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1 Title: Stable Monodisperse Nanomagnetic Colloidal Suspensions: An overview Author: Donya Ramimoghadam Samira Bagheri Sharifah Bee Abd Hamid PII: S (15) DOI: Reference: COLSUB 6893 To appear in: Colloids and Surfaces B: Biointerfaces Received date: Revised date: Accepted date: Please cite this article as: D. Ramimoghadam, S. Bagheri, S.B.A. Hamid, Stable Monodisperse Nanomagnetic Colloidal Suspensions: An overview, Colloids and Surfaces B: Biointerfaces (2015), This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

2 Stable Monodisperse Nanomagnetic Colloidal Suspensions: An overview Donya Ramimoghadam, Samira Bagheri *, Sharifah Bee Abd Hamid Nanotechnology & Catalysis Research Centre (NANOCAT), IPS Building, University of Malaya, Kuala Lumpur, Malaysia. Abstract *Author to whom correspondence: Samira Bagheri; address: Magnetic iron oxide nanoparticles (MNPs) have emerged as highly desirable nanomaterials in the context of many research works, due to their extensive industrial applications. However, they are prone to agglomerate on account of the anisotropic dipolar attraction, and therefore misled the particular properties related to single-domain magnetic nanostructures. The surface modification of MNPs is quite challenging for many applications, as it involves surfactantcoating for steric stability, or surface modifications that results in repulsive electrostatic force. Hereby, we focus on the dispersion of MNPs and colloidal stability. Keyword: Superparamagnetic iron oxide nanoparticles; Magnetic slurry; Ferrofluids; Ultrastable colloidal suspensions 1. Introduction 1.1 Iron oxide nanoparticles 1.2 Magnetic iron oxide applications 1.3 Overview of synthesis methods 2. Magnetic Iron oxide nanoparticles 2.1 Superparamagnetic iron oxide nanoparticles (SPIONs) and their applications 3. Nanoparticles aggregation 4. Surface modifications of magnetic iron oxide nanoparticles 4.1 Dispersion of magnetic iron oxide nanoparticles Size Sorting Colloidal Stability Surfactants Polymers 1 Page 1 of 70

3 4.1.5 PEGylation Biopolymers Ferrogels Hybrid materials Activated Carbon Silica Emulsions 5. Conclusion and future aspects 1. Introduction 1.1 Iron oxide nanoparticles There are various phases of iron hydroxides and oxides, e.g. (α, β, γ) Fe 2 O 3, (α, β, γ, δ) FeOOH, Fe 3 O 4, and FeO. α- FeOOH, γ-fe 2 O 3, α-fe 2 O 3, and Fe 3 O 4 are carefully studied due to their potential applications in the chemical, optical, and biomedicalfields [1 6]. Whenever the particle size is reduced from micro scale to nano scale, they display novel electrical, optical, or magnetic properties that mostly differs from their bulk counterpart [7]. Magnetite and maghemite, which are phases of iron oxide, are the most commonly employed magnetic materials, due to its high magnetic susceptibility, high saturation magnetization, and excellent biocompatibility [8, 9]. Contrary to the bulk form of iron oxide, which is a multi-domain ferromagnetic material, and shows a permanent magnetization when a magnetic field is not present, iron oxide nanoparticles in the 20 nm range is made up of a single magnetic domain, which results in superparamagnetism with a single magnetic moment [10]. It indicates that iron oxide nanoparticles are simply found using a magnetic field, but fails to maintain the residual magnetism at zero magnetic field [11, 12]. The small size of iron oxide nanoparticles is of particular interest, due to their permanent magnets, which are arbitrarily distributed from the Brownian motion in the fluid, which is consequently neutralized; which explains why the fluid does not show permanent magnetism. The Brownian motion of the nanoparticles, therefore, fortifies the stability of a magnetic fluid forming the colloidal system, which avoids precipitation and agglomeration. This particular motion improves with reduced particle size. Simultaneously, particles must not be too small, as when the particles size are less than 1 2 nm, their magnetic properties will be nonexistent [13]. Nevertheless, in the case of larger particles, agglomeration is a problem, as magnetic interactions is predominant. Nanomaterials is favored, as it prevents particles from agglomerating. Magnetic iron oxide nanoparticles (specifically maghemite and magnetite) are fast being the focus of contemporary studies due to their physico-chemical properties and potential in many scientific and technological fields. From a physical perspective, these particles might display superparamagnetism and single magnetic domain particle behaviors, however, from a chemistry standpoint; the large specific surface area and rich surface chemistry are important factors that controls phenomena related to chemical and biochemical reactivity [14]. 2 Page 2 of 70

4 1.2 Magnetic iron oxide applications Metal nanoparticles have attracted lots of attention recently due to their unique properties compared to bulk metals, such as high specific surface area and accessibility, as well as a series of energy degenerated states, which make them particularly useful for specific applications like catalysis, sensors, (bio) medicine, opto-electronics, and semiconductors. To begin with, iron oxide nanoparticles are extensively utilized as highly active catalysts due to the enhanced surface area ratio and more coordination of unsaturated sites on their surfaces. In addition, matrix supported nanoparticles which have proved advanced stability have found a broad application as catalysts. For instance, synthetic improvements which have lately resulted in iron oxide nanoparticles are significantly more effective than conventional larger-sized iron oxide for the oxidation of CO and the oxidative pyrolysis of biomass [15]. Magnetic iron oxide nanoparticles, particularly superparamagnetic iron oxide nanoparticles (SPIONs), have captivated immense interest, owing to their unique properties and diverse applications. Among different iron oxide phases, maghemite (γ-fe 2 O 3 ) and magnetite (Fe 3 O 4 ) are progressively prominent in engineering and biomedical applications. Nevertheless, magnetite (Fe 3 O 4 ) particles, due to their higher saturation magnetization compared to maghemite (γ-fe 2 O 3 ) particles, are favored for biomedical applications [8]. Magnetic iron oxide nanoparticles have a long history of biomedical applications, such as hyperthermia, bioseparation, biosensor, cell imaging and labeling, diagnostic contrast agents (CAs) for magnetic resonance imaging (MRI), and drug or gene delivery [15 17]. Biomedical applications under current investigation include retinal detachment therapy [19], cell separation methods [20], tumor hyperthermia [21], improved MRI diagnostic contrast agents [21 23], and magnetic field-guided carriers for localizing drugs or radioactive therapies [24 26]. In addition to biomedical applications, there is plenty of experimental proof indicating the practicability of SPIONs as an excellent nano-agent tailor for heavy metals removal [27, 28], colorants [30], chlorinated hydrocarbon [31], organic pollutants [32], suspended solids [32, 33], as well as aquatic organisms [35] from water resources. Normally, the cleanup procedures were performed by primary seeding the target pollutants with SPIONs via electrostatic interaction or a particular binding species, and subsequently, the pollutants-bound particles were magnetically removed. It should also be mentioned that SPIONs generate inhomogeneity in the local field, which is experienced by the surrounding water molecules, leading to faster T 2 relaxation times of the water protons and negative contrast in the MRI images [35 37]. A great number of studies have also revealed that magnetic nanoparticles are applicable in sensors based on giant magnetoresistance, magnetically controllable Single Electron Transistordevices and photonic crystals [39]. Furthermore, they have recently indicated proper sensing characteristics to CO and hydrocarbon gases which most likely enables the gas sensing applications. Magnetic nanoparticles were also explored for their potential application as highdensity magnetic data storage. Additionally, magnetic iron oxide nanoparticles have been applied in semiconductors like their metal oxide counterparts such as TiO 2 [40], [41] and ZnO [42] [44] nanoparticles. In this regard, iron ions are capable of doping into the semiconductors and generate dilute magnetic semiconductors (DMS) with the purpose of modifying the optical and 3 Page 3 of 70

5 electronic properties of magnetic nanoparticles and semiconductors. Moreover, DMS nanoparticles can function as the magneto-optical switches thanks to sensing both light and magnetic fields. Last but not the least, a huge amount of synthetic and natural iron oxides (63% and 37%, repectively) is widely used as pigment in all over the world. Diverse colors of yellow, black, brown and red are frequently generated using different iron oxides phases such as goethite, magnetite, maghemite, and hematite, respectively. The transparent iron oxide pigments could be also obtained as a consequence of reduced particle size [45]. 1.3 Overview of synthesis methods Some methods of synthesizing magnetic particles are co-precipitation, wet grinding, reduction of metal salts in aqueous solution, microemulsion and inverse microemulsion [46], and high temperature decomposition of organic precursors and oxidization of magnetite nanoparticles [39 41]. Some methods of synthesizing magnetic particles are co-precipitation [50] [52], hydrothermal [53], [54], microemulsion and inverse microemulsion [46], [55], thermal decomposition of organic precursors [56], [57], sol-gel [58], [59], and wet grinding method [60]. Moreover, for the past few decades, various methods of controllable synthesis of monodispersed SPIONs have been developed, as certain requirements for the synthesis of iron oxide nanoparticles needs to be fulfilled in order to be successfully utilized in applications, such as good dispersiblity, nano-sized distribution, highly uniformed-superparamagnetic properties, hydrophilic surface with different functional groups for covalent binding to biomolecules, and homogenous physical and chemical properties [61]. Nevertheless, no method can meet all the above mentioned requirements, except the ones that are able to form functionalized monodispersed nanoparticles by thoroughly encapsulating magnetic cores, resulting in controlled size and biocompatible surfaces [62]. The synthetic method that have been usually adopted in the literature to synthesize magnetic iron oxide nanoparticles is the co-precipitation of iron hydroxide, followed by either calcination at high temperatures (e.g. 600 C) [1], hydrothermal treatment [5, 44 46], treatment at boiling, or lower temperatures [2, 47 49]. In this method, divalent and trivalent iron salts are often condensed in the presence of hydroxide bases (ph ). The particle s crystal structure simply forms in aqueous medium. This method is reproducible, simple, cheap, and results in high yield [18]. Another privilege of the technique is that the surface of particles is kept free for any following functionalization, with the preferred molecular [69] or polymeric [70] entities. However, the particles obtained in the first step might agglomerate, because countercations such as Na + can neutralize the negative charge of Fe O groups on the surface, which are electrostatically able to stabilize the particles in an alkaline medium [71]. Moreover, the required time to accomplish the transformation process is frequently introduced over 24 h. In the case of treatment temperature of 100 C and lower, the required time to gain α-fe 2 O 3 is even longer, reaching several days [2, 49]. Furthermore, the ph value is also a crucial factor that affect the production of iron oxides. Liu et al. [66] used multiple ph values and sodium hydroxide to precipitate iron hydroxides from FeCl 3, followed by heating it to transform Fe(OH) 3 gel into α-fe 2 O 3. The results showed that a 4 Page 4 of 70

