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

The 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

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

This 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

Design, Characterization, and In Vitro Assays on Muscle Cells of Endocannabinoid-like Molecule Loaded Lipid Nanoparticles for a Therapeutic Anti-Inflammatory Approach to Sarcopenia

Inflammatory processes play a key role in the pathogenesis of sarcopenia owing to their effects on the balance between muscle protein breakdown and synthesis. Palmitoylethanolamide (PEA), an endocannabinoid-like molecule, has been well documented for its anti-inflammatory properties, suggesting its possible beneficial use to counteract sarcopenia. The promising therapeutic effects of PEA are, however, impaired by its poor bioavailability. In order to overcome this limitation, the present study focused on the encapsulation of PEA in solid lipid nanoparticles (PEA-SLNs) in a perspective of a systemic administration. PEA-SLNs were characterized for their physico-chemical properties as well as cytotoxicity and cell internalization capacity on C2C12 myoblast cells. Their size was approximately 250 nm and the encapsulation efficiency reached 90%. Differential scanning calorimetry analyses demonstrated the amorphous state of PEA in the inner SLN matrix, which improved PEA dissolution, as observed in the in vitro assays. Despite the high internalization capacity observed with the flow cytometer (values between 85 and 94% after 14 h of incubation), the Nile Red labeled PEA-SLNs showed practically no toxicity towards myoblasts. Confocal analysis showed the presence of SLNs in the cytoplasm and not in the nucleus. These results suggest the potentiality provided by PEA-SLNs to obtain an innovative and side-effect-free tool in the medical treatment of sarcopeni

Self-Assembled Lipid Nanoparticles for Oral Delivery of Heparin-Coated Iron Oxide Nanoparticles for Theranostic Purposes

Recently, solid lipid nanoparticles (SLNs) have attracted increasing attention owing to their potential as an oral delivery system, promoting intestinal absorption in the lymphatic circulation which plays a role in disseminating metastatic cancer cells and infectious agents throughout the body. SLN features can be exploited for the oral delivery of theranostics. Therefore, the aim of this work was to design and characterise self-assembled lipid nanoparticles (SALNs) to encapsulate and stabilise iron oxide nanoparticles non-covalently coated with heparin (Fe@hepa) as a model of a theranostic tool. SALNs were characterised for physico-chemical properties (particle size, surface charge, encapsulation efficiency, in vitro stability, and heparin leakage), as well as in vitro cytotoxicity by methyl thiazole tetrazolium (MTT) assay and cell internalisation in CaCo-2, a cell line model used as an indirect indication of intestinal lymphatic absorption. SALNs of about 180 nm, which are stable in suspension and have a high encapsulation efficiency (>90%) were obtained. SALNs were able to stabilise the heparin coating of Fe@hepa, which are typically unstable in physiological environments. Moreover, SALNs-Fe@hepa showed no cytotoxicity, although their ability to be internalised into CaCo-2 cells was highlighted by confocal microscopy analysis. Therefore, the results indicated that SALNs can be considered as a promising tool to orally deliver theranostic Fe@hepa into the lymphatic circulation, although further in vivo studies are needed to comprehend further potential applications

Characterization of natural clays from italian deposits with focus on elemental composition and exchange estimated by edx analysis: potential pharmaceutical and cosmetic uses

Purification processes performed on natural clays to select specific clay minerals are complex and expensive and can lead to over-exploitation of some deposits. The present study aimed to examine physicochemical (mineralogy, morphology, size, surface charge, chemical composition, cation exchange capacity [CEC], and pH) and hydration (swelling, wettability, water sorption, and rheological behavior) properties of three native clays from Italian deposits for potential pharmaceutical and cosmetic uses due to the presence of phyllosilicate minerals. Particular emphasis was placed on energy dispersive X-ray (EDX) microanalysis coupled with the ‘cesium method’ to assay clay elemental composition and CEC. One bentonite of volcanic origin (BNT) and two kaolins, one of hydrothermal origin (K-H) and another of lacustrine-fluvial origin (K-L), were evaluated in comparison with a commercial, purified bentonite. The CEC assay revealed the complete substitution of exchangeable cations (Na+ and Ca2+) by Cs+ in BNT samples and CEC values consistent with those of typical smectites (100.64 7.33 meq/100 g). For kaolins, partial substitution of Na+ cations occurred only in the K-L samples because of the interstratified mineral component which has small CEC values (11.13 5.46 meq/100 g for the K-H sample and 14.75 6.58 meq/100 g for the K-L sample). The degree of isomorphous substitution of Al3+ by Mg2+ affected the hydration properties of BNT in terms of swelling, water sorption, and rheology, whereas both of the poorly expandable kaolins exhibited significant water-adsorption properties. The EDX microanalysis has proved to be of considerable interest in terms of providing more information about clay properties in comparison with other commonly used methods and to identify the role played by both chemical and mineralogical composition of natural clays for their appropriate use in pharmaceutical and cosmetic fields

In Vivo Biodistribution of Respirable Solid Lipid Nanoparticles Surface-Decorated with a Mannose-Based Surfactant: A Promising Tool for Pulmonary Tuberculosis Treatment?

The active targeting to alveolar macrophages (AM) is an attractive strategy to improve the therapeutic efficacy of ‘old’ drugs currently used in clinical practice for the treatment of pulmonary tuberculosis. Previous studies highlighted the ability of respirable solid lipid nanoparticle assemblies (SLNas), loaded with rifampicin (RIF) and functionalized with a novel synthesized mannose-based surfactant (MS), both alone and in a blend with sodium taurocholate, to efficiently target the AM via mannose receptor-mediated mechanism. Here, we present the in vivo biodistribution of these mannosylated SLNas, in comparison with the behavior of both non-functionalized SLNas and bare RIF. SLNas biodistribution was assessed, after intratracheal instillation in mice, by whole-body real-time fluorescence imaging in living animals and RIF quantification in excised organs and plasma. Additionally, SLNas cell uptake was determined by using fluorescence microscopy on AM from bronchoalveolar lavage fluid and alveolar epithelium from lung dissections. Finally, histopathological evaluation was performed on lungs 24 h after administration. SLNas functionalized with MS alone generated the highest retention in lungs associated with a poor spreading in extra-pulmonary regions. This effect could be probably due to a greater AM phagocytosis with respect to SLNas devoid of mannose on their surface. The results obtained pointed out the unique ability of the nanoparticle surface decoration to provide a potential more efficient treatment restricted to the lungs where the primary tuberculosis infection is located

The Impact of Lipid Corona on Rifampicin Intramacrophagic Transport Using Inhaled Solid Lipid Nanoparticles Surface-Decorated with a Mannosylated Surfactant

The mimicking of physiological conditions is crucial for the success of accurate in vitro studies. For inhaled nanoparticles, which are designed for being deposited on alveolar epithelium and taken up by macrophages, it is relevant to investigate the interactions with pulmonary surfactant lining alveoli. As a matter of fact, the formation of a lipid corona layer around the nanoparticles could modulate the cell internalization and the fate of the transported drugs. Based on this concept, the present research focused on the interactions between pulmonary surfactant and Solid Lipid Nanoparticle assemblies (SLNas), loaded with rifampicin, an anti-tuberculosis drug. SLNas were functionalized with a synthesized mannosylated surfactant, both alone and in a blend with sodium taurocholate, to achieve an active targeting to mannose receptors present on alveolar macrophages (AM). Physico-chemical properties of the mannosylated SLNas satisfied the requirements relative to suitable respirability, drug payload, and AM active targeting. Our studies have shown that a lipid corona is formed around SLNas in the presence of Curosurf, a commercial substitute of the natural pulmonary surfactant. The lipid corona promoted an additional resistance to the drug diffusion for SLNas functionalized with the mannosylated surfactant and this improved drug retention within SLNas before AM phagocytosis takes place. Moreover, lipid corona formation did not modify the role of nanoparticle mannosylation towards the specific receptors on MH-S cell membrane

Short- and long-term effects of essential oils on swine spermatozoa during liquid phase refrigeration

: The application of essential oils as potential alternatives to antibiotics in swine semen storage is promising, due to their antioxidant and antibacterial properties. However, detrimental effects on spermatozoa should be clarified first. The aim of this study was to evaluate 9 essential oils (EOs; Satureja montana, Pelargonium graveolens, Cymbopogon nardus, Melaleuca leucadendron, Eucaliptus globulus, Citrus limon, Lavandula angustifolia, Lavandula hybrida, Mentha piperita) and a blend (GL mix) on key morpho-functional parameters of swine spermatozoa. Test compounds were firstly chemo-characterized and experimental doses were prepared by suspending a fixed number of spermatozoa with 3 different concentrations (0.1, 0.5, 1 mg/mL) of EOs. Experimental doses were stored at 16 °C and sampled after 3 and 120 h for analysis. Overall, S. montana, P. graveolens and L. angustifolia EOs induced the strongest alterations, with C. nardus and E. globulus EOs being the best tolerated. Swine spermatozoa represent a good preliminary testing platform to screen toxicity and its different patterns. The comprehensive overview on the potential mechanisms of action of some of the most common EOs, despite of the direct aim of the study being swine reproduction, may be exploited in other fields of research within both veterinary and human medicine

PROGETTAZIONE E SVILUPPO DI SISTEMI DI VEICOLAZIONE NANOPARTICELLARI A BASE DI LIPIDI E OLIGOSACCARIDI PER LA SOMMINISTRAZIONE DI COMPOSTI ATTIVI

I 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