**2. Biomedical applications of magnetic nanoparticles**

Magnetic nanoparticles (MNP) with dimensions ranging from a few nanometers up to tens of nanometers, thanks to their comparable or smaller size than proteins, cells or viruses, are able to interact with (bind to or penetrate into) biological entities of interest [8]. These size advan‐ tages of MNPs together with their sensing, moving and heating capabilites based on the unique nanometer-scale magnetic and physiological properties give them the possibility to be used in biomedical applications such as magnetic resonance imaging (MRI), targeted drug delivery and hyperthermia [9].

can be increased above 40-42 o

C and the infected cells could be selectively destroyed. According

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to the models describing the heat release mechanisms of MNPs, increasing the frequency and the amplitude of the alternating field promises to significantly enhance the amount of released heat, but the limitations imposed by the biological systems restrict these values under a few tens of kA/m and a few hundreds of kHz for the field strength and frequency, respectively [17]. The MNPs' heating capacity in magnetic hyperthermia is denoted by Specific Absorbtion Rate (SAR) or in another term Specific Loss of Power (SLP), which is a measure of the energy converted into heat per unit mass. As the similar case in MRI CAs, the majority of magnetic hyperthermia heat mediators investigated to date are based on iron oxide MNPs, where in these studies the typical values reported for the maximum attained SAR range between 10 and 200 W/g [18-20]. However, it has been also focused on several systems alternative to iron oxide, where in some of them SAR values 3-5 times larger than those of similar iron oxide MNPs are attained for the same field parameters [21-22]. Recently, although there are lots of in-vitro studies about magnetic hyperthermia, the therapy with hyperthermia is still in pre-clinical stage and only a few studies on human patients are reported [23,24]. However, in cancer treatment the magnetic hyperthermia is thought to be introduced as a complementary technique to chemo- and radiotherapy as increasing the effects of these therapies [25, 26].

In another major in-vivo application, the MNPs are used as drug carriers in a magnetic 'tagdrag-release' process called targeted drug delivery. In a drug delivery process the MNPs, loaded with special drug molecules or conventional chemotherapy agents, are directly vectorized to tumor cells by targeting ligands on their surfaces or they brought into the vicinity of target tissue through magnetic forces exerted on them under an applied external magnetic field. Once the drugs/carriers are concentrated at the diseased site, the drugs are released from the carriers, again through modulation of magnetic field, enzymatic activity or changes in physiological conditions such as pH, osmolality or temperature. With this approach, the tumor cells can be destroyed by concentrating only the required quantity (dose) of drugs at target specific locations with minimized side effects on healthy tissues. The performance of the application depends mainly on the drug release kinetics and the cellular uptake of MNPs in tissues. There are huge number of drug delivery studies in the literature reporting both invitro and in-vivo results on different cell cultures and different types of tumors respectively, where in these studies several kind of targeting ligands and anticancer drugs are tested and in most of them again superparamagnetic iron oxide is used as magnetic core [27,28]. Generally in the design of MNPs for targeted drug delivery, in order to monitor the effect of the therapy,

MRI contrast increament ability of the same MNP system is also investigated [29].

Actually in recent years much more interest has been concentrated on multifunctional MNPs, in which the above mentioned diagnostic (MRI) and therapeutic (hyperthemia and drug delivery) capabilities are combined [15,30,31]. Although some MNP-based MRI contrast agents are commercialy available on one side and magnetic hyperthermia is already utilized in conjunction with other kind of therapies on the other side, MNPs optimized to perform both functions (diagnostic and therapeutical) have not been developed yet. Indeed the possibility to associate therapeutic effect generated by the heat release and delivered drugs with the enhanced contrast in MRI images, is extremely appealing since it would provide the

In MRI, which is the most promising non-invasive technique for the diagnosis of diseases, MNPs are used as contrast enhancement agents [10-12]. The improved contrast in MR images permits better definition and precise locating of diseased tissues (such as tumors) together with monitoring the effect of applied therapy. The operation of MRI is based on the Nuclear Magnetic Resonance (NMR) phenomena and the image processing is realized by spatially encoding of NMR signal of water protons which comes from different volume elements in the body called voxels. The image contrast in MRI depends mainly on proton density, spin-lattice (T1) and spin-spin (T2) nuclear relaxation times, differently weighted along different parts (voxels) of the body. The so-called contrast agents (CAs) themselves do not generate any signals, yet they contribute to the nuclear relaxation of water protons by creating local magnetic fields, which are fluctuating in time through different mechanisms like magnetization reversal and water diffusion [13]. As a consequence, the CAs decrease or increase the MRI signal intensity in the tissues by shortening both the T1 and T2 relaxation times of nearby protons resulting darker or brighter points in the image. The contrast enhancement efficiency of CAs is measured by the relaxivity r1,2, which is defined as the increament of the nuclear relaxation rates 1/T1,2 of water protons induced by one mM of the magnetic ion. The CAs having a ratio r2/r1 greater than two, especially at magnetic fields mostly used in MRI tomography (0.5, 1,5 or 3 Tesla), are classified as T2-relaxing (or negative) CAs since they more effectively decrease T2 rather than T1. On the other side CAs, characterized with a ratio r2/r1 smaller than two, have more pronounced effect on T1 and hence called as T1-relaxing (or positive) contrast agents [14]. The MNPs showing superparamagnetic property at physiological temperatures generally serve as T2-relaxing CAs and they negatively improve the image contrast resulting darker spots where they are delivered. Commercially a wide variety of superparamagnetic iron oxide (SPIO) based negative CAs are available in the market like Endorem, Sinerem, Resovist, Supravist, Clariscan, Abdoscan etc., where each of them are used for different puposes or in different organs in clinical MRI application.

