| Synthetic vectors are an attractive alternative form of gene therapy with minimal or no immune responses. However, generating a therapeutic effect has been difficult to achieve. In an effort to enhance transfection efficiency, most studies have been focusing on targeting, increasing the circulation time, tracing the intracellular pathway, examining the tissue distribution, determining the DNA complex structures, designing gene expression vectors, and synthesizing gene carriers. Targeting and enhancing the circulation time involve surface modifications of synthetic vectors even though the basic formation process is not well understood. The determination of the intracellular pathway and tissue distribution are essential parts to understanding the delivery process, but the lack of physical characterizations limits the usefulness of data. Structural characterizations are important, but most undermine the work by failing to correlate with transfection efficiency. Synthesis of novel gene carriers and expression systems has improved transfection efficiency, but the current screening method is based mostly on a trial and error process that is time consuming, expensive, and arduous. Thus, this research investigates the formation process, examines stability, characterizes vectors properties, and develops a systemic characterization approach to identify potential vectors. The formation of DNA/poly-L-lysine and DNA/cationic liposomes systems is probed by time-resolved multiangle laser light scattering. Two kinetic pathways are observed and found to depend on mass concentration or charge ratio. The kinetic rates of DNA/cationic liposomes lipoplex are determined and found to increase with DNA concentration. By monitoring the physical properties changes, the experimental results indicate the lipoplex instability during long-term storage is actually due to a degradation process, as opposed to the universally assumed aggregation dilemma. Thus, the results provide new insights for designing synthetic vectors with greater stability. Experimental studies demonstrate the DNA loading correlates with gene expression efficiency, even when transfection occurs in the presence of serum. DNA loading was also investigated to determine its role in transfection efficiency for the DOTAP/DOPE lipoplex system with distinct microstructures. The varying transfection efficiencies reported for distinct microstructures are found to also correlate with DNA loading. Thus, there may be a universal characteristic parameter to facilitate the identification of promising synthetic vectors formulations. |
