<p>In this thesis, chitosan, a biocompatible polysaccharide that has been approved as a food additive was selected as a platform for the development of safe, efficient non-viral gene delivery systems to mammalian cells. Previously, chitosan-based gene formulations had been generally associated with high molecular weight chitosans, which were poorly characterised in terms of molecular weight distribution and degree of acetylation. Therefore, in order to improve the properties of chitosan-based gene formulations, the research associated with this thesis focused on establishing the structure-property relationships of well-defined, low molecular weight chitosans (chitosan oligomers) as delivery systems for nucleic acids (pDNA and siRNA)<i> in vitro</i> and after lung administration <i>in vivo</i>. pDNA dissociated more easily from chitosan oligomers than from conventional high molecular weight chitosans, resulting in a faster onset and higher levels of<i> in vivo</i> gene expression, comparable to those mediated by polyethyleneimine (PEI), one of the most efficient non-viral delivery systems. Coupling of a trisaccharide branch to the chitosan oligomers so as to target extracellular lectins resulted in a significant improvement in transfection efficiency because of enhanced cellular uptake and colloidal stability. In contrast to pDNA, longer linear chitosan oligomers were required to form physically-stable nanoparticles with siRNA that mediated efficient, sustained gene silencing <i>in vitro</i>. Finally, the use of an optimised catheter device for the nebulisation of small volumes of pDNA formulations resulted in improved dose precision and lung distribution<i> in vivo</i> compared with conventional intratracheal instillation. In conclusion, chitosan oligomers are interesting and viable alternatives to other non-viral gene delivery systems.</p>

In this thesis, chitosan, a biocompatible polysaccharide that has been approved as a food additive was selected as a platform for the development of safe, efficient non-viral gene delivery systems to mammalian cells. Previously, chitosan-based gene formulations had been generally associated with high molecular weight chitosans, which were poorly characterised in terms of molecular weight distribution and degree of acetylation. Therefore, in order to improve the properties of chitosan-based gene formulations, the research associated with this thesis focused on establishing the structure-property relationships of well-defined, low molecular weight chitosans (chitosan oligomers) as delivery systems for nucleic acids (pDNA and siRNA) in vitro and after lung administration in vivo. pDNA dissociated more easily from chitosan oligomers than from conventional high molecular weight chitosans, resulting in a faster onset and higher levels of in vivo gene expression, comparable to those mediated by polyethyleneimine (PEI), one of the most efficient non-viral delivery systems. Coupling of a trisaccharide branch to the chitosan oligomers so as to target extracellular lectins resulted in a significant improvement in transfection efficiency because of enhanced cellular uptake and colloidal stability. In contrast to pDNA, longer linear chitosan oligomers were required to form physically-stable nanoparticles with siRNA that mediated efficient, sustained gene silencing in vitro. Finally, the use of an optimised catheter device for the nebulisation of small volumes of pDNA formulations resulted in improved dose precision and lung distribution in vivo compared with conventional intratracheal instillation. In conclusion, chitosan oligomers are interesting and viable alternatives to other non-viral gene delivery systems.

<p>The subject of this thesis was to develop and optimize delivery systems for plasmid DNA (pDNA) based on biocompatible polymers, in particular chitosan, suitable for non-viral gene therapy. At the onset of this thesis, studies had reported conflicting results on the efficiency of chitosan-based gene delivery systems. Therefore, structure-property relationships of chitosans as non-viral gene delivery systems <i>in vitro</i> and after lung administration <i>in vivo</i> were established for the first time.</p><p>Polymer-pDNA complexes (polyplexes) based on conventional high molecular weight chitosans transfected cells <i>in vitro</i> and after lung administration <i>in vivo</i>. The chitosan polyplexes were, in contrast to polyplexes formed with the "golden standard" polymer polyethylenimine (PEI), essentially non-toxic at escalating doses. However, a very high physical stability of the chitosan-pDNA complexes together with a low buffering capacity of chitosan at the slightly acidic endo/lysosomal pH resulted in a slow onset of the gene expression and also in a lower efficiency of gene expression compared to PEI polyplexes. A slow and biodegradation-dependent release of pDNA from the chitosan polyplexes was concluded to be a rate limiting step for the efficiency of high molecular weight chitosan. The optimized polyplexes of high molecular weight chitosan (around 1,000 monomer units) showed aggregated shapes and gave increased viscosity at concentrations used for <i>in vivo</i> gene delivery. To improve the pharmaceutical properties and the delivery properties of chitosan polyplexes, low molecular weight chitosans were studied. Chitosans of around 18 monomer units retained the ability to protect pDNA against DNase degradation, but were more easily dissociated than those of higher molecular weight and had an efficiency comparable to that of PEI <i>in vitro</i> and <i>in vivo</i>. The pharmaceutical advantages of low molecular weight chitosan polyplexes compared to higher molecular weights are that there is less aggregation and no increased viscosity at the concentrations used for <i>in vivo</i> gene delivery. Coupling of an oligosaccharide targeting ligand to chitosan further increased the efficiency of some oligomer polyplexes. In conclusion, biocompatible chitosan is an interesting alternative to other non-viral gene delivery systems such as PEI.</p>

The subject of this thesis was to develop and optimize delivery systems for plasmid DNA (pDNA) based on biocompatible polymers, in particular chitosan, suitable for non-viral gene therapy. At the onset of this thesis, studies had reported conflicting results on the efficiency of chitosan-based gene delivery systems. Therefore, structure-property relationships of chitosans as non-viral gene delivery systems in vitro and after lung administration in vivo were established for the first time. Polymer-pDNA complexes (polyplexes) based on conventional high molecular weight chitosans transfected cells in vitro and after lung administration in vivo. The chitosan polyplexes were, in contrast to polyplexes formed with the "golden standard" polymer polyethylenimine (PEI), essentially non-toxic at escalating doses. However, a very high physical stability of the chitosan-pDNA complexes together with a low buffering capacity of chitosan at the slightly acidic endo/lysosomal pH resulted in a slow onset of the gene expression and also in a lower efficiency of gene expression compared to PEI polyplexes. A slow and biodegradation-dependent release of pDNA from the chitosan polyplexes was concluded to be a rate limiting step for the efficiency of high molecular weight chitosan. The optimized polyplexes of high molecular weight chitosan (around 1,000 monomer units) showed aggregated shapes and gave increased viscosity at concentrations used for in vivo gene delivery. To improve the pharmaceutical properties and the delivery properties of chitosan polyplexes, low molecular weight chitosans were studied. Chitosans of around 18 monomer units retained the ability to protect pDNA against DNase degradation, but were more easily dissociated than those of higher molecular weight and had an efficiency comparable to that of PEI in vitro and in vivo. The pharmaceutical advantages of low molecular weight chitosan polyplexes compared to higher molecular weights are that there is less aggregation and no increased viscosity at the concentrations used for in vivo gene delivery. Coupling of an oligosaccharide targeting ligand to chitosan further increased the efficiency of some oligomer polyplexes. In conclusion, biocompatible chitosan is an interesting alternative to other non-viral gene delivery systems such as PEI.