**3.2.1 Magnetic properties of iron oxide nanoparticles**

Particles whose unpaired electron spins align themselves spontaneously so that the material can exhibit magnetization without being in a magnetic field are called ferromagnetic particles. Materials such as iron oxide nanoparticles that exhibit ferromagnetism can be permanently magnetized. The magnetic properties of iron oxide nanoparticles can be described by the dependence of the magnetic induction *B* on the magnetic field *H*. For most materials, the relationship between *B* and *H* is linear: *B* = *µH*, where *µ* is the magnetic permeability of the particles. Iron oxide particles exhibit paramagnetism if *µ* > 1, and diamagnetism if *µ* < 1.

Usually, ferromagnetic properties arise only when a certain number of atoms are bound together in solid form; single atoms cannot exhibit ferromagnetism. When the size of particles is smaller than the ferromagnetic domain, they are no longer ferromagnetic but exhibit superparamagnetism (Bansil et al., 1998). Magnetic nanoparticles smaller than ~ 30 nm show superparamagnetic behavior without any magnetic remanence (i.e., restoration of the induced magnetization to zero upon removal of the external magnetic field), but the particles still exhibit very strong paramagnetic properties with a very large susceptibility. This is one important advantage for magnetic nanoparticles: it enables their stability and dispersion upon removal of the magnetic field, as no residual magnetic force exists between the particles.

In MRI, superparamagnetic nanoparticles made of iron oxide act as contrast enhancement agents by shortening both the T1 and T2 relaxations of the surrounding protons. The influence on the T1 relaxation depends strongly on the local MNP concentration, and the shortening processes can be hindered by the coating thickness. The effect of MNPs on T2 shortening is caused by the large susceptibility difference between the particles and surrounding medium, which results in microscopic magnetic field gradients. At low concentrations, a T1-positive contrast can be observed; at high concentrations, the susceptibility effects cause irreversible destruction of the MR signal around the particles.

NMP agglomeration tends to slightly decrease the T1 relaxation times but markedly decrease the T2 times. Therefore, superparamagnetic nanoparticles typically are used to provide negative contrast enhancement using T2-weighted pulse sequences. The effectiveness of a contrast agent can be described by its relaxivity, which is the proportionality constant of the measured rate of relaxation, or R1 (1/T1) and R2 (1/T2). The relaxivity depends on not only the composition, size, and magnetic properties of the MNP, but also depends on experimental variables such as the field strength, temperature, and medium in which the measurements are made.

### **3.2.2 Molecular imaging in cancer**

292 Biomedical Science, Engineering and Technology

MRI is a powerful noninvasive imaging modality that is utilized widely in clinical medicine. MRI is based on the property that hydrogen protons will align and process around an external alternating magnetic field. The subsequent process through which these protons return to their original state is referred to as the relaxation phenomenon. Two independent processes, longitudinal relaxation (T1-recovery) and transverse relaxation (T2-decay), are monitored to generate the MR image. Local variations in relaxation, corresponding to image contrast, arise from the proton density and the chemical and physical natures of the different tissues. Due to their ability to enhance proton relaxation and accumulation in specific tissues, MFMNPs have been actively investigated as contrast enhancement agents

Particles whose unpaired electron spins align themselves spontaneously so that the material can exhibit magnetization without being in a magnetic field are called ferromagnetic particles. Materials such as iron oxide nanoparticles that exhibit ferromagnetism can be permanently magnetized. The magnetic properties of iron oxide nanoparticles can be described by the dependence of the magnetic induction *B* on the magnetic field *H*. For most materials, the relationship between *B* and *H* is linear: *B* = *µH*, where *µ* is the magnetic permeability of the particles. Iron oxide particles exhibit paramagnetism if *µ* > 1, and

Usually, ferromagnetic properties arise only when a certain number of atoms are bound together in solid form; single atoms cannot exhibit ferromagnetism. When the size of particles is smaller than the ferromagnetic domain, they are no longer ferromagnetic but exhibit superparamagnetism (Bansil et al., 1998). Magnetic nanoparticles smaller than ~ 30 nm show superparamagnetic behavior without any magnetic remanence (i.e., restoration of the induced magnetization to zero upon removal of the external magnetic field), but the particles still exhibit very strong paramagnetic properties with a very large susceptibility. This is one important advantage for magnetic nanoparticles: it enables their stability and dispersion upon removal of the magnetic field, as no residual magnetic force exists between the particles. In MRI, superparamagnetic nanoparticles made of iron oxide act as contrast enhancement agents by shortening both the T1 and T2 relaxations of the surrounding protons. The influence on the T1 relaxation depends strongly on the local MNP concentration, and the shortening processes can be hindered by the coating thickness. The effect of MNPs on T2 shortening is caused by the large susceptibility difference between the particles and surrounding medium, which results in microscopic magnetic field gradients. At low concentrations, a T1-positive contrast can be observed; at high concentrations, the susceptibility effects cause irreversible destruction of the MR signal around the particles. NMP agglomeration tends to slightly decrease the T1 relaxation times but markedly decrease the T2 times. Therefore, superparamagnetic nanoparticles typically are used to provide negative contrast enhancement using T2-weighted pulse sequences. The effectiveness of a contrast agent can be described by its relaxivity, which is the proportionality constant of the measured rate of relaxation, or R1 (1/T1) and R2 (1/T2). The relaxivity depends on not only the composition, size, and magnetic properties of the MNP, but also depends on experimental variables such as the field strength, temperature, and

