Combined Analysis of Polycation/ODN Polyplexes by Analytical Ultracentrifugation and Dynamic Light Scattering Reveals their Size, Refractive Index Increment, Stoichiometry, Porosity, and Molecular Weight

Analytical ultracentrifugation (AUC) and dynamic light scattering (DLS) were combined to characterize polyplexes formed with 10 kDa chitosan or 10 kDa PEI and oligodeoxynucleotides (ODN). Combined analysis revealed that both polyplexes were highly porous (over 80%) and that their weight average hydrodynamic diameters were of 46 and 55 nm for chitosan/ODN and PEI/ODN complexes, respectively. Transformation of the sedimentation coefficient distribution to a size and molecular weight distribution gave an average molecular weight of 19 and 29 MDa for chitosan and PEI polyplexes, respectively. Data from AUC also allowed for the calculation of the actual dn/dc and N/P ratios of each polyplex. Additional data from scanning electron microscopy and static light scattering confirmed the conclusions that were initially derived from AUC and DLS, thus validating that the combination of AUC and DLS is a powerful approach to characterize polyplexes in terms of refractive index increment, size, and molecular weight distributions, as well as porosity.