*2.2.2. Synthesis of iron oxide nanoparticles*

The common existing methods to synthesize the iron oxide nanoparticles are physical, chemical and biological methods. Comparatively, chemical methods, especially wet chemical ones are much simpler and more efficient *(Gupta et al., 2004)*. Several synthetic procedures have been developed to synthesize iron oxide nanoparticles. The simplest, cheapest and most environmentally-friendly procedure is based on the co-precipitation wet chemical method, which involves the simultaneous precipitation of ferrous (Fe2+) and ferric (Fe3+) salts in an alkaline medium *(Kang, 1996)*. So the synthesis of iron oxide nanoparticles with an expected size distribution and stability of suspension is no longer the biggest challenge for researchers. The key issue now is how to achieve the aim of stealth of these nanoparticles in blood circulation and to attach them on desired sites for *in vivo* or *in vitro*  applications *(Sun, 2006)*.

Hydrothermal synthesis techniques are an alternative method for the preparation of highly crystalline iron oxide nanoparticles *(Wang et al, 2005)*. In this case a mixture of iron salts dissolved in aqueous media is introduced in a sealed Teflon container and heated above the boiling temperature of water, and consequently the reaction pressure is increased much above atmospheric pressure. The synergistic effect of high temperatures and pressures strongly improves the quality of the nanocrystals and hence their magnetic features. However, and in contrast to the biological technique, there is no straightforward way to control the size and the shape of the final particles and usually polydisperse samples are obtained.

Biological methods, since nanomaterials have comparable dimensions to biological aggregates, bio-related synthesis methods have been explored for novel nanoparticle synthesis. In biological methods, synthesis and assembly of crystalline inorganic materials can be regulated by biological organisms under environmentally benign conditions and desired chemical compositions and phases can be achieved. For example, the nucleation of semiconducting nanoparticles can be initiated in the presence of viruses expressing material-specific peptides. Other examples are the use of porous protein crystals, manipulation of bacteria to produce oxide nanoparticles and selection of metal-specific polypeptides from combinatorial libraries *(Reiss et al. 2004)*. In biological methods, biological entities usually serve as templates for nanoparticles formation. In all cases, the biological entities were used not only to encapsulate the nanoparticles, but to strictly regulate the dimension of the crystals. To prepare magnetic nanoparticles, ferritin can be used which consists of 24 nearly identical subunits. Self-assembly of ferritin will form a spherical cage with a 7.5–8.0 nm-diameter cavity, which can be used for the biological storage of iron in the form of ferrihydrite, an iron (III) oxy-hydroxide? The protein cage is able to withstand relatively high temperatures for biological systems (up to 65 ◦C) and various pH values (~ 4.0–9.0) for certain periods of time. Therefore this protein template is quite strong and will not cause any significant disruption of the quaternary structure.

#### *2.2.3. Classification of iron oxide nanoparticles*

104 Hyperthermia

usually water, kerosene or various oils. Due to their small size, the magnetic nanoparticles in carrier liquids neither form sediment in the gravitational field or in moderate magnetic field gradients, nor do they agglomerate due to magnetic dipole interaction. However, a stable suspension can only be achieved if the particles are protected against agglomeration due to the van der Waals interaction. Usually this protection can be achieved by one approach is the electric charge stabilization. In this approach, a thin layer of gold is coated on the surface of the nanospheres. Meanwhile, the thin gold layer can also serve as an ideal base on which chemical or biological agents can be functionalized. These molecules generate a repulsive force, preventing the particles from coming into contact and thus suppressing the destabilizing effect of the van der Waals interaction. In practical applications, this approach is often used in combination for the majority of ferrofluids, since this allows the

Magnetic iron oxide nanoparticles are the most investigated material in biomedical techniques, due to its superior biocompatibility with respect to other magnetic materials, either in form of oxides or pure metals. Several types of iron oxides exist in nature and can be prepared in the laboratory. Nowadays, only maghemite (γ-Fe2O3) and magnetite (Fe3O4) are able to fulfill the necessary requirements for biomedical applications. These requirements include sufficiently high magnetic moments, chemical stability in physiological conditions and low toxicity, not to mention the easy and economical synthetic

