Design, synthesis and characterization of multifunctional therapeutic and diagnostic agents

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

Fluorescence Sensing Using DNA Aptamers in Cancer Research and Clinical Diagnostics

Among the various advantages of aptamers over antibodies, remarkable is their ability to tolerate a large number of chemical modifications within their backbone or at the termini without losing significant activity. Indeed, aptamers can be easily equipped with a wide variety of reporter groups or coupled to different carriers, nanoparticles, or other biomolecules, thus producing valuable molecular recognition tools effective for diagnostic and therapeutic purposes. This review reports an updated overview on fluorescent DNA aptamers, designed to recognize significant cancer biomarkers both in soluble or membrane-bound form. In many examples, the aptamer secondary structure switches induced by target recognition are suitably translated in a detectable fluorescent signal using either fluorescently-labelled or label-free aptamers. The fluorescence emission changes, producing an enhancement ("signal-on") or a quenching ("signal-off") effect, directly reflect the extent of the binding, thereby allowing for quantitative determination of the target in bioanalytical assays. Furthermore, several aptamers conjugated to fluorescent probes proved to be effective for applications in tumour diagnosis and intraoperative surgery, producing tumour-type specific, non-invasive in vivo imaging tools for cancer pre- and post-treatment assessment

Diazo transfer for azido-functional surfaces

Preparation of azido-functionalized polymers is gaining increasing attention. We wish to report an innovative, novel strategy for azido functionalization of polymeric materials, coupling plasma technology and solution processed diazo transfer reactions. This novel approach allows the azido group to be introduced downstream of the material preparation, thus preserving its physicochemical and mechanical characteristics, which can be tailored a priori according to the desired application. The whole process involves the surface plasma functionalization of a material with primary amino groups, followed by a diazo transfer reaction, which converts the amino functionalities into azido groups that can be exploited for further chemoselective reactions. The diazo transfer reaction is performed in a heterogeneous phase, where the azido group donor is in solution. Chemical reactivity of the azido functionalities was verified by subsequent copper-catalyzed azide-alkyne cycloaddition

Anticancer Ruthenium(III) Complexes and Ru(III)-Containing Nanoformulations: An Update on the Mechanism of Action and Biological Activity

The great advances in the studies on metal complexes for the treatment of different cancer forms, starting from the pioneering works on platinum derivatives, have fostered an increasingly growing interest in their properties and biomedical applications. Among the various metal-containing drugs investigated thus far, ruthenium(III) complexes have emerged for their selective cytotoxic activity in vitro and promising anticancer properties in vivo, also leading to a few candidates in advanced clinical trials. Aiming at addressing the solubility, stability and cellular uptake issues of low molecular weight Ru(III)-based compounds, some research groups have proposed the development of suitable drug delivery systems (e.g., taking advantage of nanoparticles, liposomes, etc.) able to enhance their activity compared to the naked drugs. This review highlights the unique role of Ru(III) complexes in the current panorama of anticancer agents, with particular emphasis on Ru-containing nanoformulations based on the incorporation of the Ru(III) complexes into suitable nanocarriers in order to enhance their bioavailability and pharmacokinetic properties. Preclinical evaluation of these nanoaggregates is discussed with a special focus on the investigation of their mechanism of action at a molecular level, highlighting their pharmacological potential in tumour disease models and value for biomedical applications

Nanoparticle-guided brain drug delivery: Expanding the therapeutic approach to neurodegenerative diseases

Neurodegenerative diseases (NDs) represent a heterogeneous group of aging-related disorders featured by progressive impairment of motor and/or cognitive functions, often accompanied by psychiatric disorders. NDs are denoted as ‘protein misfolding’ diseases or proteinopathies, and are classified according to their known genetic mechanisms and/or the main protein involved in disease onset and progression. Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD) are included under this nosographic umbrella, sharing histopathologically salient features, including deposition of insoluble proteins, activation of glial cells, loss of neuronal cells and synaptic connectivity. To date, there are no effective cures or disease-modifying therapies for these NDs. Several compounds have not shown efficacy in clinical trials, since they generally fail to cross the blood-brain barrier (BBB), a tightly packed layer of endothelial cells that greatly limits the brain internalization of endogenous substances. By engineering materials of a size usually within 1–100 nm, nanotechnology offers an alternative approach for promising and innovative therapeutic solutions in NDs. Nanoparticles can cross the BBB and release active molecules at target sites in the brain, minimizing side effects. This review focuses on the state-of-the-art of nanoengineered delivery systems for brain targeting in the treatment of AD, PD and HD. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

An Introduction to the Project BLASCO - Blending LAboratory and Satellite techniques for detecting CyanObacteria

Algal blooms can have an impact on health care costs, on the costs associated with the treatment of water intended for human consumption and on the tourism industry. The implementation of early warning systems would reduce these costs and the efforts needed to face and control the harmful effects of an algal bloom. A system for monitoring the quality of the waters, which operates on a large scale and at high frequency, would allow to keep under control the evolution of a bloom. The observation by satellite allows such a monitoring: in particular, the project is focused on the development of techniques for the analysis of satellite images, in order to detect the specific phytoplankton species potentially responsible for bloom formation in lakes. To reach this goal, it is necessary to analyse the spectral response characteristic of cyanobacteria and to develop algorithms to be applied to the analysis of satellite images. New calibration algorithms for the interpretation of satellite images will be obtained in lab experiments, using algal cultures. The developed algorithms will be tested through the analysis of remote sensing images, with particular attention to the bloom events occurring in the lakes of Lombardy and Piedmont. Field data on water optical properties and phytoplankton samples will be also collected. Moreover, different approaches will be applied and compared to quantify the amount of cyanobacteria (HPLC, counting, in vivo fluorimetry, spectroradiometry). Among the main results there will be the creation of a dataset of spectral signatures of some cyanobacteria taxa, as well as the development of calibration curves for the qualitative and quantitative estimation of the blooms. In general, we expect that it will be possible to distinguish, in natural conditions, the spectral signatures of cyanobacteria, even at low concentrations and within mixed populations of phytoplankton