Micro-Nanostructured Polymeric Constructs for Biomedical and Pharmaceutical Applications. Preparation and Chemical & Biological Evaluation

Controlled drug delivery technology represents one of the most rapidly advancing areas of science in which several disciplines, such as chemistry, pharmaceutical technology, and medicine are contributing to human health care. The goal of drug delivery systems, is to deploy medications intact to specifically targeted organs and compartments of the body through a medium that can control administration of the active principle by means of either a physiological or chemical trigger. During the past decade, polymeric micro-nanoparticles, polymer micelles, and hydrogel-type materials have all been shown to be effective in enhancing drug targeting specificity, lowering systemic drug toxicity, improving treatment absorption rates, and providing protection for pharmaceuticals against biochemical degradation. The research activities reported in the present thesis are the result of experimental work performed at the Laboratory of Polymeric Materials for Biomedical and Environmental Applications (Biolab) of the Department of Chemistry and Industrial Chemistry of the University of Pisa and in part at SINTEF company (Trondheim, Norway). The aim of the present PhD thesis was mainly focused on the preparation of polymeric nanoparticles based on Poly(maleic anhydride–alt–butyl vinyl ether) 5% grafted with methoxyPEG2000 and 95% grafted with 2-methoxyethanol (VAM41) loaded with human Hemoglobin to be used as artificial oxygen carriers. Nanoparticles were prepared by means of the co-precipitation technique performed under controlled conditions in order to minimize oxidative phenomena; nanoparticles characterization was carried out in terms of size, morphology and surface properties analysis, highlighting the feasibility of obtaining Hemoglobin loaded polymeric nanoparticles possessing suitable features to be used as artificial oxygen carriers. The maintenance of protein functional bioactivities, once loaded inside nanoparticles, was also investigated; results showed that although protein secondary and quaternary structure seems to be maintained, Hemoglobin oxidation takes place during the formulation process. To overcome this inactivating phenomenon different strategies were investigated including the introduction of several reducing agents inside the formulation system as well as the modification of the polymeric structure by means of the introduction of a conductive moiety. Although Hemoglobin oxidation phenomenon during the formulation process was not avoided, alterations regarding nanoparticles features were not observed, highlighting the versatility of the so developed system and opening promising perspectives in the development of VAM41 based polymeric nanoparticles loaded with functional Hemoglobin. The use of Alginate as alternative polymeric matrix was also investigated; the formulation process based on the ionic gelation was optimised carrying out successfully the reduction of Alginate particles dimensions from a millimetric to a micrometric scale, without affecting particle protein contents. Results obtained during this research activity indicated Alginate as a potential polymeric matrix usable in the development of artificial oxygen carriers. Moreover, during a six months visiting period spent at SINTEF, the research activity was focused on the development of VAM41 based polymeric nanoparticles loaded with Dead Sea Salts, to be used as dermal drug delivery system. VAM41 polymer was applied in a water-in-oil mini-emulsion based formulation system, demonstrating the feasibility of obtaining polymeric nanoparticles loaded with Dead Sea Salts possessing suitable features in terms of size and morphology; a time controlled release profile of the loaded active principle was also detected, opening promising perspectives in the use of the so developed system in topical pharmaceutical applications

Hemoglobin Loaded Alginate Particles

Over the last few years medical and pharmaceutical industries have shown an increasing interest in alginate, an anionic polysaccharide widely distributed in the cell walls of brown algae. The present work aims at loading Human Hemoglobin (Hb) into alginate particles; the modification of some formulation parameters was carefully investigated by analysing changes on particles size and protein physicochemical properties. Particles were prepared by dropping alginate into an aqueous solution containing Hb and CaCl2, which permits the formation of particles through ionic cross- linking. Hb loaded alginate beads were obtained possessing an average diameter of about 2 mm and a protein loading of about 5%. Physicochemical characterizations showed that the protein maintained its functional ability of reversibly binding oxygen and its quaternary structure once loaded into alginate beads. In a refinement of the first formulation trials, Hb loaded alginate microparticles with diameter around 150 μm were obtained with a protein loading of about 50

Gene Expression and Chromatin Organization during Mouse Oocyte Growth

AbstractMouse oocytes can be classified according to their chromatin organization and the presence [surrounded nucleolus (SN) oocytes] or absence [nonsurrounded nucleolus (NSN) oocytes] of a ring of Hoechst-positive chromatin around the nucleolus. Following fertilization only SN oocytes are able to develop beyond the two-cell stage. These studies indicate a correlation between SN and NSN chromatin organization and the developmental competence of the female gamete, which may depend on gene expression. In the present study, we have used the HSP70.1Luc transgene (murine HSP70.1 promoter + reporter gene firefly luciferase) to analyze gene expression in oocytes isolated from ovaries of 2-day- to 13-week-old females. Luciferase was assayed on oocytes after classification as SN or NSN type. Our data show that SN oocytes always exhibit a higher level of luciferase activity, demonstrating a higher gene expression in this category. Only after meiotic resumption, metaphase II oocytes derived from NSN or SN oocytes acquire the same level of transgene expression. We suggest that the limited availability of transcripts and corresponding proteins, excluded from the cytoplasm until GVBD in NSN oocytes, could explain why these oocytes have a lower ability to sustain embryonic development beyond the two-cell stage at which major zygotic transcription occurs. With this study we have furthered our knowledge of epigenetic regulation of gene expression in oogenesis

Le politiche di facilitazione alle piccole e medie imprese e il sistema delle agevolazioni finanziarie

PMI, agevolazioni, sviluppo dell'impres

Il dividend discount model e il cash flow to equity model: alcune considerazioni di metodo

L'articolo esamina le differenze tra varie modalita' di deteri4mnazione del flusso da attualizzare nelle valutazioni finanziari

Sistemi Polimerici Micro/Nanostrutturati per Applicazioni Biomediche e Farmaceutiche: Preparazione e Caratterizzazione Chimico-Fisica e Biologica.

Il presente lavoro di tesi è stato incentrato sullo studio di sistemi polimerici bioerodibili e bioeliminabili per applicazioni biomediche e farmaceutiche. I materiali polimerici (naturali, sintetici o semisintetici) hanno catturato l’interesse del mondo scientifico nella formulazione di sistemi a rilascio controllato di farmaci quali micro e nanoparticelle [Chiellini, 2001 a] e come matrici bioattive nella realizzazione di supporti (scaffolds) atti a promuovere l’adesione e la proliferazione cellulare in applicazioni di ingegneria tissutale [Chiellini, 2001 b]. Strutture polimeriche solide tridimensionali sono state preparate utilizzando polimeri naturali sia commerciali (alginati) che ottenuti da fonti rinnovabili (ulvani); I preparati sono stati caratterizzati da un punto di vista morfologico mediante microscopia a scansione elettronica (SEM); inoltre è stato valutato il grado di rigonfiamento e la loro biodegradabilità sia in tampone fosfato salino (PBS) che in terreno di crescita per colture cellulari (DMEM). I risultati ottenuti sono stati correlati all’utilizzo dei diversi agenti reticolanti impiegati per produrre le strutture polimeriche. Le stesse sono state sottoposte ad una attenta valutazione in vitro della citotossicità e della bioattività, utilizzando come riferimento la linea cellulare di epatoblastoma umano HepG2; la vitalità cellulare è stata analizzata effettuando tre tipi di saggio: l’eventuale interazione dei materiali polimerici con la membrana plasmatica è stata valutata mediante dosaggio della lattato deidrogenasi (LDH), la capacità cellulare di effettuare endocitosi è stata verificata attraverso il saggio del “Neutral Red Uptake”, mentre l’attività metabolica è stata analizzata utilizzando i sali di tetrazolio (WST-1). Parallelamente è stato iniziato uno studio per la formulazione di sistemi nanoparticellari caricati con emoglobina al fine di ottenere sostituti artificiali del sangue in grado di trasportare ossigeno. Matrici polimeriche bioerodibili quali copolimeri funzionali a base di anidride maleica e butilvinil etere (VAM40) emiesterificati con 2-metossietanolo e poli(etilene glicol ) (VAM41), sono state utilizzate per la formulazione di nanoparticelle a lunga circolazione in combinazione con emoglobina, come proteina trasportatore di ossigeno, ed uno stabilizzante sterico (Glicidilisopropilidengliceril-beta-ciclodestrina), mediante una metodologia definita di co-precipitazione sviluppata presso questo laboratorio. Le nanoparticelle ottenute sono state analizzate da un punto di vista morfologico e dimensionale mediante SEM e granulometria in sospensione, e da un punto di vista chimico fisico mediante la misura del potenziale Zeta. La quantità di emoglobina caricata nelle nanoparticelle è stata valutata utilizzando il saggio di Drabkin. Sia le matrici polimeriche che le nanoparticelle preparate sono state valutate per la loro citocompatibilità in vitro utilizzando la linea cellulare balb 3T3 clone A31 mediante i saggi biologici sopraindicati

L'impatto del leveraged buyout su azionisti e stakeholder: l'evidenza empirica in Italia

Oggetto dell'articolo è una verifica empirica delle performance post buyout delle aziende acquisite col ricorso al debito. A tal fine sono state prese in considerazione le operazioni di LBO effettuate in Italia tra il 2003 e il 2005, e sono stati presi in considerazione degli indicatori (tratti dai bilanci aziendali) in grado di esprimere l'impatto di dette operazioni sia sul benessere degli azionisti che su quello degli altri stakeholder