The degree of atomic order in the iron oxide lattice, or in other words its degree of crystallinity, as well as the dispersity of the nanoparticles in terms of size and shape are critical parameters that affect their performance in diagnostic and therapeutic techniques as a contrast agent in magnetic resonance imaging (MRI) and hyperthermia, respectively. These parameters are

The common existing methods to synthesize the iron oxide nanoparticles are physical, chemical and biological methods. Comparatively, chemical methods, especially wet chemical ones are much simpler and more efficient *(Gupta et al., 2004)*. Several synthetic procedures have been developed to synthesize iron oxide nanoparticles. The simplest, cheapest and most environmentally-friendly procedure is based on the co-precipitation wet chemical method, which involves the simultaneous precipitation of ferrous (Fe2+) and ferric (Fe3+) salts in an alkaline medium *(Kang, 1996)*. So the synthesis of iron oxide nanoparticles with an expected size distribution and stability of suspension is no longer the biggest challenge for researchers. The key issue now is how to achieve the aim of stealth of these nanoparticles in blood circulation and to attach them on desired sites for *in vivo* or *in vitro* 

procedures available for the preparation of these materials *(Neuberger et al., 2005)*.

strongly correlated to the approach for their synthesis *(Maenosono et al., 2008)*.

synthesis of suspensions which are stable over years (*Could 2004*).

*2.2.1. Magnetic (iron oxide nanoparticles)* 

*2.2.2. Synthesis of iron oxide nanoparticles* 

applications *(Sun, 2006)*.

There are many categories of iron oxide nanoparticles based on their overall diameter (including iron oxide core and hydrated coating). Iron oxide nanoparticles can be distinctly classified into super-paramagnetic iron oxide nanoparticles (SPIO NPs) between 300 nm and 3.5 *μ*m; standard SPIO (SSPIO) of approximately 60–150 nm; ultra small SPIO (USPIO) of approximately 10–40 nm (*Weissleder et al., 1990)*; monocrystalline iron oxide nanoparticles (MION—a subset of USPIO) of approximately 10–30 nm and cross-linked iron oxides (CLIO) which is a form of MION with cross-linked dextran coating (*Shen et al., 1993)*.

On the other hand, the magnetic materials are characterized by the presence of magnetic dipoles generated by the spinning of some of their electrons. Each of these polarized electrons can be aligned in a parallel or antiparallel fashion with respect to the neighboring ones in the crystal lattice, and this type of interaction is what gives rise to the macroscopic magnetic effect that we can measure. Based on the magnetic response, the magnetic

materials can be classified into; diamagnetic, paramagnetic, ferromagnetic, ferrimagnetic, anti-ferromagnetic and super-paramagnetic *(Cozzoli et al., 2006)* as shown in Fig (1).

Laser and Radiofrequency Induced Hyperthermia Treatment via Gold-Coated Magnetic Nanocomposites 107

M as a function of applied field H is called a hysteresis loop or M-H curve as shown in

When nanoparticles are used for *in vivo* applications, the nanoparticles have to stay with nil or minimal side effects. Therefore, complete characterizations of the particulate system are essentially to make a decision whether the use of nanocarriers systems are appropriate for specific *in vivo* applications or not. The nanoparticles can be described by the following physicochemical properties according to their distribution within the body system: size distribution, surface charge modification ,targeting, cellular uptake, bio-stability, metabolism, toxicity, capacity for protein adsorption, surface hydrophobicity, rate of loading , release kinetics, surface characteristics, density, porosity, degeneration of carrier system, cristallinity, density, mobility and the molecular weight *(Neubergera et al., 2005)*.

