Development and Understanding of Novel Compounds Designed as Potential MRI Contrast Agents

The two last decades have witnessed the appearance and the successful development of magnetic resonance imaging (MRI) contrast agents (CAs). Though MRI, which has become an essential medical diagnostic tool, can be performed without contrast agents, its rather low sensitivity implies long and tedious examination times. This time can be considerably reduced by the use of CAs. Paramagnetic ion complexes, and particularly gadolinium (III) complexes, can thus significantly accelerate the longitudinal relaxation rate 1/T1 of the water proton, one of the most common parameters measured in MRI experiments. The relaxivity, the property describing the paramagnetic relaxation enhancement, or PRE, is mainly affected by four parameters : the number of water molecules in the inner coordination sphere of the complex, the proton exchange rate in this inner sphere, the rotational correlation times, related to the size of the molecule, and the electron spin relaxation rates. This work deals with the development of four new types of compounds designed as potential MRI T1 contrast agents. The four compounds have different properties and were therefore developed to observe different effect on the relaxivity. High-field CAs, medium-field CA aggregates, linear polymeric and nanoparticular CAs, and targeted CAs are the four different specificities studied in this work. The first compound, presented in Chapter II, is the tricephalous complex {Mes[Gd(DO3A)(H2O)2]}. This mid-size molecule was designed as a potential high-field CA. The standard complexation method does not lead to the complete complexation, most probably due to an intramolecular folding of the compound. The complexation is therefore performed in two steps : a complete pre-complexation with magnesium (II) followed by a transmetallation to replace Mg2+ by Gd3+ in the three complexation sites. This newly developed complexation method can be really useful to achieve complete complexation of mid-size molecules, where complexation shared by two neighboring chelating units can occur. Chapter III deals with another mid-size trimeric complex, the {Ph4[Gd(DTTA)(H2O)2]-3}. The central core of this compound, composed of four phenyl rings, was designed to favor aggregation. The relaxivities of the big entities formed is expected to increase significantly in the medium resonance frequency range, i.e. between 10 and 200 MHz. The dynamic aggregation is observed by the concentration dependence of the relaxivity. The parameters obtained by fitting the experimental data allow the estimation of the number of molecule in the entities, ranging from the monomer in diluted solution to a few units for more concentrated solutions. Chapter IV presents Gd3+ chelate complexes conjugated to the linear polysaccharide chitosan. The coupling reaction, performed directly with the Gd3+ complex, is achieved through the condensation of a carboxylate uncomplexing arm on the chelating unit with the free amine of the chitosan. The ratio of modified chitosan units in the compound {Chi[Gd(DTTA-N'but)(H2O)2]-} is determined by ICP-MS and elemental analysis. Porous hydrogels nanoparticles, or nanogels, are subsequently formed from the modified linear chitosan by the ionic gelation method. As the material becomes bigger and more rigid, the relaxivities of the formed nanogels were expected to become higher than that of the linear chain. This does unfortunately not occur, most probably because of a partial complexation of the anion triphosphate, used for the formation of the particles, or by limited water diffusion inside the nanogels. The final chapter of results, Chapter V, presents a family of four compounds designed as target CAs for a specific kind of malignant tumor, such as prostate, breast or some lung cancer cells, which overexpress bombesin receptors. The four studied compounds are therefore composed by the peptide bombesin conjugated to a Gd3+ chelate complexe. In order to optimize the peptide-receptor interaction, two bombesin analogues, the Lys3-bombesin and the Ahx-bombesin(4-14), are used for the synthesis of the monovalent and the divalent peptide conjugates. The relaxometric properties of the four compounds are measured and compared.

Ion Beam Deposition and STM Analysis of Macromolecules on Solid Surfaces in UHV

Nicha Thontasen

Organic and biological macromolecules have attracted great interest within nanotechnology research due to properties that can be useful in future devices. Interesting functionalities such as optical activity, host-guest binding and molecular recognition are based on effects at the single molecular level. To study the properties and functions of molecules at submolecular scale, an atomically defined environment with low contamination is of great advantage. This can for instance be provided on a clean surface in ultrahigh vacuum (UHV), where surface sensitive analytical techniques and advanced nanostructure fabrication can be applied. Atoms and small organic molecules are easily transferred to a surface in vacuum by thermal sublimation. Many large, functional molecules, however, are nonvolatile and disintegrate at an elevated temperature, which hinders their application and investigation within the UHV environment. To explore the properties of nonvolatile molecules adsorbed at surface as well as the nanostructure fabricated from such molecules, electrospray ion beam deposition (ES-IBD), a technique capable of transferring nonvolatile molecules intact to surfaces in UHV, and in situ scanning tunneling microscopy (STM) are combined. A home-built ES-IBD setup allows molecular beam deposition with an unprecedented level of control: the ion beam composition is monitored by time-of-flight mass spectrometry (TOF-MS) prior to the deposition, molecular flux and coverage controlled via the ion beam current and the beam energy can be measured and adjusted. The modified surfaces are characterized in situ by STM, which reveals structural and electronic properties with submolecular resolution. Moreover, ex situ analysis by atomic force microscopy (AFM) allows to access meso-scale structural formation. In addition, Laser Desorption Ionization (LDI), Matrix-Assisted Laser Desorption Ionization (MALDI) and Secondary Ion Mass Spectrometry (SIMS) are performed to check the chemical composition and purity of the adsorbed materials. This work first introduces the principles of our experiments and reviews of the literature on molecular ion beam deposition. In the following, the experimental method is characterized in detail using Rhodamine 6G, an organic dye, as a model system. It is shown that homogeneous layers of adsorbed, intact molecules are formed on the surface and single molecule adsorbates are observed by STM. An investigation by SIMS and LDI reveals that the kinetic energy of the molecular ions upon collision with the surface determines whether intact or fragment ions are deposited. Molecular layer growth was studied using cluster ion beams of the nonvolatile surfactant SDS, resulting in an arrangement of the molecules typical for surfactants in a head-to-head and tail-to-tail manner based on the structural motive. Crystalline structures are observed by AFM resembling inverted bilayer-membranes formed on graphite and SiOx surfaces. A similar structural feature revealed by STM is a flat-laying double row of the molecules on Cu(100) surface showing the robustness of the structural motive. The properties of single molecular functional systems are studied on two types of large functional molecules: macrocycle complexes and proteins. Macrocyclic host-guest complexes of the three cations: Na+, Cs+ and H+ trapped in the cavity of dibenzo-24-crown-8 (DB24C8) are formed in the electrospray solutions and transferred intact to the surface. Characterization by STM and density functional theory calculations shows the structure of the complexes with the alkali ion present in the cavity. The shape of the complex at the surface is also determined. Cytochrome c, an electron transfer protein is deposited on surfaces in UHV as an ion beam of high or low charge-state. STM reveals unfolded strands with distinguishable submolecular structural features after the deposition of high charge states. When low charge states are deposited, two significantly different structures are observed. Folded proteins appear as globular features of 2-3 nm diameter, while unfolded proteins are imaged as at strands. On Au(111) the unfolded strands fold into compact two-dimensional patches. Additionally, the variation in kinetic energy upon deposition and elevated surface temperature are also studied regarding the change in conformation of the adsorbed proteins. The results of this thesis open up new opportunities for the creation of functional nanostructures on surfaces. Both the preparation of layers and thin films as well as the controlled immobilization of single functional molecules were demonstrated with ES-IBD and characterized at submolecular resolution in situ by STM. A great variety of nonvolatile substances from small to large functional molecules with different binding motives like covalent, complex coordination or hydrogen bonds, is compatible with ESI and thus can be used as building blocks to construct new materials in a well-controlled manner.

EPFL2011

Ion Beam Deposition and STM Analysis of Macromolecules on Solid Surfaces in UHV

Nicha Thontasen

EPFL2011