Magnetic resonance imaging has developed into a powerful diagnostic technique characterized by a very high spatial resolution and an inherently relatively low sensitivity. In order to improve the contrast of MRI images, contrast agents are commonly injected into the patients before an examination. These substances are paramagnetic, superparamagnetic, or ferromagnetic compounds that shorten the relaxations times of the water hydrogen atoms. At present, most of the contrast enhanced clinical exams are performed with gadolinium complexes. They are particularly useful as their ability to change the relaxation rate (or relaxivity) can be very high. Several factors have a strong influence on the relaxivity of MRI contrast agents but the water exchange time τm and the rotational correlation time τr are particularly important for obtaining an increased relaxivity. These parameters can be adjusted by suitable chemical modifications of the Gd(III) complexes. For instance, decreasing the tumbling rate by linking a Gd(III) complex to a macromolecule leads to an increased relaxivity. This goal can be achieved through a covalent or a noncovalent linkage with synthetic polymers, particles or biomacromolecules. However, the covalent bonding has a detrimental effect on the clearance of the metal complexes thus exposing the patient to the toxicity of released Gd(III) ions and metabolites. This problem could be circumvented by using covalent links that are cleaved by endogenous biomolecules or after administration of exogenous compounds following the exam. In this context, our approach was to bind Gd(III) chelates to macromolecules through disulfide links as the latter are known to be reduced in vivo by thiols present in the body. Towards this aim, we have developed two bifunctional chelator agents bearing a methanethiosulfonate group (MTS) which reacts specifically with thiols, thus spontaneously establishing a disulfide bond between the Gd(III) chelate and the thiolated macromolecule. The first ligand that we have prepared (MTS-ADO3A) is a monoamide derivative of DOTA with an ethyl-MTS substituent. This compound is relatively easily synthesized but amide arms such as the one it features are known to have a detrimental effect on relaxivity through the lengthening of water exchange times. The conjugate obtained by binding Gd(III) chelates of this ligand to albumin or to polythiolated silica nanoparticles has been studied by nuclear magnetic relaxation dispersion (NMRD),17O NMR and luminescence analyses. These measurements confirm that the method is suitable to increase the relaxivity (20 mM-1s 1, 20 MHz, 25°C) but that this relaxivity increase (of 300%) is limited by a slow water exchange (660 ns). To overcome this limitation, a second ligand called MTS-CyDOTA has been synthesized. This ligand is a DOTA ligand grafted with a cyclohexyl ring featuring a MTS function. The synthesis is more demanding but faster water exchange times are expected because of a more sterically crowded coordination sphere. Moreover, this second ligand has a more rigid structure that could limit the independent rotation of the chelate from the macromolecule. As expected, the water exchange time of the Gd(III) chelate of this ligand (120 ns) is clearly lower than the one determined for Gd MTS-ADO3A. After binding to albumin or to silica nanoparticles a notable relaxivity increase was expected. Unfortunately, if the obtained relaxivity is higher (30 mM-1s 1, 20 MHz, 25°C), its not as high as it could have been expected in view of the size of the conjugate and of the water exchange time of the free chelate. Results obtained in this work suggest that fixation on silica nanoparticles or on albumin drastically decreases the water exchange rate which remains the limiting parameter. This effect has already been reported for Gd(III) chelates linked to albumin by non-covalent bonds and has been assigned to stable layers of water molecules on the macromolecule surface. Thanks to the high loading of the silica nanoparticles (10000 Gd(III) per particle), we have reached very high molecular relaxivities (>200000 mM-1s-1). Stability tests carried out on the disulfide links formed suggest that the small amount of free thiols in the circulation is not sufficient to cause a significant degradation of the disulfide bond in the conjugate within a reasonable length of time. An injection of glutathione would be necessary to achieve a complete degradation. To avoid the problem of water exchange lenghtening, we propose to increase the distance between Gd(III) chelates and macromolecules without loss of rigidity by developing double anchor chelates with substituents grafted on the side of the ring. Considerable synthetic efforts have devoted to the synthesis of such a system and are discussed in chapter VI. At present, this work is still in progress in the laboratory and recent results suggest that it should be possible to evaluate this double arms system in a near future. On the fringe of this synthesis, we present a relaxometric study on the interaction between HSA and a hydrophobic Gd(III) chelate obtained during the preparation of our double anchor chelate. Finally, a chapter of this work is devoted to the study of two compounds, phenEDTA and phenDTPA, which are ditopic chelates featuring a dihydro-1,10-phenanthroline unit that spontaneously self-assemble in the presence of a transition metal ion. The tris-complex generated by this process rotates more slowly in solution and thus presents an increased relaxivity (+130%). As part of this work, we have determined by potentiometric titration the acidity constants of phenEDTA and its stability constant with Gd(III). Moreover, the protonation scheme of this ligand has been studied by NMR titration. The particular behavior of Gd phenDTPA and Fe(Gd phenDTPA)3 in the presence of Zn(II) has also been studied by relaxometry, luminescence and EXAFS.

