**3.2 Hyperthermia HeLa cell treatment with silica-coated manganese oxide nanoparticles**

The treatment of tumours by hyperthermia is based on the killing of tumour cells by heating them. Temperatures over around 42ºC kill tumour cells. Moreover, the behaviour of normal and tumour cells is different with temperature; generally, normal cells show better resistance to temperature than the tumour ones (Hahn et al., 1974). If it is possible to heat the tumour area at temperatures that would kill tumour cells but not the normal cells, we would be able to treat tumours selectively with less damage to the body than other therapies such as chemotherapy.

As mentioned above, in the past years nanoparticles have attracted much attention for medical applications due to their small size that enables them to be inserted inside the body and transported round it. In particular, magnetic nanoparticles have been used as a heating source for magnetic hyperthermia. Under the influence of an alternating high-frequency magnetic field, they generate heat through hysteresis losses, induced eddy currents and Neel and/or Brown relaxation processes (Jordan et al., 1993). Thus, different kinds of magnetic nanoparticles have been tested as a heating source. The new materials must satisfy stringent conditions: they must be biocompatible, be stable in aqueous solution, possess high thermal efficiency as heating elements and be highly accumulating inside tumour cells, so that when applying the alternating magnetic field (AMF) the increase in temperature can induce cellular death (Kong et al., 2001). Superparamagnetic iron oxide is the most common material tested up to now due to its high biocompatibility, low synthesis cost, enhanced specific loss power and easy functionalization (Hergt et al., 1998). However, in spite of these advantages, it is not possible to control the local heating temperature, because it is not possible to measure the exact temperature or distribution of temperature in a tissue under magnetic particle hyperthermia (MPH) treatment. Therefore, the temperature reached in a tissue under MPH treatment will depend on a large number of particle parameters, such as size and concentration in the tissue of the nanoparticles, the conditions of the external applied field, and the length of the treatment (Gazeau et al., 2008).

To avoid the obstacle of temperature control, new materials of tunable Curie temperature (Tc) are being intensively investigated to achieve temperature autostabilization at the hyperthermia conditions (Pradhan et al., 2007). Magnetic particles with tuneable Tc will prevent the temperature of the whole tumour or the hottest spot around the particle raising its temperature over the Curie Temperature, so, avoiding the use of any local temperature control system.

When a ferromagnetic nanoparticle reaches its Curie Temperature it becomes paramagnetic, and its magnetic moment drastically decreases. Thus, the eddy currents and the relaxation processes decrease as well and the hysteresis losses disappear. This means that the nanoparticles will stop heating the medium and the temperature will go back below the Curie Temperature becoming ferromagnetic again and so recovering the heating process. Therefore, it becomes a self regulating system and the culture temperature will always be around the Curie Temperature.

Bearing in mind all these facts, manganese perovskites meet the requirements for magnetic hyperthermia treatments.

The treatment of tumours by hyperthermia is based on the killing of tumour cells by heating them. Temperatures over around 42ºC kill tumour cells. Moreover, the behaviour of normal and tumour cells is different with temperature; generally, normal cells show better resistance to temperature than the tumour ones (Hahn et al., 1974). If it is possible to heat the tumour area at temperatures that would kill tumour cells but not the normal cells, we would be able to treat tumours selectively with less damage to the body than other therapies

As mentioned above, in the past years nanoparticles have attracted much attention for medical applications due to their small size that enables them to be inserted inside the body and transported round it. In particular, magnetic nanoparticles have been used as a heating source for magnetic hyperthermia. Under the influence of an alternating high-frequency magnetic field, they generate heat through hysteresis losses, induced eddy currents and Neel and/or Brown relaxation processes (Jordan et al., 1993). Thus, different kinds of magnetic nanoparticles have been tested as a heating source. The new materials must satisfy stringent conditions: they must be biocompatible, be stable in aqueous solution, possess high thermal efficiency as heating elements and be highly accumulating inside tumour cells, so that when applying the alternating magnetic field (AMF) the increase in temperature can induce cellular death (Kong et al., 2001). Superparamagnetic iron oxide is the most common material tested up to now due to its high biocompatibility, low synthesis cost, enhanced specific loss power and easy functionalization (Hergt et al., 1998). However, in spite of these advantages, it is not possible to control the local heating temperature, because it is not possible to measure the exact temperature or distribution of temperature in a tissue under magnetic particle hyperthermia (MPH) treatment. Therefore, the temperature reached in a tissue under MPH treatment will depend on a large number of particle parameters, such as size and concentration in the tissue of the nanoparticles, the conditions of the external

