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

Birchall, James Caradoc ORCID: https://orcid.org/0000-0001-8521-6924, Kellaway, I. W. and Gumbleton, Mark ORCID: https://orcid.org/0000-0002-7386-311X 2000. Physical stability and in-vitro gene expression efficiency of nebulised lipid-peptide-DNA complexes. International Journal of Pharmaceutics 197 (1-2) , pp. 221-231. 10.1016/S0378-5173(00)00339-2

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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 physicalstability 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 nebulisedlipid–DNAcomplexes. 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 physicalstability 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 nebulisedcomplexes was comparatively efficient (LPD aerosol mist 26 vs. 40% for pre-nebulisedcomplex; the respective values for 12: 1 w/w DOTAP:pDNA were 12 vs. 28%). The physicalstability and biological activity of nebulisedlipid: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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Pharmacy
Subjects:	R Medicine > RM Therapeutics. Pharmacology
Uncontrolled Keywords:	Gene therapy; Cationic lipid; Plasmid DNA; Polycationic peptide; Protamine; Nebulisation; Aerosolisation; Lung
Publisher:	Elsevier
Last Modified:	20 Oct 2022 09:31
URI:	https://orca.cardiff.ac.uk/id/eprint/32362

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