The purpose of this thesis was to investigate the utility of the Ultrasmall Superparamagnetic Iron Oxide Particle (USPIO), NC100150 Injection for assessment of macro- and microvascular morphology and function using magnetic resonance imaging. The feasibility of NC100150 Injection was tested for the following applications: Coronary angiography, Thrombus detection, Cardiac function, Myocardial perfusion, Assessment of myocardial blood volume and water exchange and finally assessment of endothelial integrity of the myocardium. The test method included computer simulations, in vitro, animal and clinical experiments. The computer simulations included propagation of longitudinal magnetization in non-steady state acquisitions. Animal models used were coronary artery stenosis in pigs, thrombus formation in the jugular vein in pigs, normal pig myocardium and transplanted hearts in rats. A human study of patients with suspected coronary artery disease was also performed. The results showed that angiography using an USPIO is less efficient in the coronary arteries than in the peripheral arteries. Direct targeting to thrombus using an USPIO is possible but will be limited by the spatial resolution. An USPIO will enhance gradient echo imaging of cardiac function. T2-weighted myocardial perfusion imaging is feasible as well as assessment of myocardial blood volume and endothelial integrity. Due to physiological limitations and technical development the utility of NC100150 Injection for assessment of the macrovascular morphology is limited.However for the assessment of microvascular function, especially perfusion and permeability, NC100150 Injection may contribute to a successful implementation of these methods.

Applied cancer research is heavily focused on the development of diagnostic tools with high sensitivity and specificity that are able to accurately detect the presence and anatomical location of neoplastic cells, as well as therapeutic strategies that are effective at curing or controlling the disease while being minimally invasive and having negligible side effects. Recently, much effort has been placed on the development of nanoparticles as diagnostic imaging and therapeutic agents, and several of these nanoplatforms have been successfully adopted in both the research and clinical arenas. This thesis describes the development of a nanoparticulate liposome system for use in a number of applications including multimodality imaging with computed tomography (CT) and magnetic resonance (MR), longitudinal vascular imaging, image-based biodistribution assessment, and CT detection of neoplastic and inflammatory lesions. Extensive in vitro and in vivo characterization was performed to determine the physico-chemical properties of the liposome agent, including its size, morphology, stability and agent loading, as well as its pharmacokinetics, biodistribution, tumor targeting and imaging performance. Emphasis was placed on the in vivo CT-based quantification of liposome accumulation and clearance from healthy and tumor tissues in a VX2 carcinoma rabbit model, gaining insight not only on the spatial but also the temporal biodistribution of the agent. The thesis concludes with a report that describes the performance of liposomes and CT imaging to detect and localize tumor and inflammatory lesions as compared to that of 18F-fluorodeoxyglucose (FDG) – positron emission tomography (PET). The outcome of the study suggests that liposome-CT could be employed as a competitive method for whole body image-based disease detection and localization. Overall, this work demonstrated that this liposome agent along with quantitative imaging systems and analysis tools, has the potential to positively impact cancer treatment outcome through improved diagnosis and staging, as well as enable personalization of treatment delivery via target delineation. However, in order to prove clinical benefit, steps must be taken to advance this agent through the regulatory stages and obtain approval for its use in humans. Ultimately, the clinical adoption of this multifunctional agent may offer improvements for disease detection, spatial delineation and therapy guidance.

