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Doctor of Philosophy / Department of Chemistry / Santosh Aryal / Nanotechnology offers exciting options for the site-selective delivery of chemotherapeutics and diagnostic agents using nanoparticles. Varieties of organic and inorganic nanomaterials have been explored extensively as a delivery system either in the form of drug carriers or imaging agents. Successful stories include the clinical translation of anticancer nanomedicines such as PEGylated liposomal doxorubicin (DOXIL®), albumin-bound paclitaxel (Abraxane®), and polymeric micelle loaded paclitaxel (Genexol®), which are currently used in the clinic as one of the first lines for cancer chemotherapies. These conventional nanomedicines rely on passive-drug targeting taking advantage of leaky tumor vasculature, called the Enhanced Permeability and Retention (EPR) effect. However, delivering biologically active components selectively to the diseased cell, for example, cancer, is highly challenging due to the biological barriers in the body including blood pool cells/proteins, heterogeneous microenvironment, and intracellular degradation. Therefore, the goal of this dissertation is to develop nanoplatforms that can deliver the agents of interest in targeted fashion to cancer while bypassing or collaborating with the biological barriers. The design consideration of these nanoplatforms centralizes on using simple chemical reactions and cell biology to engineer nanoparticles. The presented nanoparticles were extensively studied and evaluated for their biological functions using in vitro and in vivo models. These nanoconstructs described herein address current limitations of conventional nanomedicine such as (1) the lack of understanding of the interaction of nanoparticle and biological system, and (2) the lack of an effective targeting strategy to deliver drugs to the cancer cell in the tumors. The significant findings of each system will be highlighted and discussed throughout this dissertation. Results obtained highlight key findings such as NP intracellular fate, maximized tumor accumulation, and unique pharmacokinetics could open the avenues for systemic investigations for personalized medicine and lay the foundation for nanomedicine design to accelerate clinical translation.

Four new potential MR imaging contrast agents were synthesised. Gadolinium-hematoporphyrin (Gd-H) was produced by inserting gadolinium into the naturally occurring porphyrin,hematoporphyrin.Gadolinium-tetra-carboranylmethoxyphenyl-porphyrin acetate (Gd-TCP)was similarly synthesised by gadolinium insertion into the synthetic porphyrin, 1, 6, 11, 16-tetra-[3-(carboranylmethoxy)phenyl] porphyrin. The monoclonal antibodies, 9.2.27 against melanoma and WM53 against leukaemia cell lines, were conjugated with cyclic anhydride gadolinium-diethylenetriaminepenta-acetic acid (Gd-cDTPAa), yielding the attachment of chelate DTPA to the antibodies. Gadolinium ion was inserted into the chelate DTPA, thus labelled both these antibodies with Gd-DTPA. Overall, with the satisfactory low levels of gadolinium in the liver, kidneys, and spleen, and good tumour uptake, gadolinium antibody conjugates has considerable potential for further diagnostic applications of MR imaging. / Doctor of Philosophy (PhD)

A main driving force behind the recent years’ immense interest in nanoscience and nanotechnology is the possibility of achieving new material properties and functionalities within, e.g., material physics, biomedicine, sensor technology, chemical catalysis, energy storing systems, and so on. New (theoretical) possibilities represent, in turn, a challenging task for chemists and physicists. An important feature of the present nanoscience surge is its strongly interdisciplinary character, which is reflected in the present work. In this thesis, nanocrystals and thin films of magnetic and ferroelectric metal oxides, e.g. RE2O3 (RE = Y, Gd, Dy), GdFeO3, Gd3Fe5O12, Na0.5K0.5NbO3, have been prepared by colloidal and sol-gel methods. The sizes of the nanocrystals were in the range 3-15 nm and different carboxylic acids, e.g. oleic or citric acid, were chemisorbed onto the surface of the nanoparticles. From FT-IR measurements it is concluded that the bonding to the surface takes place via the carboxylate group in a bidentate or bridging fashion, with some preference for the latter coordination mode. The magnetic properties of nanocrystalline Gd2O3 and GdFeO3 were measured, both with respect to magnetic resonance relaxivity and magnetic susceptibility. Both types of materials exhibit promising relaxivity properties, and may have the potential for use as positive contrast enhancing agents in magnetic resonance imaging (MRI). The nanocrystalline samples were also characterised by transmission electron microscopy (TEM), x-ray photoelectron spectroscopy (XPS), and quantum chemical calculations. Thin films of Na0.5K0.5NbO3, GdFeO3 and Gd3Fe5O12 were prepared by sol-gel methods and characterized by x-ray powder diffraction (XRPD) and scanning electron microscopy (SEM). Under appropriate synthesis conditions, rather pure phase materials could be obtained with grain sizes ranging from 50 to 300 nm. Magnetic measurements in the temperature range 2-350 K indicated that the magnetization of the perovskite phase GdFeO3 can be described as the sum of two contributing terms. One term (mainly) due to the spontaneous magnetic ordering of the iron containing sublattice, and the other a susceptibility term, attributable to the paramagnetic gadolinium sublattice. The two terms yield the relationship M(T)=M0(T)+χ(T)*H for the magnetization. The garnet phase Gd3Fe5O12 is ferrimagnetic and showed a compensation temperature Tcomp ≈ 295 K.

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.

Texaphyrins represent the vanguard of experimental anticancer drugs and also symbolize a well-known example of expanded porphyrins, a class of oligopyrrolic macrocycles with tumor localization properties and powerful metal chelating properties. Chapter 1 of this thesis describes the unique structural characteristics of this complex synthetic molecule along with the biological relevance and scientific justifications for studying its anticancer properties and powerful MRI contrast ability. This Chapter also serves to underscore the need to improve further and refine the efficacy of texaphyrins as compounds that may be applied in the struggle against cancer. Chapter 2 details the synthesis of bismuth(III) and lead(II)-texaphyrin complexes that could potentially find use as [alpha]-core emitters for radiotherapy. In principle, porphyrins would ostensibly appear to be ideal ligands for use in radiotherapy due to their tumor-localizing ability. However, Bi(III)- and Pb(II)-porphyrin complexes are extremely rare, most reflecting the vastly challenging synthesis of these compounds as well as their general lack of stability. These limitations provided an incentive for us to use texaphyrins as more versatile ligands to prepare and fully characterize stable bismuth(III) and lead(II) complexes. To be of interest in future medical applications, we needed to prepare these complexes quickly as compared to the relevant time scales set by the half-lives of the isotopes targeted for use in radiotherapy. This goal was successfully realized. As mentioned above, texaphyrin is able to form stable complexes with a large variety of metals particularly in the lanthanide series. Gadolinium(III) complexes of texaphyrin have been studied in considerable detail. Chapter 3 details the synthesis and conjugation methods used to develop a texaphyrin conjugated dual mode nanoparticle contrast agent. This project has been done in collaboration with the group of Prof. Jinwoo Cheon (Yonsei University, Seoul, Korea), who demonstrated fascinating results with the texaphyrin functionalized nanoparticles. Not only do these conjugates act as improved magnetic resonance contrast agents displaying enhanced signals in both the T1 and T2 MRI modes, but also serve to sensitize apoptotic hyperthermia. It is this latter, double effector feature, that has been most extensively studied to date. Chapter 4 of this dissertation describes work done in close collaboration with Dr. Natalie Barkey and Dr. David Morse (Moffitt Cancer Center, Tampa, FL) where a gadolinium texaphyrin complex was developed that is able to target the melanocortin 1 receptor (MC1R) when encapsulated in a micellar system. As detailed in this Chapter, these collaborateurs demonstrated that these gadolinium-texaphyrin micelles are able to target MC1R-expressing xenograft tumors in vivo. This work relied on the supply of a new set of texaphyrin derivatives that were prepared and characterized as part of this dissertation work Chapter 5 of this disseration introduces sapphyrins, another class of expanded porphyrins with tumor selectivity. This project is based on the hypothesis that a direct linkage of sapphyrin with an anticancer agent based on ruthenium(II) could improve the efficacy of both compounds. Since sapphyrins exhibit limited ability to form stable complexes with transition metals, an appended 1,10-phenanthroline unit was chosen as an efficient N-donor aromatic ligand for ruthenium(II). Therefore, extensive synthetic efforts were made to form this sapphyrin-1,10-phenanthroline construct in an effort to stabilize a mixed sapphyrin-metallo-phenanthroline complex. Finally, Chapter 6 of this dissertation demonstrates the author's efforts to synthesize a planar rosarin species. Non-aromatic and non-planar rosarins have been known for over two decades. Through structural modification of the compound, namely through linking of both [Beta] positions on the bipyrrole unit, a new planar rosarin species has been synthesized exhibiting Hückel antiaromaticity. / text