**3.2.1 Magnetic properties of iron oxide nanoparticles** 

medium in which the measurements are made.

**3.2 MRI** 

for MRI.

diamagnetism if *µ* < 1.

The generation of new molecular targets that are closely related to pathophysiology will open the way for the development of new treatment paradigms for currently untreatable diseases. In recent years, many new diagnostic technologies have been developed, including molecular diagnostic compounds and new imaging technologies such as MR molecular imaging. Molecular imaging is the noninvasive imaging of targeted macromolecules, cells, and biological or cellular processes in living organisms. Due to their ability to act as molecularly targeted imaging agents, MNPs play an integral role in the applications of early disease detection, individualized treatment, and drug development. In the clinical imaging of tumors, MNPs can be used as contrast enhancement agents to improve the detection, diagnosis, and therapeutic management of solid tumors by exploiting the unique molecular signatures of the diseases. MNPs also have been investigated to improve the delineation of the tumor position, boundaries, and volume.

The first clinical indication for iron oxide nanoparticles was the imaging of liver tumors and metastases. After intravenous injection, MNPs are taken up rapidly by hepatic specialized macrophages. This process causes a drop in MR signal intensity and generates hypointense images, mostly because of a susceptibility effect. However, tumors lack a permanent decrease in signal intensity after MNP administration; tumors are almost devoid of macrophages, which are located exclusively in the healthy hepatic parenchyma. Therefore, MNPs can markedly increase the contrast between healthy and diseased tissue. The clinical imaging of liver tumors and metastases through RES-mediated uptake of MNPs has allowed the detection of lesions as small as 2–3 mm (Semelka & Helmberger, 2001). In combination with MRI, MNPs also have been shown to be effective in the identification of lymph node metastases of 5–10 mm in diameter (Harisinghani et al., 2003). The use of MFMNPs as contrast enhancement agents provides increased lesion conspicuousness and lesion detection compared to nonenhanced imaging.

MFMNPs are currently under evaluation for use in improving the delineation of brain tumor boundaries and quantifying tumor volumes (Enochs et al., 1999; Neuwelt et al., 2004). Some recent approaches have explored utilizing iron oxide nanoparticles as drug delivery vehicles for the MRI-monitored magnetic targeting of brain tumors (Chertok et al., 2008). The accumulation of iron oxide nanoparticles in gliosarcomas is enhanced by magnetic targeting and successfully quantified by MRI (Chertok et al., 2008) (Fig. 6). Such noninvasive approaches for cancer diagnosis and therapy also have been adopted in the treatment of prostate, breast, and colon cancers.

As both drug delivery devices and MRI contrast enhancement agents, MNPs retain the ability to track the movement of drug through the body. This is significant because it allow clinicians to monitor the effectivity of injected therapeutics to reach their target sites. There remains significant flexibility in the contrast agents implemented in these constructs and the manner in which drugs are delivered. Medarova et al. (2007) recently developed cross-linked iron oxide nanoparticles modified with a NIR fluorophore, therapeutic siRNA sequences, and a cell penetrating peptide. The MNPs used passive targeting by the EPR effect to direct tumor localization. In vivo, these MNPs demonstrated therapeutic efficacy against target tissue, as determined by real time PCR and histological evaluation, while simultaneously demonstrating image contrast in both MR and optical imaging. In a study by Sun et al. (2008a) active cell targeting was shown by PEG-coated MNPs to which the chemotherapeutic, methotrexate, and targeting molecule, chlorotoxin, were attached. The selective contrast enhancement of the 9L brain tumor by these MNPs indicates preferential accumulation compared with the same MNP construct without the chlorotoxin peptide in a 3-day study.

Fig. 6. MR images of brain tumor. Change in the R2 relaxation for the tumor regions before (baseline) and 1–3 h after MNPs administration in (A) control and (B) targeted rats (Chertok et al., 2008).

