Independent versus Cooperative Binding in Polyethylenimine–DNA and Poly(L-lysine)–DNA Polyplexes

Purpose: The structure–activity relationships governing polyplex formation by PEI and PLL are not well understood, especially whether DNA binding is independent or cooperative and how pH affects the mechanism. This limits rational design of nonviral gene delivery vectors.

Hypothesis: If PEI–DNA and PLL–DNA complex formation is analyzed with both independent and cooperative binding models, then the binding mechanism will depend on pH and polymer structure: cooperative at acidic endosomal pH and mixed independent-to-cooperative at physiological pH.

Aims: Compare DNA complexation behavior of branched PEI, linear PEI, and PLL at pH 5.2 and 7.4. - Determine binding constants using an independent binding model and a cooperative Hill-type model. - Relate binding mechanism to polyplex size, charge-dependent aggregation, and transfection efficiency.

Delivery system: Platform: Cationic polymer–DNA polyplexes for nonviral gene delivery. - Polymers: Branched PEI 25 kDa; linear PEI 22 kDa (ExGen 500); poly(L-lysine) 200 kDa. - Payload: Plasmid pCMVβ (7164 bp) encoding β-galactosidase reporter gene. - Probe: Ethidium bromide intercalated into DNA to monitor free vs DNA-bound probe. - Formulation conditions: N/P ratios 0.2–8; pH 5.2 and 7.4; 50 mM MES, 50 mM HEPES, 75 mM NaCl buffer. - Targeting ligand: None; mechanistic physicochemical study.

Approach: In vitro only: No in vivo experiments. - Polyplex formation: Stepwise addition of polymer to ETI–DNA solution; fluorescence measured after each N/P adjustment. - Transfection: CHO, CV1-P, and ARPE-19 cells; polyplexes at N/P 8 for PEIs and N/P 4 for PLL; β-galactosidase ONPG assay. - Particle sizing: DLS at N/P 0.4–8, measured in triplicate from two samples.

Key methods: Time-correlated single photon counting (TCSPC) to resolve free ETI (~1.8 ns) vs DNA-bound ETI (~24 ns). - Independent binding model from \(A_2/A_{1,QY}\) vs inverse polymer concentration. - Cooperative binding model using Hill plot and cooperativity coefficient \(\alpha\). - DLS for hydrodynamic diameter and PDI. - ONPG assay for β-galactosidase transfection efficiency.

Key results: Polyplex core formation was complete at approximately N/P = 2, where nearly all DNA phosphate groups were bound and ETI was fully released. - At pH 5.2, polyplex formation was cooperative at all N/P ratios (\(\alpha = 2.63–3.78\)). - At pH 7.4, formation followed independent binding at N/P < 0.6 (\(\alpha = 0.76–1.01\)) and switched to cooperative binding at higher N/P ratios. - Overall cooperative binding constants were higher at pH 5.2 than pH 7.4; average cooperative binding constant per amine was higher at pH 7.4. - PLL showed 1.3–2.0× larger independent binding constants than PEIs, and 1.1–1.6× higher average cooperative binding constants per amine. - Particle sizes at low N/P were 300–400 nm at pH 5.2 and 400–500 nm at pH 7.4; at N/P = 2, sizes exceeded 2000 nm; at higher N/P, BPEI and PLL decreased to <300 nm with PDI <0.2, while LPEI at pH 7.4 did not decrease up to N/P = 8. - In transfection studies, 22 kDa LPEI was the most effective carrier among the tested polymers and cell lines.

Interpretation: The authors conclude that pH strongly influences whether PEI–DNA and PLL–DNA polyplex formation is independent or cooperative. Lower pH favors cooperative binding due to greater amine protonation, especially for PEIs containing secondary and tertiary amines. PEIs show higher amine density and cooperativity, while PLL binds more strongly per amine. Preparing nanoparticles at lower pH may therefore be beneficial even when transfection occurs at biological pH.

Limitations: Purely in vitro; no in vivo transfection, biodistribution, toxicity, or therapeutic efficacy. - TCSPC could not observe events after core formation at higher N/P ratios; fluorescently labeled polymers would be needed. - DLS samples were measured at different ages, which may affect aggregation and size readings. - Limited cell lines used for transfection; no systematic cytotoxicity comparison. - N/P calculation for PLL considered only side-chain amines, which may complicate direct comparison with PEIs.