phase with a Morin transition at $269 K, i.e., a transition of antiferromagnetic state from weak ferromagnetic state [76] and a curie temperature of 950 K [16]. The paramagnetic β-Fe 2 O 3 becomes magnetically ordered below the Neel temperature ($110 K) and exhibit antiferromagnetic state. Similarly, the recently identified ζ-Fe 2 O 3 exhibited an antiferromagnetic nature below Neel transition temperature of $69 K [76]. A paramagnetic transition of amorphous Fe 2 O 3 at temperatures above the Neel temperature of 80 K was concluded based on the interpretation on Mössbauer data [84]. In some cases, non-ideal magnetic behavior could be observed in iron oxides due to wide range of blocking temperatures, aroused from wide range of particle size distribution [6]. Wustite (FeO) is generally stable above 560°C and possess antiferromagnetic nature with a Neel temperature of about 200 K [79]. Among all Iron (III) oxyhydroxide, δ-FeOOH is the only polymorphic form which showed significant magnetization at room temperature with ferrimagnetic behavior (Tc-450 K) [87] and significant relaxation properties [88]. The smaller crystals of Feroxyhites (δ-FeOOH) showed speromagnetic behavior between 80 K and 300 K [87]. β-FeOOH is usually paramagnetic at room temperatures and below Neel temperature they exhibit antiferromagnetic property [89]. The bulk α-FeOOH and γ-FeOOH displayed a Neel transition at 252 K and 53 K respectively [90]. The magnetic property of Ferrihydrite is size dependant, which displayed antiferromagnetic behavior below 4 nm, and above 4 nm ferrimagnetic behavior was observed [91]. The magnetic properties of iron oxide nanoparticles are greatly influenced by the oxidation and aggregation. The oxidation of iron oxide nanoparticles could lead to the loss of magnetic properties. In contrast the aggregation of particles may lead to mutual magnetisation that is usually aroused by the interaction of magnetic field of one nanoparticle with the neighboring nanoparticle [32].

Chemical properties
Iron oxide nanoparticles are highly prone for oxidation, particularly in the atmospheric air, and hence require a thin and non-interactive protective coating that has minimal effect on its characteristic physical properties, especially its magnetic properties [32]. Many studies also concluded that the naked iron oxide nanoparticles tend to agglomerate owing to their high surface energy, surface area-to-volume ratio, magnetic interactions and van der Waals forces [32,92]. The agglomeration of the particles not only increases the particle size, but also enhances strong magnetic dipoledipole attractions, that make the particles ferromagnetic. In general, the hydrophobic character and the huge surface area-to-volume ratio render the iron oxide nanoparticles toxic, insisting the need for the modification of particle surface, to make them hydrophilic and biocompatible [32].

