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1. Introduction

1.1 Background

the western world [1], 883 cases of pancreatic cancer death have been reported in Taiwan itself from 1990 to 1994 [2]. Most patients with pancreatic cancer do not develop symptoms until after the disease has metastasized and at the time of diagnosis, > 80% of patients have locally advanced or metastatic disease [3]. After diagnosis the overall median survival time is 2–8 months and in general 5 years survival rate of pancreatic cancer patients is just only 1 % [4]. The most widely used and best validated marker for pancreatic cancer is CA 19-9 [5]. However due to inadequate sensitivity and specificity European Group on Tumor Marker [6] and American Society of Clinical Oncology [7] discourage the use of CA 19-9 as a test for pancreatic cancer, especially for early forms of the disease. Modern radiological techniques of imaging and diagnostic cannot detect the early stage of this disease. Additionally several overlapping symptomatological characteristics have been observed between pancreatic adenocarcinoma and chronic pancreatitis, thereby, it is often very difficult to perceive the distinction between both pathological cases [8]. Therefore, there remains a critical need for the development of novel targeted bioimaging probes for accurate and early detection of pancreatic cancer.

MUC4 is a high molecular weight O-glycoprotein expressed in various epithelial tissues including the trachea, colon, stomach, cervix, and lung [9]. Although, MUC4 aberrant

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expression is reported in premalignant and malignant pancreatic lesions as well as in several pancreatic cancer cell lines with no detectable expression in the normal pancreas or chronic pancreatitis [10,11] making it attractive biological marker for pancreatic cancer. Recent study has shown that MUC4 exhibits, 91% sensitivity and 100 % specificity for early diagnosis of pancreatic cancer [12].

In the last two decades magnetic resonance imaging (MRI) has emerge as an advanced and most powerful tool for clinical diagnostic imaging. It has many potential advantages that other imaging modality does not offer for instance, noninvasive, radiation free technique. The physical principle behind MRI is nuclear magnetic resonance (NMR). NMR was discovered by Felix Bloch and Edward Purcell back in 1946 [13-15]. The NMR phenomenon is based on the fact that the atomic nuclei which possess a spin angular momentum interact with magnetic fields. In MRI the resonance of water protons is primarily used for diagnostic purposes since water constitutes about 63% of our bodies and the natural abundance of 1H isotope is 99.9%.

The major disadvantage of MRI is its inherent low sensitivity. To enhance the quality of image contrast agents have been often used prior to MR imaging [16]. Primarily gadolinium-based contrast agents (GdCAs) have been used in MRI investigations which serve as a T1 agent.

Recent development in nanotechnology has made it possible to synthesize, characterize, and specifically tailor the functional property of nanoparticle for biomedical and diagnostic application. The unique properties and utility of nanoparticles arise by virtue of high surface to volume ratio, large percentage of surface atoms compare to bulk materials and the size of nanoparticles is comparable to biomolecules such as proteins

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and polynucleic acids [17]. The most extensively-studied nanomaterials include quantum dots (QDs) [18], carbon nanotubes (CNT) [19], nanoshells [20], and paramagnetic nanoparticles [21]. These nanoparticle have size approximately 100 to 10000 times smaller than human cells, hence nanoscale particles can offer extraordinary interactions with biomolecules both on the surface of and inside cells [22].

Recently, superparamagnetic iron oxide (SPIO) nanoparticles have found widespread application in medicine, in particular as contrast agents in MRI. SPIO nanoparticles are the most commonly used T2 contrast agents in clinics. SPIOs have an important advantage compared to paramagnetic ions since each vectorized particle bears a huge magnetic moment, compared to a single targeted paramagnetic ion [23].

Consequently SPION may potentially provide higher contrast enhancement in MRI than conventional paramagnetic Gd-based contrast agents. Initially SPIOs were developed as T2 agent due their large size and magnetic moment. However, recent study shows that SPIO nanoparticles with size less than 10 nm have excellent T1 enhancing properties [24].

Nanoparticles are usually nonspecifically taken up by the reticulo endothelial system (RES) [25] and the overall size of the nanoparticle can affect the specificity of nanoparticle to the organ (liver, spleen, or lymph node), non-targeted iron oxide nanoparticles have been used for liver [26], spleen [27], and lymph node imaging [28].

