Sustainable polysaccharide-poly(2-oxazoline) gene-delivery platforms enhanced by free polyethyleneimine chains

Developing safe and efficient non-viral vectors remains a major challenge in nucleic acid delivery, particularly for applications that demand minimal cytotoxicity, including ocular therapies. Here, we introduce a series of hybrid gene-delivery platforms based on permanently cationic polysaccharides (dextran and chitosan) grafted with poly(2-oxazoline) (POx) and supplemented by small amounts of free branched polyethyleneimine (BPEI). The introduction of permanent positive charges onto dextran and chitosan backbones enabled strong DNA binding and polymer-DNA complex formation, while grafted poly(2-oxazoline) segments improved the supramolecular stability of the carriers in serum medium. The further incorporation of low levels of free (non-complexed) BPEI chains markedly enhances transfection efficiency without compromising cell viability, in contrast to the well-known cytotoxicity of BPEI used alone. The free BPEI chains possibly play a dominant role in promoting endosomal escape and intracellular DNA release, whereas they are not required for initial DNA condensation. This modular and sustainable formulation strategy enables high gene expression with significantly reduced toxicity and offers a versatile platform for nucleic acid delivery. The approach is particularly promising for gene therapy where safety and performance must be simultaneously optimized.