Physical stability and in-vitro gene expression efficiency of nebulised lipid-peptide-DNA complexes

The lower respiratory tract provides a number of disease targets for gene t herapy. Nebulisation is the most practical system for the aerosolisation of non-viral gene delivery systems. The aerosolisation process represents a s ignificant challenge to the maintenance of the physical stability and biolo gical activity of the gene vector. In this study we investigate the role of a condensing polycationic peptide on the stability and efficiency of nebul ised lipid-DNA complexes. Complexes prepared from the cationic lipid 1,2-di oleoyl-3-trimethylammonium propane (DOTAP) and plasmid DNA (pDNA) at mass ( w/w) ratios of 12.1, 6:1 and 3.1, and complexes prepared from DOTAP, the po lycationic peptide, protamine, and pDNA (LPD) at 3:2:1 w/w ratio were nebul ised using a Pari LC Plus jet nebuliser. Samples from the nebuliser reservo ir (pre- and post-nebulisation) and from the aerosol mist were collected an d investigated for changes, including: particle diameter, retention of in-v itro transfection activity and the relative concentration and nature of the complexed pDNA remaining after the nebulisation procedure. The process of jet nebulisation adversely affected the physical stability of lipid:pDNA co mplexes with only those formulated at 12:1 w/w DOTAP:pDNA able to maintain their pre-nebulisation particle size distribution (145 +/- 3 nm pre-nebulis ation vs. 142 +/- 2 nm aerosol mist) and preserve significant pDNA integrit y in the reservoir (35% of pre-nebulisation pDNA band intensity). The LPD c omplexes were smaller (102 +/- 1 nm pre-nebulisation vs. 113 +/- 2 nm aeros ol mist) with considerably greater retention of pDNA integrity in the reser voir (90% of pre-nebulisation pDNA band intensity). In contrast the concent ration of pDNA in the aerosol mist for both the 12:1 w/w DOTAP:pDNA and LPD complexes were significantly reduced (10 and 12% of pre-nebulised values, respectively). Despite reduced pDNA concentration the transfection (% cells transfected) mediated by aerosol mist for the nebulised complexes was comp aratively efficient (LPD aerosol mist 26 vs. 40% for pre-nebulised complex; the respective values for 12: 1 w/w DOTAP:pDNA were 12 vs. 28%). The physi cal stability and biological activity of nebulised lipid:pDNA complexes can be improved by inclusion of a condensing polycationic peptide such as prot amine. The incorporation of the peptide precludes the use of potentially to xic excesses of lipid and charge and may act as a platform for the covalent attachment of peptide signals mediating sub-cellular targetting. (C) 2000 Published by Elsevier Science B.V. All rights reserved.