*2.2.5. Biomedical applications of iron oxide NPs*

110 Hyperthermia

*2.2.4.8. Toxicity* 

*2.2.4.9. Easy detection* 

*2.2.4.10. Magnetic manipulation* 

*2.2.4.11. Energy transfer* 

(*Pankhurst et al. 2003*).

nanotubes to get into biological entities.

regarding cytotoxicity *(Neuberger et al., 2005)*.

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

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

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

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

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

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

that no side effects occurred after 7 days of treatment *(Lacava et al., 1999)*.

develop nanoparticles satisfactory for biomedical applications *(Cho, 2009)*.

can then be used to build up a 3D image of the subject (*Koltsov 2004*).

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.

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 (superparamagnetism) 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 the field of biomedical and biological applications.

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 noninvasive behavior.

In view of the magnetic properties of magnetic nanoparticles, they can be manipulated by an external magnetic field gradient, which is described by Coulomb's law. Magnetic nanoparticles are able to transport into human tissues due to the intrinsic penetrability of magnetic fields into human bodies. This 'action at a distance' opens up many potential bioapplications including transportation of magnetically tagged biological entities, targeted drug delivery, etc. Another important property of magnetic nanoparticles is their resonant response related to a time-varying magnetic field *(Pankhurst et al. 2003)*. Hence energy transfer from the exciting field to the magnetic nanoparticles can be realized. In this way, toxic amounts of thermal energy are able to be delivered via magnetic nanoparticles to the targeted tumors resulting in malignant cell destruction. This process is named hyperthermia, which will be addressed in detail in this chapter. In addition to the sitespecific drug delivery and hyperthermic treatment, magnetic nanoparticles have found other versatile bio-applications such as magnetic bio-separation, contrast enhancement of magnetic resonance imaging, gene therapy, enzyme immobilization, magnetic manipulation of cell membranes, immunoassays, magnetic bio-sensing, etc. *(Sun et al. 2005)*. Each application depends upon the relationship between the external magnetic field and the biological system. Magnetic fields with proper field strength are not deleterious to either biological tissues or biotic environments. In a given bio-application, magnetic nanoparticles are usually injected intravenously into the human body and are transported to the targeted region via blood circulation for biomedical diagnostic or treatment. An alternative means is using magnetic nanoparticle suspension for injection *(Berry 2003)*. It has been well accepted that a desirable magnetic medium should not contain nanoparticle aggregation, which will block its own spread. For this reason, stable, uniform magnetic nanoparticle dispersion in either an aqueous or organic solvent at neutral pH and physiological salinity is required. The stability of this magnetic colloidal suspension depends on two parameters: an ultra small dimension and surface chemistry. The particle size should be sufficiently small to avoid precipitation due to gravitation forces while the charge and surface groups should create both steric and coulombic repulsions which stabilize the colloidal suspensions.

Laser and Radiofrequency Induced Hyperthermia Treatment via Gold-Coated Magnetic Nanocomposites 113

major categories: *in vivo* and *in vitro* applications. Consequently, additional restrictions apply on various magnetic nanoparticles for *in vivo* or *in vitro* biomedical applications. It is rather simple for *in vitro* applications of magnetic nanoparticles. The size restriction as well as biocompatibility/toxicity is not so critical for *in vitro* applications, when compared with *in vivo* ones. Therefore, super-paramagnetic composites containing submicron diamagnetic matrixes and super-paramagnetic nanocrystals can be used. Composites with long sedimentation times in the absence of a magnetic field are also acceptable. It was noticed that functionalities may be provided readily for the super-paramagnetic composites because of the diamagnetic matrixes *(Tartaj et al. 2003)*. On the other hand, severe restrictions must be applied for magnetic nanoparticles for *in vivo* biomedical applications. First of all, it is a requisite that the magnetic components should be biocompatible without any toxicity for the bio-systems of interest. This is predominantly determined by the nature of the material (e.g. iron, nickel, cobalt, metal alloy, etc). For instance, cobalt and nickel are highly magnetic materials. However, both of them are rarely used due to their toxic properties and susceptibility to oxidation. Currently, the most commonly employed magnetic nanoparticles in biomedical applications are iron oxides including magnetite (Fe3O4), maghemite (*γ* - Fe2O3) and hematite (*α*-Fe2O3). The second requirement for magnetic nanoparticles is their particle sizes. Ultrafine nanoparticles (usually smaller than 100 nm in diameter) have high effective surface area, thus they can be attached to ligand easily. Also the lower sedimentation rate leads to a high stability for colloidal suspensions, and the tissue diffusion can be improved by using nanoparticles in nanometer dimensions. After injection, nanoparticles would be able to remain in the circulation and pass through the capillary systems to reach the targeted organs and tissues without any vessel embolism. Further, the magnetic dipole–dipole interaction among magnetic nanoparticles can be substantially reduced. The third requisite for magnetic nanoparticles is their biocompatible polymer coating which may be done during or after the nanoparticle synthesis. There are several functions of the coating layers: 1) they will prohibit agglomeration of nanoparticles; 2) they prevent structural or elemental changes; 3) unnecessary biodegradation can be stopped; 4) the polymer layer offers a covalent binding or adsorption attachment of drugs to the nanoparticle surface. In summary, for *in vivo* biomedical applications, magnetic nanoparticles must be made of a non-toxic and non-immunogenic material with ultra small

It is no doubt that interdisciplinary research collaboration is badly needed for clinical and biological applications of magnetic nanoparticles *(Berry 2003)*. Research fields involved include chemistry, materials science, cell engineering, clinical tests and other related scientific efforts. In this chapter, an overview of cancer treatment approach as one of

Another major use of magnetic nanoparticles in therapeutic treatment is hyperthermia treatment for cancers. Gilchrist *et al*. did the experimental investigations for the first time when they heated various tissue samples with *γ* - Fe2O3 of 20–100 nm in diameter by a

biomedical applications of magnetic nanoparticles will be presented

particle sizes and high magnetization.

**3. Hyperthermia treatment** 

The magnetic properties of magnetic nanoparticles are determined by their elemental compositions, crystallinity, shapes and dimensions. Various magnetic nanoparticles have been developed. Therefore, the selection of proper magnetic nanoparticles with the desired properties is the first but crucial step for certain bio-applications. For example, ferromagnetic nanoparticles (e.g. Fe nanoparticles) have a large magnetic moment and they can be the best material candidate in magnetic biosensors because they not only produce a better signal but respond to an applied magnetic field readily. On the other hand, iron oxide nanoparticles with super-paramagnetic behavior do an excellent job when used to enhance the signals in magnetic resonance imaging examinations. With the help of iron oxide nanoparticles a sharpened image with detailed information can be achieved because of the change of behavior of nearby bio-molecules by introduced nanoparticles *(Bystrzejewski et al. 2005)*. For many biomedical applications, magnetic nanoparticles presenting superparamagnetic behavior (no remanence along with a rapidly changing magnetic state) at room temperature are desirable. Biomedical applications are commonly divided into two major categories: *in vivo* and *in vitro* applications. Consequently, additional restrictions apply on various magnetic nanoparticles for *in vivo* or *in vitro* biomedical applications. It is rather simple for *in vitro* applications of magnetic nanoparticles. The size restriction as well as biocompatibility/toxicity is not so critical for *in vitro* applications, when compared with *in vivo* ones. Therefore, super-paramagnetic composites containing submicron diamagnetic matrixes and super-paramagnetic nanocrystals can be used. Composites with long sedimentation times in the absence of a magnetic field are also acceptable. It was noticed that functionalities may be provided readily for the super-paramagnetic composites because of the diamagnetic matrixes *(Tartaj et al. 2003)*. On the other hand, severe restrictions must be applied for magnetic nanoparticles for *in vivo* biomedical applications. First of all, it is a requisite that the magnetic components should be biocompatible without any toxicity for the bio-systems of interest. This is predominantly determined by the nature of the material (e.g. iron, nickel, cobalt, metal alloy, etc). For instance, cobalt and nickel are highly magnetic materials. However, both of them are rarely used due to their toxic properties and susceptibility to oxidation. Currently, the most commonly employed magnetic nanoparticles in biomedical applications are iron oxides including magnetite (Fe3O4), maghemite (*γ* - Fe2O3) and hematite (*α*-Fe2O3). The second requirement for magnetic nanoparticles is their particle sizes. Ultrafine nanoparticles (usually smaller than 100 nm in diameter) have high effective surface area, thus they can be attached to ligand easily. Also the lower sedimentation rate leads to a high stability for colloidal suspensions, and the tissue diffusion can be improved by using nanoparticles in nanometer dimensions. After injection, nanoparticles would be able to remain in the circulation and pass through the capillary systems to reach the targeted organs and tissues without any vessel embolism. Further, the magnetic dipole–dipole interaction among magnetic nanoparticles can be substantially reduced. The third requisite for magnetic nanoparticles is their biocompatible polymer coating which may be done during or after the nanoparticle synthesis. There are several functions of the coating layers: 1) they will prohibit agglomeration of nanoparticles; 2) they prevent structural or elemental changes; 3) unnecessary biodegradation can be stopped; 4) the polymer layer offers a covalent binding or adsorption attachment of drugs to the nanoparticle surface. In summary, for *in vivo* biomedical applications, magnetic nanoparticles must be made of a non-toxic and non-immunogenic material with ultra small particle sizes and high magnetization.

It is no doubt that interdisciplinary research collaboration is badly needed for clinical and biological applications of magnetic nanoparticles *(Berry 2003)*. Research fields involved include chemistry, materials science, cell engineering, clinical tests and other related scientific efforts. In this chapter, an overview of cancer treatment approach as one of biomedical applications of magnetic nanoparticles will be presented

#### **3. Hyperthermia treatment**

112 Hyperthermia

In view of the magnetic properties of magnetic nanoparticles, they can be manipulated by an external magnetic field gradient, which is described by Coulomb's law. Magnetic nanoparticles are able to transport into human tissues due to the intrinsic penetrability of magnetic fields into human bodies. This 'action at a distance' opens up many potential bioapplications including transportation of magnetically tagged biological entities, targeted drug delivery, etc. Another important property of magnetic nanoparticles is their resonant response related to a time-varying magnetic field *(Pankhurst et al. 2003)*. Hence energy transfer from the exciting field to the magnetic nanoparticles can be realized. In this way, toxic amounts of thermal energy are able to be delivered via magnetic nanoparticles to the targeted tumors resulting in malignant cell destruction. This process is named hyperthermia, which will be addressed in detail in this chapter. In addition to the sitespecific drug delivery and hyperthermic treatment, magnetic nanoparticles have found other versatile bio-applications such as magnetic bio-separation, contrast enhancement of magnetic resonance imaging, gene therapy, enzyme immobilization, magnetic manipulation of cell membranes, immunoassays, magnetic bio-sensing, etc. *(Sun et al. 2005)*. Each application depends upon the relationship between the external magnetic field and the biological system. Magnetic fields with proper field strength are not deleterious to either biological tissues or biotic environments. In a given bio-application, magnetic nanoparticles are usually injected intravenously into the human body and are transported to the targeted region via blood circulation for biomedical diagnostic or treatment. An alternative means is using magnetic nanoparticle suspension for injection *(Berry 2003)*. It has been well accepted that a desirable magnetic medium should not contain nanoparticle aggregation, which will block its own spread. For this reason, stable, uniform magnetic nanoparticle dispersion in either an aqueous or organic solvent at neutral pH and physiological salinity is required. The stability of this magnetic colloidal suspension depends on two parameters: an ultra small dimension and surface chemistry. The particle size should be sufficiently small to avoid precipitation due to gravitation forces while the charge and surface groups should

create both steric and coulombic repulsions which stabilize the colloidal suspensions.

The magnetic properties of magnetic nanoparticles are determined by their elemental compositions, crystallinity, shapes and dimensions. Various magnetic nanoparticles have been developed. Therefore, the selection of proper magnetic nanoparticles with the desired properties is the first but crucial step for certain bio-applications. For example, ferromagnetic nanoparticles (e.g. Fe nanoparticles) have a large magnetic moment and they can be the best material candidate in magnetic biosensors because they not only produce a better signal but respond to an applied magnetic field readily. On the other hand, iron oxide nanoparticles with super-paramagnetic behavior do an excellent job when used to enhance the signals in magnetic resonance imaging examinations. With the help of iron oxide nanoparticles a sharpened image with detailed information can be achieved because of the change of behavior of nearby bio-molecules by introduced nanoparticles *(Bystrzejewski et al. 2005)*. For many biomedical applications, magnetic nanoparticles presenting superparamagnetic behavior (no remanence along with a rapidly changing magnetic state) at room temperature are desirable. Biomedical applications are commonly divided into two

Another major use of magnetic nanoparticles in therapeutic treatment is hyperthermia treatment for cancers. Gilchrist *et al*. did the experimental investigations for the first time when they heated various tissue samples with *γ* - Fe2O3 of 20–100 nm in diameter by a 1.2MHz magnetic field *(Gilchrist et al. 1957)*. Since then, studies have shown the feasibility of using the hyperthermic effect generated from magnetic nanoparticles by applying a highfrequency AC magnetic field as an alternate therapeutic approach for cancer treatment. Briefly speaking, the hyperthermic effect is generated from the relaxation of magnetic energy of the magnetic nanoparticles which is able to destroy tumor cells effectively *(Levy et al. 2002)*. Hyperthermia is a common cancer therapy in which certain organs or tissues are heated preferentially to temperatures between 41 ◦C and 46 ◦C, artificially induced hyperthermia has been designed to heat malignant cells without destroying the surrounding healthy tissue. When heated to a higher temperature (~56 ◦C), coagulation or carbonization may occur. This 'thermo-ablation' induces a completely different biological response and hence is not considered as hyperthermia. A classical hyperthermia not only causes almost reversible damage to cells and tissues, but also enhances radiation injury of tumor cells *(Jordan et al. 1999)*. For modern clinical hyperthermia trials, moderate temperatures (42–43 ◦C) are normally selected to optimize the thermal homogeneity in the target area. It is true that the heating effect will change the dose-dependent behavior of treated cells. However, the exact mechanism of thermal dose-response in hyperthermia is still unknown. There are great difficulties in identifying the individual cell as the target for hyperthermia. Instead, hyperthermia affects most bio-molecules including proteins and receptor molecules. On the other hand, DNA damage by irradiation has been well understood due to the highly specific interaction. As far as the underlying physics of the heating effect in hyperthermia is concerned, magnetic heating via magnetic nanoparticles essentially is determined by their sizes and magnetic properties *(Mornet et al. 2004)*.

Magnetic nanoparticles can be divided into two major categories: multi-domain and singledomain nanoparticles, which possess different heating effects. Multi-domain nanoparticles usually have larger dimensions and contain several sub-domains with definite magnetization direction for each. When they are exposed to a magnetic field, a phenomenon called 'domain wall displacements' occurs. This is featured by growth of the domain with magnetization direction along the magnetic field axis and shrinkage of the other. Figure 2 above, depicts this irreversible phenomenon. It can be seen that the magnetization curves for increasing and decreasing magnetic field do not coincide, and the area within the hysteresis loop represents the heating energy, named 'hysteresis loss', due to the AC magnetic field. For single-domain nanoparticles, since there is no domain wall, no hysteresis loss occurs leading to no heating. When exposed to an external AC magnetic field, rotation of magnetic moments from super-paramagnetic nanoparticles is assisted by the supplied energy which overcomes the energy barrier. Then these nanoparticles undergo N´eel relaxation in which their moments relax to their equilibrium orientation. Simultaneously, heat is generated during this relaxation by thermal dissipation. The N´eel relaxation time tN is related to the temperature, and can be described as:

$$\text{At} = \text{toe KV/kT} \tag{1}$$

Laser and Radiofrequency Induced Hyperthermia Treatment via Gold-Coated Magnetic Nanocomposites 115

This rotation is caused by the torque exerted on the magnetic moment by the AC magnetic

 *tB* = 3*ηvB*/*kT* (2)

Where *η* is the viscosity of the carrier, and vB is the frequency for maximal heating via Brown rotation, corresponding to the hydrodynamic volume of the particle, and it is given

The heating capacity of magnetic nanoparticles is expressed by specific absorption rate SAR, also called specific power loss (SPL), both of them have the same physical meaning, which determines the heating ability of magnetic nanoparticles in the presence of magnetic field, and can be defined as the amount of heat generated per unit gram per unit time. SAR values are usually expressed in watts per gram of magnetic material (W/g), also can be expressed in volumetric units (W/m3).The heat generated per unit volume can be obtained by multiplying the SAR value by the density of the nanoparticles. It has been well documented that the orientation and magnetized domains of magnetic nanoparticles are dependent on their intrinsic features (elemental composition, crystallinity, magneto anisotropy, shape, dimension, etc.) and micro-structural features (impurities, grain boundaries, vacancies, etc). In magnetic hyperthermia treatment, after heat conducts into the area with diseased tissues, the surrounding temperature can be maintained above the therapeutic threshold of 42 ◦C for about half an hour to destroy the cancer. It is of great importance for hyperthermia to minimize the heat effect on healthy cells. Assisted by magnetic nanoparticles, it is possible to heat the specific area while unacceptable coincidental heating of healthy tissue is avoided. Although the hyperthermia treatment for cancer has been demonstrated with therapeutic efficacy in animal models, however, there have been no reports of successful hyperthermia treatment for human patients. The major reasons are the necessities of an adequate amount of magnetic nanoparticles and sufficiently high magnetic field which are not safe for human treatments. To date, laboratory research efforts on hyperthermia treatment for animals have all used magnetic field conditions which are not clinically acceptable. In most instances, hyperthermia treatments with a reduced amount of magnetic nanoparticles and reduced field strength or frequency cannot be effective due to the reduction of heat generated. Simulations suggest a sufficient level with heat deposition rate of 100mWcm−3 to destroy cancer cells effectively in most circumstances. The practical frequency and strength of the external AC magnetic field are 0.05–1.2MHz and 0–15 kAm−1, respectively. On the other hand, sufficient magnetic materials are needed to enrich around the cancer tissues to generate enough heat for hyperthermia treatment. Direct injection of ferrofluid into the tumor tissues is able to introduce a large amount of magnetic materials for heat generation. Antibody targeting and intravascular administration offer better preference heating, but the problem here is the small quantity. It is estimated that about 5–10 mg of magnetic material concentrated in each cm3 of tumor tissues is able to generate enough heat for tumor cell destruction in human bodies. Magnetite (Fe3O4) and maghemite (*γ* -Fe2O3) nanoparticles are two common types used in hyperthermia treatments owing to their appropriate magnetic

properties and their excellent biocompatibilities. Several examples will be given here.

field. The Brown relaxation time tB is described as:

by the equation 2*π*vBtB = 1.

