**3.4. Hydrothermal and solvothermal methods**

obtained M-IONPs with controlled size, size distribution and composition. The most commonly used precursors employed to prepare monodispersed M-IONPs with diameter ranging from 3 to 50 nm are of the form: (I) metal acetylacetonate—[M(acac)n] (where M = Fe, Co,

> O4 ) <sup>n</sup>•2H2

[54]; (VIII) Prussian Blue—Fe<sup>4</sup>

in the presence of oleic acid at 100°C. Initially, he obtained an iron-oleic acid com-

and M(acac)2

O4

Pérez-Mirabet et al. used oleylamine both as stabilization agent (for the stabilization of the particles in solution) and as capping ligand (for the control of particles size), respectively, by

lamine. They obtained magnetic spinel ferrite nanoparticles with average size of 12 nm and a saturation magnetization Ms = 76 emu/g, very close to the bulk magnetite (92 emu/g) [58]. This method is also suitable for synthesis of nanocubes and nanospheres, which are magnetic

simple and single-step process [59]. They used various mixtures of ferrocene and polyvinylpyrrolidone (PVP) by solventless thermal decomposition. Lynch et al. obtained magnetic colloidal iron oxide nanoparticles by thermal decomposition. They generated gas bubbles (Ar) by boiling solvents. Their results illustrated that the argon bubbles had a stronger effect on the nucleation process of magnetic iron oxide nanoparticles than on their growth process [60]. Due to the nucleation process that involves boiling solvents, most often the accurate shape of the magnetic iron oxide nanoparticles is not fully reproducible using the thermal decomposition method.

A microemulsion is formed when a colloidal substance is dispersed in a solvent, that is not compatible with the substance (e.g. water and oil), through a surfactant. Finally, a microemulsion must be clear and stable, as long as it is an isotropic mixture of oil, water and surfactant. The surfactant forms a monolayer film at the oil/water interface, in which the hydrophilic head groups of the surfactant are dissolved in oil phase (consisting of a mixture of hydrocarbons and olefins) and the hydrophobic tail of the surfactant in the aqueous phase (consisting of metal salts) and vice versa, depending on the used surfactant. There are known two types of microemulsion: direct microemulsion, when the oil is dispersed in water and reversed microemulsion, when the water is dispersed in oil. Both have been used to synthesize the magnetic iron oxide nanoparticles with tailored size and shape. The most common surfactants that are widely used in the fabrication of M-IONPs by microemulsion method are bis(2-ethylhexyl) sulfosuccinate (AOT), sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB)

H5 ) 2 [57]. Using the thermal decomposition method, it can be easy to control the size and morphology of magnetic nanoparticles by controlling the ratio of the starting reagents, i.e. the ratio between the organometallic compounds, surfactant and solvent. Reaction time, temperature and aging period are equally important for the control of size and morphology. Hyeon obtained monodispersed iron oxide nanoparticles with size range from 4 to 20 nm by thermal decomposition

(cup)x

O]; (IV) metal carbonyl—Fe<sup>3</sup>

COO)n]; (VI) metal carboxylate, (VII) metal-urea

[Fe(CN)<sup>6</sup>

] (where cup = N-nitrosophen

•14H2

(M = Co, Mn, Cu and Zn) in oley-

nanocubes and nanospheres by a new

(CO)12 [53]

O] [55, 56]; (IX)

Ni, Mn, Cr; n = 2 or 3) [52]; (II) metal cupferronates—[M<sup>x</sup>

[50]; (V) metal acetate—[M(CH<sup>3</sup>

plex, which was leaved to aging at high temperature (300°C) [50].

ylhydroxylamine); (III) metal oxalate—[M(C<sup>2</sup>

H4 ) 6 ](NO3 ) 3

one-pot thermal decomposition of Fe(acac)3

**3.3. Microemulsion method**

nanoparticles as well. Amara et al. synthesized Fe<sup>3</sup>

metal chloride and (X) ferrocene—Fe(C<sup>2</sup>

or Fe(CO)<sup>5</sup>

of Fe(CO)<sup>5</sup>

complex—[Fe(CON<sup>2</sup>

238 Iron Ores and Iron Oxide Materials

In case of these methods, the reaction takes place in aqueous medium (the hydrothermal method) or in organic medium (solvothermal method), in reactors or autoclaves, at temperatures between 130 and 250°C under high vapor pressure, in the range 0.3–4 MPa [70–72]. Using this method, it can be obtained magnetic iron oxide nanoparticles with tailored properties (size and shape) by tuning the reaction conditions. The hydrothermal method is known as an environment-friendly process for the obtaining of M-IONPs, due to the raw materials used such as sulfates and chlorides—as cation source, dissolved in water [73].

