Conclusion

This study demonstrates how nonionic block copolymers of PEO and PPO from the poloxamer class influence the colloidal properties and interactions of LCN and LCN-siRNA complexes with biomembranes across molecular, cellular, and tissue scales. We successfully produced reverse hexagonal mesophase LCN with sizes below 200 nm and narrow PdI. Among them, LCN-P407 exhibited greater colloidal stability over time compared to LCN-P188. The incorporation of the cationic polymer, PAH, preserved the colloidal characteristics and hexagonal mesostructure while providing an efficient positive surface charge (10–20 mV), enabling electrostatic interactions with siRNA phosphate groups.

In Langmuir monolayers of DPPC, used as a model of biological membranes, both LCN-P407 and LCN-P188 interacted with phospholipids. Besides PPO length, the longer PEO chain of P407 likely increases the hydrated corona thickness, modifies the slipping plane, and contributes to the distinct membrane-interaction profile observed for the two poloxamers. LCN-P188 induced greater fluidization and alterations, correlating with the observed cytotoxic profile in 2D cell monolayers in vitro. In contrast, LCNp-P407 exhibited superior cytocompatibility, accompanied by significantly higher cellular uptake (1.2–1.7-fold) and enhanced cytoplasmic/perinuclear siRNA distribution without compromising cell morphology. Functionally, LCNp-P407-siTNFα induced a robust and dose-responsive reduction in TNFα secretion (1.2–3.5-fold), whereas LCNp-P188-siTNFα produced delayed and modest effects. In ex vivo porcine skin, both systems penetrated beyond the stratum corneum, but LCNp-P407 yielded markedly higher siRNA-associated fluorescence in deeper layers. For topical siRNA, delivery beyond the stratum corneum into viable epidermis is often sufficient for epidermal targets, whereas deeper dermal deposition may be advantageous for dermal targets.

Collectively, these findings underscore that stabilizer-driven interfacial organization, not surface charge alone, governs biological performance. Integrating membrane interaction studies with mechanistic cellular evaluation is therefore essential for the rational design of LCN platforms optimized for efficient and safe gene delivery. In addition, taken together, the data identify P407 as the preferred stabilizer for this platform, because it combines adequate membrane interaction with lower cytotoxicity, greater colloidal stability, and superior functional delivery.