Tracer based MRI perfusion measurements is a clinically useful tool to assess regional distributions of tissue blood flow and volume. The method may be based on any of the three relaxation mechanisms T1, T1 and T2*, the latter denoted DSC-MRI being the most common. The primary aim of this work was to study the feasibility of obtaining quantitative estimates using these methods. 1) Feasibility of DSC-MRI for kidneys using an iron oxide based contrast agent and the influence of secondary relaxation effects on the results, part of a clinical phase II trial: The method proved feasible and the underestimation induced by secondary relaxation can be corrected for by using a double echo sequence. 2) Influence of blood flow rate on risk factors for developing cerebral ischemia during cardio pulmonary bypass, measurements in pig with gadolinium based DSC-MRI: The results indicated an ischemic threshold level at a blood flow rate of approximately 6 ml/kg/min. 3) The ability of gadolinium based DSC-MRI to detect changes in global blood flow, experimental measurements in pig and numerical simulations: The results support that DSC-MRI can discriminate between global flow levels in the same subject given that all other parameters are kept constant. The results also indicate that calculated perfusion values are highly sensitive to the arterial deconvolution procedure. 4) Influence of differences in blood/tissue relaxivity and secondary relaxation for a gadolinium based contrast agent, measurements in pig and numerical simulations: The blood/tissue relaxivity ratio is not unity and the situation is complicated by secondary relaxation effects. Deconvolution regularization appears to partly counteract the overestimation induced by difference in blood/tissue relaxivity for DSC-MRI. In summary, the fundamental assumption of equal blood and tissue relaxivity is experimentally shown to be invalid and the influence of this discrepancy is substantial. Several factors contribute to measurement errors, a combination of these factors can incidentally lead to additive errors or error cancellation based on a variety of experimental and analysis conditions. Given that the differences in blood/tissue relaxivity cannot readily be accounted for in a clinical setting, absolute perfusion quantification by tracer based MRI remains challenging if not impossible.

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Angiogenesis is a fundamental driver of tumor biology and many other important aspect of human health. Dynamic Contrast Enhanced Magnetic Resonance Imaging (DCE-MRI) has been shown to be a valuable biomarker for the indirect assessment of angiogenesis. However, DCE-MRI is very specialized technique that has limitations. In this dissertation new models and contrast agents to address some of these limitations are presented. Chapter 1 presents an introduction to DCE-MRI, the rationale to asses tumor biology with this technique, the MRI pulses sequences and the standard pharmacokinetic modeling used for the analysis of DCE- MRI data. Chapter 2 describes the application of DCE-MRI to asses the response to the hypoxia-activated drug TH-302. It is shown that DCE-MRI can detect a response after only 24 hours of initiating therapy. In Chapter 3, a new model for the analysis of DCE-MRI is presented, the so-called Linear Reference Region Model (LRRM). This new model improves upon existing models and it was demonstrated that it is ~620 faster than current algorithms and 5 times less sensitive to noise, and more importantly less sensitive to temporal resolution which enables the analysis of DCE-MRI data obtained in the clinical setting, which opens a new area of study in clinical MRI. Chapter 4 describes the extension of the LRRM to estimate the absolute permeability of two fluorinated contrast agents; we call this approach the Reference Agent Model (RAM). In order to make this new model an experimental reality, a novel pulse sequence and contrast agents (CA) for ¹⁹F MRI were developed. Two contributions to the field of DCE-MRI are presented in this chapter, the first simultaneous ¹⁹F-DCE-MRI detection of two fluorinated CA in a mouse model of breast cancer, and the estimation of their relative permeability. RAM eliminates some of the physiological variables that affect DCE-MRI, which may improve its sensitivity and specificity. Finally, new potential applications of LRRM and RAM are discussed in Chapter 5.

Magnetic resonance imaging (MRI) is one of the most powerful imaging techniques for research and clinical diagnosis. To expand upon the intrinsic capabilities of MRI, new contrast agents that can detect the presence of biomarkers in vivo are being developed. My Masters thesis research focuses on the design and synthesis of a new MRI contrast agent that can detect glutathione (GSH), a biomarker that has been implicated in a number of oxidative stress diseases. This new MRI contrast agent is based on chelated dysprosium (Dy), an inorganic metal, which provides negative contrast to surrounding tissue. Preliminary data has shown that attaching a poly(ethylene glycol) (PEG) chain to the Dy chelate, effectively increasing its molecular weight, enhances the contrast ability of Dy. Using this new information, the contrast agent was designed to have a large molecular weight PEG chain attached to the Dy chelate through a disulfide, creating a thiol-sensitive linkage. In the presence of a thiol-containing molecule such as GSH, the Dy will be dePEGylated through a disulfide exchange reaction, removing the molecular weight effect of the PEG, and allowing for the detection of GSH by MRI. This new MRI contrast agent could provide insight into the progression and diagnosis of oxidative stress pathologies associated with GSH.

