Hyperbranched poly(β-amino ester) (HPAE) is identified as a unique non-viral carrier capable of sustaining high-efficiency transfection under elevated plasmid concentrations, overcoming the aggregation and toxicity limitations of conventional lipid and PEI reagents. We demonstrate that transfection enhancement is driven by concentration-dependent synergism between membrane accumulation and endosomal escape. Guided by this mechanism, a half-volume transfection strategy was established to transiently elevate plasmid concentration without compromising cell viability, enabling superior lentivirus yield and purity. These findings define plasmid concentration as a previously overlooked regulatory axis in nanoparticle-mediated gene delivery and position HPAE as a high-performance platform for scalable therapeutic vector production.