Where t0 ≈ 10−9 s, T is the temperature and k is the Boltzmann constant. For both multi- and single-domain nanoparticles, rotational Brownian motion in a carrier also generates heat. This rotation is caused by the torque exerted on the magnetic moment by the AC magnetic field. The Brown relaxation time tB is described as:

114 Hyperthermia

1.2MHz magnetic field *(Gilchrist et al. 1957)*. Since then, studies have shown the feasibility of using the hyperthermic effect generated from magnetic nanoparticles by applying a highfrequency AC magnetic field as an alternate therapeutic approach for cancer treatment. Briefly speaking, the hyperthermic effect is generated from the relaxation of magnetic energy of the magnetic nanoparticles which is able to destroy tumor cells effectively *(Levy et al. 2002)*. Hyperthermia is a common cancer therapy in which certain organs or tissues are heated preferentially to temperatures between 41 ◦C and 46 ◦C, artificially induced hyperthermia has been designed to heat malignant cells without destroying the surrounding healthy tissue. When heated to a higher temperature (~56 ◦C), coagulation or carbonization may occur. This 'thermo-ablation' induces a completely different biological response and hence is not considered as hyperthermia. A classical hyperthermia not only causes almost reversible damage to cells and tissues, but also enhances radiation injury of tumor cells *(Jordan et al. 1999)*. For modern clinical hyperthermia trials, moderate temperatures (42–43 ◦C) are normally selected to optimize the thermal homogeneity in the target area. It is true that the heating effect will change the dose-dependent behavior of treated cells. However, the exact mechanism of thermal dose-response in hyperthermia is still unknown. There are great difficulties in identifying the individual cell as the target for hyperthermia. Instead, hyperthermia affects most bio-molecules including proteins and receptor molecules. On the other hand, DNA damage by irradiation has been well understood due to the highly specific interaction. As far as the underlying physics of the heating effect in hyperthermia is concerned, magnetic heating via magnetic nanoparticles essentially is determined by their

Magnetic nanoparticles can be divided into two major categories: multi-domain and singledomain nanoparticles, which possess different heating effects. Multi-domain nanoparticles usually have larger dimensions and contain several sub-domains with definite magnetization direction for each. When they are exposed to a magnetic field, a phenomenon called 'domain wall displacements' occurs. This is featured by growth of the domain with magnetization direction along the magnetic field axis and shrinkage of the other. Figure 2 above, depicts this irreversible phenomenon. It can be seen that the magnetization curves for increasing and decreasing magnetic field do not coincide, and the area within the hysteresis loop represents the heating energy, named 'hysteresis loss', due to the AC magnetic field. For single-domain nanoparticles, since there is no domain wall, no hysteresis loss occurs leading to no heating. When exposed to an external AC magnetic field, rotation of magnetic moments from super-paramagnetic nanoparticles is assisted by the supplied energy which overcomes the energy barrier. Then these nanoparticles undergo N´eel relaxation in which their moments relax to their equilibrium orientation. Simultaneously, heat is generated during this relaxation by thermal dissipation. The N´eel relaxation time tN is related to the

Where t0 ≈ 10−9 s, T is the temperature and k is the Boltzmann constant. For both multi- and single-domain nanoparticles, rotational Brownian motion in a carrier also generates heat.

*tN = t0e KV/kT* (1)

sizes and magnetic properties *(Mornet et al. 2004)*.

temperature, and can be described as:

$$\mathbf{t} \mathbf{a} = \mathbf{3} \eta \upsilon u / kT \tag{2}$$

Where *η* is the viscosity of the carrier, and vB is the frequency for maximal heating via Brown rotation, corresponding to the hydrodynamic volume of the particle, and it is given by the equation 2*π*vBtB = 1.

The heating capacity of magnetic nanoparticles is expressed by specific absorption rate SAR, also called specific power loss (SPL), both of them have the same physical meaning, which determines the heating ability of magnetic nanoparticles in the presence of magnetic field, and can be defined as the amount of heat generated per unit gram per unit time. SAR values are usually expressed in watts per gram of magnetic material (W/g), also can be expressed in volumetric units (W/m3).The heat generated per unit volume can be obtained by multiplying the SAR value by the density of the nanoparticles. It has been well documented that the orientation and magnetized domains of magnetic nanoparticles are dependent on their intrinsic features (elemental composition, crystallinity, magneto anisotropy, shape, dimension, etc.) and micro-structural features (impurities, grain boundaries, vacancies, etc). In magnetic hyperthermia treatment, after heat conducts into the area with diseased tissues, the surrounding temperature can be maintained above the therapeutic threshold of 42 ◦C for about half an hour to destroy the cancer. It is of great importance for hyperthermia to minimize the heat effect on healthy cells. Assisted by magnetic nanoparticles, it is possible to heat the specific area while unacceptable coincidental heating of healthy tissue is avoided. Although the hyperthermia treatment for cancer has been demonstrated with therapeutic efficacy in animal models, however, there have been no reports of successful hyperthermia treatment for human patients. The major reasons are the necessities of an adequate amount of magnetic nanoparticles and sufficiently high magnetic field which are not safe for human treatments. To date, laboratory research efforts on hyperthermia treatment for animals have all used magnetic field conditions which are not clinically acceptable. In most instances, hyperthermia treatments with a reduced amount of magnetic nanoparticles and reduced field strength or frequency cannot be effective due to the reduction of heat generated. Simulations suggest a sufficient level with heat deposition rate of 100mWcm−3 to destroy cancer cells effectively in most circumstances. The practical frequency and strength of the external AC magnetic field are 0.05–1.2MHz and 0–15 kAm−1, respectively. On the other hand, sufficient magnetic materials are needed to enrich around the cancer tissues to generate enough heat for hyperthermia treatment. Direct injection of ferrofluid into the tumor tissues is able to introduce a large amount of magnetic materials for heat generation. Antibody targeting and intravascular administration offer better preference heating, but the problem here is the small quantity. It is estimated that about 5–10 mg of magnetic material concentrated in each cm3 of tumor tissues is able to generate enough heat for tumor cell destruction in human bodies. Magnetite (Fe3O4) and maghemite (*γ* -Fe2O3) nanoparticles are two common types used in hyperthermia treatments owing to their appropriate magnetic properties and their excellent biocompatibilities. Several examples will be given here.

