PURPOSE: The aim of this study was to examine the extent to which plasmid DNA (pDNA) complexed to cationic liposomes diffuse through cystic fibrosis (CF) sputum. The influence of the physical and chemical properties of the sputa was evaluated. We further investigated whether degradation of the sputa by recombinant human DNase I (rhDNase I) enhances the transport. METHODS: The transport of lipoplexes was studied through layers of CF sputa placed between the donor and acceptor compartment of vertical diffusion chambers. The content of the acceptor compartment was analyzed by confocal fluorescence microscopy, gel electrophoresis and Southern blotting. The influence of linear DNA present in the CF sputa on the size, surface charge and gene expression of the lipoplexes was evaluated by dynamic light scattering, particle electrophoresis and transfection experiments. RESULTS: Lipoplexes were observed in the acceptor compartments. However, the percent of diffused lipoplexes was low: 0.05/% +/- 0.01%. It was found that both steric obstruction by the sputa as well as the long" distance the lipoplexes have to travel were responsible for this low transport. Surprisingly, the transport occurred better through more viscoelastic sputa. The DNA in the CF sputa also retarded the transport, which was attributed to aggregation of the lipoplexes by the DNA. Finally, rhDNase I moderately enhanced the diffusion of lipoplexes. CONCLUSIONS: CF sputum drastically retards the diffusion of lipoplexes. DNA in the sputa aggregates the lipoplexes. This may lower the transport of lipoplexes through the sputa and gene expression. Pretreatment of CF patients with rhDNase I may enhance the efficiency of CF gene therapy, as it allows a better transport of the lipoplexes through the sputum and as it partly removes the sputum which will result in a thinner sputum layer on top of the epithelial cells.
PURPOSE: The aim of this study was to examine the extent to which plasmid DNA (pDNA) complexed to cationic liposomes diffuse through cystic fibrosis (CF) sputum. The influence of the physical and chemical properties of the sputa was evaluated. We further investigated whether degradation of the sputa by recombinant human DNase I (rhDNase I) enhances the transport. METHODS: The transport of lipoplexes was studied through layers of CF sputa placed between the donor and acceptor compartment of vertical diffusion chambers. The content of the acceptor compartment was analyzed by confocal fluorescence microscopy, gel electrophoresis and Southern blotting. The influence of linear DNA present in the CF sputa on the size, surface charge and gene expression of the lipoplexes was evaluated by dynamic light scattering, particle electrophoresis and transfection experiments. RESULTS: Lipoplexes were observed in the acceptor compartments. However, the percent of diffused lipoplexes was low: 0.05/% +/- 0.01%. It was found that both steric obstruction by the sputa as well as the long" distance the lipoplexes have to travel were responsible for this low transport. Surprisingly, the transport occurred better through more viscoelastic sputa. The DNA in the CF sputa also retarded the transport, which was attributed to aggregation of the lipoplexes by the DNA. Finally, rhDNase I moderately enhanced the diffusion of lipoplexes. CONCLUSIONS:CF sputum drastically retards the diffusion of lipoplexes. DNA in the sputa aggregates the lipoplexes. This may lower the transport of lipoplexes through the sputa and gene expression. Pretreatment of CF patients with rhDNase I may enhance the efficiency of CF gene therapy, as it allows a better transport of the lipoplexes through the sputum and as it partly removes the sputum which will result in a thinner sputum layer on top of the epithelial cells.
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