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Item Breast tumor microenvironment and endocrine resistance: dissecting the molecular link(Università della Calabria, 2023-07-04) Caruso, Amanda; Catalano, Stefania; Andò, SebastianoItem New strategies in the design of functional and bioinspired materials for therapeutic applications(Università della Calabria, 2022-01-27) Servidio, Camilla; Andò, Sebastiano; Cassano, Roberta; Iemma, Francesca; Stellacci, FrancescoItem Design and development of new functional materials for pharmaceutical and biomedical purposes(Università della Calabria, 2022-02-15) Curcio, Federica; Andò, Sebastiano; Trombino, SoniaItem Anticancer drugs: a detailed computational analysis of "non classical" compounds mechanism of action(Università della Calabria, 2020-02-05) Ponte, Fortuna; Andò, Sebastiano; Sicilia, EmiliaMetal containing drugs have attracted an enormous deal of interest for their use in cancer therapy. Transition metal compounds’ richness offers extraordinary opportunities for the design of anticancer compounds, possessing pharmacokinetic properties inaccessible to purely organic compounds. The most successful and evident proof of their pivotal role is represented by cisplatin that, together with its carboplatin and oxaliplatin derivatives, continues to be routinely used worldwide in clinical practice. However, it is well known that the use of such drugs for fighting cancer is accompanied by severe side effects and intrinsic or acquired resistance that drastically limit their successful action. Therefore, decades of research efforts have been devoted to the search and the synthesis of safer and more effective and selective agents, either containing platinum or alternative metals, acting with similar or different mechanisms. In order to accomplish this aim is of decisive importance the elucidation of the mechanism of action of the drugs. Molecular simulations, or in silico experiments, are able to provide detailed information at atomistic resolution, rarely accessible to experiments, that can complement laboratory experiments. The increasing accuracy of computational approaches and the growing performance of computer performance, allow to properly describe reaction paths and involved molecular orbitals, calculate electronic properties, simulate spectra without any limitation except those connected with the adopted level of theory and compuatational protocol. The main aim of the present work was the detailed investigation, in the framework of the Density Functional Theory, of the mechanism of action of “non classical” platinum and transition metal non-platinum compounds, for some of them in collaboration with experimentalists, and the rationalization of their behaviors. In the next paragraphs all the studied systems will be shortly described together with the motivations that have prompted us to study such systems. Both “non classical” platinum(IV) prodrugs, non-platinum drugs and photoactivatable Pt(II) and Pt(IV) complexes have been examined. In the development of new platinum-based anticancer drugs, is of great interest the emerging class of "dual action" Pt(IV) prodrugs that, undergoing a reductive elimination process, which is the key step for their activation, are able to release the active Pt(II) complexes and bioactive axial ligands that together lead to cell death. Indeed, the two axial ligands, in turn, can be chosen to possess physico-chemical and biopharmaceutical properties or even facilitate the incorporation into a drug delivery system. According to the research lines mentioned above, the use of drug delivery systems has also grown, and many different strategies have been examined to encapsulate platinum drugs within macromolecules, including macrocyclic species, which are responsible for creating supramolecular host-guest structures. The encapsulation slows down and prevents the drug degradation by proteins and peptides. One of the most widely studied class of synthetic supramolecular macrocycles are Calix[n]arenes (CX), whose property, as molecular hosts and delivery systems, are of increasing interest. Photodynamic Therapy (PDT) is a non-toxic therapeutic technique, clinically approved and minimally invasive, used for the treatment of several types of cancers based on the generation of reactive oxygen species (ROS), that acts as cytotoxic agents. In PDT applications three components are required: a photosensitizer (PS), a light of a specific wavelength and tissue oxygen. A