6 ph of 4.5 quicken the whole process to a total of 4 5 h. The size and morphology of the products can be fine tuned by introducing additives during the reaction, such as glycine [4], ethylene glycol [8], sodium dodecyl sulfate (SDS) [2, 44], quinine hydrogen sulfate (QHS) [68], CTAB (cetyltrimethylammonium bromide) [72], polyvinyl pyrrolidone (PVP) [14], glycine hypochlorite [16] and etc. Some of these additives easily act as surfactants or growth modifiers [4] to control the size and/or to gain a particular morphology, while others may get involved in the reaction process. Hydrothermal method is considered as well-known straightforward technique in which iron precursors in aqueous solution can be heated at high temperature at autogenous pressure. A huge number of literature have investigated the synthesis of magnetic nanoparticles using hydrothermal method [73], [74]. This technique exploits the solubility of almost all inorganic substances in water at elevated temperatures and pressures and subsequent crystallization of the dissolved material from the fluid [75]. The water can be replaced by other polar or nonpolar solvents, such as benzene, which is known as solvothermal synthesis [76]. In the hydrothermal process, parameters as temperature, solvent, and reaction time normally play very parafound roles in the formation of final products. In this regard, larger sizes of Fe 3 O 4 particles will be produced in the exsistence of higher water content in the reaction vessels [77]. Therefore, in this method, size and shape of iron oxide nanoparticles can be controlled using reaction condition and reactant s ratios and concentrations. In addition to stabilize iron oxide nanoparticles and retain the colloidal behavior of the superparamagnetic suspension various surfactants can be utilized [45]. Recently, some techniques like microwave have been applied in combination with hydrothermal method for preparation of superparamagnetic iron oxide nanoparticles. This modification is beneficial for scale up and fabrication of uniform particles [78]. The Mechano-chemical technique is a new method that is capable of synthesizing nanomaterials. It is an organic-solvent free process, ecologically clean, suitable for scale-up, simple, and cheap. Despite the report on the successful synthesis of 15 nm maghemite nanoparticles using high energy ball-milling in water medium [79], it is time-consuming (the milling process took at least 48 h), and the final maghemite undergoes extreme aggregation. Although the grinding process can be carried out in an alkali medium (ethanol), direct phase transformation of hematite to maghemite lead to a mixed-phase product [80]. Lin et al. used anhydrous ferric and ferrous chlorides as reactants to prepare the MNPs with an average particle size of 14.8 nm, but the method is still require extended milling time (more than 72 h). In the case of commonly used method of MNPs synthesis (polymer coating), the overall size of the particles was definitely increased, which disfavors many applications. Cheng et al. recently reported the preparation of well-dispersed Fe 3 O 4 nanoparticles covered by Tetramethylammonium hydroxide (N(CH 3 ) 4 OH) [2]. The surfaces of the nanoparticles were protected by hydroxyl groups interacting with + N(CH 3 ) 4 through electrostatic forces. Even though the obtained magnetite particles represented excellent biocompatibility, the native surface chemistry of the CH 3 groups does not seem to allow further biochemical modifications of the nanoparticles. Nevertheless, the techniques reported suffer from drawbacks such as the need to apply expensive organic materials as precursors, monotonous washing procedures to remove the surfactants, or severe aggregation of particles at high temperature oxidation in the air. Moreover, the particle 5 Page 5 of 70

7 sizes are normally larger than 10 nm. Therefore, the development of new, convenient, and largescale synthesis methods for magnetite nanoparticles, especially the SPIONs, is still a major challenge. Table 1 shows the most commonly used methods for magnetic iron oxide nanoparticles fabrication including their benefits and limitations. In this context, several novel methodologies have recently been developed for the preparation of advanced functional magnetic nanomaterials as alternatives to the more traditional impregnation/deposition and coprecipitation protocols. These include the use of ultrasound, laser ablation, photochemistry, electrochemistry and microwave irradiation technologies [56, 57]. Many of these procedures offer the added advantages of a tunable and somehow controllable deposition of nanoparticles on the support (in terms of particle sizes and distributions), as well as the possibility to design specific functional materials, depending on the envisaged application. 6 Page 6 of 70

8 Table 1. The comparison between various common synthetic methods of magnetic iron oxide nanoparticles. Methods Size(nm) Shape Advantages Disadvantages Refs. Coprecipitation Spherical/ rhombic -Conventional, -Low reaction temperature -Homogeneous -Moderate morphological control -Not suitable for the preparation of high pure, accurate stoichiometric phase -Not having universal experimental condition [83] [88] Hydrothermal 27 Spherical -Highly crystalline -Pure iron oxide NPs -Simple -Environmentally friendly -Good morphological control Thermal decomposition 4-20 spherical -Good size control -Narrow size distribution -Narrow size variation of 5% -Good crystallinity -Monodispersity Microemulsion 4-12 Spherical (inverted), cubic, lamellar phases, cylindrical micelles -Simple and versatile method -Reproducible -Using surfactant limits particle nucleation, growth, and aggregation -Better morphological control -Homogenous particle size distribution Sol-gel Spherical -Pure amorphous phases -Monodispersity -Homogeneity and phase purity -Low temperature procedure -Moderate morphological control -Useful for hybrid nanoparticles fabrication Sonochemical 5-30 Spherical -Shortened reaction time -Uniform particle size -Higher surface area -Better thermal stability -Improved phase purity -Moderate cost -Hydrothermal slurries are potentially corrosive -Accidental explosion of the high pressure vessel -Harsh reaction conditions -Moderate cost -Complicated and harsh preparation procedures -Surfactant (eg, oleic acid) is used in the process, which hinders subsequent surface modification -High decomposition temperature -Low crystallinity of SPIONs on a large scale due to low temperature usage -Complicated purification methods for separation of surfactants -Low yield of nanoparticles -Large amount of solvents required -High cost -Close monitoring is needed due to the several steps -Difficult to obtain monodispersed nanoparticles through hydrolytic sol-gel route -Broad size distribution -Particles agglomeration -Not energy efficient -Particle size tunability is not easily achievable -Use of organometallic precursors an cause in vivo toxicity [83] [85], [87] [89] [91] [55], [92] [86], [93] [94], [95] 7 Page 7 of 70

9 2. Magnetic iron oxide nanoparticles A requirement for successful translation is a deeper understanding on how every structural parameter of these materials affects not only their magnetic behavior, but also their longitudinal (r 1 ) and transverse (r 2 ) relaxivities. Only then will researchers be able to reliably synthesize nanoparticles of optimized physicochemical properties that is required for specific applications. Of these parameters, composition, aggregation [17, 58, 59] and surface coatings [98] have already been focused upon. The increase in the relaxation rate of water protons induced by SPIONs originates from dipolar coupling between the magnetic moment of water protons and the electron magnetic moment of the particles. Extensive theoretical modeling, especially by Gillis, Roch and Gossuin [58, 59, 61], have highlighted the relationship between the structural parameters of SPIONs and the magnetic field dependencies of the longitudinal and transverse relaxivities. In terms of transverse relaxivity, this relationship is simple: r 2 increases linearly with increasing magnetic field until a plateau is reached at around 0.5 T due to an increase in the magnetization of the particles from zero to their saturation value, following the Langevin function. This trend was confirmed by Bulte for the Ultra Small Particles of Iron Oxide [100], although a complete, systematic study related to the size of maghemite/magnetite nanocrystals in non-aggregated SPIONs to their transverse relaxivity has yet to be reported. (1) The bulk magneto-crystalline anisotropy field, which depends on the chemical composition and the crystallographic structure of the material. Both maghemite (γ-fe 2 O 3 ) and magnetite (Fe 3 O 4 ) have inverse spinel structures, although the bulk saturation magnetization of the former (73.5 emu g -1 ) is disadvantageously lower than that of the latter (92 emu g -1 ) [101]. (2) The demagnetizing field, which is determined by the shape of the crystal. This component is equal to zero for perfectly spherical crystals, and increases with the elongation of the crystals. (3) The surface anisotropy field. Surface spin canting has a net impact on the magnetism of small iron oxide nanoparticles [64, 65]. In terms of translated relaxivity, it resulted in a greater influence of the nature of the polymer coating on the smaller nanoparticles, and of the chemical functionalities anchoring the polymer coating on the surface of the iron oxide crystals [98]. (4) The mutual anisotropy induced by the dipolar coupling between nearby crystals in agglomerated structures, such as in SPIONs. This component increases in significance as the inter-crystal distance decreases. It is thus a key component in the change in relaxivity for particulate responsive CAs, which are based on biomarker induced controlled aggregation of SPIONs. The size of the iron oxide core affects the anisotropy energy, and consequently, the relaxivity of the CAs, in multiple ways. The surface spin canting induced by the organic coating has a greater impact for smaller nanocrystals, and the anisotropy energy increases with increasing particle size. This, in turn, increases the N eel relaxation time and decreases the N eel component of water relaxation, particularly in a high magnetic fields. Generally, metal oxides are subjected to complex interfacial charging processes involving H + and OH ions when they come into contact with aqueous electrolytes [66 70]. In the case of iron oxide, the surface of the particle can be charged either positively or negatively, depending on the ph of the medium, while the capping agents may also be electrostatically or even covalently bound, representing the presence of proper complexing groups, such as sulfate [109], carboxylic [110], silane [111] and phosphate groups [112]. An electrically charged interface is developed with the adsorption of H + or OH ions through the protonation and deprotonation of amphoteric surface groups based on Eq. (1): 8 Page 8 of 70

10 MOH MOH 0 0 H MO MOH _ H 2,, (1) where M is the surface metal atom, and MOH +2, MOH 0, and MO are positive, neutral, and negatively charged surface complexes, respectively. Moreover, both anions A and cations C + are presented when the ph of the electrolyte is altered using base or acid. Surface reactions containing these ions, which exist in both diffuse and the compact layers, lead to the formation of surface complexes MO C + and MOH +2 A as Eq. (2) presents: MOH MOH 0 2 C A MO C MOH 0 _ H H, A. Indeed, the protonation and deprotonation of the surfaces, along with the adsorption of electrolyte ions, could principally govern the of metal oxide/electrolyte interface. Considerable underrating of the MO C + and MOH +2 A complexes at low and high ph values, respectively, could be the result of ignoring the surface energetic heterogeneity of oxides [106]. For example, the zeta potential and adsorption isotherms of both anion and cation have been observed to be quick to respond to the energetic heterogeneity of the oxide surfaces. Van Riemsdijk et al. [113] took into consideration the surface heterogeneity using a simplified model, where the surface charge is mainly influenced by the surface reaction, as shown in Eq. (3): 1 ( ) _ 1 ( ) 2 2 MOH H MOH. (3) 2 According to the electrical interfacial layer model, characteristic potentials can be attributed to specific planes towards the interfacial layer. The surface potential is generally observed as the electrostatic potential of the inner plane of the compact Helmholtz layer, whereas the zeta potential corresponds to the electro kinetic slipping plane located within the diffused layer. As mentioned earlier, MNPs demonstrate characteristic properties, such as superparamagnetism, high field reversibility, high saturation field, extra anisotropy contributions, shifted loops after field cooling, and blocking temperature. All these characteristics are normal issues in discussions with regards to the behavior of MNPs, such as the reported anisotropy energy of some materials directly associated with superparamagnetism and/or the wide range of blocking temperature values. Some authors report higher values compared to their bulk counterparts, which is attributed to large size and surface contributions [76 78], while others determined that the contribution of the surface anisotropy in spherical particles should average to zero; that interparticle interaction can modify the energy barriers [117]; and that the blocking temperature depends on dipolar interactions and is unaffected by a moderately broad volume distribution [118]. More controversial discussions are associated with the effect of size and surface and internal disorder on the saturation magnetization [81, 82], as well as the dependence of the highfield magnetization on temperature [79, 83]. (2) 2.1 Superparamagnetic iron oxide nanoparticles (SPIONs) and their applications 9 Page 9 of 70

