POLYMERIC NANOSTRUCTURED FORMULATIONS FOR TARGETED ADMINISTRATION OF BIOACTIVE AGENTS

Chemotherapy is an effective treatment for cancer and other serious diseases such as cardiovascular restenosis and AIDS. It is a complicated procedure in which many factors are involved in determining its success or failure. Furthermore it carries a high risk due to drug toxicity, with the more effective drugs tending to be more toxic. Patients have to tolerate severe side effects and sacrifice their quality of life. The inefficiency and side effects of chemotherapy are caused mainly by the formulation, pharmacokinetics and toxicity of chemotherapeutic agents and the drug resistance. Hence, the development of effective carriers for both existing and newly developed drugs may be as important as the discovery of new anticancer drugs. All these concerns lead to the concept of sustained, controlled and targeted release of drugs. The ideal goal of a drug delivery system is to deliver high efficacy drug(s) at the right time to the desired location, at high concentration but safe enough, over a sufficiently long period. Effective drug delivery has also important implications from a market viewpoint. More effective drug delivery can provide drug companies with a means of expanding market share or of revitalizing therapeutics with previously unrealized potential because of poor pharmacokinetic profiles. Nanomedicine is not only important from the social and welfare aspects, but also for its economic potential. In the framework of a long–standing research activity ongoing in the Bioactive Polymeric Materials Group at the Department of Chemistry and Industrial Chemistry of the University of Pisa, the present work was aimed at the development of polymeric nanoparticles for the site specific and controlled delivery of drugs for cancer treatment and tissue engineering application. The primary objective of the present PhD was the advancement of knowledge and the theoretical understanding of the relations among variables that play important roles in the development and optimization of procedures for the preparation of polymeric nanoparticle suspensions. The work was conducted keeping in mind the practical aspects of preparing nanodelivery structures for bioactive agents administration in cancer therapy and tissue engineering applications. A variety of polymers including PLGA, PHB, VAM41, multi block copolymers of PCL-PLU, and Chitosan, were investigated. Retinoic Acid and Chondroitin Sulphate, were loaded into nanoparticles as hydrophobic and hydrophilic model drugs respectively. Proteins have been also co-encapsulated as stabilizing agent or as model proteic drugs. For the preparation of RA loaded nanoparticles several methods were investigated and set up: dialysis, nanoprecipitation, co-precipitation and colloidal-coating. Colloidal-coating is an original solvent-free method developed in the framework of the research activity carried out in the present PhD thesis. The first two methods were employed for the preparation of formulations based on PLGA, PHB and the multi block copolymer PCL-co-PLU, while the last two for formulations based on VAM41. The resulting nanoparticle suspensions were analyzed macroscopically in terms of homogeneity and formation of precipitates, and characterized from a dimensional and morphological point of view by means of light scattering (LS) and scanning electron microscopy (SEM). Particles were purified and storage modalities were set up. Drug release studies were carried out by an original method that might better reflect bioavailability data. Since HSA is one of the most abundant plasma protein it was added to the releasing medium overcoming problems related to the poor solubility of RA in water. Solubility problems often hamper the evaluation of drugs release kinetics of hydrophobic drugs in phosphate buffer solution (PBS) at 37°C, which is commonly used in in vitro environment and that should match the in vivo condition. The obtained results showed that HSA affected the stability and life of nanoparticle formulations. The activity of RA encapsulated by colloidal-coating method was evaluated on SK-N-SH human neuroblastoma cell line that is known to undergo inhibition of proliferation and neuronal differentiation upon treatment with RA. The data obtained suggested that the anticancer activity of RA was not impaired by incorporation and purification processes. A careful in vitro investigation of PHB and the relative nanoparticles cytotoxicity was carried out using two type of assays aimed at the evaluation of the interactions of the materials with cell metabolism (WST-1 tetrazolium salts) and cell endocytosis functions (Neutral Red Uptake). In vitro cytotoxicity of PHB resulted fairly low as highlighted by the high IC50 values obtained both with Tetrazolium Salts and Neutral Red assays. PHB based nanoparticles prepared by dialysis method exhibited as well a very good cytocompatibility and can be considered fully biocompatible It was also investigated the preparation of suitable vehicles for intra-articular injection in regenerated cartilages tissue, in form of nanoparticles, to allow Chondroitin Sulphate sustained release. Synthetic bioerodible (VAM41), biodegradable (PLGA), natural (Chitosan) polymers and nanoprecipitation or co-precipitation methods were used to develop the nanostructured formulations. The limit of nanoprecipitation method mainly concerns the possibility to achieve an efficiently loading of hydrophilic drug substances inside the nanoparticles. Drug is easily lost in water during the preparation and incompatibilities between the polymer and the drug could worse the situation. Due to the wide differences among the utilized polymers the nanoprecipitation and co-precipitation methods were adjusted and modified in relation to the physical-chemical characteristics of the utilized material. PLGA, VAM 41 and Chitosan nanoparticles were prepared. No detectable amounts of CS were loaded onto the PLGA nanoparticles indicating the not suitability of the method for the encapsulation of this drug or a strong incompatibility between the hydrophobic polymer and hydrophilic drug. CS was detected into VAM41 and Chitosan NPs. A quantitative determination of the drug encapsulation was not possible due to several occurrences. Spectrophotometric UV assays have been based on changes in the absorption spectrum of the dye 1,9-dimethylmethylene blue (DMMB) when bound to Glycosaminoglycans (GAGs). CS interacted somehow with VAM41 and the characteristic UV band of absorbance of DMMB was shifted to higher wavelengths. Another attempt to evaluate encapsulation was done by IR spectrophotometry. The ratio between the intensity of the peak characteristic for CS and the intensity of the peak characteristic for VAM41 was evaluated and related to the percentage of CS in the samples. Although the developed method was effective for evaluating the encapsulation of CS it was not possible to verify whether the observed interaction affected these results or not. Chitosan nanoparticles were insoluble in all the tested solvents. Hence, an approximate evaluation of CS content was carried out by XPS analysis. The presence of sulphur in the sample qualitatively confirmed the encapsulation of CS into the NPs. The reported results and relevant discussions, which give exhaustive theoretical understanding of the phenomena occurring during colloidal formations, contribute to both basic and applied research in biomedical science and pharmaceutical technology