Enzymes are important biomarkers for determining tumor growth and progression. We have developed two molecules to image enzyme response by catalyCEST MRI. This technology allows for non-invasive detection of enzymes. A background of importance of measuring enzyme activity and MRI agents developed for this purpose have been covered in Chapter 1. We have synthesized a responsive paramagnetic Chemical Exchange Saturation Transfer (CEST) agent, called Tm-DO3A-cadaverine. This contrast agents has been successfully cross-linked to the protein albumin by the enzyme transglutaminase leading to the appearance of CEST at -9.2 ppm. The enzyme catalysis has been validated by measuring chemical exchange rates. We have shown that the position of the CEST peak is influenced by the conformation of the molecule depending on the neighboring amino acids to glutamine. This is the first example to show the appearance of CEST due to formation of a covalent bond. We have also synthesized a diamagnetic CEST agent with a large chemical shift dispersion to detect cathespin B activity. Upon enzyme mediated cleavage of PheArgSal, the aryl amide CEST peak at 5.3 ppm disappears. Taking a ratio of the CEST effects from salicylic acid at 9.5 ppm and aryl amide at 5.3 ppm we can detect enzyme activity. The salicylic acid moiety also undergoes some slow response due to enzyme action, as evident by the disappearance of CEST at 9.5 ppm. However, this proof of concept study is the first example of a DIACEST agent designed to measure enzyme activity using a ratio of two CEST effects from the same substrate. The last chapter highlights suggests improvements to the catalyCEST research. The appendix shows the use of bulk magnetic susceptibility measurements by NMR to determine bio-distribution of lanthanides in ex-vivo tissue.

Macromolecular magnetic resonance imaging (MRI) contrast agents have unique localization and contrast enhancement properties. We have designed and studied a monodisperse paramagnetic β-cyclodextrin click cluster (Gd10) decorated with Gd-containing arms and unique contrast enhancing polymers. To synthesize Gd10, a novel alkyne-functionalized diethylenetriaminetetraacetic acid chelate was created and coupled to a per-azido-β-cyclodextrin core and chelated with Gd(III) to yield the precursor macromolecule. Luminescence measurements were carried out using an analogous structure Eu(III)-containing structure and indicated that each lanthanide has an average of 1.8 water exchange sites. Gd10 yields a high relaxivity profile (6.2 mM⁻¹ s⁻¹ per Gd(III) at 9.4 T). Gd10 shows toxicity higher than clinically used contrast agents such as Magnevist&trade in vitro in cardiomyoblast cells. No acute toxicity was observed in the rats (n = 9) and contrast enhanced image analysis indicates renal processes may be involved in clearance. The contrast enhancing polymers we developed are new macromolecular beacons that allow the delivery of nucleic acids to be visualized at different biological scales. They contain repeated oligoethyleneamines, for binding and compacting nucleic acids into nanoparticles, and Gd(III)/Eu(III) chelates. The chelated lanthanides allow the visualization of the delivery vehicle via microscopy and via magnetic resonance imaging (MRI). We demonstrate that these new delivery beacons effectively bind plasmid DNA(pDNA) and protect their cargo nucleic acids from nuclease damage. The lanthanide-chelate materials have been found to efficiently deliver pDNA into cultured cells and do not exhibit toxicity. Micrographs of cultured cells exposed to the nanoparticle complexes formed with fluorescein-labeled pDNA and the europium-chelated polymers reveal effective intracellular imaging of the delivery process. MRI of bulk cells exposed to the complexes formulated with pDNA and the gadolinium-chelated structures show bright image contrast, allowing visualization of effective intracellular delivery on the tissue-scale. Because of their versatility as imaging probes, these delivery beacons posses remarkable potential for tracking and understanding nucleic acid transfer in vitro and have promise for in vivo imaging applications. In later studies the Ln-chelating polymers were co-polymerized with dimethylgalacterate which definitively increases luciferase gene expression (up 50x enhancement) and cellular uptake (up to 2x enhancement). / Ph. D.

