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Cyclodextrins, a family of compounds formed from a cyclic array of glucose monomers, contain a hydrophobic cavity which displays selective complexation of size specific guests. This selective molecular recognition is critical to the development of successful chemosensors. A selection of cyclodextrin-conjugates have been designed to report complexation of guests into the cyclodextrin cavity by optical and electrochemical means. Alkylated cyclodextrins containing a single linker group have been synthesised allowing the development of two cyclen-appended cyclodextrin conjugates and their lanthanide complexes. The tetraamide ligand (25) displayed a high rigidity, assessed by (^1)H NMR and luminescence methods. The Tb complex of the monoamide ligand [Tb.26] displayed long lived luminescence arising from energy transfer from cyclodextrin-complexed napthalene to the lanthanide. The association constant, K(_11), between naphthalene and the cyclodextrin [Tb.26], was calculated as 10,200 M(^-1). K(_11) between the Gd complex [Gd.26] and GdDOTAPh (40) was determined to be 1740M(^-1) by NMRD. These association constants are ca 14 and 7 times greater respectively than for the corresponding complexes with β-cyclodextrin (1). Bromonaphthalenes exhibit long lived phosphorescence when complexed within cyclodextrins. Four bromonaphthalene-appended cyclodextrins (57-60) were designed to form inframolecular self-complexes. Competitive complexation of optically transparent guests was expected to reduce the lifetime of phosphorescent emission. The four conjugates were successfully synthesised, although luminescence studies revealed no evidence for complex formation and no response upon competitive guest complexation was observed. The rotaxane (95) and corresponding thread (92) of a hydrophilic ferrocene- cyclodextrin conjugate were made and structurally assessed by CD, (^-1)H NMR and MALDI-TOF MS. The hydrophilic per-O-ethyl conjugates (93 & 100) were also synthesised. A lipophilic quinoline-cyclodextrin conjugate (94) was made, which, when incorporated into an optode membrane, allowed the detection of acetylcholine at micromolar concentrations.

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.

Protein based contrast agents (ProCAs) developed by the Yang lab exhibit unique capabilities in enabling magnetic resonance imaging (MRI) with significantly improved sensitivity and targeting capabilities by utilizing biomarkers which can target various carcinomas in animals. Further clinical in vivo human applications require modifications of these designed contrast agents to further improve organ and tissue biodistribution, biomarker and cell targeting capabilities, and reduction of immunogenicity. The aim of this thesis is to develop a novel protein modification on ProCA by glycosylation to improve liver distribution by targeting liver receptor, asialoglycoprotein receptor (ASGPR). Rat and humanized first generation and humanized third generation ProCA were expressed and purified using either glutathione s-transferase (GST) tagged or taggless methods. Rat ProCA1, rProCA1, was then used to optimize glycan modification with glycosylation achieved at the highest level using a 100:1 molar ratio and three lysine residues. Similar to non-modified rProCA1 and PEGylated rProCA1, metal binding affinity of gadolinium for glycan modified rProCA1, Glyco-rProCA1, was found to be 9.49 x 10-12 M, and relaxivity was found to be greater than clinically available contrast agents with 34.08 and 42.67 mM-1s-1 for r1 and r2 respectively. Glycosylation of rProCA1 has significantly increased human serum stability, and we have achieved significant liver MRI enhancement via tail vein injection due to high ASGPR expression in the liver altering biodistribution of glycan modified ProCA, and we have also imaged uptake in the secretory glands. These biodistribution changes were noted by immunohistochemistry (IHC) staining which was found to stain liver sinusoid with spaces in between. The distribution to the liver was further confirmed via inductively coupled plasma optical emission spectrometry (ICP-OES) which shows Glyco-rProCA1 has significant uptake of gadolinium in the liver tissue. This study represents the first achievement of in vivo liver imaging by glycosylation using a lactose targeting moiety covalently bonded to protein contrast agents for MRI showing promise for future more specific targeting or whole body imaging capabilities.

No description available.

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.

