N/P-Dependent DNA Complexation, Transfection, and Cytotoxicity of Imine-Linked Low-Molecular-Weight PEI Polyplexes

Gene delivery with cationic polymers requires balancing DNA compaction, colloidal stability, and intracellular release, yet for imine-linked low-molecular-weight polyethyleneimine (PEI) vectors, quantitative relationships connecting the N/P ratio with the full property–transfection cascade remain undefined. Here, two amphiphilic non-viral vectors were prepared by linking a hydrophobic benzene–siloxane core (TAS) to hyperbranched PEI (800 or 2000 Da) through reversible imine bonds and complexed with DNA across a broad N/P range (10–600). Polyplexes were characterized by atomic force microscopy (AFM), dynamic light scattering (DLS), ζ-potential, agarose gel electrophoresis, transfection via green fluorescent protein (GFP) imaging and luciferase assay in HeLa cells. Both vectors formed spherical nano-entities (AFM diameters ~30 nm for TAS-PEI800; ~100 nm for TAS-PEI2000). TAS-PEI2000 achieved complete DNA retardation at N/P ≈ 30 versus N/P ≈ 150 for TAS-PEI800, consistent with its higher charge density (ζ = +37.59 vs. +18.35 mV). Transfection efficiency was superior for TAS-PEI2000 across most N/P ratios; however, TAS-PEI2000 displayed an optimal transfection efficiency at N/P ≈ 100 (ζ ≈ 3.84 mV), beyond which efficiency declined, indicating a binding–release trade-off. Cell viability remained >77% across the N/P range for TAS-PEI800, but dropped below 25% at N/P ≥ 400 for TAS-PEI2000. A phenomenological logistic model identified characteristic transition thresholds (θ ≈ 60 for TAS-PEI800; θ ≈ 40 for TAS-PEI2000), capturing the onset of cooperative self-assembly; however, the post-optimum decline observed for TAS-PEI2000 requires additional inhibitory terms. These findings demonstrate that PEI molecular weight governs both the N/P threshold required for efficient transfection and the width of the therapeutic window, thereby providing structure–activity descriptors for the rational design of imine-linked polyplex systems.