**4. Magnetic fluid hyperthermia-iron magnetic nanoparticles**

Thermotherapy represents a physical treatment induced by hyperthermia. Nowadays, macroscopic thermotherapy (ablative methods: microwave or radiofrequency, optical laser irradiation via fibers, focused ultrasound) is widely used to destroy focal tumors. The mech‐ anism of tumoral damage is the result of an irreparable destruction of molecular constituents of cells (mainly protein denaturation) that appears after an exposure of a few minutes at temperatures higher than 48°C. Even if it has lower side effects when compared to conventional therapy (chemo/radiotherapy) and although it has proved to be a reliable alternative to surgery, this therapy has several limits: the relative higher rate of incomplete destruction for tumors larger than 3 cm and a higher risk of destruction of the proximate healthy tissue. These deficiencies seem to disappear by using a new thermal method known as magnetic termic hyperthermia [38]. This approach uses an external alternating magnetic field applied to a target tumor where magnetic metallic particles (MNPs) have been infiltrated or injected. MNPs show distinguishing phenomena such as superparamagnetism, high field irreversibility, high saturation field, extra anisotropy contributions, or shifted loops after field cooling [39]. According to Reference [40], the distinguished phenomena noticed in MNPs are the result of the interaction between the intrinsic properties (size, distribution, and finite-size effects) and the interparticle interactions. The MNPs have the ability to absorb the energy of the alternating magnetic field energy and transform it into heat. Two factors are implicated in producing hyperthermia, the size of the magnetic material and the strength of the applied magnetic field. Larger implants (seeds) generate heat by resistance to circumferential eddy currents induced on the surface of the seeds by an alternative magnetic field [41]. Multidomain particles produce heat by hysteresis loss effects. On the contrary, nanoparticle, particularly subdomain particle, suspensions generate heat mainly by Brownian relaxation (heat is the result of friction arising from the total particle oscillations) and Neel relaxation (heat is the result of friction arising from the rotation of the magnetic moment with each field oscillation) [42, 43].

Superparamagnetic particles are particles that have sufficient high thermal motion after the magnetic field is removed, which can be randomly reoriented so as not to leave a residual magnetization [43].

Due to their properties, these particles may have several applications in clinical practice such as hyperthermia (HT), drug delivery and diagnosis (s.a nuclear magnetic resonance imaging).

HT represents a therapeutic procedure used to destroy a tumoral tissue at temperatures over 43°C [38]. It has been observed that tumoral cells have an increased thermal sensitivity in comparison to healthy cells; this feature is the result of an increased metabolism [44, 45]. Apoptosis is the result of cytotoxic effects that depend on physiological cell parameters (hypoxia or acidity) at temperatures over 43°C. 43°C is the temperature limit over which the expression of HSPs is stimulated, which leads to antitumor immunity and apoptosis [46]. The antitumor immunity increases as a result of an enhanced presentation of tumoral antigenic peptide to a major histocompatibility complex (MHC). HSP70 expression reaches its maximum 24 h after heating. The increased MHC class I surface expression is slower, so it starts 24 h after applied hyperthermia and the peak is after 48 h [38]. Two mechanisms have been suggested. One of the possible mechanisms is that the heat induces the enhancement of antigenic peptide presentation through MHC class I antigens of tumor cells. Another possible mechanism is the cross-presentation of antigenic peptides by dedicated antigen-presenting cells (APCs) [46].

**4. Magnetic fluid hyperthermia-iron magnetic nanoparticles**

230 Recent Advances in Liver Diseases and Surgery

from the rotation of the magnetic moment with each field oscillation) [42, 43].

magnetization [43].

Superparamagnetic particles are particles that have sufficient high thermal motion after the magnetic field is removed, which can be randomly reoriented so as not to leave a residual

Due to their properties, these particles may have several applications in clinical practice such as hyperthermia (HT), drug delivery and diagnosis (s.a nuclear magnetic resonance imaging). HT represents a therapeutic procedure used to destroy a tumoral tissue at temperatures over 43°C [38]. It has been observed that tumoral cells have an increased thermal sensitivity in comparison to healthy cells; this feature is the result of an increased metabolism [44, 45]. Apoptosis is the result of cytotoxic effects that depend on physiological cell parameters (hypoxia or acidity) at temperatures over 43°C. 43°C is the temperature limit over which the expression of HSPs is stimulated, which leads to antitumor immunity and apoptosis [46]. The antitumor immunity increases as a result of an enhanced presentation of tumoral antigenic peptide to a major histocompatibility complex (MHC). HSP70 expression reaches its maximum 24 h after heating. The increased MHC class I surface expression is slower, so it starts 24 h after applied hyperthermia and the peak is after 48 h [38]. Two mechanisms have been suggested.