The advancement in understanding of the molecular biology of cancer has provided an enormous range of target for drug delivery.

In general, tissue or organ-specific nanoparticle contrast agents have two components: a biocompatible magnetic nanoparticle capable of altering the MR signal intensity and biological moieties that possess lock-and-key interactions, including those

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observed in antibody-antigen and enzyme-substrate recognition. Surface modifications involving biomolecules, surface coverage is significantly important as is the ability for the immobilized molecules to retain their native conformations and binding profiles. One extremely useful route to postsynthetic modification of iron oxide nanoparticles is accessed by employing the common organosilane reagent, γ−aminopropyltriethoxy silane (APTES) [29].

Single-walled carbon nanotubes (SWNT) exhibit unique electrical and optical properties, including large Raman scattering cross-sections, near-infrared (NIR) fluorescence, and UV/visible/NIR absorption [30-32]. It is an upcoming potent candidate for the photothermal therapeutic agent since it generates significant amounts of heat upon excitation with near-infrared light (NIR, λ=700-1100 nm). Such a photothermal effect can be employed to induce thermal cell death in a noninvasive manner [33]. SWNT/iron oxide nanoparticle complexes have been used as multimodal biomedical imaging agents [34]. Recent studies have shown that quantum size effects cause finite CNTs to exhibit special properties such as magnetism [35].

The aim of this study was to develop T2-weighted MRI contrast agent for early diagnosis of pancreatic cancer. For this study SPIO nanoparticle was generously gifted by Mr. Ming Hung. Synthesis and characterization methods of SPIO nanoparticle are beyond the scope of the thesis. In addition we have successfully synthesized graphene nanosheets (GNSs) in high yield. The GNSs have been charactized by scanning electron microscope (SEM), X-ray diffraction (XRD), Transmission electron microscopy (TEM), and Raman shift. The carbon nanoscrolls (CNSs) synthesized from GNSs has been observed by SEM.

Experiments are in progress to evaluate saturation magnetization and T2 relaxation times

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of CNSs. The 8G7 (anti-MUC4 mAbs) was immobilized on the surface of SPIO nanoparticle which can specifically target abruptly expressed MUC4 mucine in pancreatic tumor. Typical hydrodynamic size distribution, relaxivity, and in vitro MRI study was conducted to evaluate sensitivity of SPIO-mPEG-8G7 nanoparticle.

1.2 MUC4 as Pancreatic Cancer Tumor Marker

1.2.1 Background

Mucins are proteins, which carry a large amount of sugar attached through oxygen (O-linked) to the protein core. The epithelial mucins are expressed by the cells that line the tubes and glands in some tissues of the body, for example the stomach, colon and ducts of the breast [36]. In general, musines are involved in the protection and lubrication of epithelial surfaces. However, recent study shows that mucins are also involved in cell signaling modulation and affect tumor cell phenotype [37].

1.2.2 MUC4 Mucin

MUC4 is a high molecular weight transmembrane mucin that is expressed by various epithelial cells (trachea, lung, stomach, colon and cervix) in normal tissues [38]. MUC4 has two

subunits, MUC4α and MUC4β. The two subunits are non-covalently

link to each other.

Schematic representation of the modular structure of MUC4 is shown in Figure 1

. The mucin type subunit MUC4α is of 850 kDa and the

membrane-bound growth factor like subunit of 80 kDa [39].

Numerous studies have established association of MUC4 with the progression of cancer and metastasis. An aberrant expression of MUC4 is reported in precancerous lesions, indicating its early

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involvement in the disease process with no detectable expression in the normal pancreas or chronic pancreatitis [40].

Figure 1 A: Schematic representation of the modular structure of MUC4.

B: Schematic representation of MUC4 protein. The representation is not drawn to scale.

[39]

The RT-PCR analysis of MUC genes expression in pancreatic adenocarcinoma and chronic pancreatitis are shown in Table 1 and Table 2 respectively. Overlapping symptomatological characteristics have been observed between pancreatic adenocarcinoma and chronic pancreatitis [8]. However from the Table 1 and Table 2 we can easily observe that, in pancreas aberrant expression of MUC4 gene exclusively occurs

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during pancreatic adenocarcinoma making it noval biomarker for diagnosis of pancreatic cancer.

