5%). Lipoplexes also increased the number of EGFP positive BGM cells, but their efficiency was not higher than that of PolyFect®. The starburst PAMAM dendrimer G5 did not enhance the plasmid transfection capacity. Transfection with both lPEI and brPEI polyplexes was most efficient at an N/P of ratio 8. The lipoplexes obtained their highest gene expression at a ± ratio of 8. Linear PEI (maximum of 16% transfected cells) ABT-263 concentration could double the transfection
efficiency compared to brPEI (maximum of 8% transfected cells). Normally, transfection efficiencies increase with increasing ratio. For lPEI and brPEI this was indeed observed when increasing the ratio from 5 to 8. However, at an N/P ratio of 10, transfection efficiencies dropped again but still remained higher than for an N/P ratio of 5. Based on the transfection results for BGM and DF-1 cells, both lPEI and brPEI polyplexes at an N/P ratio of 8 were selected for subsequent nebulisation experiments. Branched PEI and linear PEI polyplexes (N/P = 8) dissolved in HEPES buffer at a DNA concentration of 0.126 μg/μl were nebulised with a Cirrus™ nebulizer. The DNA concentrations, particle sizes and zeta potentials of the PEI polyplexes were measured before and after nebulisation. Particle size and zeta potential
of brPEI polyplexes did not significantly alter after nebulisation while the DNA concentration and the OD260/OD280 ratio slightly dropped. Particle size of the lPEI complexes increased to almost 1 μm buy BMS-354825 and the zeta potential decreased from 34.8 to 7.2 mV, close to electro neutrality. Additionally, the concentration of plasmid DNA recovered following nebulisation was extremely low (0.009 μg/ml) and the OD260/OD280 ratio decreased with 50%. These findings probably imply that lPEI polyplexes are most likely destroyed or retained in the nebulizer. To further characterise the PEI polyplexes after nebulisation, the stability of the polyplexes and the integrity of the pDNA inside the polyplexes were examined before and Sclareol after nebulisation, using agarose gel electrophoresis. Nebulisation of naked pDNA with the Cirrus™ nebulizer had a great
impact on the DNA integrity as demonstrated by the presence of a smeared band (DNA fragmentation) in lane 2 (Fig. 2A). The stability of non-nebulised polyplexes was assessed following SDS treatment. SDS clearly dissociated the lPEI polyplexes (lane 4, a clear DNA band is visible), while it was almost unable to disrupt brPEI polyplexes (lane 8, a DNA band with very low intensity was present). This indicates that the overall stability of lPEI polyplexes is much lower than of brPEI polyplexes. Moreover, particle size and zeta potential of the lPEI complexes were heavily influenced during nebulisation (see above) and thus complex stability must be affected. Therefore, we should expect a DNA fragment in lanes 5 and especially 6.