Christopher M. Wiethoff

Infrared Spectroscopic Characterization of the Interaction of Cationic Lipids with Plasmid DNA

Fourier transform infrared spectroscopy was used to characterize the interaction of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane and dioctadecyldimethylammonium bromide with plasmid DNA. The effect of incorporating the neutral colipids cholesterol and dioleoylphosphatidylethanolamine on this interaction was also examined. Additionally, dynamic and phase analysis light scattering were used to monitor the size and ζ potential of the resulting complexes under conditions similar to the Fourier transform infrared measurements. Results suggest that upon interaction of cationic lipids with DNA, the DNA remains in the B form. Distinct changes in the frequency of several infrared bands arising from the DNA bases, however, suggest perturbation of their hydration upon interaction with cationic lipids. A direct interaction of the lipid ammonium headgroup with and dehydration of the DNA phosphate is observed when DNA is complexed with these lipids. Changes in the apolar regions of the lipid bilayer are minimal, whereas the interfacial regions of the membrane show changes in hydration or molecular packing. Incorporation of helper lipids into the cationic membranes results in increased conformational disorder of the apolar region and further dehydration of the interfacial region. Changes in the hydration of the DNA bases were also observed as the molar ratio of helper lipid in the membranes was increased.

Characterization of cationic vector-based gene delivery vehicles using isothermal titration and differential scanning calorimetry.

Within the past 10 years, major advances in the design and development of differential scanning calorimeters (DSC) (1) and isothermal titration calorimeters (ITC) (2) have resulted in an unparalleled level of sensitivity, stability, and reproducibility in calorimetric measurements of large molecules. These improvements have allowed the thermal stability and ligand binding processes of biological macromolecules to be thermodynamically characterized with speed, accuracy, and convenience. With their increasing commercial availability, experiments that were previously limited to specialist calorimetry laboratories can now be routinely performed by most investigators.

Light-Scattering Techniques for Characterization of Synthetic Gene Therapy Vectors

The colloidal properties of delivery systems currently being developed for nonviral gene therapy are extremely important. The physical stability of these systems on the shelf, as well as in the biological milieu, is mostly based on their size and interfacial properties. The size and surface charge of these systems can also have dramatic effects on their biological activity (1-4). With these facts in mind, it is apparent that adequate characterization of these properties is necessary for the development of any synthetic gene delivery system.

Characterization of Synthetic Gene Delivery Vectors by Infrared Spectroscopy

For many decades, infrared (IR) spectroscopy has been used to characterize the structure of molecules. In IR spectroscopy, absorption of light, corresponding to vibrational and rotational transitions of a molecule, is measured. For a transition to be IR-active, a change in the dipole moment of a particular bond must occur upon excitation. This vibrational energy is not only dependent on the chemical nature of the particular covalent bonds, but also on the environment of these coupled atoms and bonds. IR spectroscopy has been previously employed in the study of the structure of nucleic acids, producing not only information about the individual bases, sugars, and phosphate backbone, but also providing information about the helical conformation of polynucleotides (1-3). IR spectroscopy has also been successfully applied to the analysis of lipids, as well as to numerous other polymers (4). Thus, IR spectroscopy potentially possesses the ability to obtain structural information about all of the components of most synthetic gene delivery complexes, as well as changes in the structure of polymeric or lipid components upon complex formation. In addition to the ability to gather detailed structural information, there are also some practical advantages to the use of IR spectroscopy for the study of plas-mid DNA and DNA complexes compared to other techniques, including the availability of a variety of sampling techniques, permitting the analysis of samples in a wide variety of physical states including solutions, solids, and gels. There is also no upper limit to the size of the sample molecule examined, allowing both short oligonucleotides and higher molecular weight DNA to be studied. IR spectroscopy is not a destructive technique, and requires only small amounts of material, making it ideal for the analysis of valuable samples.