To avoid the obstacle of temperature control, new materials of tunable Curie temperature (Tc) are being intensively investigated to achieve temperature autostabilization at the hyperthermia conditions (Pradhan et al., 2007). Magnetic particles with tuneable Tc will prevent the temperature of the whole tumour or the hottest spot around the particle raising its temperature over the Curie Temperature, so, avoiding the use of any local temperature

When a ferromagnetic nanoparticle reaches its Curie Temperature it becomes paramagnetic, and its magnetic moment drastically decreases. Thus, the eddy currents and the relaxation processes decrease as well and the hysteresis losses disappear. This means that the nanoparticles will stop heating the medium and the temperature will go back below the Curie Temperature becoming ferromagnetic again and so recovering the heating process. Therefore, it becomes a self regulating system and the culture temperature will always be

Bearing in mind all these facts, manganese perovskites meet the requirements for magnetic

**3.2 Hyperthermia HeLa cell treatment with silica-coated manganese** 

applied field, and the length of the treatment (Gazeau et al., 2008).

**oxide nanoparticles** 

such as chemotherapy.

control system.

around the Curie Temperature.

hyperthermia treatments.

The manganese oxides perovskite La1-x(SrCa)xMnO3 have a Curie temperature that, depending on the cation ratio, can range from 300 K to 350 K (so 42-44 ºC is within the range) and they have large magnetization values of about 30 – 35 emu/g.

The preparation of the particles is as follows. First the particles are created by the ceramic method from compounds of La2O3, CaCO3, SrCO3, and MnO2. These particles obtained by the ball milling method form agglomerates due to the dipolar magnetic interaction and the lack of surfactants. The agglomerates also have a large size distribution, with sizes greater than 1 m. Since the magnetic interaction decreases with temperature, the particles are dispersed in ethanol and heated over the Tc in order to disaggregate them and to select the smaller ones, thus obtaining an average size of 100 nm. These NPs are not biocompatible so they have to be coated with silica following the Stöber method (Stöber et al., 1968).

The magnetic properties are not significantly affected by the size selection. Since the size is around 100 nm, the selected NPs still behave quite like the as-prepared ceramic. However, the magnetic properties are affected by the coating: the total magnetization is reduced by the presence of diamagnetic silica and the Curie Temperature decreases when the nanoparticles are coated. For example, for the composition La0.56(CaSr)0.22MnO3, at low temperature the magnetization at 1 kOe decreases from 31 to 21 emu/g (about 32%) while the Tc decreases from 68 ºC to 44 ºC for the uncoated and the coated nanoparticles respectively.

Another problem that must be faced is if it is necessary to increase the temperature up to 42 – 44 ºC in the whole tumour (so needing magnetic nanoparticles with very high magnetic moment or a very high concentration of nanoparticles) (Lacroix et al., 2009) in order to induce tumour damage or if it is enough to raise the temperature locally in the cells to induce apoptotic tumour death. The study of intracellular hyperthermia can shed light on this question.

Biological tests with perovskites were performed in order to prove their validity as a heating source for tumour hyperthermia. They were put inside a culture of HeLa cells. HeLa cells are a family of tumour cells widely used by biologists. HeLa cells were incubated with a concentration of 0.5 mg/ml of perovskites for 3 h. After incubation, cells were washed 3 times with PBS and then exposed for 30 min to a 100 KHz alternating magnetic field of 15 mT.