11 The adsorption of nanosized materials onto surfaces has been the focus of intensive research activity, taking into account its impact on the investigation of the interaction of nanostructures with biological and non-biological templates, with emphasis ranging from cell labeling to coatings and films [84 87]. Additionally, the increasing growth in the production of nanosized materials nowadays, both at laboratory and industrial scales, has called special attention to the potential hazardous effects caused by discarding them into the environment, and their binding to organic matter and living organisms [126]. Therefore, investigation regarding the adsorption of nanoparticles onto surfaces, with as diverse a composition and function as possible, has become critical in realizing medical and industrial applications, as well as to unveil the path of their dissemination into the environment and possible damaging effects to living systems. In this scenario, SPIONs [127] play an important role, since their high magnetization and biocompatibility make them potential nanomaterials for use in biomedicine, drug delivery systems [128], biolabeling [129], magnetic hyperthermia [92, 93], and MRI contrast enhancers [94, 95], among others. The magnetic properties of SPIONs differ from their bulk counterparts, originating from particle size. Their size is supposed to be below a critical diameter for magnetic domain wall formation. When no external magnetic field is applied, there is sufficient thermal energy to force the magnetic moments in these single-domain particles to balance and control any preferential orientation. Nevertheless, in the presence of an external magnetic field, their magnetic moments quickly rearrange in the direction of the applied field, and the materials show a net magnetization. After removing the magnetic field, the thermal energy refers to its normal, which allows the particles vector moments to swing randomly [61]. That s why the use of SPIONs may have a profound impact on new industrial technologies, for instance, while developing insulating magnetic oil for transformers [134], structures for spin electronics [135], bioelectrochemistry [136], catalysis [137], and chemical sensing [138]. Specific applications include surface modification of SPIONs to allow covalent attachment with biomolecules, which are then used as molecular recognition sites for sensing. The large surface-to-volume ratio of SPIONs enhances reaction activity and catalytic efficiency, hence improving chemical sensitivity. Indeed, it has been reported that SPIONs have the capability to increase electron transfer between electrode and molecular recognition sites, such as enzymes [139]. SPIONs, for instance maghemite (γ-fe 2 O 3 ), magnetite (Fe 3 O 4 ) and cobalt ferrite (CoFe 2 O 4 ), display a typical cubic inverse spinel structure and form single domains of about 5 20 nm in diameter. Under wet chemical synthesis approaches, SPIONs can be prepared in large quantities with appreciable control over its size, composition, crystallinity, and physical properties (e.g. magnetic properties) [140]. Furthermore, with proper surface functionalization, SPIONs can be readily dispersed into suitable solvents, thus producing homogeneous and highly stable magnetic fluid (MF) samples [141]. Therefore, while using MF samples as a material platform for the manipulation and encapsulation of SPIONs, their adsorption energetics and kinetics on different surfaces, for instance in metal electrode [142] and polymeric matrix [143], need to be known in order to provide optimum parameters for their deposition, and also to improve the loading of recognition sites within the sensor structure. 3. Nanoparticles aggregation Iron oxide nanoparticles tend to agglomerate because of the strong magnetic dipole dipole interactions between particles in conjunction with inherently large surface energy (>100 dyn cm - 1 ). Therefore, stabilizers, such as surfactants or polymeric compounds with some particular 10 Page 10 of 70

12 functional groups have been utilized to adjust the particles properties in order to prevent the sedimentation [ ]. On the account of Van der Waals and magnetic attractions between nanoparticles, on top of the need to decrease the high surface energies caused by the high surface area to volume ratio [7], the particles tend to aggregate, which necessitates their stabilization in the carrier liquid [7, 17]. The main key restriction to the MNPs application is the affinity of the particles to agglomerate, specifically in high-ionic-strength solutions like biological environment. Therefore, an important research focus in this area is the stabilization of particles opposed to aggregation. Several strategies have been devised to achieve the stabilization of these nano-entities involving, for example, the use of ligands, capping agents, ionic liquids, and related stabilizing agents. A promising research avenue in stabilizing nanoparticles is related to the use of another nanotechnology, aptly known as nanoporous supports. High surface area nanoporous materials can offer an optimized medium for a controllable deposition and stabilization of nanoparticles, both on the surface of the support and within the porous framework [82]. The extent to which manufactured NPs agglomerate will depend on the balance between the attractive and repulsive forces among the nanoparticles, as well as between them and the environmental matrix mobilizing agents will be studied as well [147]. 4. Surface modification of magnetic iron oxide nanoparticles Iron oxide nanoparticles are prone to agglomerating into large clusters due to the anisotropic dipolar attraction, and therefore mislay the particular properties related to single-domain magnetic nanostructures. In this regard, surface modification of MNPs is a crucial and demanding step for many applications and fundamental studies [148]. The particles must either be coated with a surfactant to provide steric stability, or have their surface modified to produce a repulsive electrostatic force to prevent aggregation [13, 38, 111, 112]. Furthermore, considerable efforts were made to modify the surfaces of such MNPs with different strategies to stabilize the gravitational force and avoid strong interaction and agglomeration of the nanoparticles. The dominating interparticle forces in dispersion of MNPs systems include van der Waals, double layer (electrostatic), and steric (polymeric) forces, which properly shown in Figure 1. Figure 1. Schematic illustration of the main mechanisms for stabilization of magnetic nanoparticle dispersions, assuming positively charged surfaces (Adopted from [151]). Another advantage of surface modification is that the surface of Fe 3 O 4 MNPs can be functionalized as per applications, such as catalysis, separation with advantages of high dispersion, high reactivity, and easy separation [152]. One of the most recent interests is the tailoring of the surface of MNPs for applications in biomedicine, which require narrow size distribution or monodispersed nanoparticles with the desired chemical and biological surface functionalities [ ]. Surface modification of MNPs by surfactants has been demonstrated as a method to avoid particle agglomeration [118, 119]. Secondary bonds that were established between the nanoparticles and a polymer matrix with the aid of surfactants can provide adequate strength to a nanocomposite, but covalent bonds are generally more efficient [ ]. The surface of SIONPs must be modified to not only prevent aggregation of the particles, leading to 11 Page 11 of 70

13 colloidal stability, but also render them water-soluble, biocompatible, nonspecific adsorption to cells, and bioconjugation. Besides aggregations, SPIONs are very vulnerable to air oxidation [8, 11]. In order to prevent air oxidation and aggregation, SPIONs are often covered with surfactants, polymers, and inorganic materials [9, 17]. Among surfactants, oleic acid or sodium oleate is widely applied to disperse SPIONs due to their superior affinity to the surface of metallic oxides [123, 124]. The coating technique is carried out by co-precipitating ferric and ferrous salts with sodium hydroxide or ammonium hydroxide as base, along with oleate. It is also possible that oleate molecules is adsorbed on the surface of already prepared iron oxide particles [163], although irregular shapednanoparticles with broad size distribution are normally obtained. Furthermore, the microemulsion method have been employed to prepare hydrophobic SPIONs with a narrower size distribution, but the drawback of this system includes the requirement of a base as the precipitating agent and removal of surfactants thereafter [9]. Additionally, hightemperature decomposition of iron precursors in the existence of oleic acid facilitates us with another technique to produce homogeneous magnetic crystallites [164]. However, this method are faced with disadvantages, such as the dependency of the crystal structures upon magnetic properties of the particles on the key parameters of purity of organometallic precursors and reaction temperature and time, which makes this approach inappropriate for scale-up production. Numerous approaches have been investigated to modify the surface of SIONPs using small molecules counting biomolecules to obtain water-soluble SIONPs, namely post-addition of water-soluble ligands, including direct adsorption [ ], addition of second layer [107, 130, 131], ligand exchange [116, 132, 133], functional silica coating [ ], and ionic interaction [175]. Moreover, an approach includes in situ production of water-soluble SIONPs in the existence of stabilizing ligands such as d-mannose [176], 2-pyrrolidone [177], poly(ethylene oxide) diacids (HOOC-PEG-COOH) [178] and dextran (Dex) [179]. Polymers with more than one group are able to bond to the particles surfaces could improve colloidal stability of inorganic NPs including SPIONs, leads to their superior optical, magnetic, and electronic properties [180]. In addition, polymers afford chemical and mechanical stability for the nanomaterials. 4.1 Dispersion of magnetic iron oxide nanoparticles SPIONs can be produced using organic and aqueous solvent techniques, although the use of organic solvents generally allows for greater control over nanoparticle sizes, shapes, and crystal structures [115], this method results in hydrophobic SPIONs that are capped with organic lipid ligands. The resulting hydrophobic particles do not readily undergo phase transfer, allowing dispersion in water, which is required for biocompatibility. Hence, clinically approved SPIONs are synthesized in water and dispersed through the addition of amphiphilic polymers, such as dextran resulting in the formation of agglomerates of multiple SPIONs [181]. Recent research efforts have been aimed at developing new phase transfer chemistries to solubilize these particles for use in-vivo [182]. Traditionally, the approach used is to sterically or charge stabilized 12 Page 12 of 70

14 SPIONs with polymers or small molecules that are either bound through metal coordination of the iron oxide cores, or through hydrophobic interactions with lipid groups on the SPIONs. Another approach to address the challenges of phase transfer and dispersal of SPIONs has been the use of lipid based colloidal aggregates as the dispersants or carriers. A number of reports have investigated methods of surface functionalizing hydrophobic SPIONs with hydrophilic ligands, such as polymers and small molecules, to make them colloidally stable in water [72, 145], and occasionally buffer. Some studies have been reported for MNP modification using glycopolymers based on the grafting-onto approach. However, the full dispersion of MNPs were not possible. In the case of glycosylated polyacrylate, which was affixed onto silica-coated IONPs, the magnetic fluid was pretty dilute [184]. The application of glucosylated poly(pentafluorostyrene) derivatives assisted in enhancing the colloidal control over iron oxide suspensions within the nm size range [185]. However, the dynamic light scattering technique displayed a relatively high polydispersity index (PDI), which represents non-uniform particles. Moreover, the dispersion of magnetic nanospheres can be done by carrier fluids using particular interactions among the particle surfaces, polymeric, or selected low-molecular-weight surfactants. Fluid dispersions containing small magnetic particles are called ferrofluids [186]. Magnetic attractive forces, together with intrinsically large surface energies [163] can cause the nanospheres to aggregate in the magnetic fluid [187]. Factors that are crucial to a good dispersion are the surfactants properties, its nature, and concentration of grafting sites. The dispersion of nanoparticles in polymer matrices is essential to the optical and mechanical properties [150, 151]. Furthermore, nanocomposite s morphology is also likely to effect the magnetic properties, since MNPs can interact through various mechanisms, such as exchange, dipole dipole, magneto-elastic, and super exchange interactions [ ]. An approach for monodispersion of nanoparticles is the primary synthesis of polydisperse nanoparticle dispersion via conventional techniques, and subsequent size-selective separation of the nanoparticles through various processing methods. This approach was improved to prevail over some of the restrictions related with size-selective nanoparticles post-synthesis procedures using the gas-expanded liquid (GXL) systems [155, 156] in a solvent antisolvent route. This procedure includes a liquid solvent, along with a gaseous antisolvent (CO 2 ), which are utilized to precipitate nanoparticles of gradually smaller size from the solution by increasing CO 2 pressure [195]. It should also be pointed out that the biggest nanoparticles will precipitate earlier on deteriorated solvent conditions due to higher Van der Waals forces. This process is called GXL size-selective fractionation, and has been productively used in several studies [155, 158]. Two key factors of magnetic behavior, saturation magnetization and coercivity, are noticeably domain-size dependent [197]. Due to the correlation between magnetic behavior and size, a low polydispersity is usually favored for optimized properties. A variety of chemical techniques have also been investigated to fabricate ultrafine maghemite particles [198], including hot-injection methods on the basis of organometallic materials decomposition that lead to highly monodispersed colloids. However, the coprecipitation technique in water, which has been introduced by Massart [71], remains broadly used, due to the low cost and toxicity of the reactants. Moreover, the flexibility of this technique in controlling the particle size by altering experimental conditions such as concentration of iron salts, nature of the applied base, ph, and 13 Page 13 of 70