The history of using magnetic particles for selective heating of the tumors started in 1957 when Gilchrist et al used particles of a few mm in size for inductive heating of lymph nodes in dogs (*Gilchrist et al., 1957)*. More than 20 years later, Gordon et al used a magnetic fluid ('dextran-magnetites' with a core size of up to ~ 6 nm) for inducing hyperthermia *(Jordon et al., 1979)*. Injection of micro-scaled ferromagnetic particles into renal carcinomas of rabbits and subsequent heating was reported by Rand and co-workers *(Rand et al., 1981)*. Magnetic nanoparticles used in a different approach termed as ferromagnetic embolization. In this technique, the MNPs were injected into the main feeding artery of the tumor; this injection resulted in aggregates of MNPs which in turn embolized the feeding artery and hence necrosis of the tumor cells. This technique seems to be especially well suited for the treatment of hepatic malignancies due to the differences in blood supply between hepatic tumor cells and normal liver parenchyma *(Archer et al., 1990)*. Direct injection of dextrancoated magnetite NPs with a core size of ~ 3 nm into tumors was first reported in 1997 *(Jordan et al., 1997)*. Other groups in Japan developed "magnetic cationic liposomes" (MCLs) with improved adsorption and accumulation properties within tumors and demonstrated the efficacy of their technique in several animal tumor models: rat glioma *(Le et al., 2001)*. Hilger et al injected colloidal suspensions of coated MNPs (particle sizes of ~10 nm and 200 nm) into human carcinomas implanted into mice *(Hilger et al., 2002)*. Ohno et al inserted stick-type carboxymethyl cellulose magnetite containing NPs into gliomas and described as a three-fold prolongation of survival time *(Ohno et al., 2002)*. Moroz and co-workers concluded from their data that for a given tumor iron concentration, larger tumors heat at a greater rate than small tumors due to the poorer tissue cooling and better heat conduction in the necrotic regions of large tumors *(Moroz et al., 2002)*. Tanaka et al used MCLs in melanoma in combination with immunotherapy *(Tanaka et al., 2005)* and used for prostate cancer treatment *(Kawai et al., 2005)*. Yan *et al*. demonstrated the use of Fe2O3 nanoparticles combined with magnetic fluid for hyperthermia treatment on human hepatocarcinoma SMMC-7721 cells *in vitro* and xenograft liver cancer in nude mice (2005). Their experiments verified the significantly inhibitory effect of magnetic ferrofluid in weight and volume on xenograft liver cancer. After infiltrating magnetic ferrofluid into the target tissues, a timevaried magnetic field was applied and its energy was transformed to heat energy by the magnetic nanoparticles resulting in a temperature rise to 42–45 ◦C. This generated heat is able to kill malignant tumor cells without injuring the normal cells nearby. The growth and apoptosis of SMMC-7721 cells treated with the ferrofluids containing Fe2O3 nanoparticles at various concentrations (2, 4, 6 and 8 mg/ml) were examined by MTT, flow cytometry (FCM) and transmission electron microscopy (TEM) after 30–60 minute treatments. It was observed that Fe2O3 nanoparticles-based ferrofluid could significantly inhibit the proliferation and increase the ratio of apoptosis of SMMC-7721 cells. These dose-dependent inhibitions were 26.5 %, 33.53 %, 54.4 %, 81.2 %, and 30.26 %, 38.65 %, 50.28 %, 69.33 %, for inhibitory rate and apoptosis rate, respectively. It was also observed from animal experiments that the tumors became smaller and smaller as the dosage of magnetic ferrofluid increased. The weight and volume inhibitory ratios were 42.10 %, 66.34 %, 78.5 %, 91.46 %, and 58.77 %, 80.44 %, 93.40 %, 98.30 %, respectively. In a comparison of the control and experimental groups, each group exhibited significant difference. According to histological examination, Laser and Radiofrequency Induced Hyperthermia Treatment via Gold-Coated Magnetic Nanocomposites 117

many brown uniform spots are located at the stroma in the margin of the tumors, which are identified as iron oxide nanoparticles. Although interstitial hyperthermia following direct injection of nanoparticles has been proven successful in many animal models, nevertheless only one of these approaches has been successfully translated from research to clinical stage for prostate cancer treatment either by iron oxide against RF ,this clinical studies were

Fumiko et al 2004 developed magnetite cationic liposomes (MCLs) and applied them to local hyperthermia as a mediator. MCLs have a positive charge and generate heat under an alternating magnetic field (AMF) by hysteresis loss. In this study, the effect of hyperthermia using MCLs was examined in an in vivo study of hamster osteosarcoma. In this study, threeweek-old Syrian female hamsters were purchased from Japan SLC, Inc., Shizuoka, Japan, and used for the animal study. After that MCLs were injected into the osteosarcoma and then subjected to an AMF. The results revealed that, the tumor was heated at over 42°C, but other normal tissues were not heated as much. Complete regression was observed in 100% of the treated group hamsters, whereas no regression was observed in the control group hamsters. At day 12, the average tumor volume of the treated hamsters was about 1/1000 of that of the control hamsters. In the treated hamsters, no regrowth of osteosarcomas was observed over a period of 3 months after the complete regression. These results suggest that

One of recent and novel study was applied by El Sherbini et al (2011). The aim of this experimental study is to evaluate the effect of magnetic resonance on magnetic nanoparticles, this *in -vivo* experiments in which hyperthermia is induced in female Swiss albino mice weighing 20.0 to 29.2g median, 26.3g implanted with subcutaneous *Ehrlich* carcinoma cells under magnetic resonance imaging. The strategy of this study was based on preparation, characterization of super-paramagnetic magnetic iron oxides nanoparticles and evaluation of magnetic resonance hyperthermia (MRH) technique in presence of super-

Preparation of tumors bearing mice and iron oxide magnetic nanoparticles followed the method described by Elsherbini et al (2011). The prepared SPIO nanoparticles were suspended in glycerin medium to increase the stability especially *in vivo* conditions. The prepared suspension was stable for several months due to the high viscosity of glycerin. The influence of the SPIO nanoparticles concentration on the total amount of specific heat energy dose (SED) was studied in a preliminary study. The mean values of the total cumulative specific energy dose were monitored for different concentrations of SPIO nanoparticles in the tumors. The mean values reported of SED Jgm-1 were [282.1±13.8 for (200µg), 462.7±10.0 for (400µg), 663.7±13.0 for (600µg), 864.1±16.6 for (800µg) and 1087 ±18 for (103µg)] as shown in figure (3). The quantitative analysis revealed that, the SED values were directly

proportional to the concentrations of the injected nanoparticles inside the tumors.

The results of heat deposition rate HDR inside the tumor revealed that, the mean values of HDR were [0.157 for (200µg), 0.259 for (400µg), and 0.367 for (600µg), 0.478 for (800µg) and 0.604 for (103µg)] as shown in figure (4). These values varied considerably between the

paramagnetic nanoparticles and an alternating magnetic field (AMF).

performed by Johannsen 2005.

this treatment is effective for osteosarcoma.

many brown uniform spots are located at the stroma in the margin of the tumors, which are identified as iron oxide nanoparticles. Although interstitial hyperthermia following direct injection of nanoparticles has been proven successful in many animal models, nevertheless only one of these approaches has been successfully translated from research to clinical stage for prostate cancer treatment either by iron oxide against RF ,this clinical studies were performed by Johannsen 2005.

116 Hyperthermia

The history of using magnetic particles for selective heating of the tumors started in 1957 when Gilchrist et al used particles of a few mm in size for inductive heating of lymph nodes in dogs (*Gilchrist et al., 1957)*. More than 20 years later, Gordon et al used a magnetic fluid ('dextran-magnetites' with a core size of up to ~ 6 nm) for inducing hyperthermia *(Jordon et al., 1979)*. Injection of micro-scaled ferromagnetic particles into renal carcinomas of rabbits and subsequent heating was reported by Rand and co-workers *(Rand et al., 1981)*. Magnetic nanoparticles used in a different approach termed as ferromagnetic embolization. In this technique, the MNPs were injected into the main feeding artery of the tumor; this injection resulted in aggregates of MNPs which in turn embolized the feeding artery and hence necrosis of the tumor cells. This technique seems to be especially well suited for the treatment of hepatic malignancies due to the differences in blood supply between hepatic tumor cells and normal liver parenchyma *(Archer et al., 1990)*. Direct injection of dextrancoated magnetite NPs with a core size of ~ 3 nm into tumors was first reported in 1997 *(Jordan et al., 1997)*. Other groups in Japan developed "magnetic cationic liposomes" (MCLs) with improved adsorption and accumulation properties within tumors and demonstrated the efficacy of their technique in several animal tumor models: rat glioma *(Le et al., 2001)*. Hilger et al injected colloidal suspensions of coated MNPs (particle sizes of ~10 nm and 200 nm) into human carcinomas implanted into mice *(Hilger et al., 2002)*. Ohno et al inserted stick-type carboxymethyl cellulose magnetite containing NPs into gliomas and described as a three-fold prolongation of survival time *(Ohno et al., 2002)*. Moroz and co-workers concluded from their data that for a given tumor iron concentration, larger tumors heat at a greater rate than small tumors due to the poorer tissue cooling and better heat conduction in the necrotic regions of large tumors *(Moroz et al., 2002)*. Tanaka et al used MCLs in melanoma in combination with immunotherapy *(Tanaka et al., 2005)* and used for prostate cancer treatment *(Kawai et al., 2005)*. Yan *et al*. demonstrated the use of Fe2O3 nanoparticles combined with magnetic fluid for hyperthermia treatment on human hepatocarcinoma SMMC-7721 cells *in vitro* and xenograft liver cancer in nude mice (2005). Their experiments verified the significantly inhibitory effect of magnetic ferrofluid in weight and volume on xenograft liver cancer. After infiltrating magnetic ferrofluid into the target tissues, a timevaried magnetic field was applied and its energy was transformed to heat energy by the magnetic nanoparticles resulting in a temperature rise to 42–45 ◦C. This generated heat is able to kill malignant tumor cells without injuring the normal cells nearby. The growth and apoptosis of SMMC-7721 cells treated with the ferrofluids containing Fe2O3 nanoparticles at various concentrations (2, 4, 6 and 8 mg/ml) were examined by MTT, flow cytometry (FCM) and transmission electron microscopy (TEM) after 30–60 minute treatments. It was observed that Fe2O3 nanoparticles-based ferrofluid could significantly inhibit the proliferation and increase the ratio of apoptosis of SMMC-7721 cells. These dose-dependent inhibitions were 26.5 %, 33.53 %, 54.4 %, 81.2 %, and 30.26 %, 38.65 %, 50.28 %, 69.33 %, for inhibitory rate and apoptosis rate, respectively. It was also observed from animal experiments that the tumors became smaller and smaller as the dosage of magnetic ferrofluid increased. The weight and volume inhibitory ratios were 42.10 %, 66.34 %, 78.5 %, 91.46 %, and 58.77 %, 80.44 %, 93.40 %, 98.30 %, respectively. In a comparison of the control and experimental groups, each group exhibited significant difference. According to histological examination,

