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

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)*.

#### *2.2.4.1. Size distribution*

106 Hyperthermia

materials can be classified into; diamagnetic, paramagnetic, ferromagnetic, ferrimagnetic,

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

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

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

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

magnetization does not return to zero but retains a record of the applied field.

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

anti-ferromagnetic and super-paramagnetic *(Cozzoli et al., 2006)* as shown in Fig (1).

these materials is negative and independent on temperature.

magnetic moment is spontaneously recovered.

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

#### *2.2.4.2. Surface charge and protein adsorption*

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

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

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

a zeta potential close to zero. In contrast, Phagocytosis process is increased with a higher surface charge independent of whether the charge is negative or positive. The higher the surface charge the shorter is the residence time of nanoparticles in the circulatory system *(Neuberger et al., 2005)*.

Laser and Radiofrequency Induced Hyperthermia Treatment via Gold-Coated Magnetic Nanocomposites 109

phagocytotic activity increases with size of particles, whereas smaller particles<150 nm can be up-taken by all types of cells through pinocytosis process (cell drinking).The third is nonspecific endocytosis or receptor-mediated endocytosis process *(Neuberger et al., 2005)*.Superparamagnetic iron oxide nanoparticles have been shown to be uptake by a receptor

When nanoparticles are introduced into the body, several aspects can compromise its stability. First of all, the physiological media have different ionic strength as compared with the ultrapure water mainly used in laboratories: increasing the ionic strength of aqueous solution will suppress the electric double layer around the charged particles, resulting in a partial or total aggregation of the system. A similar behavior could be observed by the particles once they enter specific body compartments, due to a variation in pH with respect to the media in which the nanoparticles are initially dispersed. In addition, when nanoparticles are injected in the blood circulation system, a nonspecific adsorption of plasma proteins onto nanoparticles surface *"opsonization"* will occur, this phenomenon is more pronounced for nanometer size particles due to two main effects: the high surface to volume ratio, as well as the attractive forces between the nanoparticles such as magnetic nanoparticles. When this phenomenon occurs, a fast clearance of the nanoparticles is observed. To prevent such effects, several synthetic and natural polymers have been introduced to the nanoparticles surface including PEG and dextrin (*Kohler et al.,* 

The metabolism process of the nanoparticles is another issue that should be taken into account. For example, iron oxide nanoparticles can be present in two different oxidation states: the ferrous Fe (II) form which will be oxidized by endogenous molecular oxygen, resulting in the conversion of ferrous iron to ferric Fe (III). Ferric iron is the preferred physiological oxidation state of iron; Fe (III) is highly reactive and can induce catalytic activity that may result in severe oxidative cell damage. As a result, iron carrier proteins and chelates are used to allow for safe transfer of iron from cell to cell within the body, and for safe intra-cellular storage of excess iron. The natural eventual fate of Fe3O4 nanoparticles above approximately 200 nm in diameter is to reside in macrophage-rich tissue, such as the liver and spleen (peak concentration at 2 hours after contrast intake).While particles below 10 nm are removed rapidly through extravasations and renal clearance *(Gupta et al.,* 2004).

Biocompatibility is one of the most important considerations in the development of biomedical applications of nanomaterials. Most of the magnetic nanowires are compatible with living cells. They can be functionalized with biologically active molecules, and they do not disrupt normal cell functions, such as cell proliferation and adhesion, and gene

mediated endocytosis process (*Raynal et al., 2004)*.

*2.2.4.5. Bio-stability* 

*2004)*.

*2.2.4.6. Metabolism* 

*2.2.4.7. Biocompatibility* 

expression (*Hultgren et al. 2005*).

The adsorption of proteins at the particle surface is called "*opsonization".* This phenomenon results from immediate interaction between nanoparticles with plasma proteins after intravenous injection .The amount of adsorbed proteins is based on the size of the molecules, as well as the surface charge of the particle, where the capacity of protein adsorption increases by increasing size and charge of the particles. The adsorbed protein components play an important role in the biodistribution, degradation and elimination of the nanoparticles. Therefore, the treatment method of the nanoparticles surface must be addressed *(Muller et al., 1997)*.

