Radical dendrimers as magnetic resonance imaging contrast agents

Resumen

The main objective of this Thesis is to develop metal-free contrast agents (CA) for magnetic resonance imaging (MRI) to overcome the stablished toxicity of the most widely used CA in clinical diagnosis based on Gd(III) complexes. Dendrimers are monodisperse polymers with a well-defined molecular structure, which can be a perfect support for anchoring organic radicals, what is called 'radical dendrimer'. In these compounds the relaxivities can be improved and a shielding effect can be provided to the radicals offering them protection against reduction. One of the greatest challenges in the synthesis of CA for biomedical applications is to make them water-soluble. Besides, CA should present high relaxivity, low toxicity and good biocompatibility. In this Thesis we use different strategies to prepare water-soluble radical dendrimers, by playing with different types of dendrimers, linkers and supramolecular structures. Amino acids can be ideal linkers for preparing water-soluble dendrimers because they provide an amino group for radical coupling, and an acid group that can provide negative charges conferring high solubility and low toxicity. Tyrosine and lysine have been chosen as linkers between polyphosphorhydrazone (PPH) dendrimer branches and PROXYL radicals. In the group it had been started the synthesis of four generations of PPH radical dendrimers with tyrosine as linker (Gn-Tyr(PROXYL)-COOLi, n=0-3). They are fully soluble in water, present high relaxivity (from 1.39 mM-1s-1 in G0 to 12.96 mM-1s-1 in G3), and no cytotoxicity. Lysine has been properly anchored to four generations of PPH dendrimers (Gn-Lys(BOC)-COOMe, n=0-3). However, under acid conditions the generations higher than G0 showed lysine release through P-N bond cleavage, preventing the obtaining of radical dendrimers with this linker. We have been able to determine that G1-Lys(BOC)-COOLi presents lysine release at pH 7.7. Therefore, these new lysine-functionalized PPH dendrimers can be used to prepare pH controlled degradable dendrimers or pH controlled release, opening a new field of research. Another strategy has been the use of dendrimers that are water-soluble themselves, in particular oligoethylene glycol (OEG) based dendrimers. Two generations of OEG-based dendrimers fully functionalized with 5 and 20 PROXYL radicals have been synthesized and characterized. They are soluble in water, non-cytotoxic and have a good relaxivity: G1-OEG-PROXYL radical dendrimer has a similar r1 relaxivity value (3.4 mM−1s−1) than Gd-DTPA (3.2 mM−1s−1) widely used in clinics. A third possibility is the use of amphiphilic compounds to prepare self-assembled supramolecular structures such as nanoparticles. We have used dendritic-linear-dendritic polymers based on (bis-MPA) dendrons at the end of a poly(ethylene glycol) chain. Three different generations have been synthesized by click chemistry (G1-, G2- and G3-MPA-PEG20k-TEMPO) and we have obtained homogeneous suspension of G2 nanoparticles (G2NPs) in water. While G2NPs form spherical nanoparticles, G2 directly dissolved in water forms nanofibers. They are non-cytotoxic and G2NPs present slightly higher relaxivity than G2, probably because of the higher rigidity of the former. We have also explored the possibility of obtaining not only magnetic but also fluorescent dendrimers to have bimodal CA for potential MRI and fluorescence imaging applications.We have synthesized six radical dendrimers based on fluorescent oligo(styryl)benzenes. In organic solvents these compounds present simultaneously fluorescence and relaxivity. In order to obtain these compounds in aqueous solution, CTAB micelles have been prepared to encapsulate them. Such systems, with the radical dendrimers inside, maintain both magnetic and fluorescent properties. Finally, the contrast ability in vivo of G3-Tyr(PROXYL)-COONa (G3) radical dendrimer has been described in healthy and GL261 glioblastoma-bearing mice. Biodistribution studies shows enhancement mostly in kidney cortex and pelvis, as well as a selective accumulation of the radical dendrimer in the brain tumor. G3 provides similar contrast enhancement than commercial Gd-based CA and a longer circulation time.