Table 1 Expression of MUC genes in pancreatic adenocarcinoma by RT-PCR analysisa. Total RNA from 15 pancreatic tumor cell lines was isolated and subjected to semiquantitative RT-PCRa

aData obtained from reference [40]. b D.S., differentiation stage; PD, poorly differentiated; MD, moderately differentiated; WD, well differentiated; ND, not determined. c_, no expression; +, low level; ++, moderate level; +++, high level; ++++, very high level; ND, not determined.d Normal tissue samples were included as controls. S.I., small intestine; S.G., salivary gland

Expression levelc

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Table 2 Expression of MUC genes in chronic pancreatitis by RT-PCR analysisa. Total RNA from 10 chronic pancreatitis samples was isolated and subjected to semiquantitative RT-PCR.

Expression levela

a Data obtained from reference [40]. b _, no expression; +, low level; ++, moderate level; +++, high level; ++++, very high level.

1.2.3 Role of MUC4 in Cancer Development

During cancer development controlled interaction between neighboring cells and the cell and the extracellular matrix are interrupted, whereas new interactions establish due to alterations in the cell surface proteins, extracellular matrix composition, and loss of cell polarity. A favorable environment is created by these molecular and cytoarchitectural changes at the tumor site which facilitate in tumor development [41]. The MUC4 is a multifunctional protein implicated in a variety of biological functions. Under normal conditions, MUC4 is localized at the apical surface of the epithelial cells.

Genetic/epigenetic changes, alternative splicing, and biochemical modifications may result in an aberrant expression of MUC4 [12]. In addition, MUC4 can change the role of other adhesion-associated signaling molecules via steric hindrance. Lose in polarity of tumor cell, facilitate MUC4 to find new interacting partner(s). Recent studies established

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that MUC4 interacts with HER2/ ErbB2 and alter its expression [41]. Role of MUC4 in cancer development is systematically shown in Figure 2.

Figure 2 Role of MUC4 in cancer development [12]

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1.2.4 Monoclonal Antibodies Targeting MUC4

The overriding challenge in developing antibodies to specifically target mucin proteins is lies in their structure, 80% of their mass is composed of glycan chains. The Glycan chains cover greater part of the core protein epitopes. The main mucin-type antibodies available recognize glycan epitopes and therefore do not bind with a unique mucin. For instance, the DU-PAN-2 antibody is reactive with MUC1 and MUC4 [40]. Recently, a series of monoclonal antibodies (mAbs), 9H8-1F3, 13F12-2C9, 12B8-2D9, 8G7-1D1, 12C11-1G2, 11A8-1B7, and K2G6-1H6, directed against the TR region of MUC4 have been reported [38]. To generate these antibodies a 16-amino-acid sequence was chosen same as that of 16 amino acid residues repeated in tandem up to 400 times for the main MUC4 allele [42]. The mAb, 8G7 has been discovered to strongly react against the MUC4 peptide and with native MUC4 from human tissues or pancreatic cancer cells in Western blotting, immunohistochemistry, and confocal analysis [38].

1.3 Contrast Agent 1.3.1 Background

It had been known long back that atomic nuclei, which possess a spin angular momentum, will interact with magnetic field. In 1971, Raymond Damadian observed that certain mouse tumors display high relaxation times compared with normal tissue and this was the was the inception of imaging the human body [43]. MR signal is due to relaxation of water protons that are trying to realign with a static magnetic field following the application of radiofrequency (RF) pulse. In 1948, Bloch et al. reported the use of the paramagnetic ferric nitrate salt to enhance the relaxation rate of water proton. Three

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decades later Lauterbur et al. applied Mn(II) salt to distinguish between different tissue based on the differential relaxation time and thus produce the first MR image [44].

