Towards an integrated approach for red mud valorisation: a focus on titanium

AbstractIn this work red mud, a highly alkaline waste product generated during alumina production process, was valorised as a source of valuable metals and as an adsorbent material. A hydrometallurgical process was developed in order to recover titanium from red mud. By a leaching step with hydrochloric acid followed by ammonia precipitation and a further purification step by solvent extraction with Cyanex 301 using toluene as a solvent, quantitative recovery of titanium with a high purity level (> 95%) was achieved. Red mud adsorption properties were also tested for metal removal from aqueous solutions. The results showed the red mud potential in applications such as environmental remediation. The adsorption order was found to be: iron > lead > copper > manganese, zinc. Red mud can be thus potentially valorised both as a source of secondary titanium and as an adsorbent material, according to the principles of Circular Economy which promote waste reduction and the preservation of natural resources

Fighting the Huntington's Disease with a G-Quadruplex-Forming Aptamer Specifically Binding to Mutant Huntingtin Protein: Biophysical Characterization, In Vitro and In Vivo Studies

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington's disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer

Truncated Analogues of a G-Quadruplex-Forming Aptamer Targeting Mutant Huntingtin: Shorter Is Better!

Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington’s disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer

Calorimetric and spectroscopic investigation of biomolecules for therapeutic applications

Molecular recognition is the key for all biological processes. Such phenomena can be either intermolecular, as for the biding of a ligand to a macromolecule, or intramolecular, as for the denaturation of proteins or nucleic acids. In recent decades, DNA-ligand, protein-ligand or protein-DNA interactions have been the subject of numerous physico-chemical studies. The understanding of the energetics both of biomolecules stability and of their binding with other (bio)molecules is extremely interesting in biochemistry, biotechnology, and especially in the pharmaceutical field for a targeted structure-based drug design. Calorimetric and spectroscopic methodologies, combined to computational studies and biological assays, are essential for drug discovery. Indeed, thermodynamic stability of macromolecules as well as the energetics of their interaction with potential drugs are an essential complement to structural data for the optimization of lead compounds. Specifically, in this Ph.D. thesis, studies have been addressed to investigate: Physico-chemical factors affecting drug bioavailability (Chapter 3). Thermodynamic stability of G-quadruplexes (G4s) in oncogene promoters and their interactions with ligands (Chapter 4). Effects of epigenetic modifications on G4s stability (Chapter 5). Chapter 3 describes how the combination of the appropriate pH, solvent, temperature, and mixing time can improve the complexation between quercetin, a natural compound characterized by interesting pharmacological activities, and hydroxypropyl--cyclodextrin, a commonly used drug carrier. Chapter 4 focuses on the energetics of the interaction between KRAS proto-oncogene G4 and new putative anticancer drugs. This study led to the identification of a series of molecules able to selectively interact with G4s and characterized by cytotoxic activity on cancer cell lines. Finally, in Chapter 5, it was investigated, through mass spectrometry experiments, the capability of two modified sequences of KIT proto-oncogene, containing 5-methylcytosine and 5-carboxylcytosine, to fold into G4. The results proved that, despite the epigenetic modifications, these sequences were able to fold into G4, even though with a slower kinetics, as the unmodified sequence

Unravelling the role of lipid composition on liposome-protein interactions

: Upon in vivo administration of nanoparticles, a protein corona forms on their surface and affects their half-life in circulation, biodistribution properties, and stability; in turn, the composition of the protein corona depends on the physico-chemical properties of the nanoparticles. We have previously observed lipid composition-dependent in vitro and in vivo microRNA delivery from lipid nanoparticles. Here, we carried out an extensive physico-chemical characterisation to understand the role of the lipid composition on the in vivo fate of lipid-based nanoparticles. We used a combination of differential scanning calorimetry (DSC), membrane deformability measurements, isothermal titration calorimetry (ITC), and dynamic light scattering (DLS) to probe the interactions between the nanoparticle surface and bovine serum albumin (BSA) as a model protein. The lipid composition influenced membrane deformability, improved lipid intermixing, and affected the formation of lipid domains while BSA binding to the liposome surface was affected by the PEGylated lipid content and the presence of cholesterol. These findings highlight the importance of the lipid composition on the protein-liposome interaction and provide important insights for the design of lipid-based nanoparticles for drug delivery applications

