Electrostatic interactions between the polycationic chitosan and polyanionic DNA biopolymers lead to the formation of self-assembled hybrid nanostructures known as polyelectrolyte complexes (PECs). These nanocomplexes have a great potential as non-viral vectors for the effective intracellular delivery of genetic materials. In the framework of this Thesis, the self-assembly of chitosan and DNA was explored and the physical and chemical properties of the resulting polyelectrolyte complexes were investigated. For this purpose, the effect of important parameters of chitosan (degree of acetylation, DA and molecular weight, Mw) and DNA [single-stranded (ss) vs 3-dimensional DNA nanotube origami] on PEC formation were studied. Here, DNA origami is the art of folding DNA into distinct 2D/3D structures and in this study a tubular origami comprising of six helices, known as six-helix bundle (6-HB) DNA origami was used. The two medium molecular weight parent chitosan polymers, NAS-032 (high DA: 43.4%) and NAS-075 (low DA: 4.7%) and their depolymerised low molecular weight (depoly NAS-032 and depoly NAS-075) derivatives were fully characterised and used to form PECs at various amine to phosphate molar charge mixing ratios (N/P). The self-assembled nanostructures were physico-chemically characterised using various techniques. The formation of the PECs was probed by ζ-potential measurements. The charge of the PECs shifted from negative to positive potential at N/P ~ 2, as expected from the charge compensation of the polyelectrolytes that drives the electrostatic association of chitosan and ssDNA or 6-HB DNA origami. The radius of gyration (Rg) was determined via asymmetric flow field-flow fractionation (AF4) coupled with multi-angle light scattering (MALS), ultra-violet (UV) and refractive index (RI) detectors. In addition, the hydrodynamic radius (Rh) was obtained through dynamic light scattering (DLS) used in bulk mode and in flow-mode detection (fitted to AF4). First, the influence of different cross-flow rates (1 and 3 mL/min) on the AF4 elution profiles of PEC formed by chitosan-DNA was assessed with no significant impact on AF4 fractionation and characterisation. The shape factor (ρ = Rg/Rh) for these PEC was between ~ 0.77-0.85, corresponding to a spherical conformation, that was verified by transmission electron microscopy (TEM). While chitosan-6HB PEC resulted in ρ > 2 for most of the PEC, consistent with rod shape of high axial ratio conformation, in agreement with elongated fibrillary structures on TEM images. Moreover, 6-HB after complexation with chitosan showed an increase in its resistance to DNase degradation. Furthermore, chitosan-6HB hybrid structures were also assembled and characterised by complexing the chitosan (polymers/oligomers) with ssM13mp18 DNA scaffold prior to origami self-assembly/folding thus providing a new layer of control on complexation. The AF4 elution profiles for these hybrid self-assembled structures formed by depolymerised chitosan oligomers and parent chitosan polymers were considerably different to those obtained in PECs of chitosan (polymers) already formed 6-HB PECs. In a separate study, a novel DNA origami in the shape of a violin bow was designed and assembled using a customised sequence specific and M13mp18 scaffold, where well-formed structures were obtained by the latter. In general, through this Thesis we have gained insights on the structure-function relationship of PECs, which can be harnessed to improve and facilitate in optimising a potential gene delivery system. Moreover, the use of DNA in the form of an origami to form PEC with chitosan provides an additional level of high-order control on the formation and design process.
