The Spatial Distribution of Lipophilic Cations in Gradient Copolymers Regulates Polymer-pDNA Complexation, Polyplex...

Here, we demonstrate that the spatial distribution of lipophilic cations governs the complexation pathways, serum stability, and biological performance of polymer-pDNA complexes (polyplexes). Previous research focused on block/statistical copolymers, whereas gradient copolymers, where the density of lipophilic cations diminishes (gradually or steeply) along polymer backbones, remain underexplored. We engineered gradient copolymers that combine the polyplex colloidal stability of block copolymers with the transfection efficiency of statistical copolymers. We synthesized length- and compositionally equivalent gradient copolymers (G1-G3) along with statistical (S) and block (B) copolymers of 2-(diisopropylamino)ethyl methacrylate and 2-hydroxyethyl methacrylate. We mapped how polymer microstructure governs pDNA loading per polyplex, pDNA conformational changes, and polymer-pDNA binding thermodynamics via static light scattering, circular dichroism spectroscopy, and isothermal titration calorimetry, respectively. While gradient steepness is a powerful design handle to improve polyplex physical properties, augment pDNA delivery capacity, and attenuate polycation-triggered hemolysis, microstructural contrasts did not elicit differences in complement activation.