Riccardi, Claudia (2017) Design, synthesis and characterization of multifunctional therapeutic and diagnostic agents. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Lingua: English
Title: Design, synthesis and characterization of multifunctional therapeutic and diagnostic agents
Riccardi, Claudiaclaudia.riccardi@unina.it
Date: 9 April 2017
Number of Pages: 172
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Chimiche
Dottorato: Scienze chimiche
Ciclo di dottorato: 29
Coordinatore del Corso di dottorato:
Paduano, Luigiluigi.paduano@unina.it
Montesarchio, DanielaUNSPECIFIED
Date: 9 April 2017
Number of Pages: 172
Uncontrolled Keywords: multifunctional nanoparticles, Ru(III)-complexes, NOTA chelators, Aptamers, TBA, AS1411
Settori scientifico-disciplinari del MIUR: Area 03 - Scienze chimiche > CHIM/06 - Chimica organica
Date Deposited: 03 May 2017 17:22
Last Modified: 14 Mar 2018 10:05
URI: http://www.fedoa.unina.it/id/eprint/11699
DOI: 10.6093/UNINA/FEDOA/11699


My PhD program has been focused on the development of multifunctional systems for potential applications in theranostic nanomedicine, which aims at simultaneously providing the diagnosis and treatment of a disease. For the construction of these tools, different kinds of nanoparticles (NPs) have been investigated, selected as optimal nanoplatforms for the in vivo delivery of drugs and diagnostic agents thanks to their capacity to incorporate multiple functional units into their coating. Main goal of my research has been the derivatization of these scaffolds with both therapeutic and diagnostic agents, as well as with active targeting ligands. In detail, the compounds here studied are: - new ruthenium(III)-based complexes, as anticancer agents. - suitable derivatives of the macrocycle NOTA, as chelators of 68Ga radioisotope, used in PET imaging. - oligonucleotide aptamers, as the thrombin binding aptamer (TBA) and the nucleolin-targeting AS1411. All these decorations have been ad hoc designed and synthesized with suitable tethers so to allow their attachment onto specific nanoparticles (i.e., gold NPs, streptavidin-coated silica NPs, superparamagnetic NPs, liposomes or niosomes). In consideration of the kind of chosen derivatization, different recognition schemes can be exploited (i.e. hydrophobic or electrostatic interactions, covalent bonds, selective recognition). Following this general strategy, a small library of decorated multifunctional NPs, differing for the nature of the NP core, Ru(III)-complex, oligonucleotide sequence and 68Ga chelator, has been prepared. These systems have been tested in vitro as theranostic agents towards targeted pathologies, as cancer and clotting disorders. Despite many Ru(III)-based compounds, in particular NAMI-A and KP1019 - currently in advanced clinical trials - display better antitumor activity and pharmacological profile than other metal-based drugs, they are poorly stable in aqueous media. So, new amphiphilic nucleolipids have been recently proposed by the research group of prof. D. Montesarchio as innovative carriers to transport in cell Ru(III)-compounds. Following this strategy, a novel nucleolipid-based Ru(III)-complex, named LipThyRu, has been here prepared. Then, in order to expand the repertoire and chemical diversity of the available amphiphilic Ru(III)-complexes, biocompatible scaffolds alternative to nucleosides, i.e. trifunctional α-amino acids, have been also exploited to serve as carriers for the Ru(III) metal core. Within this approach, two new aminoacyl lipidic Ru(III)-complexes, i.e. TUGluRu and TOTyroRu, have been synthesized. As far as the imaging agents are concerned, a mini-library of novel lipophilic derivatives of NOTA, designed as 68Ga chelators in PET analyses, has been prepared. The synthesized analogs differ for their hydrophobic tail, i.e. an oleic acid, a lipoic acid or a biotin residue (NOTA-OL, NOTA-Lip and NOTA-Bio, respectively), which ensures the subsequent anchoring of NOTA on differently decorated nanoplatforms. Two active targeting agents have been investigated: the 15-mer thrombin-binding aptamer (TBA) - able to inhibit the activity of human thrombin, a protein with key roles in coagulation processes - and AS1411, an oligonucleotide targeting nucleolin, which is a ubiquitous, multifunctional protein involved in cell survival, growth and proliferation, overexpressed on the outer membrane of cancer cells. In a first study, a new tris-conjugated TBA (named tris-mTBA) has been prepared, characterized in solution using UV, CD and fluorescence spectroscopy and