Tesis doctoral de Koldo Urbiola Pérez
In this work, different targeted formulations have been designed and evaluated in order to improve gene delivery to cancer cells by non-viral vectors in vitro and in vivo. All the nanosystems are based on the dendrimeric carrier pamam, to which four different ligands (hyaluronic acid, transferrin, b6 and ge11 peptides) have been attached, in order to evaluate the targeting capacity of each nanocarrier. First, a novel pamam-hyaluronic acid conjugate has been synthetized by an amine bond formation between pamam and oxydized hyaluronic acid. This conjugate was able to form nanoparticles in the presence of pdna and exhibited excellent capacity to effectively bind pdna and protect it from enzymatic degradation by nucleases. In vitro evaluation of pamam-hyaluronic acid dendriplexes showed an increase in transfection activity in mda-mb231 and b16f10 cells compared to non-targeted complexes. A competition study with an excess of free ha confirmed the uptake via specific receptor-mediated mechanism. Toxicity studies showed a good cell viability and lower toxicity than the highly used pei-polyplexes. In vivo results showed an increase in luciferase expression in the liver and heart of balb-c mice compared to non-targeted complexes. These systems were also able to transfect efficiently b16f10 tumors in c57bl/6 tumor-bearing mice, although no significant differences compared to non-targeted ones were detected. Secondly, pamam-transferrin conjugates were prepared and evaluated. In vitro evaluation of this new pamam-transferrin conjugate demonstrated increased gene delivery to cancer cells (hela, hepg2 and ct26) when complexes were formulated at n/p ratio of 6. Toxicity was lower than pei-polyplexes. The uptake via receptor-mediated endocytosis was ensured by a competition assay. Finally, b6 and ge11 peptides were studied as targeting ligands in these systems. Small interfering rna (sirna) was formulated in targeted complexes containing each peptide in the presence of peg (2 kda). Pamam-peg-b6 and pamam-peg-ge11 dendriplexes formed stable nanoparticles, able to condense sirna effectively. In vitro evaluation of the gene silencing capacity of sirna encapsulated into pamam-peg-b6 or pamam-peg-ge11 complexes showed that specific sirna-luc was able to reduce luciferase expression in hela and ls174t cells, without leading to toxicity effects.
Datos académicos de la tesis doctoral «Novel targeted poly-amidoamine (pamam) nanocarriers for gene delivery: design, development and evaluation«
- Título de la tesis: Novel targeted poly-amidoamine (pamam) nanocarriers for gene delivery: design, development and evaluation
- Autor: Koldo Urbiola Pérez
- Universidad: Navarra
- Fecha de lectura de la tesis: 10/06/2014
Dirección y tribunal
- Director de la tesis
- María Concepción Tros De Ilarduya Apaolaza
- Tribunal
- Presidente del tribunal: José manuel García fernández
- Juan Antonio Palop cubillo (vocal)
- Manuel Guzmán navarro (vocal)
- Carlos mauricio Goní§alves barbosa (vocal)