Therapeutic efficacy of drugs depends on their ability to reach the treatment target. Drugs that exert their effect within cells are constrained by an inability to cross the cell membrane. Methods are being developed to overcome this barrier including biochemical and biophysical strategies. The application of ultrasound with microbubbles increases the permeability of cell membranes allowing molecules, which otherwise would be excluded, to enter the intracellular space of cells; a phenomenon known as sonoporation. This thesis describes studies aimed at improving our understanding of the mechanism underpinning sonoporation and of the exposure parameters affecting sonoporation efficiency. Cancer cells (KHT-C) in suspension were exposed to ultrasound and microbubbles – total of 97 exposure conditions. The effects on cells were assessed through uptake of cell-impermeable molecules (10 kDa to 2 MDa FITC-dextran), cell viability and microscopic observations of the plasma membrane using flow cytometry, colony assay and electron microscopy techniques. Sonoporation was a result of the interaction of ultrasound and microbubbles with the cell membrane. Disruptions (30-100 nm) were generated on the cell membrane allowing cell impermeable molecules to cross the membrane. Molecules up to 2 MDa in size were delivered at high efficiency (~70% permeabilisation). Sonoporation was short lived; cells re-established their barrier function within one minute, which allowed compounds to remain inside the cell. Following uptake, cells remained viable; ~50% of sonoporated cells proliferated. Sonoporation efficiency depended on ultrasound and microbubble exposure conditions. Microbubble disruption was a necessary but insufficient indicator of ultrasound-induced permeabilisation. The exposure conditions can be tailored to achieve a desired effect; cell permeability of ~70% with ~25% cell death versus permeability of ~35% with ~2% cell death. In addition, sonoporation depended on position in the cell cycle. Cells in later stages were more prone to being permeabilised and killed by ultrasound and microbubbles. This study indicated that sonoporation can be controlled through exposure parameters and that molecular size may not be a limiting factor. However, the transient nature may necessitate that the drug be in close vicinity to target cells in sonoporation-mediated therapies. Future work will extend the investigation into in vivo models.

Nanomateriais têm sido amplamente estudados, como resultado de suas propriedades físicas e químicas diferenciadas, que oferecem um grande número de possibilidades para aplicações em biomedicina, principalmente na terapia de câncer e no desenvolvimento de estratégias de diagnóstico não invasivo. O óxido de ferro superparamagnético (SPION) é o principal material estudado como agente de contraste para imagem por ressonância magnética, devido à sua capacidade de reduzir o tempo de relaxação transversal (T2) em diferentes tecidos e sua menor toxicidade que os complexos de Gd3+ e Mn2+ usados atualmente. Entretanto, o acumulo de SPIONs pode ser facilmente confundido com sinais referentes à calcificação, depósito de metais pesados e sangramentos, e a alta susceptibilidade magnética do material promove distorções na imagem. Assim, alguns aspectos são desejáveis em material para que este tenha potencial para substituir o SPION, tais como forma nanoparticulada, para fácil modificação de superfície e possibilidade de funcionalização com agentes biosseletivos, e contraste positivo em T1. As nanopartículas (NPs) antiferromagnéticas de MnO atendem a todos os requisitos necessários para substituir o óxido de ferro. As NPs de MnO foram sintetizadas a partir da decomposição térmica do acetilacetonato de manganês(II) em uma variação do método poliol modificado, resultando na formação de NPs com tamanho médio de 21 ± 3,9 nm. Foi realizada a substituição de ligantes de superfície para que se substituísse o ácido oleico adsorvido sobre o material por 3-aminopropiltrimetoxisilano (APTMS) e foi determinada a concentração de grupamentos amino sobre a superfície das NPs. Posteriormente, obteve-se uma estrutura do tipo \"core/shell\" dispersível em meio aquoso e biocompatível pela reação dos grupos amino livres com o carboxilato da carboximetil dextrana (CMDex). O potencial de superfície e a estabilidade coloidal das NPs funcionalizadas foram caracterizados por mobilidade eletroforética e por espalhamento de luz dinâmico em água deionizada e em condições que mimetizavam o sangue. As NPs apresentaram toxicidade em células cancerosas de carcinoma cervical humano (HeLa). Entretanto, não foi observada toxicidade significativa na linhagem de células não cancerosas NCTC clone L929. Tanto as NPs como sintetizadas quanto as recobertas com CMDex apresentaram controle de tamanho e forma, apresentando distribuição de tamanho compatível com o esperado para as aplicações em biomedicina. / Nanomaterials have been widely studied as a result of their interesting physical and chemical properties, which offer a large number of possibilities for applications in biomedicine mainly in cancer therapy and the development of strategies for non-invasive diagnosis. The superparamagnetic iron oxide nanoparticles (SPION) is the main studied material as contrast agent for magnetic resonance imaging (MRI) due to its ability to reduce the transverse relaxation time (T2) in different tissues and lower toxicity than Gd3+ and Mn2+ complexes currently used. However, this SPIONs accumulation can be confused with signals from calcification, bleeding or metal deposits, and the high magnetic susceptibility distorts the background image because its ferromagnetic behavior. Some aspects are desirable to replace SPIONs, such as nanoparticulate form for simple surface modification and labeling with targeting agents, and positive longitudinal T1 relaxation time contrast ability. The antiferromagnetic MnO NPs attend all these requirements and overcome the drawback of using SPION. In our study, MnO NPs were synthesized by the thermal decomposition of Mn(II) acetylacetonate by a variation of the modified polyol process resulting in spherical nanoparticles with average size of 21 ± 3,9 nm. The ligand-exchange step was used to replace the oleic acid adsorbed on the as-synthesized NPs surface by 3-aminopropyltriethoxysilane (APTMS) and the total free amine groups on the NPs surface was determined. After that, a biocompatible and water-dispersible core/shell structure was obtained by coating with carboxymethyl dextran (CMDex) using the free amine-terminal group from APTMS and the carboxylate groups present in the CMDex molecules conjungation. Surface potential and colloidal stability of these functionalized NPs were evaluated by electrophoretic mobility and dynamic light scattering techniques in both water and artificial blood by using the Simulated Body Fluid (SBF) medium. While the water-dispersible NPs have shown toxicity in the human cell line derived from cervical cancer (HeLa), they have not shown significantly cytotoxicity in the healthy fibroblast cells (cell line L929). Both the as-synthesized and coated NPs present controlled size and shape and the final NPs size distribution and magnetic properties are compatible with the expected for biomedical applications.