Lin et al. [74] has used hydrothermal and solvothermal methods to obtain hollow M-IONPs. Briefly, they used FeCl<sup>3</sup> (as source of iron), ethylene glycol (as reducing agent), ammonium acetate and urea were used to guide the formation of hollow magnetite spheres. After homogeneous dispersion, the mixture is transferred to a Teflon-lined stainless steel autoclave and sealed to heat at about 200°C for 8–24 h. The authors demonstrated that the Fe3+ ions on the surface of the hollow spheres exist in the form of Fe3 O4 , and the results are confirmed by the Mössbauer measurements. Tian et al. synthesized ultra-small monodisperse Fe3 O4 nanoparticles, with precise size control of 1 nm, by solvothermal method [75]. They used Fe(acac)3 as iron source, n-octanol as a solvent and n-octylamine as a reductant. The authors obtained Fe3 O4 nanoparticles with a size range from 4 to 6 nm, by varying the volume ratios of n-octylamine and n-octanol, without the need of a gas (N2 ) bubbling or reflux conditions. By comparing this method with the thermal decomposition methods for obtaining Fe<sup>3</sup> O4 nanoparticles, this solvothermal process was more convenient.

The use of metallic nitrates in a mixture with a suitable fuel has the great advantage that, following the combustion reaction, the gases are released without a high risk of toxicity: CO<sup>2</sup>

oxidizing agent/fuel ratio, it can be tailored the size of the particles, the specific surface area

Ianoş et al. reported a new combustion synthesis technique for the preparation of nanosized

gated the effect of both the reaction atmosphere (in the presence or in the absence of air) and the fuels nature on the properties of the resulted nanoparticles. Using sucrose, citric acid and glucose as fuels, the authors demonstrated that the reaction atmosphere is very important

nanoparticles in the size range of 10 (when glucose was used as fuel) to 18 nm (when citric

Mihoc et al. also investigated the effect of both the fuels nature and the reaction atmosphere for obtaining magnetic nanoparticles used as adsorbent for the removal of phenol and p-chlorophenol from wastewater [81]. The authors revealed that the working atmosphere influences the phase composition of the combustion reaction product. Using urea with ammonium chlo-

Working in the absence of air, using oxalic, tartaric and citric acid as fuel, the single phase

Using the combustion method, the magnetic oxide nanoparticles are covered with some organic residues resulting from fuel combustion. Mihoc et al. demonstrated that these materials exhibit better adsorption capacity as compared with the naked magnetic oxides [82].

However, if it is desired to remove the residual carbon resulting from the combustion process, Ianos et al. found a method in which the residual carbon was eliminated by washing

resulted magnetic nanoparticles the carbon was removed by chemical oxidation, from 32.7 to

M-IONPs proved to be versatile due to the large range board of applications in medicine. Nanomedicine is an emerging field that offers new approaches but especially new solutions for many medical problems. For example, the discovery of antibiotics has been of historic importance, but over time, antibiotic resistance has become an issue and new approaches are therefore needed. Many groups of researchers have already demonstrated that the synergic effects of the antimicrobials agents (not only the antibiotics) with nanoparticles can be promoted as a new method for the severe infection treatments, even with low antimicrobial doses. For the biomedical uses, only the M-IONPs which fulfill the following requirements are proper: superparamagnetic properties at room temperature, large saturation of magnetizations, biocompatibility and sizes around 20 nm for *in vivo* administration. To convert the pure magnetic nanoparticles in biocompatible colloidal suspensions, many researchers have pro-

O2

O4

H12O6

and residual carbon was obtained. The authors demonstrated that by H2

0.4%, and the color of the sample changed from black to reddish brown [83].

nanoparticles [80]. The authors developed a new, facile and cheap scheme of installa-

nanoparticles as a single crystalline phase. There were obtained Fe3

O3

, irrespective of the nature of the fuel.

nanoparticles in the absence of air. They also investi-

(when the reaction took place in air).

http://dx.doi.org/10.5772/intechopen.74176

. They revealed that by combustion reac-

O2

a black magnetic nanoparticle containing γ-Fe<sup>2</sup>

and the crystallinity degree of the obtained material [78, 79].