In a second biomedical application called magnetic hyperthermia, which is a thermally treatment of cancerous cells based on the fact that the cancer cells are more susceptible to high temperatures than the healthy ones, the MNPs can be used as heating mediators [15,16]. In this technique after concentrating the MNPs in the region of malignant tissue (by targeting or by direct injection), the MNPs are made to resonantly respond to a time-varying magnetic field and transfer energy from the exciting field to the surroundings as heat. By this way using an alternating field with sufficient intensity and optimum frequency, the temperature of tissue can be increased above 40-42 o C and the infected cells could be selectively destroyed. According to the models describing the heat release mechanisms of MNPs, increasing the frequency and the amplitude of the alternating field promises to significantly enhance the amount of released heat, but the limitations imposed by the biological systems restrict these values under a few tens of kA/m and a few hundreds of kHz for the field strength and frequency, respectively [17]. The MNPs' heating capacity in magnetic hyperthermia is denoted by Specific Absorbtion Rate (SAR) or in another term Specific Loss of Power (SLP), which is a measure of the energy converted into heat per unit mass. As the similar case in MRI CAs, the majority of magnetic hyperthermia heat mediators investigated to date are based on iron oxide MNPs, where in these studies the typical values reported for the maximum attained SAR range between 10 and 200 W/g [18-20]. However, it has been also focused on several systems alternative to iron oxide, where in some of them SAR values 3-5 times larger than those of similar iron oxide MNPs are attained for the same field parameters [21-22]. Recently, although there are lots of in-vitro studies about magnetic hyperthermia, the therapy with hyperthermia is still in pre-clinical stage and only a few studies on human patients are reported [23,24]. However, in cancer treatment the magnetic hyperthermia is thought to be introduced as a complementary technique to chemo- and radiotherapy as increasing the effects of these therapies [25, 26].

**2. Biomedical applications of magnetic nanoparticles**

and hyperthermia [9].

186 Modern Surface Engineering Treatments

different organs in clinical MRI application.

Magnetic nanoparticles (MNP) with dimensions ranging from a few nanometers up to tens of nanometers, thanks to their comparable or smaller size than proteins, cells or viruses, are able to interact with (bind to or penetrate into) biological entities of interest [8]. These size advan‐ tages of MNPs together with their sensing, moving and heating capabilites based on the unique nanometer-scale magnetic and physiological properties give them the possibility to be used in biomedical applications such as magnetic resonance imaging (MRI), targeted drug delivery

In MRI, which is the most promising non-invasive technique for the diagnosis of diseases, MNPs are used as contrast enhancement agents [10-12]. The improved contrast in MR images permits better definition and precise locating of diseased tissues (such as tumors) together with monitoring the effect of applied therapy. The operation of MRI is based on the Nuclear Magnetic Resonance (NMR) phenomena and the image processing is realized by spatially encoding of NMR signal of water protons which comes from different volume elements in the body called voxels. The image contrast in MRI depends mainly on proton density, spin-lattice (T1) and spin-spin (T2) nuclear relaxation times, differently weighted along different parts (voxels) of the body. The so-called contrast agents (CAs) themselves do not generate any signals, yet they contribute to the nuclear relaxation of water protons by creating local magnetic fields, which are fluctuating in time through different mechanisms like magnetization reversal and water diffusion [13]. As a consequence, the CAs decrease or increase the MRI signal intensity in the tissues by shortening both the T1 and T2 relaxation times of nearby protons resulting darker or brighter points in the image. The contrast enhancement efficiency of CAs is measured by the relaxivity r1,2, which is defined as the increament of the nuclear relaxation rates 1/T1,2 of water protons induced by one mM of the magnetic ion. The CAs having a ratio r2/r1 greater than two, especially at magnetic fields mostly used in MRI tomography (0.5, 1,5 or 3 Tesla), are classified as T2-relaxing (or negative) CAs since they more effectively decrease T2 rather than T1. On the other side CAs, characterized with a ratio r2/r1 smaller than two, have more pronounced effect on T1 and hence called as T1-relaxing (or positive) contrast agents [14]. The MNPs showing superparamagnetic property at physiological temperatures generally serve as T2-relaxing CAs and they negatively improve the image contrast resulting darker spots where they are delivered. Commercially a wide variety of superparamagnetic iron oxide (SPIO) based negative CAs are available in the market like Endorem, Sinerem, Resovist, Supravist, Clariscan, Abdoscan etc., where each of them are used for different puposes or in

