Gene therapy has emerged as a significant advancement in medicine in recent years. However, the development of effective gene delivery vectors, particularly polymer vectors, remains a significant challenge. Limited understanding of the internal structure of polymer vectors has hindered efforts to enhance their efficiency. This work focuses on investigating the impact of polymer structure on gene delivery, using the well-known polymeric vector poly(β-amino ester) (PAE) as a case study. For the first time, we revealed the distinct characteristics of individual polymer components and their synergistic effects-the appropriate combination of different components within a polymer (high MW and low MW components) on gene delivery. Additionally, artificial intelligence (AI) analysis was employed to decipher the relationship between the polymer component distribution (PCD) and gene transfection performance. Guided by this analysis, a series of highly efficient polymer vectors that outperform current commercial reagents such as jetPEI and Lipo3000 were developed, among which the transfection efficiency of the PAE-B1-based polyplex was approximately 1.5 times that of Lipo3000 and 2 times that of jetPEI in U251 cells.

The lack of safe and efficient delivery vectors remains a major bottleneck in nucleic acid therapeutics, limiting clinical translation. In this study, by incorporating the synergistic effects of guanidinium and tertiary amine functional groups, a new versatile branched poly­(β-amino ester) (PAE)-based delivery system was designed and developed, resulting in enhanced nucleic acid (DNA/mRNA) delivery. The synergistic interactions between these functional groups were systematically analyzed using 2D-NMR, AFM, TEM, and photophysical characterization. The results show that self-assembly occurs when polymers containing different functional groups are mixed, which changes the interactions between molecules and significantly affects the structure of the polyplex. This synergy was found to significantly enhance cellular uptake, contributing to improved transfection efficiency. Compared to the leading commercial reagents (Lipofectamine 3000, jetPEI, Xfect, and Lipofectamine MessengerMAX), the optimized polymeric vector formulations demonstrated superior transfection efficiency in vitro with both DNA and mRNA. Their high transfection performance was also confirmed in vivo, along with reduced cytotoxicity and tunable organ-targeting property.

Cationic modification of hyaluronic acid (HA) is challenging due to its polyanionic nature, poor reactivity in water, and the instability of conventional coupling intermediates. This limits the development of HA-based components in non-viral gene delivery systems, which already suffer from amorphous morphology and mechanical fragility that reduce their transfection efficiency. Here, we reprogram a classically unfavorable EDAC-mediated rearrangement into a productive synthetic route, enabling direct cationization of hyaluronic acid (HA) through spontaneous O-acylisourea rearrangement. This water-based, catalyst-free process achieves up to 70 % substitution of HA's carboxyl groups—introducing cationic tertiary amine functionalities in water. The resulting aminated-hyaluronic acid (HAA) scaffolds act as rigid structural backbones in virus-inspired polymer–DNA nanoparticles termed as “Skeletoplexes”, with enhanced stability and performance. When incorporated into polyplexes formed from diverse cationic systems—including poly(β-amino esters) and commercial vectors such as BrPERfect, Xfect, jetPEI, and Lipofectamine3000—HAA scaffolds improved in vitro transfection efficiency by up to 4-fold and in vivo gene expression by approximately 2-fold. These results establish a generalizable and green scaffold-based strategy that bridges the structural and functional gap between viral and non-viral gene delivery vectors.

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.