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Preparation of Dry Powder Dispersions for Non-viral Gene Delivery by Freeze-drying and Spray-drying

Seville, Peter C. ORCID: https://orcid.org/0000-0001-8546-3474, Kellaway, I.W. and Birchall, J.C. (2002) Preparation of Dry Powder Dispersions for Non-viral Gene Delivery by Freeze-drying and Spray-drying. Journal of Gene Medicine, 4 (4). pp. 428-437. ISSN Online: 1521-2254

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Abstract

BACKGROUND: Dry powder dispersion devices offer potential for delivering therapeutic macromolecules to the pulmonary epithelia. Previously, freeze-drying (lyophilisation) has been the accepted method for preparing dried formulations of proteins and non-viral gene vectors despite the respirability of such powders being inadequate without further processing. In this study we compare the utility of freeze-drying and spray-drying, a one-step process for producing dry and respirable powders, as methods for preparing non-viral respiratory gene delivery systems. METHODS: Lipid:polycation:pDNA (LPD) vectors comprising 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), protamine sulphate and pEGFP-N1 in 3% lactose solution were either snap-frozen and lyophilised or spray-dried. Lyophilised powder was used as recovered or following coarse grinding. Structural integrity of dehydrated pDNA was assessed by agarose gel electrophoresis and powder particle size determined by laser diffraction. The apparent structure of the systems was visualised by scanning and transmission electron microscopy with the biological functionality quantified in vitro (A549 human lung epithelial cell line) by Green Fluorescent Protein (GFP) associated fluorescence. RESULTS: Lyophilisation produced large, irregularly shaped particles prior to (mean diameter approximately 21 microm) and following (mean diameter approximately 18 microm) coarse grinding. Spray-drying produced uniformly shaped spherical particles (mean diameter approximately 4 microm). All dehydrated formulations mediated reporter gene expression in A549 cells with the spray-dried formulation generally proving superior even when compared with freshly prepared LPD complexes. Biological functionality of the LPD dry powders was not adversely affected following 3 months storage. CONCLUSIONS: Spray-drying has utility for producing stable, efficient and potentially respirable non-viral dry powder systems for respiratory gene delivery.

Item Type: Article
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Uncontrolled Keywords: DNA, dry powder, freeze-drying, gene delivery, non-viral, spray-drying
Divisions: Academic Departments > Institute of Science and the Environment
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Depositing User: Peter Seville
Date Deposited: 17 Jun 2019 09:42
Last Modified: 17 Jun 2019 09:42
URI: https://eprints.worc.ac.uk/id/eprint/8196

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