15 temperature should also be taken into consideration [199]. In the case of observation of any size polydispersity, a post-synthesis size selection step can be carried out, including centrifugations or selective flocculation using salts [200]. It should be noted that the synthetic technique directly affects the magnetic behavior of particles with similar mean particle size [81, 163]. Dispersion of nanoparticles, which tend to self-aggregate, can be enhanced using polymers such as polydimethylsiloxane (PDMS) [164, 165], polymethylmethacrylate (PMMA) [166, 167], polystyrene [206], polyimide [169, 170], ethyl(hydroxyethyl)cellulose (EHEC) [209] or 3,4- epoxycyclohexylmethyl-3040-epoxycyclohexanecarboxylate (CE). The composites are possibly made either by capping the nanoparticles with an organic phase, or using a solvent-based dispersion technique. Peluse et al. applied thermal decomposition technique using iron mercaptide to incorporate MNPs into polystyrene [206]. Solvent-based dispersion technique is normally carried out using a mixture of nanoparticles and polymer that were separately diluted in an organic solution like benzene, toluene, or chloroform, followed by evaporating the bulk of the solvent. Maghemite nanoparticles have been successfully dispersed into PDMS using this technique [210]. The transfer of NPs from organic solvents to aqueous solutions is normally carried out using an amphiphilic layer attached to the particles [107, 173]. Due to the successful attachment, a hydrophobic-hydrophobic interaction must be formed between amphiphilic layer and original ligands of nanoparticles, which create a lipid bilayer-like structure. This suggests the encapsulation of nanoparticle s core and ligands. However, there is possibility of multiple nanoparticles encapsulation, which may end up enlarging the particle size [107, 174]. Therefore, different techniques of separation such as filtration can be carried out to obtain smaller size and water-dispersed nanoparticles. Alternatively, another common approach is replacing the hydrophobic coatings by a hydrophilic molecule [175, 176]. A number of ligands have been investigated to substitute the hydrophobic coatings of IONPs, for example short-chain poly(acrylic acid) [215], poly(ethylene glycol) (PEG) [216], polyethylenimine (PEI) [213], dendrons [217] etc. This approach fabricates welldispersed water-soluble nanoparticles via particular surface functional groups such as -COOH, - NH 2, and -SH. The functional groups not only propose different bioconjugation possibilities, but also demonstrated enormous stability in buffers. Figure 2 shows the ligand exchange process, assuming that the primary coatings of iron oxide NPs are entirely replaced by the hydrophilic molecules such as PAA, PEI, and GSH. The functional groups of PAA molecules and carboxylic groups are moderately attached onto the NP s surfaces, while the rest of them hangs from the surface, rendering the NP soluble. Correspondingly, PEI molecules interact with iron oxide NPs through their amine groups. These anionic and cationic polymers provide not only surface charges for electrostatic repulsion, but also steric repulsion from the polymer chains. In the case of GSH molecules, the NH 2 groups are assumed to interact with the NPs because of the stronger binding affinity of amine groups to iron oxide surfaces compared to that of the carboxylic groups. 14 Page 14 of 70

16 Figure 2. Schematic illustration of the ligand exchange process using PAA, PEI and GSH. Adopted from [12] Size Sorting Preparation of monodispersed colloidal particles is of great interest to the research community. Numerous synthesis protocols have been reported to produce uniform nanoparticles. The choice of synthesis method and the control of experimental conditions are critical factors in producing monodisperse nanoparticles. However, once these nanoparticles are dispersed in an aqueous medium, the new challenge is controlling aggregation and hydrodynamic sizes. The presence of aggregates and their sizes may affect a number of properties, such as the MRI signal of iron oxide nanoparticles [218]. Even monodispersed nanoparticles can lead to highly polydispersed aggregates in aqueous dispersions. For this reason, size sorting methods are still greatly needed. Size sorting refers to decoupling of a source population into two or more subpopulations with distinctly different sizes and narrow size distributions. Thus, if we consider a polydispersed system as a mixture of aggregates of different sizes, size sorting could also be used to separate the aggregates by size. A good sorting method should be able to separate two or more subpopulations of particles or aggregates with similar sizes, in a simple way, with a large throughput and low associated costs. In this scenario, we can find many sorting methods described in literature. Most of the currently available methods are believed to be based on the destabilisation of colloidal nanoparticles in their dispersions by changes of the environmental ionic strength or ph [162, 181]. Some techniques take advantage of the special physicochemical properties of the particles associated with their sizes, such as their magnetic [182, 183] and optical [222] properties. Recently, several sorting techniques have been developed, although most of them involve expensive equipment and require time-consuming protocols, frequently with particle size limitations [223]. For instance, techniques such as flow cytometry, which was designed to sort cells, have been utilized to sort relatively large particles (500 nm to several mm) [224] with a good yield. Replacing these techniques with more sophisticated methods such as microfluidics or magnetophoresis is possible [225], although typically at the expense of throughput. Three different size sorting methods, such as ultracentrifugation, ultrafiltration, and magnetic separation are of interest in the context of simplicity, effectiveness, and cost. Its aim is to split a USPIO colloid where big particles and aggregates are in equilibrium with individual MNPs, and to carry out a comprehensive analysis of the resulting fractions to achieve a better understanding of the physicochemical behaviour of these aggregates. Previous work on particle aggregation was based on monodispersed spherical particle models that were artificially aggregated [226], allowing for an excellent comparison between theory and simulations Colloidal Stability Ferrofluids are known as colloidal dispersions, which are associated with small, single-domain magnetic particles suspended within a good continuous phase whose rheological behavior can be adjusted using a magnetic field [227]. Ferrofluids are able to convert from a liquid to a solid phase and vice versa immediately [228], making its preparation of great interest of wide 15 Page 15 of 70

17 range of aforesaid applications. This is due to the high impact of dispersed particles on matrices that they are incorporated with [115, 117]. Moreover, ferrofluids and their chemistry may be significantly extended through binding polymeric or molecular capping agents on the surface of the particles via wet chemical techniques [229], presenting characteristics that are more advanced than inherent properties of iron oxides. To control the colloidal stability of ferrofluids, equilibrium must have taken placed between repulsive and attractive forces, such as Van der Waals, steric, hydrophobic, and double layer. This balance has been widely investigated in the area of colloidal suspensions. The classical approach applied Derjaguin-Landau-Verwey- Overbeek (DLVO) theory [ ] is a way to forecast the net interaction energy implicating Van der Waals, steric, and electrostatic forces in dilute dispersions. The rate of stability and fluctuation of colloidal dispersion can be controlled by changing the main factors contributing to the aforementioned forces. For example, in the case of gold nanoparticles, the rate of aggregation has been controlled by varying the electrostatic charge on the particles surface [230] or through altering the ionic properties of the medium instead [195, 196]. The stabilities of magnetic nanoparticle dispersions have also been studied by changing the ph in the presence of external magnetic fields. This shifted the electrostatic repulsions, and led to magnetite particles flocculation in powerful magnetic fields [235]. The Van der Waals force, or dispersion, is always present, which is mostly attractive, requiring to be monitored by an adequately long-range repulsion to produce a stable colloidal dispersion. The amount and range of the Van der Waals interaction is directly associated with the value of the Hamaker constant, A, which is related to the dielectric properties of the implicated materials and the medium [236]. For instance, the VdW interaction free energy, V vdw, between two spheres of radius r at surface separation s, can be roughly equal to Eq. (4): V vdw = -(A/12)(r/s), (4) When s<<r. In a stable system, the thermal energy should be more than once or twice the maximum attractive interparticle energy for it to easily break all particle-particle bonds [ ]. In addition to the enormous impact of VdW interactions on the colloidal stability, it also affect the morphology and ordering in superlattices of nanoparticles through dewetting and segregation of nanoparticles due to size-dependent interactions, and directionality of VdW interactions between anisotropic building blocks [ ]. Massive numbers of studies investigated the preparation of hydrocarbon-based ferrofluids using oleic acid as a stabilizer [205, 206]. This is due to the observation on the formation of MNPs chain-like structure under an applied magnetic field [245]. Therefore, the preparation of magnetic fluid is essential in improving a steric stabilizer that is capable of preventing the magnetite particles from coagulating. It is also important that ferrofluids are prepared in a carrier liquid that are not capable of evaporating or degrading readily at higher temperatures [246]. In this regard, many commercial ferrofluids generally employ heavy oils with low vapor pressures as carrier liquids instead of lighter organic solvents. Furthermore, ionic liquids (ILs) have also emerged recently as a new fascinating substance with focus in chemical fields and industries [247]. ILs are totally formed of ions that melt under 100 C. They can proceed as a non-aqueous solvents to self-assemble amphiphilic biomacromolecules with no former modifications [248]. Due to their exclusive physicochemical properties, ILs are potential candidates for colloidal dispersion of nanomaterials as well. 16 Page 16 of 70

18 Different studies have lately reported using ILs for diverse applications, representing in-situ synthesis of nanoparticles in ILs [211, 212], improvement of colloidal stability in ILs [251], phase transfer from some other media to ILs [252], and catalysis using metal nanoparticles in ILs [253]. In aquatic systems, fulvic acids (FAs) are expected to play key roles on the aggregation behavior and stability of manufactured nanoparticles (NPs). The exact conditions under which aggregation or dispersion occurs will depend on the nanoparticle surface charge properties, FAs concentrations, as well as solution conditions, such as ph and ionic strength. The systematic calculation of stability (aggregation versus disaggregation) diagrams is therefore a key aspect in the prediction of the environmental outcome and behavior of manufactured nanoparticles in these systems. In a study, the responses to changes in ph and FAs concentrations on the resulting surface charge of purified iron oxide nanoparticles (53 nm nominal diameter) has been investigated. By adjusting the ph, different nanoparticle surface charge electrostatic regions are found, corresponding to positively, neutral, and negatively charged nanoparticle solutions. For each situation, the adsorption of negatively charged FAs at variable concentrations is considered by analyzing surface charge modifications and calculating experimental kinetics aggregation rates. The results showed that under the conditions being used and a range of FAs environmental relevant conditions, the nanoparticle aggregation process is promoted only when the nanoparticle positive surface charge (solution ph less than the charge neutralization point) is compensated by the adsorption of FAs. In all of the other cases, FAs adsorption and increase of FAs concentration are expected to promote not only the NPs stabilization, but also the disaggregation of NPs aggregates. The understanding of the interaction processes between manufactured nanoparticles and aquatic compounds is essential to predict nanoparticle mobility, bioavailability, and their physicochemical transformations in aquatic systems. Humic substances (HS) demonstrate an active and important part of natural organic matter (NOM) in aquatic environments [254], and play important roles in water quality for many pollutants, namely trace metals, radionuclides, and organic compounds, owing to their strong sorbent properties, small size, and high charge densities [255]. HS are also expected to adsorb on mineral and colloidal surfaces, including manufactured nanoparticles, and the resulting adsorption layers and surface modification have a significant impact on their fate and circulation [218, 219]. Stabilized nanoparticles may be transported over long distances, whereas unstable NPs may be trapped locally through aggregation and sedimentation processes. The exact composition of HS is still unclear, due to the high heterogeneity of the functional groups, such as aromatic residues, aliphatic chains, carboxylic, phenolic, and alkoxy groups, however, HS can be categorized into three groups: fulvic acids (FAs), which are the major component and smallest structures of HS and soluble at any ph, humic acids (HA), which represent bigger structures that are insoluble at ph\2, and finally Humin, which is insoluble at any ph [258]. E. Illes and coworker investigated the ph-dependent adsorption of HA on magnetite and its impact on the surface charging, along with the aggregation of oxide particles. They discovered that HA is able to modify the surface charge characteristics of magnetite completely, or to a particular level, conditional on the quantity of adsorbed polyanions. A little amount of HA roughly counterbalances the positively charged Fe OH +2 sites under the ph of PZC (point of zero charge) of oxide, and oppositely charged spots produce on the surface of particles, therefore leading to increased aggregation of magnetite particles. When huge amount of HA is present, the particle surface is capped thoroughly by the adsorption layer of organic polyanions, which made the magnetite particles charge extremely negative in acidic conditions as well. The humate-coated MNPs produce stable colloidal dispersion, with no sedimentation or aggregation in a broad range of ph. Moreover, it is 17 Page 17 of 70