The cells incubated with the perovskites but without being submitted to an alternating magnetic field do not show any change, thus, demonstrating that the coating of the nanoparticles makes them biocompatible as expected. For the cells submitted to an alternating field the cell morphology was not affected immediately after the incubation and exposition to AMF. However, the perovskite + AMF treatment provoked deep morphological alterations, 24 h after the combined treatment, which corresponds to different stages of cell death by an apoptotic process. It is known that apoptosis is a regulated process which requires the active participation of specific molecules and is a characteristic mechanism of cell death for temperature around 42 ºC. The temperature increase of the culture during the application of the AMF (controlled by an infrared thermometer) was lower than 0.5 K for the PER incubated HeLa cells. This means that the small size of the perovskites cannot heat the cell culture. However, perovskites can induce local hot spots that damage irreversibly the structure and functionality of the cell proteins triggering the cell apoptosis (Fig 12).

Magnetic Sensors for Biomedical Applications 145

biocompatibility of magnetic materials, this last option is more appropriate. Two large groups of materials can be distinguished: those used as bulk materials and the

On the one hand, among the widely established bulk biomaterials (titanium alloys, cobaltchromium alloys, noble metals, Nitinol, austenitic stainless steel, alumina, calcium phosphates, carbon and polymers like UHMW polyethylene, PMMA or silicones) there are none with ferromagnetic properties. Regarding the metallic materials (excepting the noble metals, which are not ferromagnetic) only those that develop a well attached surface oxide layer usually present a good response to corrosion. In fact, if Chromium is added above 12% weight to Fe or Co the alloys become stainless. The stainless steels used for medical applications (316L and 304L) also incorporate Ni, among other elements, in their composition, which improves their corrosion resistance and stabilizes the face centred cubic

In general, it could be said that most the ferromagnetic materials are not biocompatible or their biocompatibility is not known. Very few papers on the biocompatibility of magnetic materials can be found. Even fewer can be found on cells. Most of them are corrosion studies done with liquids that simulate the pH of different biological mediums. Magnets have attracted interest in medicine (Riley at al., 2002). In particular, rare earth magnets have been investigated in the field of dentistry to push or pull teeth or as prostheses retention systems (Noar et al., 1999). They show bad corrosion resistance and have been used

Studies on Fe-Co alloys and magnetostrictive NiMnGa and Terfenol\_D show poor cell viability, excepting the last one that presents high corrosion (Pouponneau et al. 2006). However the system Co-Pt and Fe-Pt show better corrosion response (Yiu et al. 2004)). At research level, the Fe based alloy PM2000 shows good corrosion behaviour and cell viability together with a significant saturation magnetization, especially when coated with alumina

The surface is where the first contact between the material and the biological entity takes place. This is why surface biomaterials are being researched in depth, while surface modification is one of the most widely used strategies for improving biomaterial properties. In the case where a magnetic feature can only be achieved with a material that is not biocompatible the simplest solution is to encapsulate it or to coat it with a biocompatible material. Anyway, as coatings also present their own problems (adherence, thickness,...), it would be very interesting to investigate and to develop biomaterials with good

On the other hand, there already exist some nanoparticles commercially used as contrast agents in imaging diagnostic techniques or drug targeting and magnetic separation applications, like the Iron Oxide or the Gd, because of their magnetic properties. However, an enormous effort is being made to develop biocompatible magnetic nanoparticles for their application in biomedicine due to the attractive possibilities that they offer (Pankhurst, 2003) as hyperthermia agents for coadyuvant cancer treatment, drug delivery systems, as well as

structure. This latter fact explains their paramagnetic character.

embedded in different polymeric materials or with Ti coating.

by thermal oxidation (Flores et al., 2004).

for the previously mentioned reasons.

magnetic properties.

micro/nanoparticles.

Fig. 12. Nanoparticles (black spots) inside the cell after apopthosis (White holes) (Villanueva, 2010)

These results show that perovskite nanoparticles have a high potential for cancer cell hyperthermia, working as smart mediators for self controlled heating of tumours, where the heating source is switched off when the local temperature of the tumour reaches the desired value.
