The spatial distribution of lipophilic cations in gradient copolymers regulates pDNA binding interactions, polyplex aggregation, and transgene expression

Synthetic polymers—chemically versatile and affordable materials—are promising nanocarriers for the intracellular delivery of nucleic acids. Copolymers comprising of lipophilic cations and neutral hydrophilic co-monomers effectively complex and deliver bulky nucleic acid payloads such as plasmids (pDNA). In this work, we demonstrate that the spatial distribution of lipophilic cations governs the complexation pathways, serum stability, and biological performance of polymer–pDNA complexes (polyplexes). Hitherto, investigators focused predominantly on block and statistical copolymers while largely ignoring gradient copolymers, where the density of lipophilic cations diminishes (gradually or steeply) along polymer backbones. Our goal is to engineer gradient copolymers that combine the colloidal stability of polyplexes formed from block copolymers with the high transfection efficacy of statistical copolymers. We synthesized length- and compositionally-equivalent gradient copolymers (G1–G3) via reversible addition fragmentation chain transfer polymerization in addition to equivalent statistical (S) and block (B) copolymers. We mapped microstructure-dependent differences in pDNA loading per polyplex, pDNA conformational changes, and polymer–pDNA binding thermodynamics via static light scattering, circular dichroism spectroscopy, and isothermal titration calorimetry, respectively. B exhibited vastly different pDNA complexation profiles from the other four copolymers while loading the most pDNA per polyplex. Further, we discovered that subtle modulation of gradient steepness effectively negotiates trade-offs among pDNA delivery efficiency, cytotoxicity, and colloidal stability in serum. For instance, G1 overcame the colloidal instability of S polyplexes in serum, while maintaining comparable transfection efficiency and cell viability. Microstructural contrasts did not elicit differences in complement activation but governed polycation-triggered hemolysis. Our work demonstrates that the spatial distribution of lipophilic cations is an effective, albeit underutilized, design handle to improve polyplex physical properties and pDNA delivery capacity.