Molecular Imaging provides new aspects in cancer diagnosis and treatment. With the ap-plication of imaging and biological techniques, molecular imaging can monitor molecular and cellular changes of different diseases. To interpret the mechanism of disease, more and more at-tention is focused on the development of new probes for molecular imaging. Magnetic resonance imaging (MRI) is a powerful, non-invasive clinical diagnostic tool with high spatial resolution without the limitation of the depth of tissues. Applications of MRI contrast agents can amply the MRI signal during imaging. Many studies have been devoted to developing targeted MR contrast agents. Proteins and peptides have been widely used for target-ing cancer cells in cancer diagnosis and treatments. GRP, gastrin-releasing peptide, is one of a well-characterized group of mammalian bombesin-like peptides. GRP acts through its cell surface receptors, GRP receptor (GRPR). It has been reported that there is a high density of GRP receptors in the majority of prostate carci-noma. In contrast, the GRPRs are not highly expressed in normal cells of most tissues. Thus, this tumor specific expression pattern provides an advantage for cancer targeting. A novel class of MRI contrast agent was designed by adding the Gd3+ binding sites into a stable host protein, the domain 1 of rat CD2. Due to the unique features of the designed metal binding properties, the protein contrast agent (ProCA1) exhibits more than 10-fold enhanced MRI relaxivity compared to that of the more commonly used Gd-DTPA. The high relaxivity of the designed protein contrast agent largely overcomes the major barrier of low sensitivity of MRI techniques. A peptide of ten amino acids from the C-terminal of GRP was grafted onto ProCA1. GRP-grafted protein contrast agents (ProCA1.GRPs) showed the targeting capability to the GRPRs which are over-expressed on prostate cancer cells. Cell MRI Imaging demonstrated that ProCA1.GRP(52) grafted between Lys51 and Ser52 had better targeting capability than C-terminal one. Administration of ProCA1.GRP into xenograft tumor model enhances the contrast in the GRPR+ prostate tumor under MRI and optical imaging. Study demonstrated a potential application for disease marker targeted MR imaging by using our developed protein contrast agent.

Using protein design, a series of metal binding proteins have been designed, allowing the local factors that contribute to metal affinity and thermostability to be studied. Those proteins with the highest metal binding affinities had the lowest apo-form Tm and the largest ÄTm upon metal binding. In this thesis, major steps have been taken toward applying the engineered protein to MR imaging. The progress of magnetic resonance imaging is hindered by low specificity and rapid elimination of FDA-approved MRI contrast agents. The engineered protein contrast agent has been conjugated to a cancer-specific targeting peptide and encapsulated in albumin microspheres to provide tandem passive and active tumor targeting. Also, a simple, high-yield purification method has been developed.