Fumiko et al 2004 developed magnetite cationic liposomes (MCLs) and applied them to local hyperthermia as a mediator. MCLs have a positive charge and generate heat under an alternating magnetic field (AMF) by hysteresis loss. In this study, the effect of hyperthermia using MCLs was examined in an in vivo study of hamster osteosarcoma. In this study, threeweek-old Syrian female hamsters were purchased from Japan SLC, Inc., Shizuoka, Japan, and used for the animal study. After that MCLs were injected into the osteosarcoma and then subjected to an AMF. The results revealed that, the tumor was heated at over 42°C, but other normal tissues were not heated as much. Complete regression was observed in 100% of the treated group hamsters, whereas no regression was observed in the control group hamsters. At day 12, the average tumor volume of the treated hamsters was about 1/1000 of that of the control hamsters. In the treated hamsters, no regrowth of osteosarcomas was observed over a period of 3 months after the complete regression. These results suggest that this treatment is effective for osteosarcoma.

One of recent and novel study was applied by El Sherbini et al (2011). The aim of this experimental study is to evaluate the effect of magnetic resonance on magnetic nanoparticles, this *in -vivo* experiments in which hyperthermia is induced in female Swiss albino mice weighing 20.0 to 29.2g median, 26.3g implanted with subcutaneous *Ehrlich* carcinoma cells under magnetic resonance imaging. The strategy of this study was based on preparation, characterization of super-paramagnetic magnetic iron oxides nanoparticles and evaluation of magnetic resonance hyperthermia (MRH) technique in presence of superparamagnetic nanoparticles and an alternating magnetic field (AMF).

Preparation of tumors bearing mice and iron oxide magnetic nanoparticles followed the method described by Elsherbini et al (2011). The prepared SPIO nanoparticles were suspended in glycerin medium to increase the stability especially *in vivo* conditions. The prepared suspension was stable for several months due to the high viscosity of glycerin. The influence of the SPIO nanoparticles concentration on the total amount of specific heat energy dose (SED) was studied in a preliminary study. The mean values of the total cumulative specific energy dose were monitored for different concentrations of SPIO nanoparticles in the tumors. The mean values reported of SED Jgm-1 were [282.1±13.8 for (200µg), 462.7±10.0 for (400µg), 663.7±13.0 for (600µg), 864.1±16.6 for (800µg) and 1087 ±18 for (103µg)] as shown in figure (3). The quantitative analysis revealed that, the SED values were directly proportional to the concentrations of the injected nanoparticles inside the tumors.

The results of heat deposition rate HDR inside the tumor revealed that, the mean values of HDR were [0.157 for (200µg), 0.259 for (400µg), and 0.367 for (600µg), 0.478 for (800µg) and 0.604 for (103µg)] as shown in figure (4). These values varied considerably between the different concentrations of SPIO nanoparticles with highly significant *p* value *p* <.007. The temperature changes were recorded in the intra-tumoural SPIO nanoparticles accumulation. The results revealed that the maximum temperatures achieved were [40.11±1.52°C for (200µg), 42.36±1.54°C for (400µg), 44.43±2.0°C for (600µg), 46.8±1.5°C for (800µg) and 48.6±1.0 °C for (103µg)] as shown in figure (5).The time taken to maximum temperature TMT was recorded as [40±2.5 min for 200&400µg, 30±2.0 min for 600µg, 25±5.0 min for 800µg and 20± 5.0 min for 103µg] as shown in figure (6). The statistical analysis revealed that the TMT values were inversely proportional to the concentrations of the injected nanoparticles inside the tumors. *In vivo* experiments for magnetic resonance hyperthermia demonstrated that the use of SPIO nanoparticles combined with magnetic resonance for hyperthermia treatment on Ehrlich carcinoma. The experiments revealed that after treatment sessions, magnetic resonance images verified degree of apoptotic cells presented by dark signal intensity in the center of the tumor in all mice on T1 weighted images; the centers of the lesions were asymmetrical and non-homogenous when compared to magnetic resonance images before treatment, as well as the images showed variations in signal intensity in the abdominal regions attributed to the distribution of the SPIO nanoparticles over the treatment sessions as shown in figure (7). As well as the experiments verified the significantly inhibitory effect of SPIO nanoparticles in volume on Ehrlich tumor. Significant volume differences between mice in all groups under experiments are shown in figure (8). It worth mentioning that, histopathology examination was further used to confirm MR results.

Laser and Radiofrequency Induced Hyperthermia Treatment via Gold-Coated Magnetic Nanocomposites 119

In a study by El- Sayed and co-workers conjugated gold nanoparticles of approximately 40 nm to anti-epithelial growth factors receptors (EGFR) antibodies and targeted to types of human head and neck cancer cells, the nanoparticles induce cancer cell damage at 19 W/cm2 after the irradiation with argon Ar+ laser at 514 nm for 4 min, while healthy cells do not

Huang et al also described the photo-thermal destruction of cancer cells using biofunctionalized gold nanorods. The nanorods were conjugated to anti- EGFR (specific antibody to the malignant cell types used), and then incubated with a non-malignant epithelial cell line (HaCat), as well as two malignant oral epithelial cell lines (HOC313 clone8 and HSC3). Following laser irradiation, the results revealed that the malignant cells were destroyed at about half the laser fluence needed to kill the nonmalignant cells. The efficient destruction of the malignant cells was evidently due to the preferential attachment of the anti-EGFR-gold nanorod conjugates to the over-expressed EGFR on the surface of the

In 2006, El-Sayed and co-workers conjugated gold nanorods to anti-EGFR antibodies specifically bind to the head and neck cancer cells, these labeled cells subjected to laser irradiation (Ti: Sapphire laser, CW at 800 nm) which was maximally overlapped with the surface plasmonic resonance absorption band of the nanorods. Under laser exposure for 4 min, it was found that the cancer cells required half the laser energy (10 W/cm2) to be photo-

In 2007, Huff and co-workers conjugated folate ligands with oligo-ethylene-glycol onto gold nanorods by *in situ* dithio-carbamate formation; the folate conjugated gold nanorods were selectively bound to KB cancer cells (a tumor cell line derived from oral epithelium) which led to photo-thermal damage on cell membranes following laser irradiation *(Huff et al., 2007)*. Another study from the same group showed that under laser irradiation membrane blebbing occurred due to the influx of calcium ion Ca2+ into the cells *(Tong et al., 2007)*.

Attempts using gold nanocages for PPPT have also been made recently. In the *in vitro* studies by Li and co-workers conjugated gold nanocages of approximately 30 nm to anti-EGFR to target A431 cells. At laser energy density of 40 W/cm2, almost all immunonanocage treated cells were damaged *(Li et al., 2008)*. Other *in vitro* studies by Xiaohug and co-workers using gold nanocages of approximately 45 nm conjugated to HER-2 and near infrared femtosecond Ti: Sapphire pulsed laser to treat Sk-BR-3 breast cancer cells *(Xiaohua et al.,* 

Paul and Tuan reported the application of liposome-encapsulated gold nanoshells for in vitro photo-induced hyperthermia in human mammary carcinoma cells. In addition to evaluating their effects in vitro, the authors compared the application liposomeencapsulated gold nanoshells and free-standing gold nanoshells for NanoPhotoTherapy (NPT). NPT-induced hyperthermia was performed using a 785-nm near-infrared light from a diode laser and the in vitro effects were evaluated using nucleic acid molecular probes by fluorescence microscopy. Additionally, they monitored the effectiveness of NPT by

thermally damaged as compared to the normal cells (20 W/cm2) *(El Sayed et al., 2006)*.

show the loss of cell viability under the same treatment *(El Sayed et al., 2005)*.

malignant cell *(Huang et al., 2006)*.

*2010)*.

Plasmonic photo-thermal therapy (PPTT).Gold nanoshells belong to a prospective class of optical adjustable nanoparticles with a dielectric silica core encased in a thin metallic gold shell (Hirsch, et al 2006). The absorption cross-section of a solid nanoshell is high enough to provide a competitive nanoparticle technology with application of indocyanine green dye, a typical photothermal sensitizer used in laser cancer therapy (Gupta, et al 2007).

