Analysis of Self-Assembled Cationic Lipid−DNA Gene Carrier Complexes Using Flow Field-Flow Fractionation and Light Scattering

• Published in: Analytical chemistry (Washington) Vol. 73; no. 4; pp. 837 - 843
• Main Authors: Lee, Hookeun, Williams, S. Kim Ratanathanawongs, Allison, S. Dean, Anchordoquy, Thomas J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15-02-2001
• Subjects: Biological and medical sciences; Biotechnology; Chemistry; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA - genetics; Drug Carriers; Fundamental and applied biological sciences. Psychology; Gene therapy; Gene Transfer Techniques; Genes; Geology; Health. Pharmaceutical industry; Industrial applications and implications. Economical aspects; Light; Lipids; Lipids - chemistry; Scattering, Radiation; Spectrophotometry, Ultraviolet; Cationic complex; Field flow fractionation; Transfection; Structure activity relation; DNA; Light scattering; Lipids; Gene therapy; Physical properties; Vector
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac000831n

Self-assembled cationic lipid−DNA complexes have shown an ability to facilitate the delivery of heterologous DNA across outer cell membranes and nuclear membranes (transfection) for gene therapy applications. While the size of the complex and the surface charge (which is a function of the lipid-to-DNA mass ratio) are important factors that determine transfection efficiency, lipid−DNA complex preparations are heterogeneous with respect to particle size and net charge. This heterogeneity contributes to the low transfection efficiency and instability of cationic lipid−DNA vectors. Efforts to define structure−activity relations and stable vector populations have been hampered by the lack of analytical techniques that can separate this type of particle and analyze both the physical characteristics and biological activity of the resulting fractions. In this study, we investigated the feasibility of flow field-flow fractionation (flow FFF) to separate cationic lipid−DNA complexes prepared at various lipid−DNA ratios. The compatibility of the lipid−DNA particles with several combinations of FFF carrier liquids and channel membranes was assessed. In addition, changes in elution profiles (or size distributions) were monitored as a function of time using on-line ultraviolet, multiangle light scattering, and refractive index detectors. Multiangle light scattering detected the formation of particle aggregates during storage, which were not observed with the other detectors. In comparison to population-averaged techniques, such as photon correlation spectroscopy, flow FFF allows a detailed examination of subtle changes in the physical properties of nonviral vectors and provides a basis for the definition of structure−activity relations for this novel class of pharmaceutical agents.