In a second biomedical application called magnetic hyperthermia, which is a thermally treatment of cancerous cells based on the fact that the cancer cells are more susceptible to high temperatures than the healthy ones, the MNPs can be used as heating mediators [15,16]. In this technique after concentrating the MNPs in the region of malignant tissue (by targeting or by direct injection), the MNPs are made to resonantly respond to a time-varying magnetic field and transfer energy from the exciting field to the surroundings as heat. By this way using an alternating field with sufficient intensity and optimum frequency, the temperature of tissue In another major in-vivo application, the MNPs are used as drug carriers in a magnetic 'tagdrag-release' process called targeted drug delivery. In a drug delivery process the MNPs, loaded with special drug molecules or conventional chemotherapy agents, are directly vectorized to tumor cells by targeting ligands on their surfaces or they brought into the vicinity of target tissue through magnetic forces exerted on them under an applied external magnetic field. Once the drugs/carriers are concentrated at the diseased site, the drugs are released from the carriers, again through modulation of magnetic field, enzymatic activity or changes in physiological conditions such as pH, osmolality or temperature. With this approach, the tumor cells can be destroyed by concentrating only the required quantity (dose) of drugs at target specific locations with minimized side effects on healthy tissues. The performance of the application depends mainly on the drug release kinetics and the cellular uptake of MNPs in tissues. There are huge number of drug delivery studies in the literature reporting both invitro and in-vivo results on different cell cultures and different types of tumors respectively, where in these studies several kind of targeting ligands and anticancer drugs are tested and in most of them again superparamagnetic iron oxide is used as magnetic core [27,28]. Generally in the design of MNPs for targeted drug delivery, in order to monitor the effect of the therapy, MRI contrast increament ability of the same MNP system is also investigated [29].

Actually in recent years much more interest has been concentrated on multifunctional MNPs, in which the above mentioned diagnostic (MRI) and therapeutic (hyperthemia and drug delivery) capabilities are combined [15,30,31]. Although some MNP-based MRI contrast agents are commercialy available on one side and magnetic hyperthermia is already utilized in conjunction with other kind of therapies on the other side, MNPs optimized to perform both functions (diagnostic and therapeutical) have not been developed yet. Indeed the possibility to associate therapeutic effect generated by the heat release and delivered drugs with the enhanced contrast in MRI images, is extremely appealing since it would provide the possibility before heating the tissue to track the particle distribution by MRI, and after the drug therapy or thermotherapy to have an immediate control of the efficacy of the treat‐ ment itself. In Figure.1 a summarized illustration of biomedical applications has been shown.

and uniform shape (monodispersed). This is because all the magnetic and physico-chemical properties strongly depend on the size and shape of the magnetic cores. From an applicative point of view the size, properly speaking the hydrodynamic size which is the total diameter of MNP including the coating tickness, is also important for the elongation of MNPs' circula‐ tion time in blood and for the improvement of their internalization by the cells at the target tissue such that smaller nanoparticles have bigger chance to reach the target cells and to

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In the search of suitable elements for the magnetic core of MNPs, among other magnetic materials transition metals like Fe, Ni, Co and Mn are good candidates since they offer high magnetization values which is important for high performance MRI and hyperther‐ mia applications. However they are not stable and oxidate very quickly yet in the synthesis step, if they are not specially treated. For this reason transition metal oxide compounds (also called ferrites) which are stable and have acceptable magnetizations are generally introduced in biomedical applications. Superparamagnetic iron oxide (SPION), belonging to ferrite family, is the most commonly employed one in biomedical applications. Nano‐ crystalline iron oxides have an inverse spinel crystal structures, where the oxygen atoms form face centered cubic lattices and iron ions occupy tetrahedral (Td) and octahedral (Oh) interstitial sites (Figure.2). İron oxide generally exist as two stable forms called magnetite (Fe3O4) and its γ phase maghemite (γ-Fe2O3), but there is also a α phase called hematite (α-Fe2O3), which is not stable and obtained by thermal treatment of magnetite or maghe‐ mite. In magnetite, bivalent Fe+2 ions occupy Oh sites and trivalent Fe+3 ions are equally distributed between Oh and Td sites, whereas maghemite, which can be result from the oxidation of magnetite, only contains Fe+2 ions distributed randomly over Oh and Td sites [32]. In magnetite, since there is the same number of Fe+3 ions in Oh and Td sites, which compensate for each other, the resulting magnetization arises only from the uncompensat‐ ed Fe+2 ions in Oh sites. On the other side the magnetization of maghemite originates from uncompensated Fe+3 ions. However their magnetic behaivours and other properties are

quite similar, which makes it very difficult to distinguish between them.

**Figure 2.** The cubic inverse spinel crystal structure of iron oxide showing Td and Oh sites

Alternatively other types of ferrites were also studied for biomedical applications. In these ferrites, as compared to the iron oxide nanocrystalls, Fe+2 ions are fully or partially replaced by other transition metals in spinel structure and they represented by a general formula

penetrate inside them.

**Figure 1.** Biomedical applications of magnetic nanoparticles (image has been reproduced from A. Lasicalfari et al. [9].