19 found that ph sensitivity of amphoteric magnetite can be entirely removed, and a considerable enhancement in resistance versus salt can be obtained by adding HA [259]. The effect of FAs adsorption on the aggregation kinetics and fractal dimension of hematite particle aggregates was investigated in [260] at constant ph (ph 3). It was shown that the fractal dimensions of hematite aggregates partially coated with FAs were higher than those obtained with no adsorbed FAs. Computer simulations were also used to study the interactions of FAs with hematite particles to get an insight into the global effect of the electrostatic repulsive and attractive interactions at ph 8 on the amount of adsorbed FAs and the resulting structure of the adsorbed layer. Results demonstrated that the amount of adsorbed FAs was largely controlled by the solution ionic strength, and FAs coagulation in the solution may strongly compete with FAs adsorption at the hematite surface, and that the adsorption of large quantities of FAs at the NPs surface was not necessary, leading to the formation of a homogeneous compact adsorbed layer. Despite the importance of ph in controlling the NPs surface charge density as well as NPs surface charge (positive, neutral or negative) modification, surface transformation (and thus chemical reactivity) due to the presence of NOM, several studies have been carried out to determine the stability diagram of iron oxide NPs [ ]. Furthermore, the aqueous alkanoic acid stabilized magnetic colloids are a group of model suspensions for which nanoparticle aggregation has been studied. These suspensions are stabilized by a surface bilayer coating, composed of a primary chemisorbed layer with the carboxylate groups bound to the surface, and a secondary interpenetrating physisorbed layer with the hydrophilic head-groups pointing outwards [145]. Additionally, monosaccharides propose colloidal stability via charge and steric interplays, and therefore have been applied to stabilize MNPs. Carbohydrates bring several advantages like water solubility, biocompatibility and, in some cases, specific protein-targeting properties [264]. This new pathway is based on the use of glycoconjugates, which possess a desired sugar-based moiety covalently connected to an organic spacer. On one hand, tuning the end-chain functional group (thiol, carboxylic acid, amine, etc.) allows a larger variety of functionalizable surfaces, while on the other hand, tuning the nature of the carbohydrate may influence solubility and biocompatibility. Nevertheless, despite these advantages, the synthesis of glycol-conjugates is somewhat tedious, time-consuming, expensive, and unsustainable. For instance, several functional glycol-conjugates of lactose, maltose, and glucose, reported by Barrientos et al. [265], can be obtained in no less than 5 steps, which involve, among others, highly toxic chemical compounds like azobisisobutyronitrile. Polyglycerol-grafted Fe 3 O 4 nanoparticles were fabricated by surface initiated polymerizarion of glycidol from hydroxycaproic acid stabilized nanoparticles [266]. They are produced by nonhydrolytic organic phase technique, and then functionalized with (3-glycidyloxypropyl) trimethoxysilane, a binder that avoids NPs from aggregation and is accessible for consequent coupling reactions with an extensive range of polymers and biomolecules Surfactants Surfactants are able to stabilize magnetic fluids in either oil or water, acting as dispersing agents [13]. Surfactant-coated MNPs, indeed, hinder clustering through steric repulsion. Cetyltrimethylammonium bromide (CTAB), a commonly used surfactant in nanoscience, can assist in the preparation of different kinds of controlled-morphology nanoparticles [229, 230]. Its main application is considered as phase-transfer reagent. A variety of nanoparticles have been 18 Page 18 of 70

20 carried from the organic solution to the aqueous one with the aid of CTAB [231, 232]. Nevertheless, the formation of clusters has not been reported so far with different concentrations of CTAB. Another approach to synthesize the sterically stabilized aqueous magnetic fluid is bilayer surfactant using a two-step procedure [145]. In this approach, Fe 3 O 4 nanoparticles was initially coated with a primary surfactant formed by the coprecipitation from an aqueous solution of iron chloride (II and III). Then, the extra primary surfactant should be removed from the solution, followed by coating the particles with a secondary surfactant to produce self-organized bilayers of the two surfactants on the surface of iron oxide nanoparticles. It has been proven that this approach is much more reliable when primary surfactant is present for the duration of the precipitation process. Fatty acids are some of the commonly used stabilizers in this approach. They can provide both primary and secondary surfactants with bilayer-stabilized magnetic nanofluids [271]. Fatty acids are slightly soluble in water; however, their solubility is improved using acetone, an outstanding solvent for fatty acids, and is entirely miscible with water. The solubility improvement enhances the possibility of interactions between fatty acid molecules and the crystals of iron oxide, which is particularly important for avoiding the aggregations during the crystal growth step. Acetone will quickly evaporate while stirring and heating at 80 C, creating an immense contact area between the newly precipitated particles and surfactant. In the meantime, the carboxylic group of fatty acid and iron oxide are attracted to each other, leading to surfactant orientation towards the particle/surfactant interface which can end with chemisorption reactions. Upon the surfactant s adsorption, the number of available sites for additional crystal growth is decreased, which stops the growth. Due to the fast kinetics of the precipitation process and the high affinity of uncoated-mnps to aggregate, the early existence of fatty acids supplies particles to be dispersed efficiently during the crystal growth process. Based on the open literature, biocompatible MNFs were produced using or oleic acid (OA), myristic acid (MA) and lauric acid (LA) as double layer surfactant approaches [272]. The oleate coating is desired due to producing the surface complex between carboxylate groups and Fe-OH sites [273], particularly if supplementary fictionalization of MNPs is favored [274]. Parameters such as surfactants properties and concentration and the nature of binding site are considered main keys to a welldispersed MNFs [275]. Shimoiizaka et al. reported the stabilization of MNPs using bilayer surfactants, starting with oleic acid as the first surfactant, and then dispersing in solution of sodium dodecylbenzene sulfonate (SDBS), poly(oxyethylene) nonylphenyl ethers, and di(2- ethylhexyl)- adipate as the second [276]. Moreover, Shen et al. fabricated MNFs with noticeably superior stability using c-ray-induced polymerization of an olefin-terminated surfactant bilayer coating on the surface of MNPs [169]. Afterwards, Wooding et al. prepared highly stable aqueous magnetic fluids by applying various unsaturated and saturated fatty acids as surfactants, both primary and secondary [146]. Furthermore, the synthesis of extremely crystalline and dispersed maghemite in oil using thermal decomposition technique in the company of oleic acid [155] was followed by easily transferring it into an aqueous solution by phase transfer reagents [277]. Jain et al. have also improved stable aqueous-dispersed MNPs using bilayer surfactants of oleic acid/peo PPO copolymer (Pluronic) [278]. It was presumed that physical adsorption of PPO blocks on the particle surface occurred after coating with oleic acid as its primary surfactant. In the meantime, PEO blocks equipped the aqueous medium with steric stabilization. In spite of the vast number of studies investigating the preparation of iron oxide suspensions using anionic surfactants, a few recent evidence confirmed the synthesis of stable MNPs 19 Page 19 of 70

21 applying anionic surfactant bilayers through steric and electrostatic repulsions [107, 241]. These latest results are deficient without an outline that would provide the surfactant s choices and their amounts to produce stable particle suspensions. This drawback is obvious with regards to nano scale zero valent iron (NZVI) particles. NZVI nanoparticles, with a zero valent iron core, have an iron oxide shell, which are similar to iron oxide nanoparticles [280]. Saleh et al. [31] studied the stability of NZVI suspensions formed with different polymers along with SDBS. When SDBS exists, the NZVI indicated rather low stability (30 min) compared to other polymer-based suspensions. Taking into account these observations on the surfactants effect on the formation of stable suspensions, the complications of choosing the proper formulation for surfactant-based suspensions was rather obvious. The stability of MNPs suspensions using SO and SDBS was also investigated. According to the results of this study, the structure of surfactant and its concentration control the adsorption of surfactant layer, as well as the suspension stability. It was observed that at given SO concentration, highly stable suspension was produced. Contrary to this, relatively poor-stabilized suspension could be achieved using different concentrations of SDBS. From the adsorption isotherm, this can be construed as stable suspension being obtainable when SO concentration attained its critical micelle concentration (CMC). At this point, the maximum adsorption of SO has taken place, where the zeta-potential of the suspension indicated a value of -50 mv. On the other hand, the SDBS isotherm disclosed that not only SDBS is weakly adsorbed on the surface of iron oxide, but also a patchy, loosely packed bilayer, is produced when the SDBS concentration gets to its CMC. In conclusion, the crucial element in preparing stable MNPs suspensions using anionic surfactants is the construction of surfactant bilayer. It was also proven that in the case of anionic surfactants, electrostatic repulsions are regarded as a vital parameter to initiate an energy barrier versus flocculation. Additionally to make the most out of stability of dispersion, the concentration of the surfactant in suspension at equilibrium with the adsorbed surfactant must be near or moderately above its CMC. Furthermore, the molecular structure of the surfactant should provide the configuration of closely packed bilayers [281]. It should also be mentioned that the usage of surfactant bilayer or electrostatic repulsion to thoroughly stabilize MNPs dispersions faces some limitations, such as ph- and ionic strengthsensitivity, and inflexibility against changes in the particles surface properties [282]. The ph of the metal nanoparticles generally depends on the dissociation constants of the acid groups of the surfactants [283]. For instance, according to report from Toshima and co-workers, the gold particles modified using carboxylic acids are prone to aggregate under ph 3.8, while Fiurasek and Reven reported ph=5.5 and below in which phosphonic acid modified-gold particles start to agglomerate [284]. In contrast, arsenic acid has higher acid dissociation constant (pk a1 = 4.5, pk a2 = 9.0) compared to carboxylic or phosphoric acid [285]; therefore, arsenic acid and its derivatives are predicted to be novel progression of the nanoparticles surface modifications Polymers The applications of electrostatic stabilizers have been limited due to the obtained suspensions, which are ph-sensitive, deficient in functional groups, and unstable. Therefore, applying polymers are progressively attracting more interest [ ]. A stable dispersed magnetic fluid is more likely to be obtained when polymers can be grafted onto the MNPs surface [289]. This 20 Page 20 of 70

22 attachment can be carried out through various feasible techniques. The first and the most common one includes the surface copolymerization using a monomer where the inorganic phase is integrated within the polymer chains [290]. The second technique, called Grafting-to, is involved in the interaction between a polymer with proper functional group and inorganic nanoparticles surface sites [253, 254]. In addition, in another technique, which is called grafting-from, a molecule which has been primarily linked onto the NPs surface grow and form chains [255, 256]. Upon controlling the polymer growth on the NPs surfaces [295], fascinating materials can be fabricated. However, successful assortment of grafted polymer based on length and molecular weight, surface coverage, and conformational arrangement onto the surface is required to guarantee the stability of nanoparticles in suspensions. Short length polymers, which are water soluble, has priority over longer polymers due to the contact enhancement of reactive molecule on the particles surface, which can boost the magnetic fraction in the particles [296]. Miles et al. investigated the impact of grafting PPO PEO copolymers with various molecular weights onto the iron oxide particles dispersed in phosphate buffered saline (PBS), employing ammonium groups as dispersing agent [297]. Zeta potential measurements were utilized to determine the grafting of phosphate groups to the particles after 24 h of introduction to PBS. It was discovered that the surface charge were altered on adsorption of phosphate groups compared to similar particles dispersed in deionized water. In conclusion, the grafting density and molecular weight of the applied polymer were estimated as the main factors in stopping the adsorption of phosphate groups on the particles surface. For instance, in the case of the equal number of chains per surface area, longer chains were more efficient as particle stabilizers compared to shorter chains, as showed in the rise of the hydrodynamic size of the particles. However, the stability of the NPs was dismal, due to dislocation of the ammonium grafted copolymers that were not successfully protected by the low molecular weight polymers anchored onto the surface of the nanoparticles. Silanes have been examined as a proper covalent grafting group for iron oxide nanoparticles modification [298]. A substitute technique to adjust particle size with narrow distribution is the thermal decomposition, where iron precursors decay using a non-polar surfactant through solvents with high boiling point [164]. This technique fabricates highly monodisperse MNPs with roughly no agglomeration within the synthesis procedure. Nevertheless, the obtained particles are hydrophobic, and further processing is required to make the particles hydrophilic by replacing the non-polar surfactant on the particle s surface with a water-soluble molecule. In the work of Butterworth et al. [299] and Herve et al. [300], MNPs were produced via a coprecipitation method, leading to the formation of clusters of nanoparticles with polydispersity in the size distribution. Incongruity in grafting density, particle size, and in proportion of particle size to thickness of polymer layer makes it hard to efficiently evaluate the effect of polymer molecular weight on suspension stability. Moreover, there are various fundamental techniques that let the production of a superior graft density of polymer brushes with controlled molecular weight and polydispersity. Diverse living radical polymerization routs include atom transfer radical polymerization (ATRP), reversible addition fragmentation chain transfer, and nitroxide-mediated polymerization [301]. ATRP is 21 Page 21 of 70