The surface charge and protein adsorption capability are more related to the surfactants bond to the nanoparticles surface. There is another important role of surfactants on nanoparticles, when the NPs are injected into human body as contrast agents, these nanoparticles must locate the targeting area accurately and rapidly. Appropriate surfactant could achieve such objective. Some experiments *in vitro* already proved folate-mediated nanoparticles composed of ploy ethylene glycol (PEG) / poly ε-caprolactone have potential of tumor cell-selective targeting *(Gee et al., 2003)*.

#### *2.2.4.3. Targeting*

All *in vivo* applications require that the NPs should accurately localize to therapeutic sites. All targeting methods could be classified to passive, active and physical targeting. The physical targeting is the localization of the nanoparticles with external assistance, typically by applied magnetic field from outside of the body; the physical targeting has less capability to recognize specific cells or tissues. The passive targeting based only on the disrupted endothelial lining of tumor tissues; enhanced penetration and retention (EPR) allows nanoparticles of smaller size to pass, and accumulate in the tumor. In active targeting, specific targeting functional groups, such as monoclonal antibodies, are immobilized on the particle surface to efficiently increase the chance of uptake by specific cells *(Kelly et al., 2005)*.

#### *2.2.4.4. Cellular uptake*

Cellular uptake of nanoparticles is another issue that should be taken into account, when considering their use in diagnostic and therapeutic applications. The cellular uptake of nanoparticles is strongly dependent on particle size as it was proven *in vitro* and *in vivo*. In general, small nanoparticles can go deeper into tissue than larger particles and often penetrate the cell itself *(Leslie-Pelecky, 2007)*. Lewinski et al summarized the situation for many types of nanoparticles *(Lewinski et al., 2008)*.

The cellular uptake of nanoparticles occurs through a process known as endocytosis, which can be generally classified into three processes depending on nanoparticles size. Phagocytosis process which is the predominant mechanism for uptake of large particles, phagocytotic activity increases with size of particles, whereas smaller particles<150 nm can be up-taken by all types of cells through pinocytosis process (cell drinking).The third is nonspecific endocytosis or receptor-mediated endocytosis process *(Neuberger et al., 2005)*.Superparamagnetic iron oxide nanoparticles have been shown to be uptake by a receptor mediated endocytosis process (*Raynal et al., 2004)*.

#### *2.2.4.5. Bio-stability*

108 Hyperthermia

*(Neuberger et al., 2005)*.

addressed *(Muller et al., 1997)*.

*2.2.4.3. Targeting* 

*2.2.4.4. Cellular uptake* 

of tumor cell-selective targeting *(Gee et al., 2003)*.

many types of nanoparticles *(Lewinski et al., 2008)*.

a zeta potential close to zero. In contrast, Phagocytosis process is increased with a higher surface charge independent of whether the charge is negative or positive. The higher the surface charge the shorter is the residence time of nanoparticles in the circulatory system

The adsorption of proteins at the particle surface is called "*opsonization".* This phenomenon results from immediate interaction between nanoparticles with plasma proteins after intravenous injection .The amount of adsorbed proteins is based on the size of the molecules, as well as the surface charge of the particle, where the capacity of protein adsorption increases by increasing size and charge of the particles. The adsorbed protein components play an important role in the biodistribution, degradation and elimination of the nanoparticles. Therefore, the treatment method of the nanoparticles surface must be

The surface charge and protein adsorption capability are more related to the surfactants bond to the nanoparticles surface. There is another important role of surfactants on nanoparticles, when the NPs are injected into human body as contrast agents, these nanoparticles must locate the targeting area accurately and rapidly. Appropriate surfactant could achieve such objective. Some experiments *in vitro* already proved folate-mediated nanoparticles composed of ploy ethylene glycol (PEG) / poly ε-caprolactone have potential

All *in vivo* applications require that the NPs should accurately localize to therapeutic sites. All targeting methods could be classified to passive, active and physical targeting. The physical targeting is the localization of the nanoparticles with external assistance, typically by applied magnetic field from outside of the body; the physical targeting has less capability to recognize specific cells or tissues. The passive targeting based only on the disrupted endothelial lining of tumor tissues; enhanced penetration and retention (EPR) allows nanoparticles of smaller size to pass, and accumulate in the tumor. In active targeting, specific targeting functional groups, such as monoclonal antibodies, are immobilized on the particle surface to efficiently increase the chance of uptake by specific cells *(Kelly et al., 2005)*.

