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PROGETTAZIONE E SVILUPPO DI SISTEMI DI VEICOLAZIONE NANOPARTICELLARI A BASE DI LIPIDI E OLIGOSACCARIDI PER LA SOMMINISTRAZIONE DI COMPOSTI ATTIVI
No full textI sistemi di veicolazione del farmaco sono largamente studiati come tra i più efficienti metodi per migliorare l’efficacia dei farmaci. Negli ultimi vent’anni un’enorme attenzione è stata focalizzata sui sistemi nanometrici di veicolazione che sono in grado di interagire selettivamente con organismi patogeni, cellule o tessuti. Tra i gli eccipienti utilizzabili nella preparazione di questi sistemi, lipidi e oligosaccaridi mostrano una elevata biocompatibilità, biodegradabilità e idoneità per la somministrazione di farmaci attraverso varie vie. Lo scopo della tesi è stato lo sviluppo di sistemi di veicolazione nanometrici progettati specificatamente per aumentare l’efficacia e il direzionamento di determinati composti attivi.
Nel primo progetto nanoparticelle lipidiche in grado di auto-assemblarsi (SALNs) sono state sviluppate per incorporare a loro volta nanoparticelle di ossido di ferro ricoperte da eparina (Fe@hepa) al fine di ottenere un sistema “teranostico” per via orale assorbibile mediante la circolazione linfatica. Le SALNs sono state caratterizzate e testate in vitro su modelli cellulari (CaCo-2) di assorbimento intestinale. I risultati hanno dimostrato la capacità delle SALNs di veicolare le Fe@hepa in cellule CaCo-2 senza indurre tossicità.
Nel secondo progetto, sono stati sviluppati liposomi co-caricati con due farmaci anti-tubercolosi di prima scelta, isoniazide (INH) e rifampicina (RIF) somministrabili per via inalatoria. I liposomi sono stati caratterizzati mediante la tecnica di scattering di neutroni a piccolo angolo (SANS). Le analisi hanno evidenziato che il co-caricamento di RIF e INH induce una stabilizzazione sulla struttura dei liposomi, confermata con l’aumento del loro caricamento.
Nel contesto della tubercolosi polmonare, anche nanoparticelle solido-lipidiche in cluster (SLNas) sono state sviluppate, caratterizzate e somministrate in vivo su topi. SLNas sono state preparate mediante un tensioattivo mannosilato di neo-sintesi (SLNas/MS) per il direzionamento attivo ai macrofagi alveolari (AM). Dopo la somministrazione inalatoria, SLNas/MS hanno dimostrato di raggiungere gli alveoli e di localizzarsi nei polmoni senza diffondersi nel resto del corpo. Inoltre, è stata dimostrata l’internalizzazione delle particelle da parte dei AM (raccolti dopo il trattamento) mediante microscopia a fluorescenza. Tutti i risultati suggeriscono la reale capacità delle SLNas di agire sui AM.
Nel terzo progetto, sono state studiate due strategie basate su sistemi nanometrici per una efficiente e sicura veicolazione di geraniolo (GER) per il trattamento del Morbo di Parkinson mediante la via “nose-to-brain”. Nella prima strategia nanoparticelle polimeriche(NP) e lipidiche (SLN) sono state preparate, liofilizzate per aumentarne la stabilità e ne è stato valutato il contenuto in GER-. I risultati hanno indicato che, durante la liofilizzazione, il GER non è trattenuto dalle particelle, probabilmente a causa della sua volatilità. Pertanto NP e SLN cariche del coniugato GER-acido ursodesossicolico (GER-UDCA, profarmaco di GER) sono state sviluppate e caratterizzate in termini di contenuto, rilascio in vitro, morfologia, e infine somministrate in vivo. I risultati hanno dimostrato che le SLN garantiscono alte concentrazioni fino a 3 ore del profarmaco nel cervello, senza recare nessun danno alla mucosa nasale. Per la seconda strategia, complessi di inclusione tra GER e ciclodestrine (GER-CD) sono stati preparati usando la 2-hydroxypropyl-β-CD (HPβCD) e la β-CD. I complessi GER-CD sono stati caratterizzati e i risultati hanno dimostrato la reale inclusione di GER nella cavità delle CD. La somministrazione in vivo dei complessi è attualmente in corso.Drug delivery systems (DDS) are widely investigated as one of the main tools in medicine due to their potential to treat diseases. During the last two decades, great attention has been focused on nanostructured DDS able to selectively interact with pathogens, cells or tissues. Among all the exploitable materials in formulating DDS, lipids and oligosaccharides exhibit high biocompatibility, biodegradability, and suitability for the administration of drugs through several routes. The aim of this thesis was the development of specific nanostructured DDS designed to enhance efficacy and targeting of active compounds.
