Ultra-high gene transfection efficiency in suspension cells mediated by highly branched-linear poly(β-amino ester)s

Suspension cells, particularly 293F cells, are widely used for the production of therapeutic proteins, antibodies, virus-like particles (VLPs) for vaccines, and viral vectors for gene and cell therapies. However, DNA-encapsulated polyplexes typically exhibit poor interaction with the cellular membrane of suspension cells, resulting in low cellular uptake and transfection efficiency, which underscores the need for the development of more effective gene delivery systems for suspension cells. Molecular weight (MW) and topological structure are two of the most critical structural parameters that indicate the gene transfection performance of highly branched-linear poly(β-amino ester)s (H-LPAEs). Herein, we developed a series of novel H-LPAEs with varying MWs and branched structures through a two-step linear oligomer combination and branching strategy. Results demonstrate that both MW and branched structure significantly affect the multiple mechanistic steps of gene transfection. H-LPAEs with intermediate MW exhibited strong DNA binding affinity, leading to the formation of H-LPAE/DNA polyplexes with small size, favorable positive Zeta potential, high cellular uptake, and effective endosomal escape capability. Importantly, H-LPAEs 11.5 kDa achieved gene transfection efficiency of up to 84.1% and 84.5% in 293T cells and suspended human embryonic kidney cells (293F), respectively, while maintaining excellent biocompatibility. This study highlights H-LPAEs with intermediate MW provide a strong benchmark for effective transfection of both adherent and suspension cells, making them promising candidates for gene therapy.