Cytosine epigenetic modifications and conformational changes in G-quadruplex DNA: An ultraviolet resonance Raman spectroscopy study

Epigenetic modifications of DNA are known to play important regulatory roles in biological systems, especially in regulation of gene expression, and are associated with many types of human diseases, including cancer. Alternative DNA secondary structures, such as G-quadruplexes, can also influence gene transcription, thus suggesting that such structures may represent a distinctive layer of epigenetic information. G-quadruplex structures and DNA epigenetic modifications often go side by side, and recent evidence reveals that cytosine modifications within loops of G-quadruplexes can play a role in modulating their stability and structural polymorphism. Therefore, the development and validation of experimental techniques that can easily and reliably analyse G-quadruplex structures are highly desirable. In the present study, we propose to exploit the advantages of UV resonance Raman (UVRR) spectroscopy to investigate cytosine epigenetic modifications along with conformational changes in G-quadruplex-forming DNA. Our findings show that clear and specific spectral changes occur when there is a change in a G-quadruplex structure. Moreover, UVRR spectral analysis can indirectly distinguish the spectral variations occurring because of modifications in the guanine glycosidic conformations, as well as detect changes in the loops induced by H-bond formation or hydration of nitrogenous bases. The results further underscore the utility of UVRR spectroscopy for G-quadruplex structure elucidation under biologically relevant solution conditions

Structuring and de-structuring of nanovectors from algal lipids. Part 1: physico-chemical characterization

Lipid nanocarriers are among the most employed systems for drug delivery purposes in several research and industrial sectors, since their favorable properties ensure broad applicability. The design and characterization of these nanosystems are of paramount importance to obtain controlled outcome, since the supramolecular structure and molecular interactions deeply impact the functionality of the resulting aggregates. The choice of the most appropriate formulation for the target of interest relies on in-depth physico-chemical characterization in order to optimize stability, loading rates and sustained release. Several supramolecular architectures suited for carrier development can be obtained from lipid building blocks, by varying lipid composition and packing parameter. In particular, cubosome and liposome aggregates are often used as drug vectors thanks to their high cargo capability and biocompatibility. Moreover, the possibility to employ lipids from natural sources i.e. biomasses to prepare nanosystems makes them especially attractive. In this work, two aggregate types were characterized and compared as drug vectors for poorly water-soluble antioxidants, particularly curcumin and two adjuvants (i.e. tocopherol and piperine). The nanovectors were obtained by extracting lipids from algal biomasses with different lipid composition, and characterized by advanced structural (DLS, SAXS, Cryo-TEM) techniques, spectroscopy (NMR) and calorimetry (ITC). Finally, the structural stability of both aggregate types was evaluated

Conformational plasticity of DNA secondary structures: probing the conversion between i-motif and hairpin species by circular dichroism and ultraviolet resonance Raman spectroscopies

The promoter regions of important oncogenes such as BCL2 and KRAS contain GC-rich sequences that can form distinctive noncanonical DNA structures involved in the regulation of transcription: G-quadruplexes on the G-rich strand and i-motifs on the C-rich strand. Interestingly, BCL2 and KRAS promoter i-motifs are highly dynamic in nature and exist in a pH-dependent equilibrium with hairpin and even with hybrid i-motif/hairpin species. Herein, the effect of pH and presence of cell-mimicking molecular crowding conditions on conformational equilibria of the BCL2 and KRAS i-motif-forming sequences were investigated by ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectroscopies. Multivariate analysis of CD data was essential to model the presence and identity of the species involved. Analysis of UVRR spectra measured as a function of pH, performed also by the two-dimensional correlation spectroscopy (2D-COS) technique, showed the role of several functional groups in the DNA conformational transitions, and provided structural and dynamic information. Thus, the UVRR investigation of intramolecular interactions and of local and environmental dynamics in promoting the different species induced by the solution conditions provided valuable insights into i-motif conformational transitions. The combined use of the two spectroscopic tools is emphasized by the relevant possibility to work in the same DNA concentration range and by the heterospectral UVRR/CD 2D-COS analysis. The results of this study shed light on the factors that can influence at molecular level the equilibrium between the different conformational species putatively involved in the oncogene expression

Truncated Analogues of a G-Quadruplex-Forming Aptamer Targeting Mutant Huntingtin: Shorter Is Better!

Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington's disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer