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PIGNATTO, M (2010) NANOASSEMBLATI DI AVIDINA E DNA COME STRUMENTI IN MEDICINA E DIAGNOSTICA. [Tesi di dottorato]

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Abstract (inglese)

This work originates from the recent discovery of the existence in nature of a high-affinity interaction between avidin and nucleic acids wich can be guided to give rise to discrete and solution stable polyavidin nanoassemblies.
The interest towards these systems is due to the fact that, since the latter are composed by many avidin units stably assembled, they can potentially be used in several biotechnological and biomedical applications as an improvement of the classic avidin-biotin system.
The work herein described had a two-fold purpose: a) to deepen the knowledge about the molecular basis of the interaction between avidin and DNA and b) to evaluate the potential effectiveness of the nanoassemblies that arise from such interaction.
The interaction between avidin and nucleic acids of different length, single and double stranded (plasmidic DNA, genomic DNA and synthetic oligomers) has been studied by using different analytical techniques, such as electrophoretic mobility assay, tryptophan fluorescence quench, HPLC and dynamic light scattering. The results provided clear indications on the size of the binding site, the mode of interaction with the different types of nucleic acids and their dissociation constants Kd. The results also provided valuable information to obtain optimized nanoassemblies formulations.
ELISA and dot blot assays were performed to verify, in practical terms, the effective analytical performance of these nanoassemblies in in vitro diagnostic applications. The results showed the superiority of the nanoassemblies compared to the monomeric avidin.
In addition, in view of a potential use as in vivo drug delivery and diagnostics tools, preliminary investigation on the in vivo biodistribution – conducted by means of fluorescence optical imaging – was carried out. The nanoassemblies are able to circulate in the blood stream for more than 72 hours and no preferential organ distribution was observed despite some liver accumulation.

Abstract (italiano)

Questo lavoro origina dalla recente scoperta dell’esistenza in natura di una interazione ad alta affinità tra la proteina avidina e gli acidi nucleici e dal fatto che è possibile guidare tale interazione verso la formazione di nanoassemblati discreti e stabili. L’interesse verso questi sistemi deriva dal fatto che, in quanto costituiti da diverse unita’ di avidina unite assieme in modo stabile, essi possono potenzialmente trovare impiego in svariate applicazioni biotecnologiche e biomediche in analogia e come miglioramento del sistema avidina-biotina classico.
Il lavoro descritto in questa tesi ha avuto un duplice obiettivo: approfondire le conoscenze relative alle basi molecolari dell’interazione tra avidina e DNA e al contempo valutare le effettive potenzialità in ambito applicativo dei nanoassemblati che originano grazie a tale interazione.
Utilizzando diverse tecniche analitiche quali l’electrophoretic mobility assay, il quench della fluorescenza del triptofano, l’HPLC e il light scattering dinamico è stata studiata l’interazione tra avidina e acidi nucleici di diversa lunghezza (DNA plasmidico, DNA genomico, oligomeri sintetici) a singolo e doppio filamento. I risultati hanno fornito chiare indicazioni sulla dimensione del sito di binding, la modalità dell’interazione con le diverse tipologie di acidi nucleici e le relative costanti di dissociazione Kd.
Lo studio dei meccanismi molecolari alla base dell’interazione tra la proteina e gli acidi nucleici ha permesso di ricavare informazioni preziose per l’ottenimento di formulazioni ottimizzate di nanoassemblati.
Con lo scopo di verificare in termini pratici le potenzialità in ambito diagnostico dei nanoassemblati avidina-DNA è stata valutata la loro performance analitica in saggi di tipo ELISA e dot blot. I risultati ottenuti hanno dimostrato in modo inequivocabile la superiorità dei nanoassemblati rispetto all’avidina monomerica.
In vista di un potenziale utilizzo in vivo dei nanaoassemblati come strumenti in diagnostica e nel direzionamento di farmaci è stata effettuata una prima indagine sulla loro biodistribuzione mediante imaging ottico di fluorescenza. I nanoassemblati sono in grado di circolare nel torrente sanguigno per oltre 72 ore e, a parte un lieve accumulo a livello epatico, non risulta evidente una distribuzione preferenziale verso determinati organi.

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Tipo di EPrint:Tesi di dottorato
Relatore:MORPURGO, M
Dottorato (corsi e scuole):Ciclo 22 > Scuole per il 22simo ciclo > SCIENZE MOLECOLARI > SCIENZE FARMACEUTICHE
Data di deposito della tesi:NON SPECIFICATO
Anno di Pubblicazione:25 Gennaio 2010
Parole chiave (italiano / inglese):Molecular amplifier, nanotechnology, drug delivery, avidin, DNA protein interaction
Settori scientifico-disciplinari MIUR:Area 03 - Scienze chimiche > CHIM/09 Farmaceutico tecnologico applicativo
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Farmaceutiche
Codice ID:2442
Depositato il:09 Nov 2010 16:46
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