On the Fractionation and Physicochemical Characterisation of Self-Assembled Chitosan-DNA Polyelectrolyte Complexes

Chitosan is extensively studied as a carrier for gene delivery and is an attractive non-viral gene vector owing to its polycationic, biodegradable, and biocompatible nature. Thus, it is essential to understand the chemistry of self-assembled chitosan-DNA complexation and their structural and functional properties, enabling the formation of an effective non-viral gene delivery system. In this study, two parent chitosans (samples NAS-032 and NAS-075; Mw range ~118-164 kDa) and their depolymerised derivatives (deploy nas-032 and deploy nas-075; Mw range 6-14 kDa) with degrees of acetylation 43.4 and 4.7%, respectively, were used to form polyelectrolyte complexes (PECs) with DNA at varying [-NH₃⁺]/[-PO₄^-] (N/P) molar charge ratios. We investigated the formation of the PECs using ζ-potential, asymmetric flow field-flow fractionation (AF4) coupled with multiangle light scattering (MALS), refractive index (RI), ultraviolet (UV) and dynamic light scattering (DLS) detectors, and TEM imaging. PEC formation was confirmed by ζ-potential measurements that shifted from negative to positive values at N/P ratio ~2. The radius of gyration (R) was determined for the eluting fractions by AF4-MALS-RI-UV, while the corresponding hydrodynamic radius (R), by the DLS data. We studied the influence of different cross-flow rates on AF4 elution patterns for PECs obtained at N/P ratios 5, 10, and 20. The determined rho shape factor (ρ = R/R) values for the various PECs corresponded with a sphere morphology (ρ ~0.77-0.85), which was consistent with TEM images. The results of this study represent a further step towards the characterisation of chitosan-DNA PECs by the use of multi-detection AF4 as an important tool to fractionate and infer aspects of their morphology.