In the first project, self-assembled lipid nanoparticles (SALNs) were developed for the encapsulation of heparin-coated iron oxide nanoparticles (Fe@hepa) in order to obtain a nanotheranostic tool able to be absorbed orally through the lymphatic route. SALNs were fully characterized and tested in vitro on cell models (CaCo-2 cell line) for intestinal absorption. The results demonstrated the suitability of SALNs in efficiently delivering Fe@hepa into CaCo-2 cells without causing cytotoxicity.
In the second project, co-loaded liposomes with two first-line antituberculosis drugs, isoniazid (INH) and rifampicin (RIF), were developed for inhaled therapy. Liposomes were characterized in-depth by small-angle neutron scattering technique (SANS). The analysis highlighted that the RIF-INH co-loading elicited a stabilizing effect on the liposome structure, confirmed by the increment of the drug loading capacity.
In a pulmonary tuberculosis context, RIF-loaded solid lipid nanoparticles assemblies (SLNas) were also developed, fully characterized in vitro and administered in vivo on mice. SLNas were formulated with the employment of a newly synthesized mannosylated surfactant (SLNas/MS) for the active targeting to the alveolar macrophages (AM). After administration, SLNas/MS demonstrated the ability to reach the alveolar region and to be retained in the lungs without broad distribution in the body. Furthermore, fluorescence microscopy analysis was performed on AM (collected after the treatment) showing cell internalization of the particles. All the results suggested the suitability of SLNas/MS in efficiently targeting AM.
In the third project, two different strategies based on nanostructured DDS were investigated for an efficient and safe delivery of Geraniol (GER) via nose-to-brain for the treatment of Parkinson’s Disease. In the first strategy, polymeric (NP) and lipid-based (SLN) nanoparticles were prepared. In order to obtain long-term stable formulations, the samples were freeze-dried and characterized regarding GER loading. The results indicated that no GER was retained in the nanoparticles, probably due to its volatility during the freeze-drying process. Therefore, GER-ursodeoxycholic acid conjugate (GER-UDCA, a GER prodrug) was used instead of GER. NP and SLN were developed, characterized regarding drug content, in vitro release and morphology, and finally administered in vivo. The results demonstrated the suitability of GER-UDCA-loaded SLN for the in vivo administration, which guaranteed high concentrations of the prodrug up to 3 hours in the brain without causing any damage to the nasal mucosa.
For the second strategy, inclusion complexes between GER and cyclodextrins (CD) were prepared by using 2-hydroxypropyl-β-CD (HP-βCD) and β-CD. The inclusion complexes were characterized in-depth and the results confirmed the real inclusion of GER into CD cavities. In vivo administration of both the inclusion complexes will be further investigated
Efficacy and Synergistic Potential of Cinnamon (Cinnamomum zeylanicum) and Clove (Syzygium aromaticum L. Merr. & Perry) Essential Oils to Control Food-Borne Pathogens in Fresh-Cut Fruits
Full text linkThe presence of microbial pathogens in ready-to-eat produce represents a serious health problem. The antibacterial activity of cinnamon (Cinnamomum zeylanicum) and clove (Syzygium aromaticum L. Merr. & Perry) essential oils (EOs) was determined toward food-borne pathogens by agar disk diffusion and minimum inhibitory concentration (MIC) assays. The growth kinetics of all strains, both in a buffer suspension assay and “on food” in artificially contaminated samples, were also investigated. The two EOs demonstrated a good antibacterial effect both alone and in combination (EO/EO). The use of EO/EO led to a synergistic antibacterial effect, also confirmed by the growth kinetics studies, where the EOs were active after 10 h of incubation (p < 0.0001) at significantly lower concentrations than those when alone. In the “on food” studies performed on artificially contaminated fruit samples stored at 4 °C for 8 days, the greatest killing activity was observed at the end of the trial (8 days) with a reduction of up to 7 log CFU/g compared to the control. These results confirm the good antibacterial activity of the EOs, which were more effective when used in combination. Data from the "on food" studies suggest cinnamon and clove essential oils, traditionally used in the food industry, as a possible natural alternative to chemical additives.