promising approach to increase the effectiveness of anticancer therapy is the combination of multimodal treatment methods into a single system. Recently, a strategy has been proposed providing the possibility to combine the classical Pt-based chemotherapy with photodynamic therapy (PDT) treatment. This approach involves the functionalization of a photosensitizer (PS) with a therapeutic drug such as cisplatin-like compounds.Item Contribution of flavonoids and iridoids to health properties of edible plants of Arbutas, Cornas, and Vuccinium genera from Mediterranean area(Università della Calabria, 2020-06-19) Tenuta, Maria Concetta; Tundis, Rosa; Andò, Sebastiano; Deguin, Brigitte; Lallemand, Marie-ChristineAim of the study: Natural compounds produced by the “nature laboratory” continue to play a leading role in the process of discovering and developing of new drugs, food supplements and/or functional foods. Today, in fact, we are witnessing a renewed interest in these products for the prevention and treatment of numerous diseases, including those associated with oxidative stress such as diabetes and inflammatory diseases. In this context, the present research project aimed at identifying the best techniques that allow the extraction of two classes of active compounds, flavonoids and iridoids, from the fruits and leaves of Arbutus unedo L., Vaccinium corymbosum L., Cornus mas L., and Cornus sanguinea L. Their edible fruits but also their leaves (considered byproducts) represent rich sources of bioactive compounds. The work continued with the evaluation of in vitro biological activity and the fractionation of the most active extracts in order to envisage their potential use as a source of bioactive compounds for the development of functional foods, nutraceuticals and/or food supplements . Materials and methods: The chemical profile of extracts was evaluated by using LC/ESI/QTOF/MS. Four different in vitro tests (DPPH, ABTS, -carotene bleaching test and FRAP), were performed to investigate the antioxidant activity. The potential hypoglycaemic activity was investigated by the inhibition of -amylase and -glucosidase enzymes. The inhibitory effects of the extracts on the production of nitric oxide have been studied by Griess assay. The most active extracts of C. mas and C. sanguinea were subjected to fractionation in order to obtain a separation of flavonoids and iridoids using XAD-16 resin. Obtained fractions were tested to evaluate their ability to reduce the activation of the NF-kB factor. Results: LC/ESI/QTOF/MS analyses detected for the first time in the A. unedo the presence of ellagic acid 4-O--D-glucopiranoside, kaempferol 3-O-glucoside, myricetin, myricetin 3-O-rhamnopiranoside, naringenin 7-O-glucoside, isovitexin 7-O-glucoside, and norbergenine and V. corymbosum the presence of the iridoids scandoside, vaccinoside, geniposide, and dihydromonotropein. The complete chemical characterization of C. sanguinea fruits and leaves was conducted herein for the first time, reporting the presence of flavonoids and iridoids. Worthy of note are the results obtained from the hypoglycaemic activity. The extracts of both Cornus species showed a high inhibition of -glucosidase and a moderate inhibition of -amylase. Of particular note were the results obtained with the hydroalcoholic maceration of leaves and dried fruits of C. mas (TDB and MDB), the ethanol maceration of fresh leaves (PF1) and the hydroalcoholic maceration of dry leaves (SD2) of C. sanguinea, which for this reason were subjected to fractionation using XAD-16 resin. C. sanguinea PF1 (II) and SD2 (II) fractions showed the best antioxidant and NF-KB inhibition activity. Conclusion: All the investigated extracts showed a promising antioxidant, hypoglycaemic and anti-inflammatory potential, confirming the positive contribution of the two classes of compounds under study, flavonoids and iridoids, suggesting their potential use as a rich source of useful bioactive compounds for the formulation of new products to prevent diseases associated with oxidative stress such as inflammatory diseases and diabetes. In particular, results obtained with the C. sanguinea fractions PF1 (II) and SD2 (II) encourage researchers to continue the study with further in vivo studies. Scopo dello studio: I composti naturali prodotti dal “laboratorio natura” continuano ad avere un ruolo di primo piano nel processo di scoperta e sviluppo di nuovi farmaci, integratori alimentari e/o alimenti