Tarallo, Rossella (2013) NEW BIOTECHNOLOGICAL METHODS FOR INTRACELLULAR DELIVERY. [Tesi di dottorato]

[img]
Preview
Text
Thesis_Tarallo.pdf

Download (54MB) | Preview
[error in script] [error in script]
Item Type: Tesi di dottorato
Lingua: English
Title: NEW BIOTECHNOLOGICAL METHODS FOR INTRACELLULAR DELIVERY
Creators:
CreatorsEmail
Tarallo, Rossellarossellatarallo@hotmail.it
Date: 26 March 2013
Number of Pages: 226
Institution: Università degli Studi di Napoli Federico II
Department: Farmacia
Scuola di dottorato: Biotecnologie
Dottorato: Scienze biotecnologiche
Ciclo di dottorato: 25
Coordinatore del Corso di dottorato:
nomeemail
Sannia, Giovannisannia@unina.it
Tutor:
nomeemail
Galdiero, Stefaniasgaldier@unina.it
Sannia, Giovannisannia@unina.it
Date: 26 March 2013
Number of Pages: 226
Uncontrolled Keywords: nanovehicles, peptides, drug delivery
Settori scientifico-disciplinari del MIUR: Area 03 - Scienze chimiche > CHIM/03 - Chimica generale e inorganica
Aree tematiche (7° programma Quadro): BIOTECNOLOGIE, PRODOTTI ALIMENTARI E AGRICOLTURA > Scienze della vita, biotecnologia e biochimica per prodotti e processi non-alimentari sostenibili
Date Deposited: 03 Apr 2013 14:31
Last Modified: 23 Jul 2014 09:41
URI: http://www.fedoa.unina.it/id/eprint/9116
DOI: 10.6092/UNINA/FEDOA/9116

Abstract

Biological membranes represent a critical hindrance for administering active molecules which are often unable to reach their designated intracellular target. In order to overcome this barrier-like behavior, not easily circumvented by many pharmacologically-active molecules, and to promote cellular uptake, synthetic peptide-based transporters have been exploited. In fact, linking or complexing therapeutic molecules to peptides that can translocate through the cellular membranes could enhance their internal delivery, and consequently, a higher amount of active compound would reach the site of action. Use of Cell Penetrating Peptides (CPPs) is one of the most promising non invasive strategies to efficiently translocate macromolecules through the plasma membrane, and have, recently, attracted a great attention. A new viral traslocator peptide (gH625) is described in this thesis, which is derived from the Herpes simplex virus type 1 (HSV-1) glycoprotein H (gH). gH625 has proved to be a useful delivery vehicle due to its intrinsic properties of inducing membrane perturbation. Quantum dots (QDs), Liposomes, Dendrimers and Polystyrene Nanoparticles (NPs) have been used as powerful platforms for studying the behavior of gH625 as a nano-vehicles. QDs are a new class of fluorescent probes under intense research and development for broad application in molecular, cellular and in vivo imaging. QDs do not significantly traverse the membrane bilayer on their own. On the contrary, when functionalized with gH625, are able to translocate through cellular plasma membranes by a mechanism which seems to be only relatively dependent on the endocytic route of entry. Liposomal aggregates have been successfully used as in vivo carriers of active principles; they display some unique pharmacokinetic properties and can be adapted to a wide range of therapeutic agents. An easy and versatile synthetic strategy, based on click chemistry, has been used to bind, in a controlled way, several copies of gH625 peptide on the external surface of DOPG based liposomes. Liposomes have been also loaded with the cytotoxic doxorubicin drug and their ability to penetrate inside cells has been evaluated by confocal microscopy experiments. Results suggest that liposomes functionalized with gH625 may act as promising intracellular targeting carriers for efficient delivery of drugs, such as chemotherapeutic agents, into tumor cells. Dendrimers are perfectly hyperbranched macromolecules with a well-defined structure, which exhibit properties very different from linear polymers with the same composition and molecular weight. A poly(amide)-based dendrimer was synthesized and conjugated with gH625. The attachment of the peptide sequences to the termini of a dendrimer allows the conjugate to penetrate into the cellular matrix, whereas the unfunctionalized dendrimer is excluded from translocation. The peptide functionalized dendrimer is rapidly taken into the cells mainly through a non-active translocation mechanism. The surface of fluorescent aminated polystyrene nanoparticles (NPs) was functionalized with gH625 via a covalent binding procedure, and the NP uptake mechanism and permeation across in vitro Blood Brain Barrier (BBB) models were studied. At early incubation times, the uptake of NPs with gH625 by brain endothelial cells is greater than that of the NPs without the peptide, and their intracellular motion is mainly characterized by a random walk behavior. Most importantly, gH625 peptide decreases NP intracellular accumulation as large aggregates and enhances their BBB crossing. In summary, these results establish that gH625 may represent a good choice for the design of promising carriers to deliver drugs for the treatment of human diseases.

Actions (login required)

View Item View Item