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. Lipophilic polycations with incorporated hydrophilic co-monomers effectively complex and deliver bulky nu- cleic acid payloads such as plasmids (pDNA). The spatial distribution of lipophilic cations and neutral, hydrophilic repeat units governs the pDNA binding, serum stability, pDNA delivery efficiency, and cytocompatibility of polymer–pDNA complexes, 1 or polyplexes. Yet, investigators have focused predominantly on block and statistical copolymers while largely ignoring gradient copolymers, where the density of lipophilic cations diminishes gradually along polymer backbones. Seeking to obtain gradient copolymers that combine the colloidal stability of block copolymers with the high trans-fection 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) copolymer references). We mapped microstructure-dependent differences in polymer–pDNA, pDNA loading per polyplex, pDNA conformational changes, and binding thermodynamics via static light scattering, circular dichroism spectroscopy, and isothermal titration calorime- try, respectively. B exhibited vastly different binding profiles compared to the other four copolymers while having the highest pDNA loading capacity. Further, we discovered that subtle modulation of gradient steepness is an effective strategy to negotiate 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. Similarly, G1 showed the highest TxV value (from Pareto front analysis), indicating that lipophilic cation distribution in copolymer microstructure promotes cell viability along with high transfection. Microstructural con- trasts did not elicit differences in complement activation, while S triggered the highest hemolysis. Our work demonstrates that the spatial distribution of lipophilic cations is an effective, albeit underutilized, design handle to improve the physical properties and biological performance of polymeric gene carriers.