Theranostic applications of magnetic iron oxide nanoparticles in cancer 4.1 Tumor imaging
Among various imaging modalities, magnetic resonance imaging acquires a rapid image of invivo tumor environment owing to their high spatial resolution. The unique magnetic properties with shorten relaxation times of iron oxide nanoparticles could enhance the sensitivity of T2 and T2* contrasting ability in magnetic resonance imaging (MRI) [9]. The imaging sensitivity and specificity of iron oxide nanoparticles could be enhanced by modifying the surface of iron oxides with small molecules, peptides and antibodies, whose receptors are overexpressed on the surface of tumor cells. Yang et al., modified the poly(amino acid)-coated iron oxide nanoparticle surface with Her-2/neu antibody for the detection of HER2/neu positive breast cancer cells and observed an enhanced MRI contrasting ability with significant T 2 relaxation time. Over many years, a well-known antibody herceptin is used clinically against the HER2/neu receptor [93]. Lee et al., demonstrated the detection of small tumors using the herceptin conjugated iron oxide nanoparticles [94]. Though antibodies show promising future for onco-imaging and therapy, their steric effects limits their conjugation with iron oxides. Further, the decrease in specificity of antibody-iron oxide conjugate was observed due to the interaction of Fc receptors of normal tissues with the antibody. Recently, single chain antibodies (scFv) have gained more traction owing to their small molecular weight, antigen binding ability, non-immunogenicity and low cost. Some scFv's such as scFvEGFR10 and sm3E have been conjugated to the iron oxide nanoparticles for the reduction of T 2 relaxation time and enhanced MRI capability. Yang and his co-workers conjugated amino-terminal fragment (ATF) peptide and near infrared dye Cy5.5 into the iron oxide-amphiphillic polymer conjugate to target the urokinase plasminogen activator receptor that is over expressed in human tumor cells and stromal cells where the tumor is associated. Herein, the study reported that the imaging probe is capable to detect small tumors ranging from 0.5 to 1 mm 3 . The other peptides that are generally used to achieve multi-modal imaging capability include EPPT 1 peptide, Luteinizing hormone release hormone, Folic acid, Vitamin FA and Arg-Gly-Asp [95][96][97] and chlorotoxin peptide [98]. The iron oxides could also possess T 1 relaxation, when the particle size is greatly reduced, i.e. less than 5 nm. This phenomenon is observed due to the decrease in magnetic moment exerted with decreased particle size. The Iron (II, III) oxides below 5 nm are considered to be responsive MRI contrast agents due to its ability to be used as both T 1 and T 2 contrasting ability. Under tumor environment, the iron oxides are released from the carrier providing the T 2 -T 1 switching ability during imaging [95][96][97]. Despite of the establishment of in vitro imaging potential of tumor targeted iron oxides, they are not widely used in clinical practice owing to their low specificity and sensitivity in vivo. Also, for successful clinical use, the targeted iron oxides must be able to monitor tumor metastasis and therapy response. The low sensitivity of single imaging modality has evolved the multimodal imaging technique which involves the combination of MRI contrast imaging with photothermal imaging [95].

Cancer labelling and sorting
Cell labelling and sorting is a key technique in the field of oncology and stem cell research. Usually, cells are labeled using ferro or superpara or paramagnetic materials in the process of invivo cell separation, and the labeled cells are visualized using the magnetic resonance imaging based protocol. In principle, two approaches are employed for cell labelling using iron oxides; (i) one by directly attaching the magnetic iron oxide nanoparticles to the cell, and (ii) the other by internalizing the iron oxide nanoparticles into the cytosol of cell using receptor-mediated endocytosis or fluid phase endocytosis or phagocytosis [99][100][101]. Cell labelling must not compromise the proliferating capability, motility, and cellular functions for effective stem cell trials or therapies. Arbab et al. labeled CD34+ hematopoietic and mesenchymal stem cells with ferrumoxides-protamine sulfate complex and reported the unaltered expression of phenotypic markers and differentiating capacity [102]. The cell viability remained intact when labeling concentration of iron oxide nanoparticles was used. In contrast, at high concentrations, the cellular viability decreased. The uptake of iron oxide into stem cells can be enhanced by modifying the core with dextran, citrates, HIV tat peptide, unfractionated heparin, and aminosilane. The modification of the iron oxide core exerts various biological activities in labeled cells. For instance, there is an increased cell proliferation in iron oxide-ferucarbotran labeled mesenchymal stem cells. The target-specific molecules/Iron oxide nanoconjugate can precisely label the cells in vivo [103]. Chen et al. demonstrated the MRI imaging potential of Herceptin bound-dextran-coated iron oxides in HER2/neu receptors expressed cancer cell lines [104]. In another study, the contrasting ability of folic acid bound-PEG coated iron oxides in the tumor cells expressing folate receptors [105].