23 one of the most used techniques to build polymer brushes, where the atom transfer step is the key elementary reaction responsible for the homogeneous growth of the polymer chains [302]. In the preparation of adjusted brushes using ATRP, two approaches are viable. In the first approach, a free initiator is applied, while in the second approach, a resistant radical should be used for making the balance and therefore leading to the controlled polymerization. Physisorption is another route in developing dispersion, where physisorption of an end-functionalized polymer or a block copolymer onto the NPs surface occurs [303]. In this regard, a study investigated the preparation of MNPs using polystyrene (PS) brushes oriented onto the surface of iron oxide MNPs employing the ATRP technique through a free initiator to adjust the polymerization that had ability to create an adequate concentration of persistent radicals, as shown in Figure 3 [304]. The main challenge in the fabrication of magnetic polymer nanocomposites is to provide a high quality nanocomposite. The interaction between nanoparticles and polymer matrix needs to be strong enough to prevent gas voids and deleterious effect on properties of the nanocomposites. The interphase is an inhomogeneous region between nanoparticles and a polymer matrix, which is affected by the surface properties of the nanoparticles and the polymer matrix. Several polymers have been used as matrices for the fabrication of magnetic polymer nanocomposites, among which, polyurethane is preferred, because of several advantages such as chemical stability, excellent mechanical properties, blood and tissue compatibility, good handling, and its low cost [267, 268]. There are a few study related to fabrication of Fe 3 O 4 /polyurethane magnetic elastomer nansocomposites. Recently, Ashjari et al. reported dispersion of oleic acid surface modified Fe 3 O 4 nanoparticles in the polyurethane matrix through Solution Mixing and investigated the properties of the nanocomposite [307]. Also in another study, Xu et al. synthesized polyurethane film reinforced with pure Fe 3 O 4 nanoparticles [301]. They synthesized Fe 3 O 4 /Polyurethane nanocomposites films by directly mixing Fe 3 O 4 nanoparticles, which were dispersed in ethanol with dissolved polyurethane in THF. Guo et al. also prepared magnetic polyurethane nanocomposites as microwave absorbers using magnetic carbonyl iron nanoparticle via the SIP (surface initiated polymerization) method [308]. Figure 3. ATRP procedure for growing PMMA brushes on nanoparticles (adopted from [304]). To the best of our knowledge and based on literature, the commonly used polymers for the preparation of stabilized-mnps are including PEG [271, 272], PVA [311], poly (ethylene imine) (PEI) [312], dextran and its derivatives [ ], starch [316], alginate [317], poly(d,l-lactideco-glycolide [318], poly (ε- caprolactone) [319], polylactide [320] and dendrimers [321]. Several studies have also introduced techniques involving physical grinding in order to modify the surface of iron oxide nanoparticles [322]. Furthermore, functional polymers have attracted interest in recent years, since they facilitate various ligands in being grafted on the IOPs surface. Among polymers in this category, Chitosan and its derivatives have been vastly discussed [323], especially in controlled drug delivery and tissue engineering, due to its biodegradability and biocompatibility [324]. For this purpose, hydrophilic polymers, namely poloxamines and poloxamers, have also been applied to prepare stabilized-iops [287, 288]. 22 Page 22 of 70

24 Polymeric shells have some exceptional rewards due to the pliability in the control of chemical compositions and polymers functions. A number of researchers have reported the preparation of the polymeric core/ shell MNPs, such as Fe 2 O 3 /PS 1, MnFe 2 O 4 /PS 2 and Fe 3 O 4 /P 3 VP 3 and etc. Polymer-stabilized MNPs were synthesized using two biocompatible polyelectrolytes: N- carboxyethyl chitosan (CECh) and poly(2-acrylamido-2-methyl propane sulfonic acid) (PAMPS). The stability and particle size of the dispersions could be efficiently governed by the nature of polyelectrolyte. Transmission Electron Microscopy (TEM) can determine the existence of polyelectrolyte shell, followed by reconfirmation from thermogravimetric analyses (TGA). MNPs, depending on nature of polyelectrolyte, may appear in various magnetic states, including superparamagnetic or a state in-between ferromagnetic and superparamagnetic, which has been proven by Mossbauer analyses and magnetization measurements [327]. Currently, no work has reported the synthesis of MNPs with smart polymer chains, such as poly-n-isopropylacrylamide (PNIPAM). PNIPAM is a polymer that is known to undergo dramatic temperature-induced changes in chain conformation [328]. Numerous reports describing the synthesis and the use of PNIPAM in diverse areas are available. For instance, Fu et al. coat crosslinked PNIPAM on silica encapsulated MNPs to synthesize thermo-responsive magnetic microspheres with core/shell structure [329]. However, the PNIPAM shell can only change the diameter of microspheres in water when the temperature fluctuates between 32 C, and has no effect on the magnetic separation behavior. Polymer coating of MNPs can be obtained using various approaches including in situ, postsynthesis adsorption, and post-synthesis end grafting, as shown in Figure 4. In situ and post synthesis modification with polysaccharides and copolymers lead to coatings that homogeneously encapsulate cores. Alternatively, end grafted polymers, such as PEG, are anchored onto the NP surface by the polymer end groups, forming brush like extensions. Liposome and micelle-forming molecules create a shell around the MNPs core. Each technique comes with advantages and drawbacks, depending on the applied polymer. Figure 4. Illustration depicting the assembly of polymers onto the surface of MNPs cores (Adopted from [296]). One of the approaches in preparing monodispersed IONPs is ligand exchange using polymers. For instance, a research study reported the preparation of MNPs applying the ligand exchange approach using poly(allylamine) to modify the surface of previously-prepared 10 nm Fe 3 O 4 NPs. The Fe 3 O 4 nanoparticles were initially stabilized using diethanolamine (DEA) and diethylene glycol (DEG). It should be noted that the dispersion of nanoparticles into the cluster form needs to take place prior to the formation of polymer shell on the surface of nanoparticles. Interestingly, the ligand exchange approach could enhance the dispersibility of stabilized IONPs using DEA-DEG and poly(allylamine). In fact, polymer was efficiently able to produce a charged brush under the conditions as shown in Figure Page 23 of 70

25 Figure 5. Ligand exchange reaction of poly(allylamine) with DEA stabilized Fe 3 O 4 NPs (Adopted from [330]). Polymer coating can be replaced by a substitute procedure called insertion, which is defined as initially preparing carrier microparticles, followed by incorporation with magnetic and fluorescent components (for example magnetic IONPs) [331] or their ions [332]. The latter process facilitates the final particles with monodispersity and favorable size. In order to carry out this procedure successfully, a suitable carrier materials, as well as sufficient incorporation of functional moieties into the carrier microspheres are required. In this regard, swellable and reactive polymers are certainly appropriate carrier materials, owing to their ability of diffusing the fluorescent and magnetic components and performing reactions to fabricate the ultimate functional components. Poly(glycidyl methacrylate) (PGMA) has been selected as a carrier material in a study. The preparation procedures in forming homogenized microspheres have been already acknowledged [333]. Ethylenediamine was utilized to interact with the epoxy groups of PGMA to present more reactive amino groups for the later syntheses, which is the production of SPIONs and the incorporation of fluorescein isothiocyanate (FITC) fluorophores PEGylation PEG is one of the most frequently used synthetic polymers for surface modifications of MNPs, especially for biomedical applications, since it isbiocompatible and hydrophilic [334]. The hydrophilic nature of PEG is attributed to its hydration with bound water molecules, which increases the molecular weight of the polymer [335]. The conformation of the PEG at low and high molecular weights has been documented as coil and helix, respectively. Therefore, the coated-polymer thickness, as well as stability and hydrodynamic size of the nanoparticles, may vary depending on the molecular weight of the grafted-peg. Several studies investigated the influence of the coated-peg thickness when physically adsorbed onto the MNPs surface, as well as its surface conformation based on the restriction of the access of reactant s molecules to MNPs surface. Xie et al. examined the PEG-grafted iron oxide suspension s stability using hydroxyl groups to attach the polymer onto the surface of the particles [336]. The results revealed that polymers with higher molecular weight led to the development of a dense coating layer, which intercepts the polymer from desorption throughout incubation for 24 h. Butterworth et al. investigated the colloidal stability of PEG-silane coated-mnps with different molecular weights of PEG polymer in water as a function of ph using dynamic light scattering and zeta potential [299]. Captivatingly and unexpectedly, no considerable changes in the hydrodynamic diameter of the particles ( 50 nm) occurred when PEG molecular weight varied between g/mol. PEG-coated particles possessing low molecular weight of polymer indicated reversible flocculation when saturated salt was added to the solution. The observation of poor stability in suspension was assigned to low PEG coverage on the particles surface. Low surface coverage, together with polydispersity of iron oxide cores, can possibly be taken into account for the lack of significant difference in hydrodynamic diameter while using different molecular weights. Herve et al. also reported the investigation of PEG-coated MNPs stability, which has been initially modified with silan-group using molecular weight of 5000 g/mol PEG polymer as a function of ph [300]. They claimed that magnetic particles were stable in the ph range of 4 to 10 with an isoelectric point at ph=5 and a zeta potential of -11 mv at ph 7. The stability of the 24 Page 24 of 70