Poly(hydroxyalkanoates)-Based Polymeric Nanoparticles for Drug Delivery

Poly (hydroxyalkanoates) (PHAs) have recently attracted a great deal of academic and industrial interest for their biodegradability and biocompatibility making them suitable for environmental and biomedical applications. Poly(3-hydroxybutyrate-) (PHB-) and Poly(DL-lactide-co-glycolide) (PLGA-) based nanoparticles were prepared using the dialysis method as yet unreported for the preparation of nanoparticles based on PHB. Processing conditions were varied in order to evaluate their influence on morphology, drug encapsulation, and size of nanoparticles. The relevant results obtained give a theoretical understanding of the phenomenon occurring during colloidal formation. The adopted procedure allows for a relatively small diameter and homogeneity in size distribution of the PHB nanoparticles to be obtained compared to other methods like the one based on solvent evaporation which leads to particles on microscale. The biocompatibility of PHB and relative nanoparticles was investigated and both exhibited very good cytocompatibility

Modern and ancient migrants’ narratives through ELF. An Experiential-Linguistic project in Responsible Tourism

Abstract – This article explores the emotional experience of Italian seaside resorts whose geographical position in the Southern Mediterranean coasts has always determined their destiny as places of hospitality and hybridization of languages and cultures. A Cognitive-pragmatic Model of Experiential Linguistics (Lakoff, Johnson 1999; Langacker 1991; Sweetser 1990) and some strategies of Experiential Place Marketing (Hosany, Prayag 2011; Jani, Han 2013; Prayag et al. 2013) will be employed to ‘emotionally promote’ Responsible Tourism (Lin et al. 2014; Ma et al. 2013) in order to enquire into the effects of emotions upon the tourists’ experience of the holiday as a path towards their ‘personal and cultural growth’. The case study illustrated in this article represents precisely an instance of ELF communication developing from tourists’ and migrants’ appraisal of: (a) the contemporary non-Western migrants’ dramatic sea-voyage narratives reported in their ELF variations (Guido 2008, 2012), and (b) the epic narratives of Mediterranean ‘odysseys’ towards ‘utopian places’ belonging to the Western cultural heritage, translated from Ancient Greek and Latin into ELF. The subjects of this case study under analysis are tourists playing the role of ‘intercultural mediators’ with migrants in one of the seaside resorts of Salento affected by migrant arrivals. To facilitate tourists’ and migrants’ processes of ‘experiential embodiment’ of past and present dramatic sea voyages, they will be introduced to an ‘Ethnopoetic analysis’ (Hymes 1994, 2003) of two corpora of modern and ancient oral journey narratives – the former collected during ethnographic fieldworks in reception centres for refugees, and the latter including extracts from Homer’s Odyssey and Virgil’s Aeneid. The purpose is to make tourists and migrants play the roles of ‘philologists’ and ‘ethnographers’ as they realize how such ancient and modern oral narratives are experientially organized into spontaneous ‘verse structures’ reproducing the sequences and rhythms of human actions and emotions in response to the traumatic experience of violent natural phenomena which, through the use of ergative syntactic structures (Talmy 1988), become metaphorically personified as objects and elements endowed with an autonomous, dynamic force capable of destroying the human beings at their mercy. The Ethnopoetic analysis and translation, together with the subsequent multimodal rendering of such journey narratives into ‘premotional videos’ for place-marketing purposes (Kress 2009), aim at making both tourists and migrants aware of their common experiential roots, as well as of the socio-cultural values of the different populations that have produced them

A novel Electrospinning Procedure for the Production of Straight Aligned and Winded Fibers

An electrospinning procedure allowing the spinning of a straight jet of polymer solution was developed. By using proper collector devices, it enables to collect winded and aligned fibers and to prepare polymeric constructs developing along the Z axis. The reported results are expected to provide basic understandings on which parameters are controlling the stability/instability of the process and implement new applications of electrospinning with specific reference to the preparation of well defined three-dimensional structure

High-density ZnO nanowires for cellular biointerfaces: a new role as myogenic differentiation switch

The design of artificial platforms for expanding undifferentiated stem cells is of tremendous importance for regenerative medicine [1]. We have recently demonstrated that a ZnO nanowires (NWs) decorated glass support permits to obtain a differentiation switch during proliferation for mesoangioblasts (MABs)– i.e. multipotent progenitor cells which are remarkable candidates for the therapy of muscle diseases [2]. We have optimized the ZnO NWs synthesis on glass surfaces by numerical simulations and experimental systematic investigations, considering zinc speciation and supersaturation [3]. In particular, we demonstrated by numerical simulations that the ligand ethylenediamine, at the isoelectric point of the ZnO NWs tips, can effectively control – at 1:1 stoichiometric ratio with zinc – both speciation and supersaturation of zinc in the nutrient solution. In this regard, we employed ethanolamine (a safer precursor) for in-situ producing ethylenediamine by means of a zinc-catalysed amination reaction of ethanolamine by ammonia. The obtained highquality ZnONWs-cells biointerface allows cells to maintain viability and a spherical viable undifferentiated state during the 8 days observation time. Simulations of the interface by theoretical models [4] and our experimental investigations by SEM and confocal microscopy demonstrate that NWs do not induce any damage on the cellular membrane, whilst blocking their differentiation. More specifically, the myosin heavy chain, typically expressed in differentiated myogenic progenitors, is completely absent. Interestingly, the differentiation capabilities are completely restored upon cell removal from the NW-functionalized substrate and regrowing onto a standard culture glass dish. These results open the way towards unprecedented applications of ZnO NWs for cell-based therapy and tissue engineering [5]. References [1] G. Cossu, P. Bianco, Curr. Opin. Genet. Dev. 2003, 13, 537-542. [2] V. Errico, G. Arrabito, E. Fornetti, C. Fuoco, S. Testa, G. Saggio, S. Rufini, S. M. Cannata, A. Desideri, C. Falconi, C. Gargioli, ACS Appl. Mater. Interfaces, 2018, 10, 14097- 14107. [3] G. Arrabito, V. Errico, Z. Zhang, W. Han, C. Falconi, Nano Energy, 2018, 46, 54-62. [4] N. Buch-Månson, S. Bonde, J. Bolinsson, T. Berthing, J. Nygård, K.L. Martinez, Adv. Funct. Mater. 2015, 25, 3246-3255. [5] Y. Su, I. Cockerill, Y. Wang, Y.-X. Qin, L. Chang, Y. Zheng, and D. Zhu, Trends in Biotechnology, 2019, 37, 428-441