The magnetic behavior of iron oxides allows the isolation of specific cells from the biological suspensions like blood, apart from its diagnostic ability. In non-invasive magnetic-activated cell sorting, the antibody-bound iron oxides bind specifically to the specific antigens present on the surface of target cells. The bound fraction can be separated from the unbound fraction by applying a magnetic field. For instance, circulating tumor cells are captured, and analyzed for staging cancer, selection of therapy and monitoring treatment [106]. Mi et al. developed a low cytotoxic Herceptin-functionalised conjugated oligomer-based Iron oxide-silica nanoparticle system for magnetic-activated sorting and fluorescence-activated detection of circulating tumor cells at its metastatic stage [107]. Du et al. bioengineered D-tyrosine phosphate decorated iron oxides that can be dephosphorylated to tyrosine coated iron oxides by the overexpressed alkaline phosphatases in the surface of cancer cells. The tyrosine-coated iron oxides upon dephosphorylation have been attached to the tumor cells and captured using a small magnet [108].

Cancer immunotherapy
Immunotherapy is a robust strategy to eliminate cancer cells. For effective cancer immunotherapy, the immune system must be activated against tumor antigens. The immunotherapeutic approaches include cell-based immunotherapy, monoclonal antibodies (mAb) for checkpoint blockade and cancer vaccines. In cell-based immunotherapy, the migration, expansion, and depletion of immune cells are tracked to understand the complex cellular and molecular mechanism involved in the immune system [100]. The schematic representation of various applications of iron oxide nanoparticles is depicted in Figure 2.

Cell-based immunotherapy
The T cells have the ability to differentiate into various forms based on the interaction of specific tumor antigens on the antigen-presenting cells. Real-time T-cell trafficking using MRI can improve the T cell-based immunotherapies by achieving better localization [109]. For instance, the three-dimensional MRI visualization of in vivo T-cell trafficking to target tumors and the temporal regulation of T-cells within the tumors has been demonstrated using ova-specific CD8+ T cells labeled crosslinked iron oxides (highly derivatized). In addition, a similar proliferating potential, cellular interaction, and cytotoxic profiles was observed for both labeled and unlabelled cells [110]. Liu et al. developed PEG conjugated fluorescent dyes coated iron oxide for achieving dual-mode imaging such as MRI and Fluorescence. The human and murine T cellular functions were not altered following the injection of these nanoparticles [111]. The labeling of T cells is the biggest challenge due to their non-phagocytic nature. However, significant internalization of Iron oxides can be achieved using the transfection agents such as polyethyleneimine, poly-L-lysine, lipofectamine [100].
Macrophages eliminate foreign particles and cellular wastes by secreting cytokines and initiating phagocytosis. Reprogramming or polarizing the tumor-associated macrophages (TAM) in the tumor environment shall overcome the difficulty of penetration of M1 macrophages from the outside environment. Iron oxides on their own activate the macrophages through metabolic degradation. In a study, the response to CD47 monoclonal antibodies by tumor-associated macrophages (TAM) has been monitored using ferumoxytol-enhanced MRI. The study showed that phagocytosis of TAM had been activated due to the inhibition of the interaction between SIRPα and CD47 by CD47 mAb [112]. In another study, 3-methyladenine was incorporated into hollow iron oxide nanoparticles to promote the immune response by reprograming the TAM into M1-type macrophages. The nanoparticle system upregulates the NF-κB p65 while inhibiting the expression of P13Kγ to promote an immune response. The mouse model revealed a synergistic effect of polarization of macrophages by 3methyladenine and iron oxide nanoparticles [113]. An artificial reprogramming of macrophages was reported in a study using hyaluronic acid-coated iron oxide nanoparticles. In this study, the iron oxide nanoparticles and macrophages from RAW 264.7 mouse were incubated together and injected into 4 T1 tumor-bearing mice. The results indicated the capability of these artificially programmed macrophages in polarization of TAM and enhanced tumor effect [114].