On the other hand, there are several *in vitro* experiments concerning application of gold nanoparticles and core shell NPs to PPTT of cancer cells, while number of *in vivo* studies is quite limited (Loo, et al, 2005). The first account of the use of gold nanoparticles in hyperthermal therapy was published in 2003. Halas et al used gold -on-silica nanoshells to treat breast carcinoma cells using the HER2 antibody *(Hirsch et al., 2003)*. Another study using pulsed laser and gold nanospheres was performed in 2003 by Lin and co-workers for selective and highly localized photothermolysis of targeted lymphocytes cells. Lymphocytes incubated with gold nanospheres conjugated to anti-bodies were exposed to nanosecond laser pulses (Q-switched Nd: YAG laser, 565 nm wavelength, 20 ns duration) .The results showed that 100 laser pulses at an energy of 0.5 J/cm2 were sufficient to induce cell death. While adjacent cells just a few micrometers away without nanoparticles remained viable *(Pitsillides et al., 2003)*. In the same year, Zharov et al performed similar studies on the photothermal destruction of K562 cancer cells. They further detected the laser induced- bubbles and studied their dynamics during the treatment using a pump–probe photo thermal imaging technique *(Zharov et al., 2003)*. O'Neal et al 2004 reported *in vivo* impressive results by showing selective photo-thermal ablation in mice using near infrared-absorbing NPs *(O*, *Neal et al., 2004)*. Another *in vivo study was applied on* a murine model using NIR light against gold nanoshells (*Loo et al., 2004*).

In a study by El- Sayed and co-workers conjugated gold nanoparticles of approximately 40 nm to anti-epithelial growth factors receptors (EGFR) antibodies and targeted to types of human head and neck cancer cells, the nanoparticles induce cancer cell damage at 19 W/cm2 after the irradiation with argon Ar+ laser at 514 nm for 4 min, while healthy cells do not show the loss of cell viability under the same treatment *(El Sayed et al., 2005)*.

118 Hyperthermia

different concentrations of SPIO nanoparticles with highly significant *p* value *p* <.007. The temperature changes were recorded in the intra-tumoural SPIO nanoparticles accumulation. The results revealed that the maximum temperatures achieved were [40.11±1.52°C for (200µg), 42.36±1.54°C for (400µg), 44.43±2.0°C for (600µg), 46.8±1.5°C for (800µg) and 48.6±1.0 °C for (103µg)] as shown in figure (5).The time taken to maximum temperature TMT was recorded as [40±2.5 min for 200&400µg, 30±2.0 min for 600µg, 25±5.0 min for 800µg and 20± 5.0 min for 103µg] as shown in figure (6). The statistical analysis revealed that the TMT values were inversely proportional to the concentrations of the injected nanoparticles inside the tumors. *In vivo* experiments for magnetic resonance hyperthermia demonstrated that the use of SPIO nanoparticles combined with magnetic resonance for hyperthermia treatment on Ehrlich carcinoma. The experiments revealed that after treatment sessions, magnetic resonance images verified degree of apoptotic cells presented by dark signal intensity in the center of the tumor in all mice on T1 weighted images; the centers of the lesions were asymmetrical and non-homogenous when compared to magnetic resonance images before treatment, as well as the images showed variations in signal intensity in the abdominal regions attributed to the distribution of the SPIO nanoparticles over the treatment sessions as shown in figure (7). As well as the experiments verified the significantly inhibitory effect of SPIO nanoparticles in volume on Ehrlich tumor. Significant volume differences between mice in all groups under experiments are shown in figure (8). It worth mentioning that,

Plasmonic photo-thermal therapy (PPTT).Gold nanoshells belong to a prospective class of optical adjustable nanoparticles with a dielectric silica core encased in a thin metallic gold shell (Hirsch, et al 2006). The absorption cross-section of a solid nanoshell is high enough to provide a competitive nanoparticle technology with application of indocyanine green dye, a

On the other hand, there are several *in vitro* experiments concerning application of gold nanoparticles and core shell NPs to PPTT of cancer cells, while number of *in vivo* studies is quite limited (Loo, et al, 2005). The first account of the use of gold nanoparticles in hyperthermal therapy was published in 2003. Halas et al used gold -on-silica nanoshells to treat breast carcinoma cells using the HER2 antibody *(Hirsch et al., 2003)*. Another study using pulsed laser and gold nanospheres was performed in 2003 by Lin and co-workers for selective and highly localized photothermolysis of targeted lymphocytes cells. Lymphocytes incubated with gold nanospheres conjugated to anti-bodies were exposed to nanosecond laser pulses (Q-switched Nd: YAG laser, 565 nm wavelength, 20 ns duration) .The results showed that 100 laser pulses at an energy of 0.5 J/cm2 were sufficient to induce cell death. While adjacent cells just a few micrometers away without nanoparticles remained viable *(Pitsillides et al., 2003)*. In the same year, Zharov et al performed similar studies on the photothermal destruction of K562 cancer cells. They further detected the laser induced- bubbles and studied their dynamics during the treatment using a pump–probe photo thermal imaging technique *(Zharov et al., 2003)*. O'Neal et al 2004 reported *in vivo* impressive results by showing selective photo-thermal ablation in mice using near infrared-absorbing NPs *(O*, *Neal et al., 2004)*. Another *in vivo study was applied on* a murine model using NIR light against

histopathology examination was further used to confirm MR results.

gold nanoshells (*Loo et al., 2004*).

typical photothermal sensitizer used in laser cancer therapy (Gupta, et al 2007).

Huang et al also described the photo-thermal destruction of cancer cells using biofunctionalized gold nanorods. The nanorods were conjugated to anti- EGFR (specific antibody to the malignant cell types used), and then incubated with a non-malignant epithelial cell line (HaCat), as well as two malignant oral epithelial cell lines (HOC313 clone8 and HSC3). Following laser irradiation, the results revealed that the malignant cells were destroyed at about half the laser fluence needed to kill the nonmalignant cells. The efficient destruction of the malignant cells was evidently due to the preferential attachment of the anti-EGFR-gold nanorod conjugates to the over-expressed EGFR on the surface of the malignant cell *(Huang et al., 2006)*.

In 2006, El-Sayed and co-workers conjugated gold nanorods to anti-EGFR antibodies specifically bind to the head and neck cancer cells, these labeled cells subjected to laser irradiation (Ti: Sapphire laser, CW at 800 nm) which was maximally overlapped with the surface plasmonic resonance absorption band of the nanorods. Under laser exposure for 4 min, it was found that the cancer cells required half the laser energy (10 W/cm2) to be photothermally damaged as compared to the normal cells (20 W/cm2) *(El Sayed et al., 2006)*.

In 2007, Huff and co-workers conjugated folate ligands with oligo-ethylene-glycol onto gold nanorods by *in situ* dithio-carbamate formation; the folate conjugated gold nanorods were selectively bound to KB cancer cells (a tumor cell line derived from oral epithelium) which led to photo-thermal damage on cell membranes following laser irradiation *(Huff et al., 2007)*. Another study from the same group showed that under laser irradiation membrane blebbing occurred due to the influx of calcium ion Ca2+ into the cells *(Tong et al., 2007)*.

Attempts using gold nanocages for PPPT have also been made recently. In the *in vitro* studies by Li and co-workers conjugated gold nanocages of approximately 30 nm to anti-EGFR to target A431 cells. At laser energy density of 40 W/cm2, almost all immunonanocage treated cells were damaged *(Li et al., 2008)*. Other *in vitro* studies by Xiaohug and co-workers using gold nanocages of approximately 45 nm conjugated to HER-2 and near infrared femtosecond Ti: Sapphire pulsed laser to treat Sk-BR-3 breast cancer cells *(Xiaohua et al., 2010)*.

Paul and Tuan reported the application of liposome-encapsulated gold nanoshells for in vitro photo-induced hyperthermia in human mammary carcinoma cells. In addition to evaluating their effects in vitro, the authors compared the application liposomeencapsulated gold nanoshells and free-standing gold nanoshells for NanoPhotoTherapy (NPT). NPT-induced hyperthermia was performed using a 785-nm near-infrared light from a diode laser and the in vitro effects were evaluated using nucleic acid molecular probes by fluorescence microscopy. Additionally, they monitored the effectiveness of NPT by

detecting apoptosis via capase-9 activity. The experiments clearly showed that liposomal delivery enhanced the intracellular bioavailability of gold nanoshells and thus is able to induce a higher degree of cell death more effectively than free-standing gold nanoshells.