funzionali. Oggi, infatti, si assiste a un rinnovato interesse nei riguardi di tali prodotti nella prevenzione e nel trattamento di numerose patologie, tra cui quelle associate allo stress ossidativo come diabete e malattie infiammatorie. In questo ambito si inserisce il presente lavoro di ricerca, volto all’identificazione delle migliori tecniche che consentano l’estrazione di due classi di principi attivi, i flavonoidi e gli iridoidi, dai frutti e dalle foglie di Arbutus unedo L., Vaccinium corymbosum L., Cornus mas L. e Cornus sanguinea L. I loro frutti commestibili ma anche le foglie (considerate prodotti di scarto) rappresentano ricche fonti di composti bioattivi. Il lavoro ha quindi riguardato la valutazione dell’attività biologica in vitro e il frazionamento bio-guidato degli estratti risultati più attivi al fine di prospettare un loro potenziale impiego come fonte di composti bioattivi per lo sviluppo di alimenti funzionali, nutraceutici e/o integratori alimentari. Materiali e metodi: Il profilo fitochimico degli estratti è stato valutato mediante LC/ESI/QTOF/MS. Per la determinazione in vitro della capacità antiossidante sono stati eseguiti quattro diversi test (DPPH, ABTS, FRAP e-carotene bleaching test). La potenziale attività ipoglicemizzante è stata investigata mediante l’inibizione degli enzimi -amilasi e -glucosidasi. Gli effetti inibitori degli estratti sulla produzione di ossido nitrico sono stati studiati mediante saggio di Griess. Gli estratti maggiormente attivi di C. mas e C. sanguinea sono stati sottoposti a frazionamento per la separazione di flavonoidi e iridoidi mediante l’impiego della resina XAD-16. Le frazioni ottenute sono state saggiate per valutare la capacità di ridurre l’attivazione del fattore NF-kB. Risultati: L’analisi LC/ESI/QTOF/MS ha rilevato per la prima volta nell’A. unedo la presenza di acido ellagico 4-O--D-glucopiranoside, kaemferolo 3-O-glucoside, miricetina, miricetina 3-O-ramnopiranoside, naringenina 7-O-glucoside, isovitexina 7-O-glucoside, e norbergenina e nel V. corymbosum la presenza degli iridoidi scandoside, vaccinoside, geniposide e diidromonotropeina. La caratterizzazione chimica completa di frutti e foglie del C. sanguinea è stata condotta per la prima volta, riportando la presenza flavonoidi ed iridoidi. Degni di nota sono i risultati ottenuti dall’attività ipoglicemizzante. Gli estratti di entrambe le specie di Cornus hanno mostrato un’alta inibizione dell’-glucosidasi e una moderata inibizione dell’-amilasi. Di particolare rilievo sono stati i risultati ottenuti con la macerazione idroalcolica di foglie e frutti secchi di C. mas (TDB e MDB), la macerazione in etanolo delle foglie fresche (PF1) e la macerazione idroalcolica delle foglie secche (SD2) di C. sanguinea, che per tale motivo sono stati sottoposti a bio-frazionamento mediante l’impiego della resina XAD-16. Le frazioni PF1 (II) e SD2 (II) del C. sanguinea hanno mostrato la migliore attività antiossidante e di inibizione dell’NF-KB. Conclusioni: Tutti gli estratti investigati hanno mostrato un promettente potenziale antiossidante, ipoglicemizzante ed antinfiammatorio, confermando il positivo contributo delle due classi di composti oggetto di studio, i flavonoidi e iridoidi prospettando il loro potenziale impiego come ricca fonte di composti bioattivi utili per la formulazione di nuovi prodotti per prevenire le malattie associate allo stress ossidativo come malattie infiammatorie e diabete. In particolare, i risultati ottenuti con le frazioni PF1 (II) e SD2 (II) del C. sanguinea incoraggiano i ricercatori nel proseguire lo studio con eventuali studi in vivo. Résumé Objet de l’étude : Les substances naturelles produites par le laboratoire « Nature » continuent de jouer un rôle de premier plan dans le processus de découverte et de développement de nouveaux médicaments, compléments alimentaires et/ou aliments fonctionnels. Aujourd’hui, en effet, nous assistons à un regain d'intérêt pour ces produits pour la prévention et le traitement de nombreuses maladies, notamment celles associées au stress oxydatif, telles que le diabète et les maladies inflammatoires. Le présent travail de recherche vise à identifier les meilleures techniques permettant d’extraire deux classes de principes actifs, les flavonoïdes et les iridoïdes, des fruits et des feuilles d’Arbutus unedo L., Vaccinium corymbosum L., Cornus mas L. et Cornus sanguinea L. Leurs fruits comestibles, comme leurs feuilles pourtant actuellement considérées comme des