Keywords: cinnamon and clove essential oils; synergistic activity; food-borne pathogens; fresh-cut fruit; bio-preservatio
Effects of Melaleuca alternifolia Chell (Tea Tree) and Eucalyptus globulus Labill. Essential Oils on Antibiotic-Resistant Bacterial Biofilms
Full text linkIn the present investigation, the anti-biofilm potential of two essential oils (EOs), Melaleuca alternifolia Chell (Tea-Tree) (TTO) and Eucalyptus globulus Labill. (EEO) was characterized and tested “in vitro” against both mature biofilms and biofilms in the process of formation, produced by strains belonging to three main categories of antibiotic resistant bacteria (ARB): Vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA) and broad-spectrum β-lactamase-producing Escherichia coli (ESBL). The study was carried out in 96-well microtiter-plates using EOs alone, in association with each other and in combination with antibiotics against both single and multi-species biofilm. The study demonstrated the ability of TTO and EEO to counteract the ARB strains in sessile form, with promising results in particular against the biofilm in formation. Mature biofilm by ESBL E. coli was the most sensitive in the results from the quantification study of viable cells performed in multi-species biofilms. Lastly, in all tests, carried out using TTO/EEO associations and EOs/antibiotic combinations, the synergistic effect which emerged from the FIC-index has been confirmed, and both the reduction of biofilm in formation, and the removal of mature structure was obtained at very low concentrations, with values from 4 to >512-fold lower than the minimum inhibitory concentration (MIC) of the single compounds
Multivariate Statistical Models for the Authentication of Traditional Balsamic Vinegar of Modena and Balsamic Vinegar of Modena on 1H-NMR Data: Comparison of Targeted and Untargeted Approaches
Full text linkThis work aimed to compare targeted and untargeted approaches based on NMR data for the construction of classification models for Traditional Balsamic Vinegar of Modena (TBVM) and Balsamic Vinegar of Modena (BVM). Their complexity in terms of composition makes the authentication of these products difficult, which requires the employment of several time-consuming analytical methods. Here, 1H-NMR spectroscopy was selected as the analytical method for the analysis of TVBM and BVM due to its rapidity and efficacy in food authentication. 1H-NMR spectra of old (>12 years) and extra-old (>25 years) TVBM and BVM (>60 days) and aged (>3 years) BVM were acquired, and targeted and untargeted approaches were used for building unsupervised and supervised multivariate statistical modes. Targeted and untargeted approaches were based on quantitative results of peculiar compounds present in vinegar obtained through qNMR, and all spectral variables, respectively. Several classification models were employed, and linear discriminant analysis (LDA) demonstrated sensitivity and specificity percentages higher than 85% for both approaches. The most important discriminating variables were glucose, fructose, and 5-hydroxymethylfurfural. The untargeted approach proved to be the most promising strategy for the construction of LDA models of authentication for TVBM and BVM due to its easier applicability, rapidity, and slightly higher predictive performance. The proposed method for authenticating TBVM and BVM could be employed by Italian producers for safeguarding their valuable products
SURFACE ENGINEERING OF SOLID LIPID NANOASSEMBLIES FOR INHALED INTRAMACROPHAGIC ANTI-TB THERAPY
No full textFor an inhaled tuberculosis (TB) treatment, antibiotic aerosolization has to be produced by using drugs in their solid state administered by means of Dry Powder Inhaler (DPI) devices. In this regard, untreated drugs generally fail to reach alveolar epithelium and penetrate alveolar macrophages (AM) as the primary site of the infection.1 Therefore, the urgency to treat TB disease effectively may be addressed with approaches consisting of micro- or nanoparticulate carriers redeveloping existing drugs to reach the intended goal.2, 3 Specific modifications of the particulate carrier surface by conjugation with molecules that can specifically bind the receptors (active targeting) are expected to boost the particle avidity to cells increasing accumulation and intracellular uptake. Macrophages possess mannose-specific membrane receptors (MR) that can recognize and facilitate the internalization of carriers bearing mannose residues. In particular, the