O4

tion for combustion synthesis of Fe3

O4

resulted in combustion reaction was Fe3

ride as fuels, the final product of reaction was α-Fe<sup>2</sup>

the magnetic nanoparticles several times with H2

O and C<sup>6</sup>

)3 •9H2

O [77]. By using a proper fuel, proper auxiliary additives, as well as an appropriate

Preclinical Aspects on Magnetic Iron Oxide Nanoparticles and Their Interventions as Anticancer…

N2

Fe3 O4

and H2

for obtaining Fe3

acid was used as fuel).

tion between Fe(NO3

,

241

O4

O3

treatment of the

Stoia et al. [14] synthesized Fex Oy and Fex Oy /C nanocomposite by solvothermal method, with the purpose of using these nanocomposites as adsorbents for methylene blue removal from aqueous solutions. The authors used FeCl<sup>3</sup> as iron source, 1,2-propanediol as solvent and diethylamine as precipitating agent. The activated carbon was introduced into system in order to obtain homogenous Fex Oy /C composites with high specific surface area and magnetic properties. Some researchers have attempted to modify the hydrothermal process. Ahmadi et al. obtained Fe3 O4 nanoparticles at low temperature (140°C) without having to autoclave. They have studied kinetics of the reaction, but the magnetic properties of the resulted nanoparticles are inadequate in short reaction time (below 2 hours) [76].

As advantages, the hydrothermal and solvothermal methods are suitable for obtaining shapecontrolled M-IONPs. As a disadvantage, in the case of hydrothermal technique, the reaction takes place for a long time and the amounts of resulting products are low [76].

### **3.5. Combustion method**

The combustion method is an alternative to the currently used methods, being barely mentioned in the literature for the synthesis of M-IONPs. The combustion method have a lot of advantages due to the simplicity of the working technique, short reaction time and low energy consumption, being in the same time environmentally friendly.

The combustion method involves the strong exothermic redox reaction between an oxidizing agent (iron nitrate) and various reducing agents (fuels) of organic nature. The initiation of the combustion process takes place by rapidly heating the mixture of raw materials at relatively low temperatures below 500°C (**Figure 4**). The reaction stoichiometry has a decisive role in the characteristics of the reaction product, especially in the granule size, since a combustion reaction does not occur for any molar fuel/oxidizing agent ratio.

**Figure 4.** The general scheme for obtaining iron oxide magnetic powders using the combustion method.

The use of metallic nitrates in a mixture with a suitable fuel has the great advantage that, following the combustion reaction, the gases are released without a high risk of toxicity: CO<sup>2</sup> , N2 and H2 O [77]. By using a proper fuel, proper auxiliary additives, as well as an appropriate oxidizing agent/fuel ratio, it can be tailored the size of the particles, the specific surface area and the crystallinity degree of the obtained material [78, 79].

Ianoş et al. reported a new combustion synthesis technique for the preparation of nanosized Fe3 O4 nanoparticles [80]. The authors developed a new, facile and cheap scheme of installation for combustion synthesis of Fe3 O4 nanoparticles in the absence of air. They also investigated the effect of both the reaction atmosphere (in the presence or in the absence of air) and the fuels nature on the properties of the resulted nanoparticles. Using sucrose, citric acid and glucose as fuels, the authors demonstrated that the reaction atmosphere is very important for obtaining Fe3 O4 nanoparticles as a single crystalline phase. There were obtained Fe3 O4 nanoparticles in the size range of 10 (when glucose was used as fuel) to 18 nm (when citric acid was used as fuel).

Mihoc et al. also investigated the effect of both the fuels nature and the reaction atmosphere for obtaining magnetic nanoparticles used as adsorbent for the removal of phenol and p-chlorophenol from wastewater [81]. The authors revealed that the working atmosphere influences the phase composition of the combustion reaction product. Using urea with ammonium chloride as fuels, the final product of reaction was α-Fe<sup>2</sup> O3 (when the reaction took place in air). Working in the absence of air, using oxalic, tartaric and citric acid as fuel, the single phase resulted in combustion reaction was Fe3 O4 , irrespective of the nature of the fuel.

Using the combustion method, the magnetic oxide nanoparticles are covered with some organic residues resulting from fuel combustion. Mihoc et al. demonstrated that these materials exhibit better adsorption capacity as compared with the naked magnetic oxides [82].

However, if it is desired to remove the residual carbon resulting from the combustion process, Ianos et al. found a method in which the residual carbon was eliminated by washing the magnetic nanoparticles several times with H2 O2 . They revealed that by combustion reaction between Fe(NO3 )3 •9H2 O and C<sup>6</sup> H12O6 a black magnetic nanoparticle containing γ-Fe<sup>2</sup> O3 and residual carbon was obtained. The authors demonstrated that by H2 O2 treatment of the resulted magnetic nanoparticles the carbon was removed by chemical oxidation, from 32.7 to 0.4%, and the color of the sample changed from black to reddish brown [83].

M-IONPs proved to be versatile due to the large range board of applications in medicine. Nanomedicine is an emerging field that offers new approaches but especially new solutions for many medical problems. For example, the discovery of antibiotics has been of historic importance, but over time, antibiotic resistance has become an issue and new approaches are therefore needed. Many groups of researchers have already demonstrated that the synergic effects of the antimicrobials agents (not only the antibiotics) with nanoparticles can be promoted as a new method for the severe infection treatments, even with low antimicrobial doses.

For the biomedical uses, only the M-IONPs which fulfill the following requirements are proper: superparamagnetic properties at room temperature, large saturation of magnetizations, biocompatibility and sizes around 20 nm for *in vivo* administration. To convert the pure magnetic nanoparticles in biocompatible colloidal suspensions, many researchers have pro-

**Figure 4.** The general scheme for obtaining iron oxide magnetic powders using the combustion method.

of n-octylamine and n-octanol, without the need of a gas (N2

nanoparticles, this solvothermal process was more convenient.

Oy

and Fex

Stoia et al. [14] synthesized Fex

240 Iron Ores and Iron Oxide Materials

to obtain homogenous Fex

O4

**3.5. Combustion method**

obtained Fe3

aqueous solutions. The authors used FeCl<sup>3</sup>

Oy

are inadequate in short reaction time (below 2 hours) [76].

By comparing this method with the thermal decomposition methods for obtaining Fe<sup>3</sup>

the purpose of using these nanocomposites as adsorbents for methylene blue removal from

diethylamine as precipitating agent. The activated carbon was introduced into system in order

erties. Some researchers have attempted to modify the hydrothermal process. Ahmadi et al.

have studied kinetics of the reaction, but the magnetic properties of the resulted nanoparticles

As advantages, the hydrothermal and solvothermal methods are suitable for obtaining shapecontrolled M-IONPs. As a disadvantage, in the case of hydrothermal technique, the reaction

The combustion method is an alternative to the currently used methods, being barely mentioned in the literature for the synthesis of M-IONPs. The combustion method have a lot of advantages due to the simplicity of the working technique, short reaction time and low

The combustion method involves the strong exothermic redox reaction between an oxidizing agent (iron nitrate) and various reducing agents (fuels) of organic nature. The initiation of the combustion process takes place by rapidly heating the mixture of raw materials at relatively low temperatures below 500°C (**Figure 4**). The reaction stoichiometry has a decisive role in the characteristics of the reaction product, especially in the granule size, since a combustion

takes place for a long time and the amounts of resulting products are low [76].

energy consumption, being in the same time environmentally friendly.

reaction does not occur for any molar fuel/oxidizing agent ratio.

Oy

) bubbling or reflux conditions.

/C nanocomposite by solvothermal method, with

as iron source, 1,2-propanediol as solvent and

/C composites with high specific surface area and magnetic prop-

nanoparticles at low temperature (140°C) without having to autoclave. They

O4

posed the use of different polymers like covering agents or surfactants like starch, heparin, chitosan, dextran, oleic acid, polyethylene glycol (PEG), etc.

damage are aging, cancer and neurodegenerative diseases [91]. Another mechanism of inducing cell death by the iron ions is the apoptotic pathway via mitochondria, as follows: a high amount of iron ions into the mitochondria determine the opening of the mitochondrial transition pore, release of Ca2+ and cytochrome c and activation of apoptotic cascade [91, 92].

Preclinical Aspects on Magnetic Iron Oxide Nanoparticles and Their Interventions as Anticancer…

http://dx.doi.org/10.5772/intechopen.74176

243

Based on these data, concerning the toxicity induced by iron ions, it is imperative to study the possible toxic effects induced by M-IONPs, mainly since these particles present a higher reactivity as compared with the normal sized ones. The magnetic character of iron oxide nanoparticles offers some advantages, including the capacity of this nanosized compounds to be driven to targeted sites by an external magnetic field, even to tissues and organs that are difficult to reach in normal conditions (blood brain barrier and central nervous system). M-IONPs penetrate into the cells via receptor-mediated endocytosis and settle into the lysosomes, organelles characterized by the presence of an acidic medium, where it takes place the metabolization of

In a previous study, it was demonstrated that M-IONPs penetrate differentially into the neural cells (glial cells, primary neurons of the cerebellum, microglia, astrocytes, oligodendrocytes and Schwann cells), based on their dimensions: large size nanoparticles were absorbed by endocytosis, whereas small sized ones via pinocytosis [91, 93]. It was also proved that exposure to M-IONPs has an impact on iron homeostasis by upregulating the proteins responsible for iron storage or export from the cell and by downregulating the proteins expression

Besides these positive features, application of an external magnetic field leads to accumulation of M-IONPs in target cells and potential toxicity. The accumulation of iron into the cells after exposure to M-IONPs seems to be dependent on several factors, such as (i) concentration and dose of M-IONPs (high concentrations require a longer period for elimination—several months, whereas M-IONPs in low concentrations can be eliminated within 3 weeks), size (small size nanoparticles cumulate in increased concentrations as compared to large size nanoparticles), shape (spherical nanoparticles present a longer degradation process due to a small contact surface), coating (some coating agents may prolong the degradation process or may increase it), the functional groups (the positively charged functional groups present in M-IONPs structure increase their uptake by the cells) and cell type (microglia have a higher affinity for M-IONPs,

A significant number of studies sustained that M-IONPs exerted *in vitro* and *in vivo* toxicity. The main players responsible for toxic effects are considered to be the iron ions released from M-IONPs at lysosomal level, which react with hydrogen peroxide and lead to ROS generation [91]. Exposure of neural cells to M-IONPs was associated with a low concentration of ROS, but a reduced level of glutathione and mitochondrial membrane hyperpolarization [95]. Other studies conducted on healthy cell lines (both human and animal origin) pointed out that bare M-IONPs may induce cytotoxic effects via ROS generation, leading to cell death [96–98].

The oxidation state of iron (Fe2+ or Fe3+) plays a major role in determining the nanoparticles

O3

is more toxic than Fe2+ in Fe3

O4

the nanoparticles and free iron ions are released into the cell [91].

whereas into the brain endothelial cells penetrate less nanoparticles) [91].

toxicity according to the studies that affirm that Fe3+ in Fe2

and causes more DNA oxidation [91, 99].

involved in iron uptake [94].

Polymer coating can be accomplished during or after the synthesis of magnetic nanoparticles. Polyethylene glycol (PEG) is a water-soluble, biocompatible hydrophilic polymer that can be used successfully in the synthesis of biocompatible nanoparticles with increased resistance to blood circulation [84]. Another alternative to covered magnetic nanoparticles is the use of copolymers that produce core-shell nanoparticles with possible applications in drug transport (drug vector) [85].

The use of inorganic compounds such as gold, silver, silica gel and carbon as surfactants not only provides good stability to the nanoparticles but also allows functionalizing their surface by grafting certain biological ligands. Covering of magnetic nanoparticles with gold seems to be ideal because of its low reactivity; however, coating the magnetic nanoparticles directly with gold is very difficult due to the different nature of the two surfaces [86–89]. The silica gel is the most widely used compound in the preparation of functionalized iron oxide nanoparticles surface, because it has several advantages: excellent biocompatibility, hydrophilicity, the feasibility of integrating other functional groups on the surface due to terminal silanol groups that can react with different coupling agents, provides good stabilization of the magnetic iron oxide nanoparticles in the solution, prevents the interaction between the nanoparticles thus preventing the agglomeration of the particles over time and ensures better encapsulation [90]. A very good coating of carbon layers provides an effective barrier against oxidation and acidic erosion of magnetic nanoparticles. It is therefore possible to synthesize carbon-coated magnetic nanoparticles that are thermally stable, biocompatible and also have high oxidation stability, which is crucial for certain applications [89].