The comparative analysis of Mediterranean coastal communities: six case studies

The aim of this study is to promote cooperation and actions for the benefit of coastal communities on the Southern and Eastern shores of the Mediterranean region by adopting an approach that integrates environmental, economic and social dimensions. These areas are traditionally based on Fisheries, especially small-scale fisheries (SSFs), which contribute to strengthen social cohesion, in that the seafood value chains still represent the backbone of the coastal economy. The six coastal communities analysed in this paper are located in Algiers port - Casbah (Algeria), Marsa Matrouh (Egypt), Tricase (Italy), Tyre (Lebanon), Nador Lagoon (Morocco), Zarzis (Tunisia). Conclusions emphasise the need to develop a comprehensive reference system for dialogue, cooperation and capacity building both at national and regional level. The cluster approach can help create a favourable cooperation and competition environment, generating income and employment opportunities for local communities.Le but de cette \ue9tude est de promouvoir la coop\ue9ration et des actions en faveur des communaut\ue9s c\uf4ti\ue8res sur les rives Sud et Est de la M\ue9diterran\ue9e, en adoptant une approche qui int\ue8gre les dimensions environnementale, \ue9conomique et sociale. Cette r\ue9gion est traditionnellement ax\ue9e sur la p\ueache, et notamment la petite p\ueache, qui contribue \ue0 renforcer la coh\ue9sion sociale, car les cha\ueenes de valeur des produits de la mer constituent encore la colonne vert\ue9brale de l\u2019\ue9conomie c\uf4ti\ue8re. Les six communaut\ue9s c\uf4ti\ue8res examin\ue9es dans ce travail sont situ\ue9es \ue0 la Casbah- port d\u2019Alger (Alg\ue9rie), \ue0 Marsa Matrouh (Egypte), \ue0 Tricase (Italie), Tyre (Liban), dans la lagune de Nador (Maroc) et \ue0 Zarzis (Tunisie). Dans les conclusions, l\u2019accent est mis sur l\u2019importance de structurer un syst\ue8me de r\ue9f\ue9rence global pour encourager le dialogue, la coop\ue9ration et le d\ue9veloppement des capacit\ue9s \ue0 l\u2019\ue9chelle nationale et r\ue9gionale. Le mod\ue8le du cluster pourrait contribuer \ue0 cr\ue9er un environnement de coop\ue9ration et comp\ue9tition favorable, g\ue9n\ue9rant des revenus et des emplois au niveau des communaut\ue9s locales

A Novel Method for the Preparation of Retinoic Acid-Loaded Nanoparticles

The goal of present work was to investigate the use of bioerodible polymeric nanoparticles as carriers of retinoic acid (RA), which is known to induce differentiation of several cell lines into neurons. A novel method, named “Colloidal-Coating”, has been developed for the preparation of nanoparticles based on a copolymer of maleic anhydride and butyl vinyl ether (VAM41) loaded with RA. Nanoparticles with an average diameter size of 70 nm and good morphology were prepared. The activity of the encapsulated RA was evaluated on SK-N-SH human neuroblastoma cells, which are known to undergo inhibition of proliferation and neuronal differentiation upon treatment with RA. The activity of RA was not affected by the encapsulation and purification processes

Measurement of the top quark forward-backward production asymmetry and the anomalous chromoelectric and chromomagnetic moments in pp collisions at √s = 13 TeV

Abstract The parton-level top quark (t) forward-backward asymmetry and the anomalous chromoelectric (d̂ t) and chromomagnetic (μ̂ t) moments have been measured using LHC pp collisions at a center-of-mass energy of 13 TeV, collected in the CMS detector in a data sample corresponding to an integrated luminosity of 35.9 fb−1. The linearized variable AFB(1) is used to approximate the asymmetry. Candidate t t ¯ events decaying to a muon or electron and jets in final states with low and high Lorentz boosts are selected and reconstructed using a fit of the kinematic distributions of the decay products to those expected for t t ¯ final states. The values found for the parameters are AFB(1)=0.048−0.087+0.095(stat)−0.029+0.020(syst),μ̂t=−0.024−0.009+0.013(stat)−0.011+0.016(syst), and a limit is placed on the magnitude of | d̂ t| < 0.03 at 95% confidence level. [Figure not available: see fulltext.

Measurement of t(t)over-bar normalised multi-differential cross sections in pp collisions at root s=13 TeV, and simultaneous determination of the strong coupling strength, top quark pole mass, and parton distribution functions

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