infected AM have an overexpression of MR.4 In the present study, surface engineered Solid Lipid Nanoparticle assemblies (SLNas) were developed as potential carriers of rifampicin, a first choice antitubercular drug, intended to maximize drug concentration at the primary site of TB infection. To increase specificity for macrophages and internalization potential, SLNas surface was functionalized by a mannosylated derivative to induce AM active targeting. Biocompatible lipid components such as fatty acids and their derivatives, diglycerides and triglycerides were processed by means of the melt emulsifying technique using biocompatible surfactants (sodium taurocholate and methyl mannopyranoside). Mannosylated SLNas were examined for their intrinsic properties (size and size distribution, shape, surface charge, bulk and tap density, aerodynamic diameter, porosity, flowability, physical state of the components). Powder breathability in terms of Emitted Dose and Fine Particle Fraction was assayed by Next Generation Impactor (NGI). This information on powder interparticle adhesion and deaggregation ability influencing powder dispersion and deposition onto alveolar epithelia. SLNas mannosylation was investigated by means of X-ray Photoelectron Spectroscopy for Chemical Analysis and Energy Dispersive X-ray Analysis. Prototypes of SLNas in terms of successful functionalization, optimal breathability and chemico-physical stability, were examined for cytotoxicity by MTT test on murine macrophages J774 cell lines
Medicinal Chemistry drives drug targeted delivery: a successful interplay
No full textTargeted drug delivery is object of an intense research. A medicinal chemistry approach allowed us to modify an opioid peptide in order to remove the opioid activity and retain the ability to cross the blood-brain barrier. Polyester-based nanoparticles (Np) surface-decorated with this peptide were shown to be able to deliver loperamide (a model drug) into CNS, as well as cholesterol (for the treatment of Huntington’s disease) and albumin (a model of a cargo protein). With a view of clinical translation, however, polyester Np seems to be not well suited for CNS diseases. Thus, we decided to use our peptide in alternative ways other than as a conjugate with the carboxyl group of the polyester PLGA, starting material for the production of the Np. We develop a new synthetic procedure that allow the conjugation of the peptide with substrates containing hydroxyl groups, less reactive than the carboxy group of the polyester (we considered as a substrate the poly(vinyl alcohol)), as well as a linker that could be inserted between a cargo and the peptide targeting moiety. At the same time, we moved towards lipidic carriers, a kind of delivery agents already clinically available. To gain experience on these carriers, we designed rifampicin-loaded Solid Lipid Nanoparticle assemblies (SLNas), for a direct intramacrophagic antitubercular therapy using Dry Powder Inhaler (DPI) devices and, in the first instance, methyl mannopyranoside as targeting ligand, able to interact with mannose receptors present on macrophages. Results obtained will be presented and discussed
Novel application of 1H NMR spectroscopy coupled with chemometrics for the authentication of dark chocolate
No full textThe applicability of 1H NMR spectroscopy coupled with chemometric in the quality control of dark chocolate was investigated for the first time to detect cocoa-butter equivalents (CBEs) above the allowed limit by European regulation. Blends of chocolate-fats with CBEs in the range 0-50 % were prepared and analyzed by 1H NMR spectroscopy. Datasets composed of peaks' areas or spectral variables (fingerprinting) in glycerol region were tested for the creation of multivariate statistical models. Partial least-squares discriminant analysis (PLS-DA) and regression (PLS-R) methods were used to correctly identify the type of CBE and quantify its concentration respectively. The performances of the models created on the two datasets were evaluated in terms of chemo-metric indicators and compared. The robustness of models was investigated through the analysis of test sets and random permutation tests. Fingerprinting models revealed fruitful results in classifying and quantifying CBEs in blends demonstrating the applicability of NMR in chocolate quality control
Ulvan as a source of oligosaccharides for biological applications: enzymatic hydrolysis in a biocompatible medium
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