Citrate-coated gadolinium trifluoride (Cit-GdF3) and poly(acrylic acid)-coated nanoparticles (PAA-GdF3 NPs) were synthesized, the former reproduced from literature (though using more refined conditions), the latter through a new, two-step, ligand exchange method. Diamagnetic nanoparticle analogs (Cit-YF3 NPs) were prepared to investigate citrate interactions with the nanoparticle surface using NMR. Citrate was found to bind in numerous conformations, with a total of between 29 – 46 % bound at 0 ºC. Exchange studies revealed short residence lifetimes of one and twelve seconds respectively for bound and free forms of citrate (0 ºC), perhaps explaining the colloidal instability of these nanoparticles. PAA-GdF3 NPs were synthesized by first producing their Cit-GdF3 counterparts, and then exchanging citrate for PAA. The impetus behind this latter synthesis was the relative enhancement in stability and relaxivity attainable by these nanoparticles. The displacement of citrate by PAA was verified using diffusion NMR studies.

Purpose: To compare hemodynamic of malignant and benign SRMs on CT and CEUS. Method: Seventy biopsy proven SRM underwent CEUS. Sixty-three had CT. After injection of 0.2 ml of Definity, 3min and after 0.9 ml infusion, 30 sec of data were acquires. Lesion hemodynamics relative to the cortex was evaluated both qualitatively and quantitatively. Results: Considering 15 and 20 HU as enhancement threshold, 10% to 13% of patients did not enhance on CT, while all lesions enhanced on CEUS. Papillary RCCs showed hypovascularity with 100% specificity. In other RCCs, PI, WI slope 5 to45%, 50 to100%, 10 to 90%, WO slope 100 to 50%, 100 to 10%, WO intensity at peak+30 seconds were statistically higher than benign SRMs. Conclusion: All solid SRMs enhance on CEUS, while CT does not show vascularity in 10-13% of solid SRMs. CEUS can differentiate malignant from benign SRMs by evaluating their hemodynamics.

Citrate-coated gadolinium trifluoride (Cit-GdF3) and poly(acrylic acid)-coated nanoparticles (PAA-GdF3 NPs) were synthesized, the former reproduced from literature (though using more refined conditions), the latter through a new, two-step, ligand exchange method. Diamagnetic nanoparticle analogs (Cit-YF3 NPs) were prepared to investigate citrate interactions with the nanoparticle surface using NMR. Citrate was found to bind in numerous conformations, with a total of between 29 – 46 % bound at 0 ºC. Exchange studies revealed short residence lifetimes of one and twelve seconds respectively for bound and free forms of citrate (0 ºC), perhaps explaining the colloidal instability of these nanoparticles. PAA-GdF3 NPs were synthesized by first producing their Cit-GdF3 counterparts, and then exchanging citrate for PAA. The impetus behind this latter synthesis was the relative enhancement in stability and relaxivity attainable by these nanoparticles. The displacement of citrate by PAA was verified using diffusion NMR studies.

Calmodulin (CaM) is a ubiquitous intracellular protein that regulates biological activities of numerous enzymes and ion channels. Upon responding Ca2+ concentration change, Ca2+- dependent CaM activates the hydrolyzation of cGMP by PDE and Ca2+ releasing channel activity of ryanodine receptor. In this dissertation, a series of CaM variants were engineered to enhance Ca2+ binding affinities by increasing the number of negative charged residues in individual EF-hand. The capability of shifting the biphasic Ca2+-activation profile of RyR1 is significantly altered by changing Ca2+ binding affinity of CaM at the C-terminal. This indicates that examining Ca2+-CaM affinity is a valid strategy to tune the activation profile of CaM-regulated ion channels. To further understand interactions between CaM and RyR1, NMR was used to determine their binding mode. To dissect roles of structural components of CaM in metal binding and regulation of biological functions of target proteins, we created half-CaMs and Del-CaM. Binding affinities of these variants to Ca2+, Tb3+ and Gd3+ were determined by fluorescence spectroscopy; functional studies were conducted using single channel analysis and PDE function assay. Another objective of my dissertation is to design a protein based contrast agent for molecular imaging. CaM was selected as the scaffold protein for designing Gd3+ based MRI contrast agent by modifying metal binding sites as well as grafting a biomarker peptide into the linker region to specifically target cancers with efficient and optimized modifications. The physical kinetic properties and animal imaging effects of these designed contrast agents were investigated by various methods.