Laser and Radiofrequency Induced Hyperthermia Treatment via Gold-Coated Magnetic Nanocomposites 121

subcutaneous Ehrlich carcinoma cells. The first called Optical Resonance Hyperthermia (ORH) technique in presence of gold nanospheres and green diode laser, as shown in fig (9). While the second technique called Magneto-Optical Resonance Hyperthermia (MORH), in presence of gold-iron oxide core shell nanoparticles with green, near infra-red diode laser, and magnetic field, as shown in fig (10). This approach was performed under magnetic resonance imaging guidance. The results revealed that, all mice treated by the first technique, the tumors were still as the same as before the treatments, as well as the rate of tumors growth were very slow if compared with the control mice. In contrast more than 50% of the mice treated with the second technique revealed a complete disappearance of the tumor, as shown in figure (11). So the study have demonstrated that a pair of synthetic nanospheres can work together more effectively for inducing hyperthermia than individual nanospheres, whereby more than .So, this simple, non-invasive method shows great

There are two main advantages of the plasmonic photothermal therapy technique. Firstly, there is the benefit of photostability compared with the photosensitizer dyes used in photodynamic therapy, which suffer from photobleaching as well as diffusion under laser irradiation. Secondly, there is the advantage of absorption and scattering cross-sections of gold nanoparticles, which are significantly superior to the absorbing dyes conventionally used in biological systems. Mie theory estimates that the optical cross-sections of gold nanospheres are typically four to five orders of magnitude higher than those of conventional

In spite of much progress having been made using the plasmonic photothermal therapy technique for cancer treatment in a laboratory setting, there are still many factors which must be taken into account before this method may be taken to a clinical setting, and they need to be studied further. First of all, the distribution of the elevated temperature under plasmonic photothermal therapy treatment is related to absorption of light by nanospheres acting as point wise local heat sources and by thermal diffusion over surrounding tissues. At the practical level, plasmonic photothermal therapy needs to provide an appropriate temperature increment, *Δ*T, gold nanosphere concentration, laser power density, duration of laser exposure, optimization of absorption and scattering cross-sections of nanospheres, as well as penetration of the laser light into the area of interest. It should be noted that the biological effects have a nonlinear dependence on changes in particle concentration and laser power density, which is defined by the type of tissue and thermoregulation ability of

Although interstitial hyperthermia following direct injection of nanoparticles has been proven successful in many animal models, nevertheless only one of these approaches has been successfully translated from research to clinical stage for prostate cancer treatment either by iron oxide against RF ,this clinical studies were performed by Johannsen 2005. The aim of this pilot study was to evaluate whether the technique of magnetic fluid hyperthermia can be used for minimally invasive treatment of prostate cancer. This paper presents the first clinical application of interstitial hyperthermia using magnetic

promise as a treatment technique for clinical setting.

dyes.

the living organism.

Single-walled carbon nanotubes (SWNTs) have a high optical absorbance in the nearinfrared (NIR) region. In this special optical window, biological systems are known to be highly transparent. The optical properties of SWNTs provide an opportunity for selective photo thermal therapy for cancer treatment. Specifically, SWNTs with a uniform size about (0.81 nm) and a narrow absorption peak at 980 nm are ideal candidates for such a novel approach. In a study by Feifan et al, SWNTs are conjugated to folate, which can bind specifically to the surface of the folate receptor tumor markers. Folate- SWNT (FA-SWNT) targeted tumor cells were irradiated by a 980 nm laser. Results in *vitro* and *in vivo*  experiments revealed that FA-SWNT effectively enhanced the photo thermal destruction on tumor cells and noticeably spared the photo thermal destruction for non targeted normal cells. Thus, SWNTs, combined with suitable tumor markers, can be used as novel nanomaterials for selective photo thermal therapy for cancer treatment. The authors used the mammary tumor model with EMT6 cells in the female Balb/c mice to investigate the *in vivo* effects of FA-SWNT. The mouse tumors with or without FA-SWNT were treated by the 980-nm laser. To determine the effects of NIR optical excitation of SWNTs inside tumors, the authors measured the temperature on the tumor surface during the irradiation by the 980 nm laser with an infrared thermal camera. In one experimental mouse, irradiation of tumors with a power density of 1 W/cm2 with FA-SWNT (1 mg/kg) for 5 min caused a surface temperature elevation of 63 °C. Without FASWNT, the tumor irradiated at the same light dose caused a surface temperature elevation of 54 °C. Experiments with other animals yielded similar results. These findings clearly show that FA-SWNT could effectively enhance the tumor photo thermal therapy.

Kim et al achieved close to 90% cancer cell destruction in vitro using FeNi@Aumagneticvortex microdiscs (MDs), on the application of only a few tens of hertz AMF for just 10min. This confirms that operation of MFH at lower frequencies is possible and for effective heat generation can be achieved using core–shell type of structures. Likewise, in yet another demonstration, a gold coating of approximately 0.4 to 0.5 nm thickness around SPIONs resulted in a four- to five-fold increase in the amount of heat released (the highest value of 976W/g in ethanol at 430 Hz frequency) in comparison with SPIONs on application of low frequency oscillating magnetic fields (44–430 Hz). This study was done by Mohamed et al 2010. In addition, the SPIONs@Au were found to be not particularly cytotoxic to mammalian cells. (MCF-7 breast carcinoma cells and H9c2 cardiomyoblasts) in *in vitro* studies were done by Pollert et al 2010. When similar heating experiments were carried out using stable water suspensions of La0.75Sr0.25MnO3 cores covered by silica (conc. of Mn=3.39 mg/ml), highest SAR of 130 W/g Mn at 37 °C was reached for the applied amplitude and frequency of 8.7 kAm−1, 480 kHz respectively.

In this context, a study was done by Elsherbini and co-workers, 2011. The group evaluated two different approaches in the nanotechnology era for inducing hyperthermia in

enhance the tumor photo thermal therapy.

amplitude and frequency of 8.7 kAm−1, 480 kHz respectively.

detecting apoptosis via capase-9 activity. The experiments clearly showed that liposomal delivery enhanced the intracellular bioavailability of gold nanoshells and thus is able to induce a higher degree of cell death more effectively than free-standing gold nanoshells.

Single-walled carbon nanotubes (SWNTs) have a high optical absorbance in the nearinfrared (NIR) region. In this special optical window, biological systems are known to be highly transparent. The optical properties of SWNTs provide an opportunity for selective photo thermal therapy for cancer treatment. Specifically, SWNTs with a uniform size about (0.81 nm) and a narrow absorption peak at 980 nm are ideal candidates for such a novel approach. In a study by Feifan et al, SWNTs are conjugated to folate, which can bind specifically to the surface of the folate receptor tumor markers. Folate- SWNT (FA-SWNT) targeted tumor cells were irradiated by a 980 nm laser. Results in *vitro* and *in vivo*  experiments revealed that FA-SWNT effectively enhanced the photo thermal destruction on tumor cells and noticeably spared the photo thermal destruction for non targeted normal cells. Thus, SWNTs, combined with suitable tumor markers, can be used as novel nanomaterials for selective photo thermal therapy for cancer treatment. The authors used the mammary tumor model with EMT6 cells in the female Balb/c mice to investigate the *in vivo* effects of FA-SWNT. The mouse tumors with or without FA-SWNT were treated by the 980-nm laser. To determine the effects of NIR optical excitation of SWNTs inside tumors, the authors measured the temperature on the tumor surface during the irradiation by the 980 nm laser with an infrared thermal camera. In one experimental mouse, irradiation of tumors with a power density of 1 W/cm2 with FA-SWNT (1 mg/kg) for 5 min caused a surface temperature elevation of 63 °C. Without FASWNT, the tumor irradiated at the same light dose caused a surface temperature elevation of 54 °C. Experiments with other animals yielded similar results. These findings clearly show that FA-SWNT could effectively

Kim et al achieved close to 90% cancer cell destruction in vitro using FeNi@Aumagneticvortex microdiscs (MDs), on the application of only a few tens of hertz AMF for just 10min. This confirms that operation of MFH at lower frequencies is possible and for effective heat generation can be achieved using core–shell type of structures. Likewise, in yet another demonstration, a gold coating of approximately 0.4 to 0.5 nm thickness around SPIONs resulted in a four- to five-fold increase in the amount of heat released (the highest value of 976W/g in ethanol at 430 Hz frequency) in comparison with SPIONs on application of low frequency oscillating magnetic fields (44–430 Hz). This study was done by Mohamed et al 2010. In addition, the SPIONs@Au were found to be not particularly cytotoxic to mammalian cells. (MCF-7 breast carcinoma cells and H9c2 cardiomyoblasts) in *in vitro* studies were done by Pollert et al 2010. When similar heating experiments were carried out using stable water suspensions of La0.75Sr0.25MnO3 cores covered by silica (conc. of Mn=3.39 mg/ml), highest SAR of 130 W/g Mn at 37 °C was reached for the applied

In this context, a study was done by Elsherbini and co-workers, 2011. The group evaluated two different approaches in the nanotechnology era for inducing hyperthermia in subcutaneous Ehrlich carcinoma cells. The first called Optical Resonance Hyperthermia (ORH) technique in presence of gold nanospheres and green diode laser, as shown in fig (9). While the second technique called Magneto-Optical Resonance Hyperthermia (MORH), in presence of gold-iron oxide core shell nanoparticles with green, near infra-red diode laser, and magnetic field, as shown in fig (10). This approach was performed under magnetic resonance imaging guidance. The results revealed that, all mice treated by the first technique, the tumors were still as the same as before the treatments, as well as the rate of tumors growth were very slow if compared with the control mice. In contrast more than 50% of the mice treated with the second technique revealed a complete disappearance of the tumor, as shown in figure (11). So the study have demonstrated that a pair of synthetic nanospheres can work together more effectively for inducing hyperthermia than individual nanospheres, whereby more than .So, this simple, non-invasive method shows great promise as a treatment technique for clinical setting.