Cellular uptake of nanoparticles is another issue that should be taken into account, when considering their use in diagnostic and therapeutic applications. The cellular uptake of nanoparticles is strongly dependent on particle size as it was proven *in vitro* and *in vivo*. In general, small nanoparticles can go deeper into tissue than larger particles and often penetrate the cell itself *(Leslie-Pelecky, 2007)*. Lewinski et al summarized the situation for

The cellular uptake of nanoparticles occurs through a process known as endocytosis, which can be generally classified into three processes depending on nanoparticles size. Phagocytosis process which is the predominant mechanism for uptake of large particles, When nanoparticles are introduced into the body, several aspects can compromise its stability. First of all, the physiological media have different ionic strength as compared with the ultrapure water mainly used in laboratories: increasing the ionic strength of aqueous solution will suppress the electric double layer around the charged particles, resulting in a partial or total aggregation of the system. A similar behavior could be observed by the particles once they enter specific body compartments, due to a variation in pH with respect to the media in which the nanoparticles are initially dispersed. In addition, when nanoparticles are injected in the blood circulation system, a nonspecific adsorption of plasma proteins onto nanoparticles surface *"opsonization"* will occur, this phenomenon is more pronounced for nanometer size particles due to two main effects: the high surface to volume ratio, as well as the attractive forces between the nanoparticles such as magnetic nanoparticles. When this phenomenon occurs, a fast clearance of the nanoparticles is observed. To prevent such effects, several synthetic and natural polymers have been introduced to the nanoparticles surface including PEG and dextrin (*Kohler et al., 2004)*.

### *2.2.4.6. Metabolism*

The metabolism process of the nanoparticles is another issue that should be taken into account. For example, iron oxide nanoparticles can be present in two different oxidation states: the ferrous Fe (II) form which will be oxidized by endogenous molecular oxygen, resulting in the conversion of ferrous iron to ferric Fe (III). Ferric iron is the preferred physiological oxidation state of iron; Fe (III) is highly reactive and can induce catalytic activity that may result in severe oxidative cell damage. As a result, iron carrier proteins and chelates are used to allow for safe transfer of iron from cell to cell within the body, and for safe intra-cellular storage of excess iron. The natural eventual fate of Fe3O4 nanoparticles above approximately 200 nm in diameter is to reside in macrophage-rich tissue, such as the liver and spleen (peak concentration at 2 hours after contrast intake).While particles below 10 nm are removed rapidly through extravasations and renal clearance *(Gupta et al.,* 2004).

#### *2.2.4.7. Biocompatibility*

Biocompatibility is one of the most important considerations in the development of biomedical applications of nanomaterials. Most of the magnetic nanowires are compatible with living cells. They can be functionalized with biologically active molecules, and they do not disrupt normal cell functions, such as cell proliferation and adhesion, and gene expression (*Hultgren et al. 2005*).

### *2.2.4.8. Toxicity*

The non-cytotoxic, non-immunogenic and biocompatible properties of nanoparticles are important issues for the potential application in nanoimmunology, nanomedicines and nanobiotechnology. When discussing the toxicity of nanoparticles, generalization becomes difficult because their toxicity depends on numerous factors including the dose, chemical composition, method of administration, size, biodegradability, solubility, pharmacokinetics, biodistribution, surface chemistry, design, shape and structure. In general, size, surface area, shape, composition and coating of nanoparticles are the most important characteristics regarding cytotoxicity *(Neuberger et al., 2005)*.

Laser and Radiofrequency Induced Hyperthermia Treatment via Gold-Coated Magnetic Nanocomposites 111

Nanotechnology, dealing with nanoscale objects, has been developed at three major levels: nanomaterials, nanodevices and nanosystems. At present, the nanomaterials level is the most advanced of the three. Nanomaterials are of great importance both in scientific investigations and commercial applications due to their size-dependent physical and chemical properties. Nanomaterials with various shapes have been developed successfully. Common morphologies are quantum dots, nanoparticles/nanocrystals, nanowires, nanorods, nanotubes, etc. It is desirable to have a full range within the nanomaterial family

because many applications demand particular nanomaterials with special structures.

paramagnetism) with an extremely large magnetic moment and large susceptibilities.