Dendritic cells play a key role in activating cytotoxic T lymphocytes and regulating adaptive immune response by presenting the tumor antigens into the draining lymph nodes. The immune response is highly dependant on the migration of activated dendritic cells to lymph nodes [103,115]. The incorporation of iron oxides into a dendritic cell does not impair the viability of cells [103] and demonstrate the tracking of dendritic cells with MRI [100]. The antigens are loaded into immature dendritic cells, as the phagocytic ability is higher than the mature dendritic cells. The internalization of iron oxides takes place via endocytosis for the particles ranging from 20 to 200 nm [100]. Iron oxide nanoparticles bound to oval albumin showed increased expression of TNFα, IL-6, and IFN-γ in murine dendritic cells. Further the assessment of immunotherapeutic capability in mice revealed a dramatic reduction in the tumor [116,117]. De Vries et al. reported the enhanced accuracy of magnetic resonance tracking of iron oxides-loaded dendritic cells over scintigraphic imaging. Herein, the iron oxide nanoparticles are phagocytized into the autologous ex vivo cultured dendritic cells without altering the phenotype and functional properties [115]. Dendritic cells loaded with iron oxide/zinc oxide core-shell nanoparticles exhibited a T 2 -weighted signal reduction in the lymph node of C57BL/6 mice. However, the dendritic cells loaded with zinc oxide showed a migration towards the lymph nodes [100].

Cancer vaccines
Cancer vaccines play a crucial role in presenting the tumor antigens to activate specific T cells against the tumor cells. Recently, biomimetic nano vaccines encompassing tumor cell membranes with tumor antigens are used for targeting the immune or tumor cells. The vaccination capacity can be increased by introducing iron oxides with intrinsic magnetic properties. For instance, Zhang et al. developed a biomimetic magnetosome to expand and stimulate antigen-specific cytotoxic T-cell and to track and effectively guide them into tumor tissues [118]. Further, Wang et al., demonstrated antitumor activity and immunogenic cell death with low systemic toxicity using Ce6-loaded magnetic/mesoporous organosilica nanoparticles concealed with cancer cells [119]. In another study, Long CM et al. developed magnetovaccination using iron oxide labeled GM-CSF secreting cells mixed with tumor to stimulate the immune system by inducing the T cell tumor targeting factors proliferation [120].

Checkpoint blockade
The immune checkpoint is the molecular interactions between cancer cells and immune cells. The ability of tumor cells to evade the surveillance of the immune system is the common problem associated with the T cell-mediated approach, as many healthy cells like certain tumor cells encompass inhibitory checkpoint programmed cell death protein 1 ligand 1 (PD-L1) that can inactivate the T cells by binding to the inhibitory checkpoint PD1 protein expressed on T cell surface [100]. The immune response shall be enhanced by targeting inhibitory checkpoint molecules such as proteins and antibodies. Under physiological conditions, the checkpoint inhibitory molecules are highly prone to degradation and hence required to be encapsulated into a robust delivery system. In a study, the immunoswitch design was demonstrated using antibodies-loaded dextran-coated iron oxide nanoparticles for the inhibition of immune checkpoints. The antibodies against PD-L1 and 4-1BB were used to stimulate T cells. In this study, the tumor-bearing mice (C57BL/6) was initially treated with adoptively transferred T cells, followed by the administration of antibodies-loaded dextran-coated iron oxide nanoparticles or free individual antibodies. The results suggest the targeting of multiple checkpoints, as targeting only one checkpoint did not result in size reduction of tumors. Also, highlights the multifunctional utility of iron oxides for checkpoint inhibition [121]. In another study, the Iron oxide-coated folic acid-functionalised-disulphide-polyethylene glycol-conjugated polyethylenimine nanoparticles were developed for the delivery of siRNA to inhibit PD-L1 protein. The nanoparticles exhibited higher transfection ability, MRI contrasting ability, and the high cellular uptake downregulate the PD-L1, which in turn affected the T-cells cytokine-secretion level [122]. The schematic representation of immune checkpoint regulation using immunoswitch nanoparticles is shown in Figure 3.

