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The delivery of nucleic acids into the lung is still one major bottleneck and relies on the development of efficient non-viral carriers that can be nebulized for their direct administration. Liposomal formulations, including those comprising different phospholipids, have been intensively studied. Likewise, polyethylenimines (PEI) have been widely explored due to their ability to protect nucleic acids from degradation and mediate cellular uptake and endosomal/lysosomal release. The combination of PEIs and liposomes is a particularly intriguing concept, leading to enhanced transfection efficiencies, lower cytotoxicities, and protection from aggregation. We have previously established lipopolyplexes, comprising PEI F25-LMW and the neutral phospholipid DPPC with various colipids. However, nebulization shear forces may be problematic for lipid formulations and require the testing of lipopolyplexes. In this paper, we demonstrate that DNA- or siRNA-containing PEI-based lipopolyplexes are indeed stable toward nebulization and retain their physicochemical integrity and biological activity. DNA transfection efficacies, but not siRNA knockdown, of certain lipopolyplexes are even enhanced upon nebulization, and nebulized lipopolyplexes also allow storage. This is due to the preservation of lipopolyplex size and zeta potential upon nebulization. More detailed analyses also reveal a dependence on the lipid, the medium used for preparation, the PEI and the nucleic acid. Practical applications: Our data provide a basis for the tailor-made preparation of lipopolyplexes for nebulization, by analyzing different PEIs, different lipids, different media for lipopolyplex preparation, different nucleic acids, and directly comparing nebulized versus non-nebulized lipopolyplexes, with or without storage, with regard to biological and physiochemical properties. Polyethylenimine (PEI)-based nanoscale complexes protect nucleic acids from degradation and mediate cellular delivery. Their combination with liposomes to lipopolyplexes is a particularly intriguing concept, leading to enhanced transfection efficiencies, lower cytotoxicities and protection from complex aggregation. We show that lipopolyplexes are stable towards nebulization and provide a basis for tailor-made lipopolyplexes for nebulization, by analyzing different PEIs, lipids, media and nucleic acids for lipopolyplex preparation. [ABSTRACT FROM AUTHOR]