Extensive application of MRI in clinical imaging and biomedical research has promoted the development of a new class of pharmacological product, called contrast agent. It acts as catalyst in shortening the longitudinal and transverse (i.e., T1 and T2) relaxation time of water protons in tissue in which the agent accumulates, consequently, enhances the image contrast between normal and diseased tissue and indicate the status of organ function or blood flow. Currently, 30% of all MRI examinations worldwide are performed with contrast agents [45].

The efficiency of an MRI contrast agent can be quantified by its relaxivity, r1, which is defined as the increase of the longitudinal relaxation rate of the water protons per mM of the paramagnetic compound. Relaxivity can be expressed as function of two sets of parameters. surrounding and second set of parameters in equation is responsible for the dynamic part.

Most important interactions which influence the paramagnetic relaxation enhancement

are, Fermi contact (Aiso) and the dipolar (

T ) hyperfine interactions (HFIs), i.e. the

magnetic interaction between the spins of nuclei and electrons. Coupling tensor,

Q, i.e.

The interactions of nuclear quadrupoles with the electric field gradient (EFG) are

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important for the description of relaxation of nuclei with a spin number larger than 1/2, for instance 17O nuclei [46]. The important dynamic parameters (second part of eq. 4) that influence the paramagnetic relaxation are the exchange rate between the bound water molecules and the bulk water, kex, the longitudinal and transverse electronic relaxation times, T1e and T2e, and the rotational correlation time τ R [46]. These parameters are schematically summarized in Figure 3.

Figure 3 Important interaction and dynamic parameters defining the efficiency of an MRI contrast agent.

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1.3.2 Paramagnetic Agents

Metal ions with one or more unpaired electrones are paramagnetic, paramagnetism is due to the spin and orbital angular momentums of unpaired electrons, and therefore have a permanent magnetic moment. However, the magnetic moments of individual paramagnetic atoms in a material are only weakly coupled to each other, and room temperature thermal energy is sufficient to overcome these interactions to eliminate any net magnetic moment [47]. A small fraction of the atomic moments aligns parallel to the field in accord with the Boltzmann distribution when placed in a magnetic field. The paramagnetic interaction with the field is weak and disappears in the absence of an applied field [48]. In an aqueous solution of paramagnetic metal, there is a dipolar magnetic interaction between the electronic magnetic moment of the paramagnetic atom and much smaller magnetic moments of the protons in its vicinity belonging to water molecules. Random fluctuations in this dipolar magnetic interaction, mainly a result of molecular motions, reduce both the longitudinal (T1) and transverse (T2) relaxation of water proton [49]. Gadolinium (Gd(III)) and manganese (Mn(II)) are paramagnetic ions which have been successfully used in MR contrast agents. The Gd(III) ion is an ideal paramagnetic metal ion for MRI contrast agent. The Gd(III) is an ideal choice as a paramagnetic ion is for several reasons. First of all, this ion is characterized by a large magnetic moment due to the half-filled 4f shell (7 unpaired electrons!). Secondly, due to the symmetric S state this ion has a relatively long electron spin relaxation time which is one of the necessary requirements for efficient paramagnetic relaxation enhancement.

However Gd(III) cannot be used as contrast agents in their ionic form due to an undesirable biodistribution and the relatively high toxicity [50]. Therefore, ligands have

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been designed that can form stable complexes with paramagnetic metal ions to form strong chelates which can remain stable in the body and thereby significantly reducing toxicity. The Gd(III) coordinate strongly to diethylenetriamine backbones modified with carboxylic acids. In complexes with such ligands the ion is normally nine-coordinate, were introduced. Since the approval of [Gd(DTPA)(H2O)]2− in 1988, it is estimated that over 30 metric tons of gadolinium have been administered to millions of patients worldwide [45]. Mn(II) complexes have also been investigated for its potential application in MRI. However due to poor stability it is not used as widely as Gd(III) complexes. Consequently so far, MnDPDP (Teslascant) in which Mn(II) is coordinated by dipyridoxyl diphosphate, is the only clinically approved agent [51].

