Biodistribution and transgene expression with nonviral cationic vector/DNA complexes in the lungs

Abstract

Biodistribution of nonviral cationic vector/DNA complexes was studied after systemic or intratracheal administration to the lungs and correlated with transgene expression. Intravenous injection in C57Bl/6 mice gave maximal and significant luciferase expression in the lungs with the cationic polymer PEI 22K/DNA complexes at the highest ratios of positive/negative charges versus DNA alone. While DOTAP/DNA complexes with high charge ratio determined lower but still significant luciferase activity versus uncomplexed DNA, GL-67A and PEI 25K mediated negligible luciferase expression. Labelled PEI 22K and DOTAP complexes were evenly distributed in the alveolar region, where GFP expression was revealed, while PEI 25K and GL-67A complexes were not detected, suggesting a different interaction of these complexes with the plasma membrane of endothelial cells. Following an intratracheal injection, the highest and significant levels of transfection were obtained with

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“…38 We could not follow the distribution of correspondingly labeled PEI polyplexes due to quenching, but a recent publication on the lung distribution of the same PEI formulation (25 kDa, 5:1) showed a comparable distribution to the mid-airways. 25 In general, the X-gal staining pattern after administration of PEI and UPC polyplexes of a pLacZ reporter gene agreed with the distribution studies using psoralen-labeled polyplexes. Large patches were found in which virtually every epithelial cell lining the mid-airways expressed the reporter gene.…”

“…23,24 Therefore, all in vivo comparisons were performed against the low molecular weight PEI at a charge ratio of 5:1 (+/−). 25 In vitro studies: In order to investigate whether UPC protects the pDNA against degradation by endogenous enzymes, the charge-dependent stability and transgene expression in 293 cells were investigated in the presence of serum. When naked DNA or UPC polyplexes were incubated with 10% serum, the pDNA was fully retained on the agarose gel at charge ratios of 3 (+/−) or larger, indicating that the polyplexes gave significant protection against the serum nucleases.…”

“…However, recent studies of PEI polyplexes of an identical composition indicate a similar lung distribution to that of the UPC polyplexes in the present study. 25 A qualitative assessment of the gene expression in the lungs was obtained using a new efficient plasmid (pBK-SFVB7-LacZ) that contains a translational enhancer element. 26 The gene expression was surprisingly strong in the mid-airways 72 h after administration of a 50 g dose of pLacZ in both UPC and PEI polyplexes, indicating that both polyplexes were highly effective as compared with naked DNA (Figure 7).…”

“…This makes it possible to compare previous results with the results in the present study. PEI and UPC were used at optimal charge ratios of 5:1 and 3:1, respectively, based on literature data 25,37 and on our preliminary in vivo experiments. It is also worth noting that intratracheal administration of PEI polyplexes at a charge ratio of 5:1 did not result in an inflammatory response, as had occurred at higher charge ratios.…”

“…27 Optimal charge ratios of 5:1 (+/−) and 9:1 (+/−) for PEI 25 kDa and 800 kDa, respectively, were used throughout. 25,27,37 Physicochemical characterisation of polyplexes The diameters of the polyplexes were determined by photon correlation spectroscopy (Zetasizer 4000, Malvern Instruments, Malvern, UK) and their zeta potentials (which indicate the surface charge) were determined by electrophoretic light scattering (Zetasizer IV, Malvern Instruments). The physical stability of the polyplexes was studied by agarose gel electrophoresis (0.8% agarose in TAE buffer).…”