then immobilized onto silica NPs commercially available as Sicastar®. This aptamer has been equipped with a biotin tag at the 5’-end, allowing its incorporation onto streptavidin-coated Sicastar® NPs, and further conjugated with a dansyl group and a β-cyclodextrin at the 3’- and 5’-end, respectively. This represents an efficient host-guest system useful to monitor the NP functionalization and the correct aptamer folding onto the nanoparticles, thanks to the fluorescence enhancement of the dansyl probe observed when it is inserted in the hydrophobic cavity of β-cyclodextrin. Sicastar® NPs have been thus functionalized with the tris-mTBA and characterized using DLS, SLS, gel electrophoresis and fluorescence analysis. DLS experiments nicely demonstrated that the tris-mTBA inhibited human α-thrombin ca. 10-fold more efficiently than unmodified TBA. Notably, under the same experimental conditions, the tris-mTBA/Sicastar® NPs completely blocked thrombin activity, which was restored upon addition of the TBA antidote, i.e. its complementary sequence. In a successive study, with the aim of exploiting AS1411 as an active targeting agent for multifunctional, theranostic nanoplatforms, a set of AS1411 derivatives have been selected (i.e.: 5’-stearyl-AS1411, 5’-cholesteryl-C6-AS1411 and 5’-cholesteryl-TEG-AS1411) with lipophilic tails at their 5’-end allowing their subsequent insertion into liposomes or lipid coated-NPs. The solution behavior of the AS1411 derivatives has been investigated using different techniques, in comparison with the unmodified aptamer. In particular, CD, CD-melting, UV-melting, gel electrophoresis and size exclusion chromatography analysis have been carried out to get information on their secondary structure and the thermal stability of their preferred conformations. This study was carried out in two different solutions, mimicking the extracellular and intracellular media, and at different oligonucleotide concentrations; special attention was devoted to the investigation of possible effects due to the nature of the lipophilic tail, or of the specific linker, on the overall structure and conformation of the aptamers. All these experiments consistently showed a high polymorphism for AS1411 and its derivatives, which are able to form form higher order G4 structures or large aggregates, particularly at high concentrations. These decorations (Ru complexes, aptamers and NOTA chelators) have been then combined on suitable platforms, realizing to date mainly two multifunctional systems: DOTAP-based liposomes, functionalized with TUGluRu and NOTA-OL, and niosome-based formulations, decorated with LipThyRu and AS1411. DOTAP-based liposomes functionalized with TUGluRu and NOTA-OL represent a good model for the realization of theranostic nanoparticles, offering several advantages, such as the possibility of monitoring their half-life, distribution, and tissue release. DOTAP/TUGluRu (70:30), DOTAP/NOTA-OL (95:5) and DOTAP/TUGluRu/NOTA-OL (65:30:5) systems have been prepared and fully characterized. DLS data indicated that these species form stable, monodisperse vesicles. In cell bioactivity assays, these formulations showed a good antiproliferative activity on MCF-7, C6 and HeLa cancer cell lines, with IC50 values in the low M conc., in turn not evidencing any toxicity on normal human cells even at 250 µM concentration. Niosome formulations including the nucleolipidic Ru(III)-complex LipThyRu and the nucleolin-selective aptamer AS1411 have been prepared and characterized using several techniques, i.e. gel electrophoresis, Zeta potential and DLS measurements. Preliminary cellular experiments on HeLa cells showed a small, but detectable cell viability decrease for the niosome_LipThyRu system, and a relevant antiproliferative effect for the formulation containing both AS1411 and the nucleolipidic Ru(III)-complex, indicating a marked synergic effect for these niosome-based anticancer agents. Future work, starting from the results obtained in this PhD program, will be directed to incorporating the amphiphilic Ru(III)-complexes (i.e., LipThyRu, TUGluRu, TOTyroRu or previously developed Ru(III) derivatives), the imaging agents (i.e., NOTA chelators) and the targeting agents (i.e., the lipophilic AS1411 derivatives) in combination into various kinds of other nanoplatforms, particularly in superparamagnetic NPs, so to obtain a mini-library of multifunctional systems to be tested in vitro and in vivo and identify the best anticancer theranostic agents.

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