déchets représentent de riches sources de composés bioactifs. Les travaux ont donc porté sur l'évaluation de l'activité biologique in vitro et le fractionnement bio-guidé des extraits les plus actifs afin d'envisager leur utilisation potentielle comme source de composés bioactifs pour le développement d'aliments fonctionnels, et nutraceutiques. Matériels et méthodes : Le profil phytochimique des extraits a été évalué par LC/ESI/QTOF/MS. Pour la détermination in vitro de la capacité antioxydante, quatre tests différents ont été réalisés (DPPH, ABTS, FRAP et-carotene bleaching test). L’activité hypoglycémique potentielle a été étudiée par inhibition des enzymes -amylase et -glucosidase. Les effets inhibiteurs des extraits sur la production d'oxyde nitrique ont été étudiés par le test de Griess. Les extraits les plus actifs de C. mas et C. sanguinea ont été soumis à un bio-fractionnement pour la séparation des flavonoïdes et des iridoïdes au moyen de la résine XAD-16. Les fractions obtenues ont été testées pour évaluer l'aptitude à réduire l’activation du facteur NF-kB. Résultats : L’analyse par LC/ESI/QTOF/MS détectée pour la première fois dans l’A. unedo la présence d’acide ellagique 4-O--D-glucopiranoside, de kaempférol 3-O-glucoside, de myricétine, de myricétine 3-O-rhamnopyranoside, de naringénine 7-O-glucoside, d'isovitexine 7-O-glucoside et de norbergénine, et V. corymbosum la présence d'iridoïdes scandoside, vaccinoside, géniposide et dihydromonotropeine. La caractérisation chimique complète des fruits et des feuilles de C. sanguinea a été réalisée pour la première fois, en indiquant la présence de flavonoïdes et d'iridoïdes. Il convient de noter les résultats obtenus à partir de l’activité hypoglycémique. Les extraits des deux espèces de Cornus ont montré une forte inhibition de l’-glucosidase et une inhibition modérée de l'-amylase. Résultats intéressants ont été obtenus avec la macération hydro-alcoolique de feuilles et de fruits séchés de C. mas (TDB et MDB), la macération en éthanol des feuilles fraîches (PF1) et la macération hydro-alcoolique de feuilles sèches (SD2) de C. sanguinea qui, pour cette raison, ont été soumis à un fractionnement avec la résine XAD-16. Les fractions PF1 (II) et SD2 (II) de C. sanguinea présentaient la meilleure activité inhibitrice des antioxydants et du NF-kB. Conclusion: Tous les extraits étudiés ont montrés un potentiel antioxydants, hypoglycémiants et anti-inflammatoire, confirmant l’apport positif des deux classes de composés étudiés, les flavonoïdes et les iridoïdes, suggérant leur utilisation potentielle comme source riche de composés bioactifs utiles pour la formulation de nouveaux produits pour prévenir les maladies associées au stress oxydatif, telles que les maladies inflammatoires et le diabète. En particulier, les résultats obtenus avec les fractions PF1 (II) et SD2 (II) de C. sanguinea incitent les chercheurs à poursuivre l’étude en proposant éventuellement des études in vivo.Item Effect of bergamot essential oil and its constituent linalool on myogenic and neuronally-mediated contractions of human and rat isolated colon: potential benefits in complementary treatment of intestinal diseases(Università della Calabria, 2020-07-01) Straface, Marilisa; Andò, Sebastiano; Morrone, LuigiIntroduction Bergamot essential oil (BEO) is used in aromatherapy and as an additive to food and drink to promote a citrus flavour. In animal models, BEO can modulate the synaptic functions within the Central Nervous System. However, it is not known if BEO can affect the functions of the gastrointestinal tract, despite being widely used in the food industry. BEO and its components linalool, limonene and linalyl-acetate were therefore examined for their ability to influence neuromuscular contractions of human and rat isolated colon. Material and Methods Human colon was obtained at surgery for bowel cancer following informed consent; mucosa-free strips were cut parallel to the circular muscle. Rat colon (Sprague-Dawley) strips were also cut as circular muscle preparations. In most experiments, each strip was suspended between platinum wire electrodes in tissue baths containing Krebs solution (5% CO2 in O2; 37°C) under tension (1 or 2g of tension for rat and human muscle strips, respectively) for recording of isometric contractions in response to stimulation of cholinergic nerves using electrical field stimulation (EFS) or to the application of exogenous stimulants of smooth muscle contraction (acetylcholine (ACh), 5-hydroxytryptamine (5-HT), substance P (SP) or KCl). Cumulative concentration-response curves were obtained for BEO (10-6 - 10-3 % v/v) and its major components linalool, limonene and linalyl-acetate (10-9 - 10-4 M). The inhibition of the amplitude of the contractions by each agent was expressed in percentage terms as the mean s.e.m of the numbers of patients or animals. Results In preliminary experiments, BEO and its components reduced contractions of rat colon caused by ACh, 5-HT or SP. Subsequently concentration-dependent inhibition of both KCl-evoked contractions and neuronally-mediated contractions were demonstrated in response to BEO or its components, with greater potency when tested on the latter. The inhibitory effect of BEO on myogenic and neuronally-mediated contractions was associated largely via the actions of linalool (apparent pIC50 5.6 ± 0.4, n= 4 on KCl-evoked contractions; apparent pIC50 6.7 ± 0.2, n = 4 on neuronally-mediated contractions) in human colon. Similar but less potent activity of linalool was obtained in rat colon (apparent pIC50 5.4 ± 0.3, n = 4 on KCl-evoked contractions; apparent pIC50 5.8 ± 0.1 %, n = 4 on neuronally-mediated contractions). Conclusion The results indicated that BEO, largely via the actions of linalool, inhibited both human and rat enteric neurotransmission. Some species differences were found in the ability of these substances to inhibit neuronally-mediated contractions; the rank order in terms of potency (apparent pIC50) in human was: linalool > limonene >> linalyl acetate = BEO, and in rat was: linalyl acetate > limonene = linalool >> BEO. Both BEO and linalool were more potent in human muscle strips, acting at least partly by directly inhibiting muscle contractility. These data provide a potential mechanism for their use as a complementary treatment of gastrointestinal diseases related to increased intestinal motility.Item Evaluation of a PH-sensitive mesoporous silca -based nanosystem as a vehicle for targetedcancer therapy(Università della Calabria, 2020-04-17) Nigro, Alessandra; Andò, Sebastiano; Sisci, DiegoItem Advanced and smart nanocarriers for pharmaceutical applications(Università della Calabria, 2020-04-17) Mazzotta, Elisabetta; Andò, Sebastiano; Muzzalupo, RitaThe application of nanotechnology in drug formulation has made significant headway over the last decade. Nanoscale controlled release systems allow to revalue and reformulate old drugs through targeted delivery to the desired pathological site, improving therapeutic efficiency and reducing side effects. This innovative modality of drug delivery aims to create personalized, safer and effective treatments. A great interest of the academic and industrial research is focused on the design and development of advanced nanocarriers since is a profitable way in terms of costs, times and risks than the discovery of new drugs. Herein, an extensive variety of nanocarriers systems composed of different materials including lipids, polymers and non-ionic surfactants have been proposed for different pharmaceutical applications. Specifically, the carriers have been tailor-made designed to improve drug photostability, enhance skin permeation and realize smart tools for tumor target therapy.Item New perspective for the use of citrus by-productus in food and nutraceutical industries(Università della Calabria, 2020-07-06) Leporini, Mariarosaria; Andò, Sebastiano; Loizzo, Monica RosaAim of the study: Citrus is the most produced tree fruit crop in the world. Almost 33% of the Citrus fruits are industrially processed for juice production, however, a great amount of Citrus wastes including peels, segment membrane and seeds are produced. Referring to the concept of circular economy, the present research project aimed to investigate a model for the reuse of C. × clementina Hort. by-products, in order to propose their utilization for the development of functional drink useful in prevention and treatment of prediabetes condition, hyperglycaemia and hyperlipidaemia. Materials and methods: The chemical profile of C. × clementina Hort. (Clementine di Calabria, PGI) juice and extracts was evaluated using HPLC-DAD and HPLC-UV. For determination of antioxidant activity four different in vitro tests (DPPH, ABTS, -carotene bleaching test and FRAP), were performed. The in vitro hypoglycaemic potential was tested by the inhibition of -amylase and -glucosidase enzymes. The lipase inhibitory activity was also assessed. Results: Juice obtained from fruits collected in Corigliano Calabro (JF) was characterized by a higher content of bioactive compounds with consequent higher bioactivity as confirmed by PCA. For this reason, JF was chosen as a matrix to be enriched with C. × clementina leaves and peel extracts. Hesperidin, tangeritin, sinensetin, and quercetin-O-glucoside were the most abundant constituents in all investigated samples. Considering that the content of coumarins is strictly regulated in foods, the absence of these phytochemicals represents an additional value for the potential industrial application of our samples. Among applied extraction techniques, ultrasound-assisted maceration resulted the most promising procedure to obtain extracts characterized by the highest of bioactivity in terms of antioxidant and enzymes inhibition. Indeed, leaves (from Corigliano Calabro) hydroalcoholic ultrasound-assisted maceration and peels (from Cetraro) ethanol ultrasound-assisted maceration were chosen as phytocomplex to be added to JF at different concentrations (20, 15, 10, and 5% w/v of extracts). Generally, the increase in phytochemicals content and bioactivities were significantly higher in juices enriched with leaves and leaves + peels (1:1) extracts. In order to estimate the effect of pasteurization process on our functional drink we have decided to pasteurize the 20% w/v enriched juice samples. As expected, the process decreased the bioactivity of samples in comparison to unpasteurized juice. Nevertheless, enrichment with Citrus by-products, especially with leaves extract, resulted in an increase of total phytochemicals content and bioactivity compared to the pasteurized control juice. Conclusion: On the basis of obtained data regarding the interesting bioactivity of enriched juices and comforted by sensory analysis data we can propose C. × clementina by-products as a promising source of bioactive compounds useful for formulation of functional drink or foods for preventing diseases associated to oxidative stress with particular reference to hyperglycaemia and hyperlipidaemia conditions. Moreover, the high antioxidant potential of these extract allows us to propose their use as food additives able to extent the shelf-life of different foods products. The obtained data encourage researchers to continue their work to assess the impact of functional juice on pathological models, and evaluated its bioaccessibility, bioavailability as well as possibility of hypovitaminoses due to fat reduction.Item Synthetic strategies towards short-chain peptides for potential biomedical applications and tumor-targeted mesoporous silica-based drug delivery systems development(Università della Calabria, 2020-04-17) Comandè, Alessandra; Andò, Sebastiano; Leggio, AntonellaMy PhD research project was focused on the design and development of short-chain peptides with biomedical applications and on the identification of new peptide synthesis strategies. Briefly, the specific objectives of my research work were the following: · Design and synthesis of novel methylated tetrapeptides acting as leptin antagonists with potentially improved stability and pharmacokinetic properties compared to the non-methylated analogue leptin antagonist LDFI. · Identification and synthesis of a novel peptide ligand able to recognize CD38, a marker overexpressed on multiple myeloma (MM) cells, for the targeted delivery of bortezomib, the drug of choice for this type of cancer. · Design and synthesis of new peptide entities to use as inhibitors linked to chelators to produce new molecular probes for prostate cancer imaging via PET. · Identification of new and efficient peptide synthesis strategies aimed at obtaining small peptides in the solution phase. The developed strategies can prove useful for obtaining biologically active peptides. · Development of a targeted MSN-based nanocarrier bearing the anticancer drug doxorubicin and grafted, on the external surface, with folic acid (FOL-MSN-DOXO). The system developed in this study provides an attractive template to develop more selective DOXO delivery systems by using peptides as targeting ligands. The present thesis consists of published work (papers) and work in progress (ongoing works) that must still be completed in order to be published in the future. Peptide sequences and their structural characterizations have been omitted in ongoing work 1 and in ongoing work 2 since a patent application is going to be filed for both products.