Most intravenous administrated nanoparticles are recognized as ''foreign'' from the body system and are eliminated immediately through macrophages of the mononuclear phagocytosis system (MPS) depending on the size. The size of particles usually refers to the total diameter of the particles including the core and the coating layer. It is well known that, the smallest diameter of capillaries in the body is 4 µm. So, NPs smaller than 4µm are taken up through cells of the reticuloendothelial system (RES) mainly in the liver (60–90%) and spleen (3–10%). While small particles up to 150 nm will be phagocytosed through liver cells. There is a tendency for particles larger than 200 nm to be filtered by the venous sinuses of the spleen, as well as will be captured and withheld in the lungs. In general, the large particles are eliminated faster from the blood, and have short plasma half-life-period

Particles with large sizes and/or aggregations of small particles such as magnetic nanoparticles (MNPs) may be trapped causing emboli within the capillary bed of the lungs. Therefore, it is important to know the surface charge and aggregation behavior of the

All bare nanoparticles are unsuitable for *in vivo* applications, where the particle surface would be exposed to a biological environment and oxidized during application. Using bare nanoparticles directly, this could damage its structures and its properties. On the other hand, nanoparticles in solid phase cannot be injected into human body. So, before injection NPs have to be dispersed to hydrophilic solvent via specific interaction between the

The surface charge also plays an important role during endocytosis process. There should be a slower uptake for negatively charged particles due to the negative ''rejection'' effect of the negatively charged cell membrane. However, the endocytosis index *in vitro* is minimal with

*2.2.4. Characteristics of magnetic nanomaterials for in vivo bio-applications* 

figure (2).

*2.2.4.1. Size distribution* 

compared to the small particles (*Muller et al., 1997)*.

particles in the blood circulation system *(Neuberger et al., 2005)*.

nanoparticles surface and surfactants *(Harisinghani et al., 2003)*.

*2.2.4.2. Surface charge and protein adsorption* 

Diamagnetic materials are characterized by coupled or paired magnetic dipoles, so there is no permanent net magnetic moment per atom. That is to say that these materials have not any interactions or slightly repelled with the magnetic field. The magnetic susceptibility of these materials is negative and independent on temperature.

Paramagnetic materials characterized by randomly oriented (or uncoupled) magnetic dipoles, this can be aligned only in the presence of an external magnetic field along its direction. This type of material has neither coercivity nor remanence, which means that when the external magnetic field is switched off the internal magnetic dipoles randomize again. No extra energy is required to demagnetize the material and hence the initial zero net magnetic moment is spontaneously recovered.

Ferromagnetic materials characterized by individual magnetic dipoles in a crystal, those can align parallel one to the other, hence exhibiting an enhanced collective response even in the absence of an external magnetic field. This is what is known as *ferromagnetism*. Beside strong intensity of magnetization, the fundamental property of ferromagnetic solids is their ability to record the direction of an applied magnetic field. When the magnetic field is removed, the magnetization does not return to zero but retains a record of the applied field.

Ferrimagnetic and anti-ferromagnetic materials, in contrast to the ferromagnetic situation, neighboring magnetic dipoles can align antiparallel in the lattice, which means that they will cancel each other i.e. repulsion of magnetic dipoles . This type of magnetic exchange can lead to two different situations. The first is *ferrimagnetism;* when the two coupled spins show different values, and in that case a net magnetic dipole different than zero will still magnetize the material even in the absence of an external magnetic field. While the second is *anti-ferromagnetism;* when the magnetic dipoles or interacting spins have the same value and hence the material shows a net zero magnetization. The latter case lacks of interest for biomedical applications due to zero net magnetic moment arising in such materials.

Super-paramagnetic materials, bulky sized particles of magnetic materials such as (Fe), (Co) or (Ni), as well as some of their alloys (FePt & FeCo) have ferromagnetic properties due to their multi-domain structures of the particles. In contrast, at the nanometer scale of approximately 14 nm, the multi-domain combined together forming a single domain crystal, which is classified as super -paramagnetic *(Schmidt, 2001)*. Super-paramagnetic iron oxide nanoparticles are special class of paramagnetic materials which combine ferromagnetic and paramagnetic properties due to high magnetic moments which are observed under the effect of a magnetic field, but no remanent magnetic moment will be present when the external magnetic field is removed. This property translates into a significant advantage especially *in vivo* experiments, where the absence of coercivity or in other words the zero net magnetic moment of the nanoparticles after concluding the diagnostic measurement or the therapy will prevent the potential aggregation of the particles that could easily cause the formation of embolisms in the blood vessels (*Thorek et al., 2006)*.The path of magnetization M as a function of applied field H is called a hysteresis loop or M-H curve as shown in figure (2).
