Lee, Li Kim; (2003) Biophysical studies of synthetic gene delivery systems. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Driven by the rapid development of therapeutic applications for synthetic gene delivery systems, specifically formulations of plasmid DNA with cationic lipids and/or peptides, the characterisation of DNA complexes has become an important area of research. Current efforts to increase their transfection efficiency has led to the development of analytical techniques for a better understanding of how the structure, stability and biological activity of DNA complexes are affected by their environment. The effects of ionic strength and pH on the colloidal stability and structure of synthetic gene delivery systems were investigated through salt-induced aggregation of poly-L-lysine/DNA complexes. A two-syringe pump flow system was used to reproducibly prepare the polyplexes. Analysis of the mean hydrodynamic diameter using dynamic light scattering showed that the aggregation rate and polydispersity of the polyplexes increased with ionic strength and pH. Polyplexes with an average diameter of 100-150 nm aggregated to 1500-3000 nm over 1.5 h. Computer simulations using equations based on the DLVO theory were found to adequately describe the stability of the polyplexes. These simulations form the basis for further work on theoretical predictions of the stability of DNA complexes. The concept of DNA complexes as aggregate structures with fractal dimensions was introduced. Static light scattering revealed fractal dimensions of 2.22 and 1.44 for polyplexes at 50-150 and 1000 mM NaCl, respectively. These values correspond to slow and rapid aggregation regimes, respectively. Biophysical characterisation of a novel receptor-mediated system, Lipofectin/integrin- targeting peptide/DNA, with potential for targeted gene therapy in vivo, was conducted. Under physiological ionic strength, lipopolyplexes at 6.8 charge ratio demonstrated instability (average size > 1000 nm) and decreased in vitro transfection activity. A PicoGreen assay showed complete binding of the DNA at a charge ratio of 3.0-4.0, corresponding to maximum expression. Lipopolyplexes at high DNA concentrations (150 μg/ml) in distilled water and 5% dextrose possessed small sizes (110 ± 35 nm and 145 ± 35 nm, respectively), but aggregated upon contact with salt or serum albumin.

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