1.3.3 Superparamagnetic Agents 1.3.3.1 Superparamagnetism

In general, macroscopic ferromagnetic materials are divided up into domains of parallel magnetic moments for minimization of their energy. Within a magnetic domain, the magnetic moments orient in one direction, while the alignment of spins in neighboring domains is usually antiparallel. The oppositely aligned magnetic domains are separated

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from each other by a domain wall (Bloch wall). As the particle size decreases below some critical value, the formation of domain walls become energetically unfavorable and the ferromagnetic particle can support only a single domain structure. The critical diameter for a magnetic particle to reach the single domain limit is equal to [52]

36 2 saturation magnetization. Magnetic particles of nanometer size are usually in a single domain structure [53]

The amount of energy required to reverse the magnetization of a single domain particle, over the energy barrier from one stable magnetic configuration to the other is proportional to KV/kBT where V is the particle volume, kB is Boltzmann’s constant and T is temperature [54]. In zero magnetic fields, the energy barrier ∆E has to be overcome to rotate the magnetization of a single-domain particle as shown in Figure 4. Because the height of the barrier, ∆E = KV, is proportional to the particle volume V, ∆E may become comparable to the thermal energy (kBT, where kB shows the Boltzmann’s constant) when the particle size decreases. If the thermal energy is large enough to overcome the anisotropy energy so that the energy barrier can no longer pin the magnetization to the time scale of observation, the magnetization is no longer stable due to thermal fluctuations and the particle is said to be superparamagnetic (SPM) [55].

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Figure 4 Energy barriers from one stable magnetic configuration to the other

1.3.3.2 SPIO Nanoparticle in Molecular Imaging

Molecular imaging, in general, refers to the study of cellular and molecular events through noninvasive investigation. In MRI, molecular imaging depends on induced changes in proton relaxivity of in vivo water molecules on the molecular and cellular level. In last one decade, biocompatible iron oxide particles conjugated with targeting moiety for targeted molecular imaging applications has been extensively investigated.

Monoclonal antibodies labeled with superparamagnetic nanoparticles are expected to be good tumor-specific contrast agents because of their high specificity against some cancers. Superparamagnetic iron oxide nanoparticles (SPION) are currently used for clinical imaging of liver tumors and prostate, breast and colon cancers as well as for the delineation of brain tumor volumes and boundaries.

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1.3.3.3 Recent Advancement in Superparamagnetic Agents

Magnetic nanoparticle probes are emerging as a next generation contrast medical imaging. Super paramagnetic iron oxide (SPIO) particles were suggested as potential liver specific MR contrast agents as early as the mid 1980s [56]. Recent research shows SPIO particles have potentially to generate higher contrast enhancement in MRI than conventional paramagnetic Gd-based contrast agents. Numerous chemical methods have been reported for synthesize of SPIO nanoparticles for medical imaging applications:

microemulsions [57], sol-gel syntheses [58], sonochemical reactions [59], hydrothermal reactions [60], hydrolysis and thermolysis of precursors [61], flow injection syntheses [62], and electrospray syntheses [63]. Aqueous co-precipitation process in the presence of the coating material has been frequently employed for the synthesis of SPIO and USPIO.

The main advantage of this process is that a large amount of nanoparticles can be synthesized. However, the control of particle size distribution is limited, because only kinetic factors are controlling the growth of the crystal [64-66]. The saturation magnetizations (Ms) Values of nanoparticles obtained by these methods are in the range of 30–50 emu/g, which is lower than the 90 emu/g reported for their bulk form. The low Ms value is due to incorporation of impurities hampering the crystal structure and surface effect [67]. Monodisperse magnetite nanoparticles has been synthesized at high-temperature by decomposition reaction of iron(III) acetylacetonate with 1,2-hexadecanediol in the presence of oleic acid and oleylamine to. The particle diameter can be tuned from 4 to 20 nm by varying the reaction temperature [68]. However, use of hydrophobic oleic acid and oleylamine surfactants in the process results in a hydrophobic coating on the particle surface, amphiphilic polymer or surface surfactant exchange have been utilized to overcome this problem [69]. In order to meet the demand of excellent

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magnetic property for the applications such as, molecular imaging, metal doped iron oxide nanoparticles have drawn much attention due to their enhanced magnetic

magnetic property for the applications such as, molecular imaging, metal doped iron oxide nanoparticles have drawn much attention due to their enhanced magnetic

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