Revisit complexation between DNA and polyethylenimine — Effect of length of free polycationic chains on gene transfection

Purpose: PEI is one of the best nonviral gene delivery vectors, but why it works so well remains unclear. In particular, the role of free, uncomplexed PEI chains and their length in transfection, cellular uptake, and endosomal escape has not been quantitatively resolved, and the “proton sponge” mechanism is increasingly questioned.

Hypothesis: If DNA is fully complexed by PEI at N:P ≈ 3, then excess PEI chains remain free in solution; these free chains—especially long ones—are the major contributors to efficient gene transfection by increasing cellular uptake, preventing endolysosome maturation, and promoting endosomal escape, with the proton sponge effect playing only a partial role.

Aims: Re-examine DNA/PEI complexation as a function of N:P ratio and PEI chain length. - Separate the effects of bound versus free PEI chains on transfection efficiency. - Determine how free chain length affects cellular uptake kinetics. - Investigate intracellular trafficking, endosomal escape, and the contribution of the proton sponge effect. - Propose a mechanism linking free PEI chain length to transfection and cytotoxicity.

Delivery system: Platform: DNA/PEI polyplexes for nonviral gene delivery. - Polymers: Branched PEIs: bPEI-0.8K, bPEI-2K, bPEI-25K. Linear PEIs: lPEI-2.5K, lPEI-25K. - Payload: Plasmid pGL3-control vector encoding firefly luciferase; Cy5-labeled pGL3 for uptake; FITC/Cy5 double-labeled pGL3 for intracellular pH. - Formulation: DNA/PEI polyplexes formed at various N:P ratios in water or PBS. Key condition: N:P ≈ 3 for complete DNA condensation; free PEI added to reach total N:P = 10. - Targeting ligand: None.

Approach: In vitro only: 293T cells used for transfection, cytotoxicity, uptake, and pH measurements; HeLa cells used in supplementary confirmation. - Transfection: 0.4 µg DNA per well in 48-well plates; luciferase expression measured 48 h post-administration. - Free PEI addition: 7 portions of free PEI chains added at t = 0 or delayed after polyplex administration. - Proton pump inhibition: Bafilomycin A1 (200 nM) added simultaneously or at different times. - No in vivo experiments.

Key methods: Gel-shift assay and POPO-3 exclusion assay for DNA complexation. - Laser light scattering (LLS) and zeta-potential measurement for polyplex size, molar mass, density, and surface charge. - Luciferase assay for transfection efficiency. - MTT assay for cytotoxicity. - LDH release assay for membrane integrity. - Flow cytometry with Cy5-labeled DNA for cellular uptake kinetics. - Intracellular pH measurement using FITC/Cy5 double-labeled DNA and pH clamping calibration. - FITC-labeled PEI fluorescence for chain-exchange studies.

Key results: Complexation: Over 90% of DNA was condensed into polyplexes at N:P ≈ 3, regardless of PEI chain length. Additional PEI beyond N:P ≈ 3 remained free in solution and did not change polyplex size, molar mass, density, or zeta potential. - Transfection: Free PEI chains enhanced transfection by ~10–10³-fold depending on chain length. Long free bPEI-25K was ~10²-fold more effective than short free bPEI-0.8K or bPEI-2K. - Uptake: With long free bPEI-25K chains, ~95% of cells were Cy5-positive; with short bPEI-2K, ~75%; without free PEI, ~65%. Long free chains increased the median Cy5-DNA fluorescence per positive cell ~10-fold. - Endosomal escape/pH: After 6 h, intracellular pH near polyplexes dropped to ~4.3 without free PEI, ~6.6 with short bPEI-2K, and ~7.1 with long bPEI-25K, indicating escape or prevention of late endolysosome maturation. - Bafilomycin A1: Reduced transfection ~4-fold (N:P = 3, no free chains), ~6-fold (short free bPEI-2K), and ~16-fold (long free bPEI-25K). Even with proton pump shut-off, long free chains still gave ~20-fold higher transfection than without free chains. - Membrane disruption: Long bPEI-25K caused much higher LDH release than short bPEI-2K at concentrations ≥2.7 µg/mL, corresponding to N:P ≥ 10.

Interpretation: The authors conclude that free PEI chains, not the physical properties of the bound polyplex core, are the dominant factor promoting PEI-mediated transfection. Long free chains increase uptake, destabilize membranes, prevent endolysosome development, and facilitate endosomal escape. The “proton sponge” effect contributes only partially. They propose that cationic chains of ~15–20 nm length can embed in anionic membranes, shield signaling proteins, and delay endosome–lysosome fusion, but this must be balanced against cytotoxicity.

Limitations: Purely in vitro; no in vivo biodistribution, efficacy, or toxicity data. - Mechanism is partly speculative; endosome–lysosome fusion prevention and membrane protein shielding were not directly visualized. - Chain exchange between free and bound PEI complicates interpretation, especially for short bound chains. - Long free PEI chains are more cytotoxic, and the therapeutic window is not fully defined. - Most experiments used 293T cells; broader cell-type validation is limited. - No disease model or therapeutic gene payload; only reporter gene expression was assessed.