Tumor ablation therapy
In tumor ablation therapy, the use of non-contact magnetic heating (above 42°C) has gained a special attention and are being utilized in clinical practice in few hospitals as an adjuvant therapy. The magnetic heating is achieved through hysteresis loss and relaxation losses under varying alternating magnetic field and radio frequencies. The efficiency of magnetic heating is directly linked to the size, shape and concentration of the magnetic nanoparticles, the strength and frequency of alternating magnetic field and cooling rate in biological tissues. The major obstacle in the use of Iron oxides for non-contact heating of tumor tissues is their low heating power. Magnetite and maghemite nanoparticles are widely employed for magnetic hyperthermia applications due to their well-established biocompatibility. However, these nanoparticles possess relatively low coercivity and require high applied frequency, usually ranging from 400 kHz to 900 kHz, to effectively heat the media in which it is dispersed. In contrast, studies indicate that ε-Fe 2 O 3 nanoparticles exhibit hyperthermia potential at low applied frequency of about 20 kHz to 100 kHz. Though many magnetic materials reported to have high heating power, their concern over safety has limited their use in clinical practice, and encourages the optimisation of structural features of iron oxide nanoparticles for enhanced clinical hyperthermia potential [123].
Kolosnjaj-Tabi et al. demonstrated a mild hyperthermia efficacy of PEG-coated iron oxide nanocubes in a magnetic field of 23.8 kA/m and 111 kHz until the particle resides in the interstitial extracellular location. The hyperthermia efficiency of the nanoparticles was lost after cellular internalization and capture in the liver and spleen. However, the hyperthermia effect destabilize the tumor stroma to enhance the drug penetration [124]. The cancer theranostic agents are widely employed for the effective control of tumor owing to their potential diagnostic cum treatment approach. The magnetic hyperthermia efficiency and MRI T 2 contrasting ability of iron oxide nanoparticles were demonstrated using the fourth-generation dendrimer coated iron oxides [125]. Hayashi et al., developed iron oxide nanoclusters for combined MRI cum hyperthermia, as the individual iron oxides (<10 nm) are prone to leakage from capillaries. In this study, the surface of Iron oxide nanoclusters is modified with polyethylene glycol and folic acid to enhance their accumulation within the tumor environment of mice, following the intravenous administration. The mice that underwent local heating for 20 minutes reduced the tumor volume to about 1/10th of the control mice, indicating the hyperthermia efficiency of the iron oxide nanoclusters [126]. Lin et al. developed a multifunctional pegylated albumin nanocomplex comprising Iron oxide and a hydrophobic dye (IR780). The photothermal effect and MR imaging of nanocomplex were demonstrated on a cancer colon model and tumor-bearing mice, respectively [127]. The combined photothermal effect (NIR-induced) and cancer imaging (MRI and fluorescence) were demonstrated using a novel dumbbell-like Gold-Iron oxide nanoparticle by Kirui et al. [128]. A similar kind of bimodal cancer imaging with a photothermal effect was reported in hyaluronan-targeted iron oxides to bring out the photothermal efficiency and cell staining potential of the iron oxides apart from its MRI contrasting ability [129]. Espinosa et al. demonstrated a complete tumor regression using the dual-mode hyperthermia and photothermal therapeutic potential of iron oxide nanocubes [130].