Contrast enhanced ultrasound offers a unique method to measure the blood flow, perfusion, vascular volume and morphology of microvascular networks. This is achieved by exploiting the ability of microbubble contrast agents to be disrupted and preferentially detected with contrast specific imaging techniques – using a method known as disruption-replenishment. In its current form, disruption-replenishment suffers from poor reproducibility and accuracy, largely due to the inappropriate application of a mono-exponential model of microbubble replenishment, and an incomplete understanding of the dependencies of the measurement. In this thesis, we hypothesize that disruption-replenishment measurements can be improved by applying a perfusion model that considers the physical elements of the measurement, including the haemodynamics and morphology of the vascular system, the ultrasound field distribution and microbubble properties. We present a flexible, theoretical framework to model microbubble replenishment within the microvasculature. The replenishment model is further developed by in vitro and in vivo validation, and clinical translation in a trial of anti-angiogenic therapy in patients, resulting in a proposed clinical protocol. The presented formalism was shown to be more robust and demonstrated better agreement of both fitting quality and estimates of flow velocity when compared to the established model (accuracy to within 3-9%). The reproducibility of repeated in vivo disruption-replenishment flow measurements was 11.9% using the proposed perfusion model compared to 24% using the established model. Variability of clinical perfusion measurements was also reduced with a method that discards the contribution of flow from larger arteries. Excluding the large vessel component in clinical measurements of tumour blood volume decreased the inter-plane variability by up to 20%. The proposed perfusion model can be used to generate parametric maps of vascularity through which additional quantitative parameters become available. These improvements will help translate the method of disruption-replenishment into routine clinical practice and clinical trials.

Contrast enhanced ultrasound offers a unique method to measure the blood flow, perfusion, vascular volume and morphology of microvascular networks. This is achieved by exploiting the ability of microbubble contrast agents to be disrupted and preferentially detected with contrast specific imaging techniques – using a method known as disruption-replenishment. In its current form, disruption-replenishment suffers from poor reproducibility and accuracy, largely due to the inappropriate application of a mono-exponential model of microbubble replenishment, and an incomplete understanding of the dependencies of the measurement. In this thesis, we hypothesize that disruption-replenishment measurements can be improved by applying a perfusion model that considers the physical elements of the measurement, including the haemodynamics and morphology of the vascular system, the ultrasound field distribution and microbubble properties. We present a flexible, theoretical framework to model microbubble replenishment within the microvasculature. The replenishment model is further developed by in vitro and in vivo validation, and clinical translation in a trial of anti-angiogenic therapy in patients, resulting in a proposed clinical protocol. The presented formalism was shown to be more robust and demonstrated better agreement of both fitting quality and estimates of flow velocity when compared to the established model (accuracy to within 3-9%). The reproducibility of repeated in vivo disruption-replenishment flow measurements was 11.9% using the proposed perfusion model compared to 24% using the established model. Variability of clinical perfusion measurements was also reduced with a method that discards the contribution of flow from larger arteries. Excluding the large vessel component in clinical measurements of tumour blood volume decreased the inter-plane variability by up to 20%. The proposed perfusion model can be used to generate parametric maps of vascularity through which additional quantitative parameters become available. These improvements will help translate the method of disruption-replenishment into routine clinical practice and clinical trials.