Applied cancer research is heavily focused on the development of diagnostic tools with high sensitivity and specificity that are able to accurately detect the presence and anatomical location of neoplastic cells, as well as therapeutic strategies that are effective at curing or controlling the disease while being minimally invasive and having negligible side effects. Recently, much effort has been placed on the development of nanoparticles as diagnostic imaging and therapeutic agents, and several of these nanoplatforms have been successfully adopted in both the research and clinical arenas. This thesis describes the development of a nanoparticulate liposome system for use in a number of applications including multimodality imaging with computed tomography (CT) and magnetic resonance (MR), longitudinal vascular imaging, image-based biodistribution assessment, and CT detection of neoplastic and inflammatory lesions. Extensive in vitro and in vivo characterization was performed to determine the physico-chemical properties of the liposome agent, including its size, morphology, stability and agent loading, as well as its pharmacokinetics, biodistribution, tumor targeting and imaging performance. Emphasis was placed on the in vivo CT-based quantification of liposome accumulation and clearance from healthy and tumor tissues in a VX2 carcinoma rabbit model, gaining insight not only on the spatial but also the temporal biodistribution of the agent. The thesis concludes with a report that describes the performance of liposomes and CT imaging to detect and localize tumor and inflammatory lesions as compared to that of 18F-fluorodeoxyglucose (FDG) – positron emission tomography (PET). The outcome of the study suggests that liposome-CT could be employed as a competitive method for whole body image-based disease detection and localization. Overall, this work demonstrated that this liposome agent along with quantitative imaging systems and analysis tools, has the potential to positively impact cancer treatment outcome through improved diagnosis and staging, as well as enable personalization of treatment delivery via target delineation. However, in order to prove clinical benefit, steps must be taken to advance this agent through the regulatory stages and obtain approval for its use in humans. Ultimately, the clinical adoption of this multifunctional agent may offer improvements for disease detection, spatial delineation and therapy guidance.

Ultrasound contrast agents (UCA) have tremendous potential for in vivo molecular imaging because of their high sensitivity and great spatial resolution of ultrasound imaging. However, the diagnostic potential of UCAs has been difficult to exploit because current contrast agents are based on pre-formed microbubbles, which can only detect cell surface receptors. In this work, we demonstrated that chemical reactions that generate gas forming molecules can be used to perform molecular imaging by ultrasound in vivo. This new approach for generating ultrasound contrast agents was demonstrated by imaging reactive oxygen species (ROS) in vivo with allylhydrazine, a compound that is converted into nitrogen and propylene gas after reacting with radical oxidants. We demonstrated that allylhydrazine encapsulated within liposomes (termed APLs) can detect a 10 uM concentration of radical oxidants by ultrasound, and can image oxidative stress in mice, induced by lipopolysaccharide (LPS), using a clinical ultrasound machine. We showed that a 1-2% increase in gas concentration above saturation can be detected acoustically and suggest that numerous biological targets can be imaged via appropriately designed gas forming reactions. This work was the first demonstration of in vivo imaging of ROS using ultrasound, and this work presented a new strategy to generate gas bubbles from reactions involving radical oxidants. We anticipate numerous applications of chemically generated microbubbles, given the excellent spatial resolution of ultrasound imaging, its widespread clinical use and its high sensitivity to detect gas bubbles.

The recent increase in survival rates of most cancers is due to early detection greatly aided by medical imaging modalities. Combined ultrasound and photoacoustic imaging provide both morphological and functional/molecular information which can help to detect and diagnose cancer in its earliest stages. However, both modalities can benefit from the use of contrast agents. The objective of this thesis was to design, synthesize, and test a nano-sized, dual contrast agent for combined ultrasound and photoacoustic imaging named Photoacoustic nanoDroplets. This agent consists of liquid perfluorocarbon nanodroplets with encapsulated plasmonic nanoparticles. These dual contrast agents utilize optically triggered vaporization for photoacoustic signal generation, providing significantly higher signal amplitude than that from the traditionally used mechanism, thermal expansion. Upon pulsed laser irradiation, liquid perfluorocarbon undergoes a liquid-to-gas phase transition generating giant photoacoustic transients from these dwarf nanoparticles. Once triggered, the gaseous phase provides ultrasound contrast enhancement. Demonstrated in this work are the design, synthesis, characterization, and testing of Photoacoustic nanoDroplets in phantom and animal studies, and preliminary work into adapting these agents into targeted, drug delivery vehicles for simultaneous detection, diagnosis, and treatment of diseases. / text