Precise control of branch unit location and its impact on gene delivery

Controlling the internal structure of polymeric gene vectors remains a significant challenge, posing a critical barrier to understanding structure–performance relationships and advancing the design of efficient gene delivery systems. In this study, we report the synthesis of three types of branched poly(β-amino ester) polymers (KB1, KB2, and KB3), each exhibiting distinct distributions of branched monomer units, using a kinetic branching control strategy. The different polymer topologies were systematically characterized and confirmed through 2D-NMR, AFM, and photophysical analyses. Their topology-induced interactions with DNA were revealed using both experimental methods and molecular dynamics (MD) simulations. The effect of these structural variations on gene delivery performance was elucidated through in vitro assessments across various cells. Among the polymers, KB2—featuring a densely compact structure with branched units primarily concentrated at the macromolecular center—demonstrated superior DNA binding efficiency, nanoparticle stability, and transfection capability. Notably, KB2 outperformed the widely used transfection reagent Lipofectamine 3000 in transfection efficiency across multiple cell types.