This paper presents the results of a continuing investigation of several functionalized SPION MR contrast agents and the solid state NMR studies of plasma polymerized Allyl Alcohol thin films. Several species of functionalized SPIONs were tested; the most successful SPIONs were the melamine dendron, polyimidazole, and conjugated nucleic acid SPIONs. The study of the MR responses of the melamine dendron SPIONs determined that these SPIONs undergo reversible clustering and that their pH sensitive MR responses are due to increased clustering at pH> 4. The MR responses of the polyimidazole functionalized SPIONs (both the dopamine and carboxylate linked) indicate a pH sensitive MR response well within the physiological regime (inflection point pH ~6) as well as excellent baseline relaxivities. However, perhaps due to the low polyimidazole loading onto the SPION core, these agents were very sensitive to ionic environment. CPMAS studies of pulsed plasma polymerized allyl alcohol thin films indicated that the hydrophobic films had a more ordered structure than the hydrophilic films; however, all poly-allyl alcohol thin films had a highly amorphous structure. The use of synthetic mica as a substrate for CPMAS studies of polymer thin films is also discussed.

L’Imagerie par Résonance Magnétique est une des techniques de diagnostic les plus performantes. Parmi les perspectives d’avenir, les applications en imagerie moléculaire avec l’utilisation d’agents de contraste intelligents sensibles à divers paramètres physico-chimiques sont particulièrement attrayantes.Dans cette thèse, nous présentons la synthèse et la caractérisation physico-chimique de nouveaux complexes de lanthanide dans le but de développer des agents de contraste sensibles à l’activité enzymatique ou la présence d’ions calcium. Les premiers complexes sont basés sur le concept de coupler un substrat spécifique à une enzyme par l’intermédiaire d’un bras auto-immolable, à un chélate de lanthanide macrocyclique. Les modifications de structure qui suivent le clivage du substrat et l’autodestruction du bras sont sensées induire des variations de relaxivité ou des propriétés CEST des complexes. Même si nous n'avons pas pu créer des agents de GdIII avec une réponse T1, plusieurs complexes d’YbIII ou d’EuIII montrent un important changement de leurs propriétés CEST après réaction enzymatique. Certains d'entre eux portant un bras dérivé pyridinique peuvent également agir comme des sondes optiques émettant dans le visible ou le proche infrarouge.Nous avons également développé un agent sensible au calcium en combinant un chélate de lanthanide macrocyclique avec une unité d’iminodiacetate permettant la coordination du calcium. Les complexes d’EuIIIet d’YbIII montrent une importante diminution de l’effet CEST en présence de Ca2+. L’utilisation en parallèle des deux complexes permet une approche ratiométrique où la réponse IRM détectée est indépendante de la concentration de l’agent. / Today, Magnetic Resonance Imaging is one of the most powerful diagnostic techniques in the clinics. Amongfuture perspectives, molecular imaging applications based on smart contrast agents which are responsive to various physico-chemical parameters, are particularly attractive. In this work, we present the synthesis and physico-chemical characterisation of novel lanthanide complexes with the aim of developing smart contrast agents for the detection of enzyme activity or calcium concentration.The complexes designed to give an MRI response to an enzyme are based on the original concept of coupling an enzyme-specific substrate to a macrocyclic LnIII chelate via a self-immolative linker. The structural changes following enzymatic cleavage of the substrate and destruction of the self-immolative armare expected to induce variation of the relaxivity or the CEST properties of the LnIII complexes. Though we failed creating GdIII agents with a T1 response upon enzymatic reaction, several YbIII or EuIII complexes were synthesized that provide an important change in their CEST properties. Some of them, bearing a pyridine-derivative arm which is an efficient sensitizer of lanthanide luminescence, act also as enzyme-responsive NIR or visible emitting optical probes.We have developed a Ca-responsive agent combining a DOTA-tetraamide LnIII chelator with animinodiacetate unit for calcium coordination. The EuIII and YbIII complexes show an important decrease in the CEST effect in response to Ca2+. The parallel application of the two complexes allows for ratiometric approaches where the detected MRI response is independent of the concentration of the agent.