26 particles as a function of ionic strength was only examined by suspending the particles in a 0.9% (0.15 M) NaCl solution at ph=7.4. Stability was assigned to residual free silanols on the particles surface, which conferred stability via electrostatic interactions. In spite of all the aforementioned advantages, the PEG-coated MNPs preparation is complicated to some extent due to the requirements of PEG modifications and particular atmospheric conditions. Therefore, the surface of the nanoparticles are primarily modified by applying a functional group, such as silane [292, 299] or sodium oleate (as surfactant), followed by the addition of PEG as a secondary surfactant [338]. PEG was occasionally conjugated to a lipid [339] or PEI [340]. PEG-containing, comb-like polymer (CL-PEG) which is composed of relatively hydrophilic groups anchored to a hydrophobic backbone, which makes it suitable for coating hydrophobic nanoparticles, where the backbone interacts with the hydrophobic surface of the particle and the hydrophilic PEG chains facilitate water solubility and enhance the colloidal stability. The polymer hydrophilicity can be modified by adjusting either PEG s chain size or chemical functionalization [341]. The preparation of water dispersible MNPs was investigated using oleic acid and polyethylene glycol methyl ether poly(3-caprolactone) (mpeg PCL) amphiphilic diblock copolymer as polymeric stabilizers [342]. Hydrophobic PCL blocks were hypothetically adsorbed onto MNPs, which have been initially coated with oleic acid, while hydrophilic mpeg blocks protruded particle surfaces to grant steric stabilization and dispersibility in aqueous fluids. The copolymers, having an M n of 5000 g/mol mpeg-5000 g/mol PCL have been used as a stabilizer for this purpose [343]. Additionally, the literature reports studies where PEG molecules were physically grafted onto the SPION surface by sonication [306, 307]. To improve the colloidal stability of SPIONs, covalent attachment of PEG is performed by functional groups in PEG s modified molecules. Amstad et al. employed catechol derivatives to graft PEG chains to MNPs [346]. Furthermore, Park et al. utilized the PEG diacid to prepare a highly surface-modified water dispersed ultra small SPIONs [347]; Amici et al. reported the preparation of Fe 3 O 4 coated with PEG-diacrylate via UV-curing in water [348]; and Lu et al. reported a new direct way to introduce a phosphoric acid anchoring group and carboxyl functional group into PEG, leading to strong bonds to iron oxide nanoparticle surfaces [349]. Moreover, Viali et al. reported a new route of PEGylation of SPIONs by polycondensation reaction with carboxylate functional groups [350]. The SPIONs were superparamagnetic at room temperature, with a saturation magnetization (M s ) from 36.7 to 54.1 emu g -1. The colloidal stability of citrate- and PEG-coated SPIONs was examined by means of DLS measurements. Figure 6a,b shows the magnetization against magnetic field curves at 5 and 300 K, respectively. At 5 K, all samples showed magnetic hysteresis. No significant increase in M s values was noticed from bare SPION to citrate-coated SPION (36.7 emu/g at 300 K and 47.8 and 48.2 emu- /g at 5 K), indicating negligible magnetic contribution from surface carboxylate groups. The PEG-coated particles exhibited M s values ranging from 39.5 up to 43.2 emu/g at 300 K and from 50.5 up to 54.1 emu/g at 5 K. The differences in M s values at 5 and 300 K between the bare, citrate-coated, and the PEG-coated SPIONs can be attributed to the increase in crystallinity of magnetic cores of PEG-coated particles, due to the PEGylation step. All M s values obtained were lower than the values for bulk iron oxides like magnetite (92 emu/g) and maghemite (83.5 emu/g), as reported in literature [351]. Decrease in M s values of synthesized SPIONs with respect to the M s of bulk counterparts is often observed in NPs, and is attributed to the surface 25 Page 25 of 70

27 contribution of spin canting, surface disorder, stoichiometric deviation, cation distribution, and adsorbed species [312, 314]. Figure 6. Equilibrium magnetization curves for SPIONs, bare, citrate, and PEG-coated in powder (a) 5 K and (b) 300 K. (Adopted from [350]) Biopolymers When it comes to applications associated with biosystems, conjugation and encapsulation of MNPs with biopolymer are highly recommended in order to gain biocompatibility and dispersibility simultaneously. However, the most important challenge for these suspensions are their non-biodegradability and lack of superparamagnetism from the thick shell or relatively hydrophobic layer [353], toxic reagents usage [18], colloidal aggregations [354], and difficulty in the removal of polymeric micelles [355]. Among biopolymers, there were attempts to use polysaccharides to hybridize MNPs through secondary forces or the covalent bonds from hyaluronic acid [356], alginate [357], and chitosan [4, 5, 10, 17]. Chitosan is a natural plentiful amino-polysaccharide incorporating reactive functional groups, such as amino and hydroxyl groups. The exceptional aminopolysaccharide structure leads to chitosan becoming cationic, biocompatible, biodegradable, non-toxic in solution at biological phs, and chemically flexible. Owing to its amino group, many studies have succeeded in improving chitosan via different morphologies and sizes for use in drug delivery systems [358]. Numerous studies also reported the changes in morphology of chitosan from flakes or powder to gel form, films, and even nano-sized whiskers ensuing functional materials [359]. In addition, chitosan can be modified to organo-soluble moieties, including iodochitosan [360], N-pthalimido chitosan [361], and water-soluble moieties like phosphorylated chitosan [362], O-succinyl chitosan [363] and carboxymethyl chitosan [364], which are the derivatives for materials on purposes. Lately, successful modification of chitosan for the purpose of maintaining the hydrophobic and hydrophilic groups balanced, has introduced chitosan nanospheres with corecorona structure (CSNS) [365]. The stability and size of these CSNSs were confirmed to depend on solvent polarity and ph. In other words, the size of CSNS was reduced from 300 to 20 nm when the ph dropped from 4 to 12 [366]. The distinctive properties of CSNS facilitate the feasibility of developing MNPs with responsive properties using CSNS as a biopolymer backbone for hybridization with MNPs. Indeed, utilizing CSNS will not only appeal to the simple non-covalent bond hybridization, but may also realize the ph and solvent responsive materials. In the case of hybridized MNPs using chitosan, the procedure can be obtained via secondary interactions such as hydrophobic-hydrophobic Van der Waals forces, hydrogen bond, ionic interaction or the covalent bond. Even though the covalent bond system enables us to specifically control the structure, drawbacks, such as multi-step preparations, the time-consumption purification, quality control, and the yields fluctuation always prevent it from being convenient. Chitosan-hybridizing MNPs using chitosan-salt in an acidic medium has been demonstrated to be 26 Page 26 of 70

28 quite simple [367]. Nevertheless, due to the acidic conditions, the influence of acids on MNPs production and the possible disorders creating by salts or acids and the stability of MNPs are challenges that needs to be overcome. It is necessary to mention that the majority of studies used chitosan as a biopolymer to homogeneously disperse MNPs, while the minority applied chitosan to commence MNPs with unique property of chitosan, particularly ph sensitivity Ferrogels Generally, hydrogels can be employed as matrices for the advancement of novel functional materials, like ferrogels with new and fascinating mechanical and magnetic properties [368]. Ferrogels are composed of soft gel matrices dispersing MNPs, regarded as nanostructured magnetic systems. Ferrogels are vastly applied in the fields of biomedical and pharmaceuticals, such as drug delivery systems, artificial muscles, and sensors [369]. Various techniques are used to prepare ferrogels, including photo polymerization, chemical cross-linking, freezing thawing (F T), and gamma or electron irradiation. Moreover, MNPs can be incorporated with a particular shape, size and coating dispersed in a polymer solution, enabling it to cross-link [370]. MNPs distribution in the matrix considerably governs the final magnetic properties of ferrogels. The interaction of magnetic dipolar among nearest neighbors were significantly developed in concentrated media; the macroscopic magnetic properties of the material will be definitely affected. Therefore, it is essential to propose plans that adjust the level of MNPs dispersion in the matrix, the impact of aggregates and assemblies formation, and the effect of potential interactions between MNPs on the final performance of materials, all throughout the production and afterwards. PVA is known as a biocompatible polymer, and is capable of forming high quality gel, therefore, it is a supreme candidate for the preparation of ferrogels, and has been formerly employed as a matrix to disperse both metallic and MNPs for multiple applications [333, 334]. PAA is also a biocompatible, ph-sensitive polyelectrolyte that stimulates slight antigenic reaction in vivo [373]. Due to the carboxylic acid ionization, charge density of PAA chains is highly reliant on ph. Actually, acrylic acid has previously been applied for the fabrication of inter-penetrating networks (IPNs) and copolymers that reveal ph-, electro- and thermo-, sensitive performance [373]. Additionally, PAA is suitable for coating of MNPs, owing to its ability to absorb the basic proteins and dyes [374], to improve the MNPs mobility with possible applications in groundwater modification applications [375], for the preparation of controlled magnetic assemblies [376], and for the formation of Janus NPs that can be reversibly selfassemble by adjusting ph [377]. In conclusion, PAA is undoubtedly a remarkable choice to coat MNPs and ferrogels formation. Interestingly, the production of ferrogels by dispersion of PAAcoated MNPs using PVA aqueous solutions, with subsequent physical cross-linking of the polymer chains through F T method has been reported. This study attempts to take advantage of PAA s presence on the surface of NPs to provide strong hydrogen bond between PVA matrix and NPs resulting in well-dispersed ferrogels. Francois et al. integrated an aqueous ferrofluid in scleroglucan hydrogels and produced the ferrogel for loading and in vitro release of a theophylline drug [378]. Figure 7 indicates the magnetization loop of magnetic particles after 27 Page 27 of 70

29 drying the sample of ferrofluid against an applied magnetic field at 25 C. The obtained ferrogels demonstrated a superparamagnetic behavior, as proven by zero coercivity and remanance on the magnetization loop. Figure 7. Magnetization versus applied magnetic field at 25 C for the magnetic particles after drying the ferrofluid sample. Arrows indicate the sweep of the magnetization curve. : first step, : second step, and : third step of this curve. (Adopted from [378]). Frimpong et al. produced the magnetically sensitive hydrogel networks based on the MNPs composites and temperature sensitive hydrogels, which is a potential dynamic component of micro- and nanoscale biomedical devices [379]. The formation of the magnetic hydrogel nanocomposites through in-situ embedding of MIONPs into the porous chitosan hydrogel networks has been recently reported and their possible application was examined in the magnetically assisted bioseparation for bovine serum albumin [380]. So far, the techniques to produce magnetic hydrogel nanocomposites mostly included the integration of MNPs into the hydrogel matrix by combining, constitution of the MNPs and hydrogel matrix simutaneously, as well as in-situ synthesis of MNPs in the pre-formed hydrogel network. In spite of being a frequently used technique, its scope is rather restricted. Thus, the advancement of straightforward and efficient synthesis strategies with regards to such functional hydrogel materials is a momentous challenge [381] Hybrid materials Hybrid materials can be developed through doping metallic nano-oxides/hydroxides using traditional porous adsorbents with big particle size and porous structure, such as activated carbon [382], silica [383], zeolite [384] and macroporous polymer [385]. Such hybrid materials incorporate the outstanding handling and flow properties of the porous adsorbent supports with the particular tendency of nano-oxides/hydroxides toward targeted pollutants. However, the hybrid adsorbents for heavy metal modification cannot be applied due to two main problems. Firstly, nanoparticles encapsulated by macroporous supports are likely to agglomerate or develop into bulk particles, and subsequently, their decontamination efficiency for heavy metal will be significantly reduced [386]. Yavuz et al. reported that 12 nm-magnetite NPs showed improved 100-fold sorption capacities to arsenic compared with its bulk counterpart measured at 300 nm [28]; Zhang et al. also confirmed that the enhanced iron (III) loadings onto porous polymer cannot increase the arsenate adsorption at all times, and the most favorable ferric content is about 6% [387]. These achievements recommend that parameters, such as size and dispersion of the functional particles within the resultant adsorbent, might be a main aspect assigned to the heavy metals preservation efficiency. To obtain well-dispersed NPs, some researchers used chemical reagents, for instances amino acids [388], polymer [389], metal ions [390], and phosphate [382] when modifying the inorganic particles. Nevertheless, the aforementioned chemicals can be undesirable for environmental applications. Secondly, molecular sieve and activated carbon, owing to their microporous template structure, could develop the size and morphology of NPs as host matrix. However, improper incorporation of nanoparticles might lead to pore blockage, and deficient diffusion kinetics still needs to be overcome. 28 Page 28 of 70