In another recent study by Yang et al. (2007) simultaneous targeted drug delivery and MR imaging of breast cancer tumors were demonstrated through the use multifunctional magneto-polymeric nanohybrids composed of magnetic nanocrystals and doxorubicin which were simultaneously encapsulated within an amphiphilic block copolymer shell. The surfaces of these micelles were additionally functionalized with the breast cancer targeting/therapeutic ligand, anti-Herceptin antibody. In vivo evaluations of this nanoparticle system were performed in nude mice bearing NIH3T6.7 breast cancer tumors. The quantitative evaluation of MR images revealed preferential accumulation of the targeted MNPs compared to the control MNPs. The therapeutic functionality of the MNPs developed in this study were additionally evaluated and it was determined that the HER-MMPNs which were decorated with targeting ligands and loaded with doxorubicin were most effective in inhibiting tumor growth. Combined, these findings illustrate the functionality and efficacy of targeted multifunctional MNPs for simultaneous MR imaging and drug delivery.

### **3.3 Hyperthermia**

Hyperthermia is the method of using heat as a treatment for malignant tumors. It is based on the observation that tumor cells are more susceptible to heat than normal cells, due to the

Fig. 6. MR images of brain tumor. Change in the R2 relaxation for the tumor regions before

In another recent study by Yang et al. (2007) simultaneous targeted drug delivery and MR imaging of breast cancer tumors were demonstrated through the use multifunctional magneto-polymeric nanohybrids composed of magnetic nanocrystals and doxorubicin which were simultaneously encapsulated within an amphiphilic block copolymer shell. The surfaces of these micelles were additionally functionalized with the breast cancer targeting/therapeutic ligand, anti-Herceptin antibody. In vivo evaluations of this nanoparticle system were performed in nude mice bearing NIH3T6.7 breast cancer tumors. The quantitative evaluation of MR images revealed preferential accumulation of the targeted MNPs compared to the control MNPs. The therapeutic functionality of the MNPs developed in this study were additionally evaluated and it was determined that the HER-MMPNs which were decorated with targeting ligands and loaded with doxorubicin were most effective in inhibiting tumor growth. Combined, these findings illustrate the functionality and efficacy of targeted multifunctional MNPs for simultaneous MR imaging

Hyperthermia is the method of using heat as a treatment for malignant tumors. It is based on the observation that tumor cells are more susceptible to heat than normal cells, due to the

(baseline) and 1–3 h after MNPs administration in (A) control and (B) targeted rats

(Chertok et al., 2008).

and drug delivery.

**3.3 Hyperthermia** 

higher rates of metabolism of cancer cells. Cancer cells typically show signs of apoptosis and necrosis when heated to 41-47 °C, whereas normal cells can survive at higher temperatures (Milleron & Bratton, 2007). Hyperthermia with targeted nanoscale heaters is recognized as a useful therapeutic modality to kill cancer by essentially "cooking" malignant cells from the inside out.

Magnetic nanoparticle hyperthermia is actualized by the exposure of cancer tissues to an alternating magnetic field. The magnetic field cannot be absorbed by the living tissues and can be applied to deep regions in the living body. When MNPs are injected into an organ with a tumor, they tend to accumulate in the tumor due to passive and active targeting strategies (as described above). Subsequent exposure to an alternating magnetic field causes heat to be generated in the tumor tissue due to magnetic hysteresis loss. This process effectively destroys the tumor but not the surrounding healthy tissue. The amount of heat generated depends on the nature of the MNPs and magnetic field parameters used.

The use of MFMNPs for targeted hyperthermia has shown a therapeutic effect in several types of tumors. Using dextran-coated MNPs conjugated to breast cancer–targeting chimeric L6 mAb, DeNardo et al. (2005) demonstrated the feasibility of this method for treating breast cancer cells. Kobayashi et al. constructed a novel therapeutic tool of magnetite nanoparticleloaded anti-HER2 immunoliposomes that was applicable to the treatment of HER2 overexpressing cancer (Ito et al., 2004).

A clinical breakthrough in MNP use was made in 2007, when Maier-Hauff et al. (2007) reported the results of using heated implanted MNPs for therapeutic hyperthermia in humans. In that study, 14 patients with recurrent glioblastoma multiforme, a type of severe brain cancer, received an intratumoral injection of aminosilane-coated MNPs. The tumor sites were located by several comprehensive MRI scans, and the patients were exposed to an alternating magnetic field to induce particle heating. The nanoparticle deposits were stable for several weeks, and all patients tolerated the nanoparticles without any complications. These findings indicate that MNP hyperthermia may be an effective therapeutic method to cure human brain cancer.

As a potential approach for the treatment of malignant tumors, MNP hyperthermia has the following advantages: it provides a noninvasive way to raise cell temperatures to a therapeutic level; MNPs can be visualized using MRI, thus combining diagnostic and therapeutic approaches in one type of particle; and the particles can be functionalized and combined with other types of treatment, such as chemotherapy or radiotherapy.