Magnetic nanoparticles have found many successful industrial applications. Recently, tremendous research efforts have been stimulated on the usage of magnetic nanoparticles in

Understanding of biological processes and hence developing biomedical means have been continuously pursued. These aims are one of strong driving forces behind the development of nanotechnology. The interests on magnetic nanoparticles for bio-applications come from their comparable dimensions to biological entities coupled with their unique magnetic behaviors. Though common living organisms are composed of cells of about 10 µm size, the cell components are much smaller and generally in the nanosize dimension. Examples are viruses (20–450 nm), proteins (5–50 nm) and genes (2 nm wide and 10–100 nm long). Synthetic magnetic nanoparticles have controllable dimensions and just a few nanometerdiameter nanoparticles can be synthesized by carefully designing experimental procedures and controlling experimental conditions. With such a nanoscale dimension, it would be possible for magnetic nanoparticles to get close to a biological entity of interest. Moreover, the interaction between magnetic nanoparticles and biological entities can be adjusted by coating nanoparticles with biological molecules, called bio-functionalization. This offers a controllable means of 'tagging' or addressing the binding at nanoscale. The comparable dimensions and magnetic properties of magnetic nanoparticles have prompted the idea of using them as very small probes to spy on the biological processes at the cellular scale without introducing too much interference. Actually, optical and magnetic effects have been treated as the most suitable approaches for biological applications owing to their non-

Magnetic nanoparticles, being a sub-family of nanomaterials, exhibit unique magnetic properties in addition to other specific characteristics. Their remarkable new phenomena include super-paramagnetism, high saturation field, high field irreversibility, extra anisotropy, and temperature-depended hysteresis, etc. Research investigation has revealed that the finite size and surface effects of magnetic nanoparticles determine their magnetic behavior. For instance, a single magnetic domain forms when the size of a ferromagnetic nanoparticle is less than 15 nm. In other words, an ultrafine ferromagnetic nanoparticle displays a state of uniform magnetization under any field. Thus, at temperatures above the blocking temperature, these nanoparticles show identical magnetization behavior to atomic paramagnets (super-

*2.2.5. Biomedical applications of iron oxide NPs* 

the field of biomedical and biological applications.

invasive behavior.

Several *in vivo* studies on animals had shown that, with a large dosage of 3,000 µmol Fe based nanoparticles per kg body weight, the histology and serologic blood tests indicated that no side effects occurred after 7 days of treatment *(Lacava et al., 1999)*.

To minimize the risks posed by nanoparticles, there are two basic avenues. One is to develop new highly biocompatible nonmaterials with low toxicity such as silica nanoparticles. Another one is the surface modification of nanoparticles with biocompatible chemicals such as PEG, dextrin and chitosan. Thus many great efforts are being made to develop nanoparticles satisfactory for biomedical applications *(Cho, 2009)*.

### *2.2.4.9. Easy detection*

As almost all biological entities are non-magnetic, magnetic nanoparticles in biological systems can be easily detected and traced. One typical example is the enhancement of the signal from magnetic resonance imaging (MRI) using magnetic nanoparticles. In this technique, a subject is placed in a large, external magnetic field and then exposed to a pulse of radio waves. Changes to the spin of the protons in water molecules are measured after the pulse is turned off. Tiny differences in the way that protons in different tissues behave can then be used to build up a 3D image of the subject (*Koltsov 2004*).

#### *2.2.4.10. Magnetic manipulation*

Magnetic nanoparticles will rotate under an external uniform magnetic field, and will make translational movements under an external magnetic field gradient. Therefore, magnetic nanoparticles, or magnetically tagged molecules, can be manipulated by applying an external magnetic field. This is important for transporting magnetically tagged drug molecules to diseased sites. The magnetic manipulation of magnetic nanowires and nanotubes is important for applying forces to biological entities, and for nanowires or nanotubes to get into biological entities.

### *2.2.4.11. Energy transfer*

Magnetic nanoparticles can resonantly respond to a time-varying magnetic field, transferring energy from the exciting magnetic field to the nanoparticles and the tagged biological entities. This property has been used in hyperthermia treatment of cancer tumors (*Pankhurst et al. 2003*).