30 4.1.9 Activated Carbon Diverse carriers have been utilized for the deposition and immobilization of iron or iron oxide nanoclusters; carbon-containing supports, called activated carbon, graphite, carbon black, and carbon microspheres, are fast gaining attention [391]. Activated carbon (AC) is highly favored due to high sorption ability towards organic pollutants and its superior surface properties, which make it an ideal choice of organizing iron into preferred directions. Furthermore, AC is ecofriendly, accessible, and economical. AC s pore system makes it simple to disperse iron nanoclusters by immobilization. For high subsurface mobility, the best possible grain size for low-density particles like AC colloids (ACC) is normally between 0.5 to 2 µm (derivable by the model of Tufenkji et al. [392]). The preferred particle size can be controlled by milling. ACC, with a particle size of d50 = 0.8 µm and d90 = 1.6 µm, demonstrated their high mobility in porous beds in column studies [393]. Moreover, the ACC carrier facilitates the NZVI with hydrophobic vicinity, enabling the enhancement of the concentration of groundwater pollutants in the neighborhood of the reactive iron surface. Furthermore, the improved hydrophobicity of the reagent allows enhanced wettability through residual non-aqueous phase liquids (NAPL) [394] Silica Various synthetic techniques were recently developed to prepare biocompatible and hydrophilic superparamagnetic particles through coating MNPs with silica [395]. Lu et al. reported the preparation of SPIONs coated by amorphous silica with a uniform shell using the sol gel method [396]. The nanoparticles demonstrated weak magnetic response due to this fact that each of them are only composed of single or a few iron oxide nanoparticles (about 0.5 wt%). Figure 8A-C indicates the TEM images of IONPS, whose surfaces had been coated with silica shells at multiple concentrations of TEOS. It is important to mention that the silica shell was thoroughly uniform on every single iron oxide particle, apart from of its original morphology. Therefore, the shape of each IONPs was basically kept intact during silica coating, particularly when the shell was fairly thin (Figure 8A). Moreover, the original nanoparticles also showed the same polydispersity as before. When the silica coating was thickened, the core-shell nanoparticles were found to be monodispersed, due to the reduction in the respective size distribution. Figure 8. (A-C) TEM images of IONPs which have been coated using silica shells with different thicknesses. The silica coating s thickness was controlled by added precursor amount (TEOS): (A) 10, (B) 60, and (C) 1000 mg. (D) A HRTEM image of the IONPs using homogenous coating of silica shell with thickness of 6 nm. (Adopted from [396]). Kim et al. fabricated monodispersed MNPs with high remnants of magnetization using a mesoporous silica shell for drug delivery systems [395]. However, they were not superparamagnetic, which is inappropriate for biomedical applications. Figure 9 showed that the 29 Page 29 of 70

31 mesoporous silica spheres are rather homogeneous in size, with 150 nm-average particle diameter, which is an acceptable size for biomedical applications. Every individual silica sphere carried a few monodispersed magnetite nanocrystals. It seems that ethyl acetate was trapped in the hydrophobic section throughout the sol-gel reaction, and creates the hollow-like core structure. Figure 9d shows the magnetization loop versus magnetic field for mesoporous silicacoated MNPs at 300 K, with no hysteresis appointed superparamagnetic property that are favorable for drug delivery applications. Figure 9. (a) FE-SEM, (b, c) TEM, (d) HRTEM images and (f) field-dependent magnetization at 300 K of magnetic-mesoporous silica spheres (Adopted from [395]). To fabricate thicker coatings on the NPs surface, covalent bonds through grafted layers of silica can be used [397]. The coatings of silica can be obtained using a sol gel precursor via hydrolysis and condensation, where the silica is produced in situ on the particles surface [329]. Nevertheless, hydroxyl-functional silica coatings require being end-capped with carbon functionalities to prevent nanoparticles from aggregation, because of the condensation reactions, and to develop the polymer-solubility of the coatings. Carbon-functional silsesquioxane coatings are potential substitute to silica coatings, since these coatings involve carbon functional moieties in a way that the hydroxyl end-capping process can be removed, with the coatings solubility relying significantly on the molecular structure of the functionalized-carbon [398]. The silsesquioxane coatings are usually produced from triethoxy/trimethoxy-functional silanes; however, it was not able to form a coating as thick as silica s [399]. The thickness of the coating is a vital parameter, as it determines both the mechanical strength and magnetic properties of the composites. To prepare the magnetic fluids with high stability using silicon, hydrophobic polymers can be utilized as stabilizers. These polymers must be composed of a functionalized part that is capable of binding to the surface of the particles, and a hydrophobic non-reactive fraction that is able to dissolve in the dispersion medium or carried fluid. The majority of the studies recently reported in the open literature employed oleic acid as stabilizer for forming hydrocarbon-based magnetite fluids [243]. Additionally, the synthesis of capped MNPs dispersed in PDMS carrier fluids applying anionic surfactants [400] or nitrile, including PDMS triblock copolymers [401], were lately reported Emulsions In order to prepare the aqueous-based suspensions with colloidal stability, surface coatings were more numerously applied in recent years [18]. Additionally, the coatings interfacial properties on the surface of the particles may be stabilized using Pickering emulsions of oil and water [402]. MNPs in the size range of micron and below have been set aside to adsorb at oil water interfaces [403], as well as silica NPs for adsorption at carbon dioxide water interfaces [404]. The emulsion polymerization is typically based on suspending the MNPs in the dispersion, followed 30 Page 30 of 70

32 by polymerization of the monomer in the presence of the MNPs to fabricate magnetic polymeric particles. The emulsion polymerization technique has been improved lately, and resulted in many new advanced techniques, including the conventional micro-emulsion polymerization, soap-free emulsion polymerization, micro-emulsion polymerization, and mini-emulsion polymerization [405]. Among all these approaches, and based on the mechanism of emulsion polymerizations, the mini-emulsion polymerization is highly recommended for the preparation of magnetic nanocomposites. The procedure of magnetic polymeric particle s formation can be productively divided into three steps, from which two represent dispersion, while one appoints mini-emulsion polymerization [406]. This process begins with the dispersion of MNPs in monomer phase using a stabilizer (surfactant) [407], followed by mixing the aqueous and monomer phases using high shear forced-dispersing device, as well as sonication to fabricate the mini-emulsion containing the monomer droplets with diameters of nm; and finally finishes with mini-emulsion polymerization. Figure 10 demonstrates the general emulsion polymerization procedure. As seen in Figure 10, MNPs can be either hydrophilic in aqueous phase (Emulsion A), or hydrophobic in organic phase (Emulsion B), which need to be mixed with the organic phase afterwards. The drawback of this technique is the presence of the oxidizing initiator, which may result in the loss of magnetization. In fact, some polymerization factors, such as the initiator type, concentration of surfactant, and dose of monomers also influence the morphologies and properties of magnetic nanocomposite [407]. For example, when the water-soluble persulfate initiator was used, 300 nm MNPs with uniform dispersion was obtained; when oil-soluble-azobiisobutyronitrile (AIBN) was used instead of potassium persulfate, magnetite nanoparticles under 100 nm were located on the outer layer of nanocomposite spheres [408]. In the direct mini-emulsion polymerization, the surface of hydrophilic inorganic nanoparticles needs to be modified in order to alter their hydrophilic property [409], since it is enormously hard for hydrophilic inorganic nanoparticles to be dispersed into non-polar monomer phase [85]. Replacing the encapsulation process with inverse mini-emulsion polymerization, where the dispersant is a polar monomer, could elude the obligation of surface modification procedure. Figure 10. Schematic diagram showing the preparation of magnetic polymeric particles using Emulsion Polymerization with hydrophilic and hydrophobic MNPs (Adopted from [410]). 5. Conclusion and Future Aspects A critical obstacle of dispersing magnetic iron oxide nanoparticles and maintaining its stability is the tendency of MNPs to agglomerate. Generally, nanoparticle dispersion or aggregation is mainly associated with the Van der Waals attraction and electrostatic double-layer repulsion interaction between particles according to the Derjaguin Landau Verwey Overbeek (DLVO) theory. This theory describes the stabilization mechanism which are either electrostatic or steric. Mainly due to lipophilic chains, sterically stabilized particles are dispersible in non-polar aprotic solvents, such as cyclohexane, toluene, and dibenzyl ether. If an aqueous sol is needed, particles are often stabilized electrostatically. Because OH, as well as H 3 O + ions, are capable of 31 Page 31 of 70

33 neutralizing the surface charge on the particles up to the isoelectric point, stability is restricted to a certain range of ph values, beyond which flocculation occurs. In this review, we comprehensively explained various techniques to prepare colloidal MNPs suspension using polymers and organic and inorganic materials as stabilizers and surfactants, respectively. Additionally, ionic liquids, ferrogels and different types of emulsions play a vital role in generating task-oriented MNPs. As previously mentioned, choice of synthesis method and the control of experimental conditions are critical factors in producing monodispersed MNPs suspension. Thus, any size heterogeneity or polydispersity is clearly a disadvantage, because it may result in a large difference in response to the magnetic field. However, to achieve much higher sensitivity and efficiency for new generation of applications, more efforts should be broadly focused on the synthesis of hydrophilic magnetic spheres with high saturation magnetization. Owing to widespread applications of magnetic iron oxide suspensions, modification of the current techniques along with advancing novel synthetic approaches are extremely sought after. The direction of synthetic methods improvements should lie in developing environmentally friendly approaches simultaneously with using aqueous media, which may lead to more green chemistry synthetic methods. Furthermore, there is a possibility to take advantage of multifunctional systems in order to apply MNPs for the purpose of tracking optical probes, manipulation, functional groups for bioconjugation, and more advanced drug targeting. Such improvements help us with diagnostics and disease remedies. It should be noted that designing the structure and composition of MNPs to enhance their properties for particular applications is also one accessible goal within the context of this work. Acknowledgment This work was supported by the University of Malaya through HIR Grant (No. H F0032). References [1] K. A. Halim and M. Khedr, Factors affecting CO oxidation over nanosized Fe< sub> 2</sub> O< sub> 3</sub>, Mater. Res., [2] M. Ristic, S. Music, and M. Godec, Properties of γ-feooh, α-feooh and α-fe2o3 particles precipitated by hydrolysis of Fe3+ ions in perchlorate containing aqueous solutions, J. Alloys Compd., [3] P. Majewski and B. Thierry, Functionalized magnetite nanoparticles synthesis, properties, and bio-applications, Crit. Rev. Solid State, [4] A. Christensen and T. Jensen, Nano size crystals of goethite,< i> α</i>-feooh: Synthesis and thermal transformation, J. Solid State, Page 32 of 70

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63 [410] X. Liang, H. Wang, X. Jiang, and J. Chang, Development of monodispersed and functional magnetic polymeric liposomes via simple liposome method, J. Nanoparticle Res., Figure caption Figure 1. Schematic illustration of the main mechanisms for stabilization of magnetic nanoparticle dispersions, assuming positively charged surfaces (Adopted from [151]). Figure 2. Schematic illustration of the ligand exchange process using PAA, PEI and GSH. Adopted from [12]. Figure 3. ATRP procedure for growing PMMA brushes on nanoparticles (adopted from [304]). Figure 4. Illustration depicting the assembly of polymers onto the surface of MNPs cores (Adopted from [296]). Figure 5. Ligand exchange reaction of poly(allylamine) with DEA stabilized Fe 3 O 4 NPs (Adopted from [330]). Figure 6. Equilibrium magnetization curves for SPIONs, bare, citrate, and PEG-coated in powder (a) 5 K and (b) 300 K. (Adopted from [350]). Figure 7. Magnetization versus applied magnetic field at 25 C for the magnetic particles after drying the ferrofluid sample. Arrows indicate the sweep of the magnetization curve. : first step, : second step, and : third step of this curve. (Adopted from [378]). Figure 8. (A-C) TEM images of IONPs which have been coated using silica shells with different thicknesses. The silica coating s thickness was controlled by added precursor amount (TEOS): (A) 10, (B) 60, and (C) 1000 mg. (D) A HRTEM image of the IONPs using homogenous coating of silica shell with thickness of 6 nm. (Adopted from [396]). 62 Page 62 of 70

64 Figure 9. (a) FE-SEM, (b, c) TEM, (d) HRTEM images and (f) field-dependent magnetization at 300 K of magnetic-mesoporous silica spheres (Adopted from [395]). Figure 10. Schematic diagram showing the preparation of magnetic polymeric particles using Emulsion Polymerization with hydrophilic and hydrophobic MNPs (Adopted from [410]). Figures Figure 1 63 Page 63 of 70

65 Figure 2 Figure 3 64 Page 64 of 70

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69 Figure Page 68 of 70

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