Simeone, Luca (2011) SYNTHESIS, CHARACTERIZATION AND APPLICATIONS OF NOVEL AMPHIPHILIC DERIVATIVES OF NUCLEOSIDES. [Tesi di dottorato] (Unpublished)
Download (3MB) | Preview
|Item Type:||Tesi di dottorato|
|Uncontrolled Keywords:||nucleolipids, amphiphiles, nucleosides, Ruthenium, nanovectors.|
|Date Deposited:||07 Dec 2011 08:26|
|Last Modified:||30 Apr 2014 19:47|
In a first part of my work, preliminary to all the successive investigations, novel thymidine- or uridine-based nucleolipids, containing one hydrophilic oligo(ethylene glycol) chain and one or two oleic acid residues (called ToThy, HoThy, DoHu and ToThyChol), have been synthesized with the aim to develop biocompatible nanocarriers for drug delivery and/or produce pro-drugs. Studies of microstructural characterization of the corresponding aggregates have been carried out in pure water and in pseudo-physiological conditions through DLS and SANS experiments. For ToThy, HoThy and DoHu stable vesicles, with mean hydrodynamic radii ranging between 120 nm and 250 nm have been revealed. In the case of ToThyChol, stable micelles were observed under the same experimental conditions, with mean hydrodynamic radii of 100 nm. Biological validation of the nucleolipidic nanocarriers was ensured by evaluation of their toxicological profiles, performed by administration of the nanoaggregates to a panel of different cell lines. ToThy exhibited a weak cytotoxicity and, at high concentration, some ability to interfere with cell viability and/or proliferation. In contrast, DoHu, HoThy and ToThyChol exhibited no toxicological relevance, behaving similarly to POPC-based liposomes, widely used for systemic drug delivery. Taken together, these results show that the here synthesized nucleolipid-based nanocarriers are finely tunable, self-assembling materials, potentially suitable for the in vivo transport of biomolecules or drugs. In a successive study, these nucleosidic nanovectors have been exploited as multifunctional ligands to obtain the corresponding ruthenium(III) salts, of interest as potential anticancer drugs. The obtained amphiphilic nucleosidic complexes were then studied in their self-aggregation properties by DLS and SANS techniques in aqueous solutions and in pseudo-physiological conditions. In analogy with the behaviour reported in the literature for the known Ru(III) complexes, the studied complexes ToThyRu, HoThyRu, DoHuRu and ToThyCholRu showed a limited stability in aqueous solutions, producing in few hours green precipitates. Therefore we studied how to achieve the complete stabilization in physiological media of these compounds. This goal was accomplished by use of the nucleosidic Ru(III) complexes in formulation with POPC in molar ratio 15:85. Under these conditions, the complexes were completely stable in pseudo-physiological solutions for several weeks. A complete and comprehensive study on the in vitro bioactivity was performed on the synthesized complexes in POPC formulation, particularly examining their growth inhibition ability on MCF-7 and WiDr cell lines. Most remarkably, very promising results were observed on ToThyRu/POPC and HoThyRu/POPC formulations, showing IC50 values of 9 and 15 M, respectively, on MCF-7 cell lines. In vitro bioscreening studies on other cancer cell lines are currently in progress, with the peculiar aim to evaluate specificity effects. To investigate the in vivo mechanism of action of the synthesized compounds, a novel amphiphilic nucleosidic Ru(III)-complex, bearing the fluorescent dansyl group, was designed and synthesized, essentially built around the same basic skeleton present in HoThyRu, for applications in fluorescence microscopy. In a successive work, I have then investigated a novel design for the amphiphilic nucleosidic complexes starting from a highly functionalized uridine-based nucleolipid. In this optimized scaffold, the pyridine ligand for the metal complexation was attached on the sugar skeleton, in lieu of the N-3 position on the nucleobase, as in the case of ToThyRu, HoThyRu, DoHuRu and ToThyCholRu complexes. A modified nucleoside, 3-azido-3-deoxy-1--D-xylofuranosyluracil - here prepared following a new, simple and very convenient synthetic procedure - was also here exploited as a suitable key intermediate to obtain a cationic aminoacylnucleolipid, of interest per se, as a model compound of a valuable class of novel biocompatible, highly functionalized nucleolipids, and to be used in mixture with the naturally negatively charged ruthenium(III) complexes, thus producing catanionic vesicles. For all the latter compounds, including the nucleolipid-ruthenium(III) complexes and the cationic aminoacynucleolipid, a detailed microstructural characterization as well as biological activity assays are currently underway, in collaboration with specialized laboratories, and the related results will be presented in due course. In the frame of the design, synthesis and characterization of novel nucleolipids, a relevant part of my efforts have been then addressed to the study of guanosine-containing amphiphiles, almost unexplored compounds of interest in the development of smart, novel self-assembling materials as well as for their potential biological activity. A small library of sugar-modified guanosine derivatives has been prepared, starting from a common intermediate, fully protected on the nucleobase. Insertion of myristoyl chains and of diverse hydrophilic groups, such as an oligoethylene glycol, an amino acid or a disaccharide chain, connected through in vivo reversible ester linkages, or of a charged functional group provided different examples of amphiphilic guanosine analogs, named G1-G7 herein. All of the sugar-modified derivatives were positive in the potassium picrate test, showing a marked ability to form G-tetrads. CD spectra demonstrated that, as dilute solutions in CHCl3, distinctive G-quadruplex systems may be formed, with spatial organisations dependent upon the structural modifications. Two compounds, G1 and G2, proved to be good low-molecular-weight organogelators in polar organic solvents, such as methanol, ethanol and acetonitrile. Ion transportation experiments through phospholipid bilayers were carried out to evaluate their ability to mediate H+ transportation, with G5 showing the highest activity within the investigated series. Moreover, G3 and G5 exhibited a significant cytotoxic profile against human MCF-7 cancer cells in in vitro bioassays with IC50 values in the 20 M range, while no cytotoxic activity was observed on normal, control cells. Finally, to expand the knowledge about the available protective groups for nucleosides, the use of Boc as a thymine protecting group in the synthesis of sugar-alkylated (or, more generally, sugar-modified) thymidine analogs was here described. Boc was easily inserted at the 3-N position in high yields and found to be stable to standard treatments for the removal of acetyl and TBDMS groups, as well as to ZnBr2-mediated DMTr deprotection. Boc protection proved to be completely resistant to the strong basic conditions required to selectively achieve 3’-O-alkylation. This acid-labile group, unprecedentedly used for the masking of pyrimidine nucleobases, was then here exploited to allow the synthesis of 3-azido-3-deoxy-1--D-xylofuranosyluracil in six, high yielding steps from uridine, previously mentioned as a valuable starting material for the synthesis of novel, highly functionalized nucleolipids. From the full comprehension of the structure-activity relationships of the here described compounds, a relevant contribution to the knowledge of nucleolipid ruthenium(III)-complexes, as promising anticancer agents, and more generally of novel nucleoside-based amphiphiles as attractive, bioinspired building blocks of use in the production of innovative, smart materials for biomedical applications is expected.
Actions (login required)