Dissecting the barriers to PDMAEMA-mediated gene delivery: molecular weight drives efficiency while endosomal escape limits potential

Efficient intracellular delivery remains a major bottleneck in nucleic acid–based therapeutics. Among non-viral vectors, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) offers tunable cationic properties and synthetic versatility, yet its limited endosomal escape capacity constrains transfection efficiency (TE). In this work, PDMAEMA-based polyplexes were systematically investigated to elucidate how molecular weight (MW), macromolecular architecture (linear vs. star-shaped), and hydrophobic modification with poly(ε-caprolactone) (PCL) influence gene delivery performance. To this end, a library of cationic polymers was synthesized via Atom Transfer Radical Polymerization (ATRP) and characterized in terms of DNA condensation ability, polyplex physicochemical properties, cytotoxicity (CT), and TE. Linear PDMAEMA (L-PDMAEMA) in the medium- and high-MW ranges (10-20 kDa) provided the optimal balance between charge density and colloidal stability, achieving TE comparable to the benchmark 25 kDa branched polyethyleneimine ( b PEI) while exhibiting significantly lower CT. Conversely, star-shaped and micellar architectures exhibited reduced TE, likely due to steric hindrance affecting charge accessibility. To overcome endosomal entrapment, chloroquine (CQ) was employed as an endosomal escape enhancer, resulting in up to a 30-fold increase in TE for L-PDMAEMA without measurably affecting cell viability. Overall, this study demonstrates that rational tuning of polymer topology and MW enables PDMAEMA-based vectors to combine high TE with minimal CT, emphasizing the potential of integrating intrinsic endosomal escape functionality in future polymer designs.