title: Physical stability and in-vitro gene expression efficiency of nebulised lipid-peptide-DNA complexes.
abstract: The lower respiratory tract provides a number of disease targets for gene therapy. Nebulisation is the most practical system for the aerosolisation of non-viral gene delivery systems. The aerosolisation process represents a significant challenge to the maintenance of the physical stability and biological activity of the gene vector. In this study we investigate the role of a condensing polycationic peptide on the stability and efficiency of nebulised lipid-DNA complexes. Complexes prepared from the cationic lipid 1, 2-dioleoyl-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 polycationic peptide, protamine, and pDNA (LPD) at 3:2:1 w/w ratio were nebulised using a Pari LC Plus jet nebuliser. Samples from the nebuliser reservoir (pre- and post-nebulisation) and from the aerosol mist were collected and investigated for changes, including: particle diameter, retention of in-vitro 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 complexes with only those formulated at 12:1 w/w DOTAP:pDNA able to maintain their pre-nebulisation particle size distribution (145+/-3 nm pre-nebulisation vs. 142+/-2 nm aerosol mist) and preserve significant pDNA integrity in the reservoir (35% of pre-nebulisation pDNA band intensity). The LPD complexes were smaller (102+/-1 nm pre-nebulisation vs. 113+/-2 nm aerosol mist) with considerably greater retention of pDNA integrity in the reservoir (90% of pre-nebulisation pDNA band intensity). In contrast the concentration 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 comparatively 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 physical stability and biological activity of nebulised lipid:pDNA complexes can be improved by inclusion of a condensing polycationic peptide such as protamine. The incorporation of the peptide precludes the use of potentially toxic excesses of lipid and charge and may act as a platform for the covalent attachment of peptide signals mediating sub-cellular targetting.
authors.affiliation: Welsh School of Pharmacy, Redwood Building, Cardiff University, King Edward VII Avenue, Cardiff, UK. birchalljf@cf.ac.uk
authors.forename: J C
authors.initials: JC
authors.lastname: Birchall
authors.forename: I W
authors.initials: IW
authors.lastname: Kellaway
authors.forename: M
authors.initials: M
authors.lastname: Gumbleton
mesh_terms: D004247:DNA / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics*; D004337:Drug Carriers; D004355:Drug Stability; D004586:Electrophoresis; D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008055:Lipids; D008081:Liposomes; D008854:Microscopy, Electron; D009330:Nebulizers and Vaporizers; D010316:Particle Size; D010455:Peptides; D011479:Protamines / Q000737:chemistry; D000644:Quaternary Ammonium Compounds; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D004337:Drug Carriers; D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D008055:Lipids; D008081:Liposomes; D010455:Peptides; D011479:Protamines; D000644:Quaternary Ammonium Compounds; D004247:DNA; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane