Biophysical characterization of PEI/DNA complexes.

The main goal of this study was to determine the effects of polyethylenimine (PEI) molecular weight and structure (750 kDa, 25 kDa, 2 kDa branched, and 25 kDa linear PEI) and the nitrogen/phosphate (N/P) molar ratio on the physical properties and transfection efficiencies of PEI/DNA complexes. Fourier transform infrared spectroscopy revealed that DNA remained in the B conformation when complexed to all PEIs. Unique alterations in the circular dichroism spectra of DNA were observed in the presence of each PEI, whereas differential scanning calorimetry measurements showed that all PEIs examined destabilized supercoiled DNA at N/P < 3/1, but not at higher ratios.

The structure of DNA within cationic lipid/DNA complexes.

The structure of DNA within CLDCs used for gene delivery is controversial. Previous studies using CD have been interpreted to indicate that the DNA is converted from normal B to C form in complexes. This investigation reexamines this interpretation using CD of model complexes, FTIR as well as Raman spectroscopy and molecular dynamics simulations to address this issue.

An infrared spectroscopic study of the effect of hydration on cationic lipid/DNA complexes.

Infrared spectroscopy was used to examine the effect of dehydration on the structure of DNA and cationic lipid/DNA complexes (CLDCs). Information regarding the effect of hydration on the interface between the cationic lipids and DNA was obtained by following subtle but reproducible changes in vibrational bands arising from the DNA bases and phosphate backbone as well as bands from the lipid ester groups within the interfacial region of the bilayer. Dehydration of supercoiled plasmid DNA induces a transition from a B-conformation in solution to a mixed conformation in the dried state.

Enhancements in gene expression by the choice of plasmid DNA formulations containing neutral polymeric excipients.

Formulations containing maltodextrin (2% w/v) were identified to facilitate intramuscular (im) delivery of plasmid DNA in mice using the reporter genes luciferase and chloramphenicol acetyltransferase (CAT) and the therapeutic gene of erythropoietin (EPO) as monitors of transfection efficiency. Even though considerable variability in gene expression was observed in animals, a 5-8-fold enhancement of reporter gene expression was observed with this excipient compared with saline formulations of DNA. In a therapeutically significant experiment, a single im injection of an EPO plasmid formulation containing 2% (w/v) maltodextrin resulted in a significant and prolonged elevation of the hematocrit levels of mice compared with control DNA in saline.

Differential scanning calorimetric studies of the thermal stability of plasmid DNA complexed with cationic lipids and polymers.

The thermal stabilities of supercoiled (SC) and linear/open circular (LIN/OC) forms of plasmid DNA when complexed with cationic lipids or cationic polymers used for cellular transfection were assessed using differential scanning calorimetry. Differences in the stability of SC DNA produced by the cationic lipids DOTAP (1,2-dioleoyltrimethyl ammoniumpropane chloride), DSTAP (1,2-distearyltrimethyl ammoniumpropane chloride), and DDAB (dimethyldioctadecylammonium bromide) upon complexation suggest possible effects of headgroup structure on the stability of SC DNA and minimal effects of lipid acyl chain saturation/unsaturation. Complexation of DNA with the cationic polymers polyethylenimine (PEI) or poly-L-lysine (PLL) (but not poly-L-arginine) resulted in a decreased stability of SC DNA when the DNA was in charge excess, although all polymers stabilized SC DNA when the polymer was in charge excess.