Drug delivery applications
The successful delivery of therapeutic agents into the tumor environment with minimal toxicity to surrounding tissues is the biggest challenge, as it is often limited 13 Iron Oxide Nanoparticles and Nano-Composites: An Efficient Tool for Cancer Theranostics DOI: http://dx.doi.org /10.5772/intechopen.101934 by tumor heterogeneity, dense fibrotic stromal barriers and various vascular barriers including abnormal tumor blood vessels, tumor cells proliferating nests, normal blood vessels, positive intratumoural pressure [131,132]. Iron oxide nanocarrier systems are often believed to overcome these biological barriers by altering the pharmacokinetics and tissue distribution profile via enhanced permeability and retention effect [9]. However, for clinical success, EPR effect may not be sufficiently enough as it offers only certain level of tumor targeting and non-specific biodistribution. Thus, the logical choice for tumor therapy shall be actively tumor-targeted iron-oxide nano-carrier system, which can be minimally toxic to normal tissues while having enhanced bioavailability, intracellular bio-distribution, and potent cyto-toxic effects against the tumor cells. [131,132]. Recently, the tumor-targeted iron oxide nanoparticles are used to monitor the accumulation of drugs in the tumor site, while simultaneously estimating the drug level in the tumor tissues. The detection of MRI signal changes of drug loaded iron oxides can provide a track over drug delivery, estimated drug levels in tissue and therapeutic response invivo [9].
Li et al. developed the iron oxide nanoclusters with photothermal mediated synergistic chemotherapy and chemodynamic therapy. In this study, the core iron oxides were surface modified using the paclitaxel-loaded human serum albumin and conjugated to the Arg-Gly-Asp peptides for tumor specific targeting [133]. Several studies indicate that insufficient penetration through BBB reduces the efficiency in treating glioblastoma multiforme. Norouzi developed pH-sensitive doxorubicin-loaded Iron oxide nanoparticles stabilized with trimethoxysilylpropyl-ethylenediamine triacetic acid to demonstrate their uptake in brain-derived using mouse model. The cellular uptake of nanoparticles was increased by 2.8 folds and provided an enhanced antitumor effect than free doxorubicin. Further, the study indicated that the penetration of nanoparticles into the brain was augmented due to the combination of cadherin binding peptide and external alternating magnetic field [134]. Hussein-Al-Ali et al. developed iron oxide/chlorambucil/chitosan nanocomposite with a particle size of about 15 nm. Controlled release of drug chlorambucil from the nanocomposite with significant anti-tumor effects on leukemia cancer cell lines was observed [135].

Concluding remarks and future directions of iron oxides
Since several decades, many studies have been conducted to evaluate the potential use of functionalised iron oxide nanoparticles for the delivery of anticancer drugs, yet there are several obstacles that need to be overcome for increased adoption of these nano-carrier systems into clinical practice. The various challenges that needs to be considered while developing the newer targeted iron oxide nanoparticles includes the synthesis of conjugated iron oxide nanoparticles without inducing a change in the chemical and magnetic properties, the high drug loading efficiency, regulation of circulation time [113], specificity and selectivity of towards tumor tissues, and controlled release of drug with the tumor region. The other challenges that are least explored in the development of iron oxide nanocarrier systems includes tumor uptake, biodistribution and bioelimination. The biological distribution is highly dependent on the nanoparticles size, morphology and surface characteristics, as these properties can strongly influence the particle interaction with serum proteins and cells [32]. The major obstacle in achieving the effective tumor therapy is the tumor heterogeneity resulted from the genetic mutations. This emphasis the need for personalized medicine involving both imaging and targeted drug delivery simultaneously, signifying the concept 'we observe what we treat'. The potent MRI capability of the iron oxide nanoparticles shall allow the visualization of events such as delivery of drug and other cargo molecules, efficacy of the undergoing treatment, gene expression and metastasis, bioelimination of iron oxides. Though, iron oxides exhibit many distinctive properties, the long-term fate of iron oxides, PK/PD studies, toxicology studies and the toxicity criteria are yet to be clearly defined. Recently, there is an intense focus on the development of multifunctional tumor-targeted drug-loaded iron oxide nano-carrier systems, as it can offer many benefits such as, tumor-specific targeting, MRI contrasting ability (track and monitor the accumulation of the nano-carrier system), combined hyperthermia and chemotherapy to tumor cells and stimulusinduced drug release (control over the drug release at the tumor-specific site