510. In Vivo-Gene Expression of Aerosolized PEI Gene Vector Complexes Is Solvent-Dependent

Abstract

It has previously been shown that the in vitro -gene transfer efficiency of nebulized polyethylenimine (PEI) 25 kDa based gene vector complexes was affected by the solvent (distilled water, glucose 5%, or hepes buffered saline (HBS)) which was used for complex formulation. When PEI polyplexes were formulated in distilled water neither the complex parameters such as particle size and zeta potential nor the transfection efficiency were markedly affected by the nebulization process using a standard jet nebulizer (PARI GmbH, Starnberg, Germany). These data suggested that water would be the best choice of solvent for the in vitro application. In this study we examined the effect of the solvent on PEI-mediated gene transfer in vitro. PEI-based gene vector complexes (1 mg of pCMVLuc) were formulated either in distilled water, glucose 5%, HBS, or PBS at an N/P-ratio of N/P=10 using an optimized whole body nebulization device and luciferase gene expression in the lungs was measured after 24 hours. Luciferase gene expression mediated by complexes which were formulated in distilled water was 57- and 185-fold higher as compared with complexes formulated in 5% glucose or HBS, respectively. No luciferase gene expression was observed when complexes were formulated in PBS. In contrast to complexes formulated in PBS which resulted in strong particle aggregation (847±142 nm), complexes formulated in distilled water (98±2 nm), glucose 5% (89±10 nm), or HBS (153±10 nm) were within the same particle size which could not explain the differences in gene expression. In addition, solvent-dependent differences in gene expression could not be explained by different deposition rates of gene vectors. Quantification of lung deposition of radioactively-labeled plasmid DNA did not result in a different pattern with respect to the solvent used. A possible reason to explain the solvent-dependent differences of gene expression could be the effect of nebulized distilled water on the airway epithelium. Nebulized distilled water induces airway swelling, widening of the intercellular spaces, and increased cell permeability which could lead to facilitated uptake of the complexes by the airway epithelium. Such effects can be inhibited by pretreatment of mice with nebulized indomethacin. Indeed pretreatment of mice with nebulized indomethacin reduced gene expression 2.9-fold. Histological analyses demonstrated deposition of FITC-labeled PEI gene vectors both in the alveolar epithelium and in the bronchial epithelium. In particular, gene vector uptake by alveolar macrophages was observed. Reporter gene expression of β-galactosidase was detected in the epithelium of the large airways as well as in isolated alveolar macrophages. Taken together these data demonstrate that PEI-mediated aerosol gene delivery is strongly solvent-dependent and distilled water represents the most efficient solvent for complex formulation. Increased cell permeability and paracellular diffusion which facilitates cellular gene vector uptake in the presence of water could be suggested as a possible mechanism for such solvent-dependent effect.