There are two main advantages of the plasmonic photothermal therapy technique. Firstly, there is the benefit of photostability compared with the photosensitizer dyes used in photodynamic therapy, which suffer from photobleaching as well as diffusion under laser irradiation. Secondly, there is the advantage of absorption and scattering cross-sections of gold nanoparticles, which are significantly superior to the absorbing dyes conventionally used in biological systems. Mie theory estimates that the optical cross-sections of gold nanospheres are typically four to five orders of magnitude higher than those of conventional dyes.

In spite of much progress having been made using the plasmonic photothermal therapy technique for cancer treatment in a laboratory setting, there are still many factors which must be taken into account before this method may be taken to a clinical setting, and they need to be studied further. First of all, the distribution of the elevated temperature under plasmonic photothermal therapy treatment is related to absorption of light by nanospheres acting as point wise local heat sources and by thermal diffusion over surrounding tissues. At the practical level, plasmonic photothermal therapy needs to provide an appropriate temperature increment, *Δ*T, gold nanosphere concentration, laser power density, duration of laser exposure, optimization of absorption and scattering cross-sections of nanospheres, as well as penetration of the laser light into the area of interest. It should be noted that the biological effects have a nonlinear dependence on changes in particle concentration and laser power density, which is defined by the type of tissue and thermoregulation ability of the living organism.

Although interstitial hyperthermia following direct injection of nanoparticles has been proven successful in many animal models, nevertheless only one of these approaches has been successfully translated from research to clinical stage for prostate cancer treatment either by iron oxide against RF ,this clinical studies were performed by Johannsen 2005. The aim of this pilot study was to evaluate whether the technique of magnetic fluid hyperthermia can be used for minimally invasive treatment of prostate cancer. This paper presents the first clinical application of interstitial hyperthermia using magnetic

nanoparticles in locally recurrent prostate cancer. Treatment planning was carried out using computerized tomography (CT) of the prostate. Based on the individual anatomy of the prostate and the estimated specific absorption rate (SAR) of magnetic fluids in prostatic tissue, the number and position of magnetic fluid depots required for sufficient heat deposition was calculated while rectum and urethra were spared. Nanoparticle suspensions were injected transperineally into the prostate under transrectal ultrasound and flouroscopy guidance. Treatments were delivered in the first magnetic field applicator for use in humans, using an alternating current magnetic field with a frequency of 100 kHz and variable field strength (0–18 kAm-1). Invasive thermometry of the prostate was carried out in the first and last of six weekly hyperthermia sessions of 60 min duration. CT-scans of the prostate were repeated following the first and last hyperthermia treatment to document magnetic nanoparticle distribution and the position of the thermometry probes in the prostate. Nanoparticles were retained in the prostate during the treatment interval of 6 weeks. Using appropriate software (AMIRA), a non-invasive estimation of temperature values in the prostate, based on intra-tumoural distribution of magnetic nanoparticles, can be performed and correlated with invasively measured intra-prostatic temperatures. Using a specially designed cooling device, treatment was well tolerated without anesthesia. In the first patient treated, maximum and minimum intraprostatic temperatures measured at field strength of 4.0–5.0 kAm-1 were 48.50C and 40.00C during the 1st treatment and 42.50C and 39.40C during the 6th treatment, respectively. These first clinical experiences prompted us to initiate a phase I study to evaluate feasibility, toxicity and quality of life during hyperthermia using magnetic nanoparticles in patients with biopsy-proven local recurrence of prostate cancer following radiotherapy with curative intent. To the authors' knowledge, this is the first report on clinical application of interstitial hyperthermia using magnetic nanoparticles in the treatment of human cancer.

Laser and Radiofrequency Induced Hyperthermia Treatment via Gold-Coated Magnetic Nanocomposites 123

patients in Op group (P = 0.0042). But when compared between HT group and Op + RT group, there were no significant difference (P = 0.412). This first series of clinical hyperthermia using magnetic materials achieved good local control of metastatic bone

Study by Manfred et al 2007, or by using laser against gold nanoparticles, for instance Yusheng et al 2009 used core shells (silica as a core with a diameter 110nm and an outer gold shell with thickness 15 nm) to mediate laser surgery stimulation for prostate cancer treatment. The goal of this paper is to present an integrated computer model using a socalled nested-block optimization algorithm to simulate laser surgery and provides transient temperature field predictions. In particular, this algorithm aims to capture changes in optical and thermal properties due to nanoshell inclusion and tissue property variation during laser surgery. Numerical results show that this model is able to characterize variation of tissue properties for laser surgical procedures and predict transient temperature field comparable to that measured by in vivo magnetic resonance temperature imaging (MRTI) techniques. Note that the computational approach presented in the study is quite

In conclusion, this is a brief review on different approaches for inducing hyperthermia

Albert B, Bray D, Hopkin K, Johnson A, Lewis J, Raff M, Roberts K and Walter P: *Essential* 

Akihiko M, Katsuyuki K, Takao M, Ken S, Haruhiko S,Toru W, Shinichi M, Katsuya M, Kenji T, Atsumasa U: *Novel hyperthermia for metastatic bone tumors with magnetic materials by generating an alternating electromagnetic field*. Clin Exp Metastasis, 2007, 24:191–200

Archer S and Gray B: *Comparison of portal vein chemotherapy with hepatic artery chemotherapy in the treatment of liver micro- metastases*. J. Am. Surgery, 1990; vol159: pp325–329. Berry C: *Progress in functionalization of magnetic nanoparticles for applications in biomedicine.* 

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*Cell Biology*, Second Ed. New York and London: Garland Science, 2004.

Journal of Physics D: Applied Physics, 2009, 42(22): p. 224003.

general and can be applied to other types of nanoparticle inclusions.

*National Institute of Laser Enhanced Science (NILES), Cairo University, Egypt* 

cancer treatment relevant to nanomedicine

*Children Cancer Hospital, Cairo, Egypt* 

DOI 10.1007/s10585-007-9068-8.

**Author details** 

El-Sayed El-Sherbini

Ahmed El-Shahawy

**4. References** 

81–5.

lesion.

Akihiko et al have developed a novel hyperthermic treatment modality using magnetic materials for metastatic bone tumors. The purpose of this study is to show the results of novel hyperthermia for metastatic bone tumors. This novel hyperthermic treatment modality was used for 15 patients with 16 metastatic bone lesions. In seven lesions, after curettage of the metastatic lesion followed by reinforcement with a metal intra-medullary nail or plate, calcium phosphate cement (CPC) containing powdery Fe3O4 was implanted into the cavity. In one lesion, prosthetic reconstruction was then performed after an intralesional tumor excision. For the remaining eight lesions, metal intra-medullary nails were inserted into the affected bone. Hyperthermic therapy was started at 1 week postoperatively. To comparatively evaluate the radiographic results of patients who underwent hyperthermia (HT group), the authers also assessed eight patients who received a palliative operation without either radiotherapy or hyperthermia (Op group), and 22 patients who received operation in combination with postoperative radiotherapy (Op + RT group). In HT group, all patients had an acceptable limb function with pain relief without any complications. On radiographs, 87, 38, and 91% were, respectively, considered to demonstrate an effective treatment outcome in HT group, Op group, and Op + RT group. The patients in HT group showed a statistically better radiographic outcome than the patients in Op group (P = 0.0042). But when compared between HT group and Op + RT group, there were no significant difference (P = 0.412). This first series of clinical hyperthermia using magnetic materials achieved good local control of metastatic bone lesion.

Study by Manfred et al 2007, or by using laser against gold nanoparticles, for instance Yusheng et al 2009 used core shells (silica as a core with a diameter 110nm and an outer gold shell with thickness 15 nm) to mediate laser surgery stimulation for prostate cancer treatment. The goal of this paper is to present an integrated computer model using a socalled nested-block optimization algorithm to simulate laser surgery and provides transient temperature field predictions. In particular, this algorithm aims to capture changes in optical and thermal properties due to nanoshell inclusion and tissue property variation during laser surgery. Numerical results show that this model is able to characterize variation of tissue properties for laser surgical procedures and predict transient temperature field comparable to that measured by in vivo magnetic resonance temperature imaging (MRTI) techniques. Note that the computational approach presented in the study is quite general and can be applied to other types of nanoparticle inclusions.

In conclusion, this is a brief review on different approaches for inducing hyperthermia cancer treatment relevant to nanomedicine