Thermotherapy represents a physical treatment induced by hyperthermia. Nowadays, macroscopic thermotherapy (ablative methods: microwave or radiofrequency, optical laser irradiation via fibers, focused ultrasound) is widely used to destroy focal tumors. The mech‐ anism of tumoral damage is the result of an irreparable destruction of molecular constituents of cells (mainly protein denaturation) that appears after an exposure of a few minutes at temperatures higher than 48°C. Even if it has lower side effects when compared to conventional therapy (chemo/radiotherapy) and although it has proved to be a reliable alternative to surgery, this therapy has several limits: the relative higher rate of incomplete destruction for tumors larger than 3 cm and a higher risk of destruction of the proximate healthy tissue. These deficiencies seem to disappear by using a new thermal method known as magnetic termic hyperthermia [38]. This approach uses an external alternating magnetic field applied to a target tumor where magnetic metallic particles (MNPs) have been infiltrated or injected. MNPs show distinguishing phenomena such as superparamagnetism, high field irreversibility, high saturation field, extra anisotropy contributions, or shifted loops after field cooling [39]. According to Reference [40], the distinguished phenomena noticed in MNPs are the result of the interaction between the intrinsic properties (size, distribution, and finite-size effects) and the interparticle interactions. The MNPs have the ability to absorb the energy of the alternating magnetic field energy and transform it into heat. Two factors are implicated in producing hyperthermia, the size of the magnetic material and the strength of the applied magnetic field. Larger implants (seeds) generate heat by resistance to circumferential eddy currents induced on the surface of the seeds by an alternative magnetic field [41]. Multidomain particles produce heat by hysteresis loss effects. On the contrary, nanoparticle, particularly subdomain particle, suspensions generate heat mainly by Brownian relaxation (heat is the result of friction arising from the total particle oscillations) and Neel relaxation (heat is the result of friction arising

The advantage of magnetic hyperthermia is that it restricts the heating to the tumoral area, which presents both grand opportunities and challenges for the non-invasive treatment of tumors. Therefore, by combining this characteristic of the tumoral tissue with the MNPs property, it is obvious that the administration of MNPs (with the purpose of delivering toxic amounts of thermal energy to the tumoral tissue) will produce a more effective destruction of the tumoral tissue.

For clinical practice, MNPs must meet several criteria: they must be small enough to remain in the circulation after injection and pass through the capillary; they must not be an embolic agent; they must be non-toxic and non-immunogenic; they must maintain the initial structure; and they must be biodegradable. Another important property of these particles is to be highly magnetized in order for their movement to be controlled with a magnetic field so that they can be immobilized near the targeted tumoral area [47]. The most important factors, which determine the biocompatibility and toxicity of these materials, are the nature of the magneti‐ cally responsive component, the final size of the particles, their core, and their coatings [39]. The most utilized MNPs are magnetite (Fe3O4) or its oxidized form, maghemite (γ-Fe2O3). Magnetite is easier to obtain than maghemite; therefore, most of the studies utilized magnetite [38]. In order to avoid the constitution of large aggregates, the modification from the original form and biodegradation, the MNPs are coated with a biocompatible polymer during or after the synthesis [39]. The particles' size influence the stability, tissular diffusion, effective surface areas (easier attachment of ligands), and the power of absorption at tolerable altering current magnetic fields. Therefore, only subdomain magnetic particles (nanometer-sized), especially particles smaller than 100 nm (so-called nanoparticles), can be utilized [48, 49]. Also, it is important to highlight that the heating potential is dependent on particle size and shape, and thus the use of uniform particles is essential for a rigorous control in temperature [39]. Therefore, the magnetic particles used may modify the energy, absorption rate, mode of energy deposition, application, and focusing. For this technique, the sizes of the particles are as follows: seeds (rods of several millimeter size), multidomain particles (1–300 mm), nanopar‐ ticles (1–100 nm), and subdomain particles (below 20 nm) [41].