The application of magnetic resonance imaging (MRI) to non-invasively assess disease biomarkers has been hampered by lack of desired contrast agents with high relaxivity, targeting capability, and optimized pharmacokinetics. We developed a novel MRI probe which targets HER2, a biomarker for various cancers and a target for anti-cancer therapies. This multimodal HER2-targeted MRI probe integrates a rationally designed protein contrast agent with a high affinity HER2 affibody and near IR dye. Our probe can differentially monitor tumors with different HER2 levels in both cells and xenograft mice. In addition to its 10-fold higher dose efficiency compared to clinically-approved agent DTPA, our developed agent also exhibits advantages in crossing the endothelial boundary, tissue distribution, and tumor tissue retention as demonstrated by even distribution of the imaging probe across the entire tumor mass. Additionally, a second series of protein contrast agents that included affibody against EFGR developed with the capability to specifically target EGFR. These contrast agents have been utilized to monitor drug treatments and quantitatively analyze biomarker expression level. Furthermore, we anticipate these agents will provide powerful tools for quantitative assessment of molecular markers, and improved resolution for diagnosis, prognosis and drug discovery.

Magnetic Resonance Imaging instruments rely on a contrast agent to provide high-resolution images of tissues in vivo. However, current clinical contrast agents are hindered by low relaxivity and fast correlation time, necessitating high injection dosages. These concerns, among others, have driven the development of a class of protein-based contrast agents (ProCAs), by design of lanthanide binding sites into a scaffold protein. ProCA1 has a higher reported relaxivity and dosage efficiency than current contrast agents. In this study, expression and Glutathione-S-Transferase purification procedures were optimized, and a refolding method for rapid production of ProCA1 has been developed to enable studies of conformation, metal binding, relaxivity, and in vivo applications. Several ProCA1 variants with 4-5 charged ligand residues were shown to have strong gadolinium binding affinity (Kd of 10-12 M) and metal selectivity. Several options to improve ProCA1 have been explored, including addition of a polyethylene chain or a bombesin tag.

A new class of polymer stabilized gadolinium trifluoride nanoparticles (NPs) have been developed as contrast agents for magnetic resonance imaging (MRI) and computed tomography (CT), with potential long term goals in targeted imaging and anti-cancer therapy. The NPs are comprised of a 90/10 mixture of GdF3/EuF3 and are coated with linear polyacrylic acid (PAA) chains consisting of 25 repeating units. The resulting aggregates are stable in serum and possess unprecedented mass relaxivities [i.e. ~100-200 s-1(mg/mL)-1]. Electron microscopy images reveal various NP morphologies which depend on the exact synthesis protocol. These include highly cross-linked oblong clusters with 30-70 nm cross sections, extensively cross-linked aggregates with 100-300 nm cross sections, and distinct polymer stabilized nanocrystals with 50 nm diameters. Their application as contrast agents in T1-weighted MRI studies, CT imaging at various X-ray energies, and preliminary rat brain perfusion studies was also tested. NP contrast enhancement was compared to Gd-DPTA (Magnevist®) and iopramide (Ultravist 300®) to demonstrate their high contrasting properties and potential as multimodal contrast agents.

A new class of polymer stabilized gadolinium trifluoride nanoparticles (NPs) have been developed as contrast agents for magnetic resonance imaging (MRI) and computed tomography (CT), with potential long term goals in targeted imaging and anti-cancer therapy. The NPs are comprised of a 90/10 mixture of GdF3/EuF3 and are coated with linear polyacrylic acid (PAA) chains consisting of 25 repeating units. The resulting aggregates are stable in serum and possess unprecedented mass relaxivities [i.e. ~100-200 s-1(mg/mL)-1]. Electron microscopy images reveal various NP morphologies which depend on the exact synthesis protocol. These include highly cross-linked oblong clusters with 30-70 nm cross sections, extensively cross-linked aggregates with 100-300 nm cross sections, and distinct polymer stabilized nanocrystals with 50 nm diameters. Their application as contrast agents in T1-weighted MRI studies, CT imaging at various X-ray energies, and preliminary rat brain perfusion studies was also tested. NP contrast enhancement was compared to Gd-DPTA (Magnevist®) and iopramide (Ultravist 300®) to demonstrate their high contrasting properties and potential as multimodal contrast agents.