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. Isothermal titration calorimetry revealed the existence of protonation changes at low ionic strength due to possible shifts in pK(a) of the ionizable groups of PEI during complex formation. Twenty-five kilodalton branched and 25 kDa linear PEI complexes showed the highest transfection efficiencies at an N/P ratio of 6:1 in COS-7 and CHO-K1 cells, respectively. These investigations have detected alterations in the physical and colloidal properties of the complexes that were sensitive to polymer structure, molecular weight, and polymer/DNA ratio, but these properties did not directly correlate with their transfection efficiencies. To further probe any possible relationship between these parameters and activity, a more refined biophysical analysis of any subpopulations in these samples that may differ in transfection activity is suggested, although the existence of such species remains unknown.