Gilchrist was the first author that showed promising results obtained after selective heating that followed the direct injection of a suspension of magnetic particles into draining lymph nodes from colon cancer [50]. In 2001, Moroz showed that hepatic arterial infusion of lipiodol containing ferromagnetic particles could result in an excellent targeting of liver tumors with hyperthermia on the subsequent application of an external alternating magnetic field [51]. The following years, encouraged by the results of the use of MNPs in animal studies (on mouse mammary carcinoma, glioblastoma, and prostate cancer), some authors focused on the improvement of HT techniques for clinical applications [52–56]. For in vivo delivery, the authors used thermosensitive liposomes, direct injection into the tumor, or the intravenous route.

An important progress has been made in improving the quality of the MNPs; therefore, for construction, high temperature crystallization or different coatings were used, such as dextran, polyethylene glycol (PEG), dopamine, silanes and gold [43].

Several authors introduced MNPs either in the core or in between the lipid bilayer of thermosensitive liposomes and, on alternating magnetic field AMF heating, the encapsulat‐ ed drugs were released [43]. Shinkai utilized liposomes where he introduced magnetite nanoparticles (with a diameter of 10 nm). After administration, these nanoparticles in‐ creased the temperature of the tissue [57]. In another study, Ito injected magnetite cationic liposomes (MCLs) into the tumor tissue. They heated the tissue above 43°C and obtained a complete regression of mammary carcinomas in all mice [58]. Also, Jimbow [52] developed a particle with N-propionylcysteaminylphenol (NPrCAP) conjugated onto the surface of magnetite nanoparticles (NPrCAP/M). The result was the inhibition of melanoma cells growth as a result of the production of cytotoxic free radicals. In another study, a thermosensitive polymer was layered onto MNPs covalently coupled to doxorubicin with an acid-labile hydrazine bond that showed release on heating with AMF and a pH of 5.3 (the pH of endosomes) [59]. The authors combined via emulsification MNPs with a polyvinyl alcohol polymer and encapsulated hydrophobic/ hydrophilic drugs. The drugs were released after the heating with an alternative magnetic field [60].

Direct intratumoral injection was used in the first MNP HT clinical trial treating a patient with a recurrent prostatic tumor [61]. Through the use of transrectal ultrasound and fluoroscopy guidance, the authors performed a transperineal injection of the MNPs into the prostate. After the administration of MNPs, the particles were selectively heated in an externally applied alternative magnetic field. The conclusions of these trials were encouraging. Due to the low clearance of MNPs from tumors, serial heat treatments were possible after a single magnetic fluid injection. Another positive aspect was the fact that a low magnetic field was used to produce the necessary temperatures. Furthermore, this treatment does not cause discomfort or serious side effects. In these studies, the CT exam had an accuracy rate of 85% in evaluating the treatment-related parameters. The same good results were obtained later in human glioma trials [62, 63].

In 2008, Takamatsu et al. combined the intra-arterial selective HT with the transcatheter arterial embolization technique in a rabbit model for renal carcinoma [64]. For injection, they utilized a mixture of commercially available nano-sized magnetic particles (Ferucarbotran) and lipiodol as embolic material. The mixture was injected into the renal artery under fluoroscopic guidance. The intratumoral temperatures of 45ºC were obtained after the area was exposed to an external alternating-current magnetic field. Even the result was not spectacular (the treated tumor was hypovascular) the authors speculated that this method can be used only in hypervascular tumors. In another study, Huang HS injected IV MNPs (1.9 mg Fe/g tumor) in a subcutaneous squamous cell carcinoma mouse model. After the injection, they applied a field of 38 kA/m at 980 kHz; therefore, the tumors could be heated to 60°C in 2 min. The results were encouraging, showing an ablating with millimeter (mm) precision and a surrounding tissue intact [43].

Intravenous administration has several advantages compared to sowing such as: it assures a more precise cover even for an irregular tumor and small tumors; it can be used for the treatment of metastasis (after one injection more than one tumor can be treated simultaneous‐ ly); the distribution is more overall (rather than the dotted distribution from sowing); and it is minimally invasive [43, 48].

The evaluation of the iron concentrations can be mapped with high accuracy by MRI, com‐ puted tomography or magnetorelaxometry [43, 65, 66].

The science of MNPs is still in its early stages. The recent results of magnetic HT in cancer therapy are very encouraging; but it is necessary to traverse the experimental stages into clinical practice to see the real applicability of this new technique.
