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Biotechnology and Applied Biochemistry (2003) 37, (73–81) (Printed in Great Britain)

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Preparation of small unilamellar vesicles (SUV) and biophysical characterization of their complexes with poly-L-lysine-condensed plasmid DNA

Leigh A. Maguire, Hu Zhang and P. Ayazi Shamlou¹

Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, U.K.

Key words: DNA vaccines, gene therapy, homogenization, liposomes, poly-L-lysine–DNA complexes.

Abbreviations used: MLV, multilamellar large vesicles; SUV, small unilamellar vesicles; PLL, poly-L-lysine; pDNA, plasmid DNA; PDL, PLL-condensed pDNA (PLL-DNA)–liposome complex; CFD, computational fluid dynamics.

¹To whom correspondence should be addressed (e-mail p.shamlou@ucl.ac.uk).

Liposomes have numerous applications in the (bio)pharmaceutical industries as agents in the synthesis of new biomaterials for use in areas including gene delivery. There is currently a need to establish efficient scaleable methods for the manufacture of liposomes, and in the present paper we describe the operation of a new high-velocity jet homogenizer for downsizing of multilamellar large vesicles to produce small unilamellar vesicles (SUV). Measurements of size distribution of SUVs are presented and compared with mathematical simulations based on the solution of a population balance equation combined with computational-fluid-dynamics analysis of flow in the homogenizer. Anionic SUVs are produced by the new method and incubated with poly-L-lysine (PLL)-condensed plasmid DNA (pDNA) to generate complexes under different physico-chemical conditions. The colloidal properties of the resulting complexes, including their size and charge, are measured using a Zetasizer and the encapsulation efficiency is obtained experimentally using a Pico Green assay. The results show that between 85 and 95% of the PLL-pDNA condensed plasmids were encapsulated by the liposomes, the smaller liposomes being more effective in encapsulating the complexes.

Received 5 November 2002/12 December 2002; accepted 16 December 2002

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