Introduction

In recent years, advances in nanomedicine and materials engineering have produced multifunctional nanoplatforms with improved potency and biocompatibility for therapeutic applications (Shan et al., 2022). Among these, non-lamellar liquid-crystalline nanoparticles (LCN), particularly reverse hexagonal and bicontinuous cubic mesophases, have attracted attention due to their highly ordered internal nanostructure, structural robustness, and ability to promote membrane fusion and cargo release (Fong et al., 2016; Mezzenga et al., 2019; Tan et al., 2019; Yu Helvig et al., 2021). The internal curvature, aqueous nanochannels, and tunable surface organization of these mesophases allow modulation of nano-bio interactions across multiple length scales (Rodrigues et al., 2019; Tan et al., 2019; Yap et al., 2024; Zhen et al., 2012).

These features are particularly relevant for nucleic acid delivery, including small interfering RNA (siRNA) (Silvestrini et al., 2024). siRNAs can transiently silence otherwise undruggable targets, and the clinical value of RNA interference has been validated by the recent approval of RNAi-based therapeutics (Corydon et al., 2023; Naeem et al., 2025). However, siRNA clinical translation remains limited by its high molecular weight and polyanionic nature, which hinder cellular uptake due to electrostatic repulsion from cell membranes, and by rapid degradation by nucleases (Dong et al., 2019; Dowdy, 2017). These barriers emphasize the need for nanoscale carriers that combine biocompatibility with controlled interfacial properties and efficient cytosolic delivery (Corydon et al., 2023; Naeem et al., 2025).

Although previous studies demonstrate that non-lamellar lipid mesophases enhance membrane fusion and facilitate endosomal escape of therapeutic RNAs, a critical knowledge gap persists (Dyett et al., 2019; Kim et al., 2018; Kim and Leal, 2015; Leal et al., 2010; Sarkar et al., 2020; Shen et al., 2011; Strachan et al., 2020; Yanez Arteta et al., 2018). The impact of steric stabilizer architecture and cationic polymer incorporation on interfacial mechanics and functional gene silencing remains poorly defined. Incorporating polycations is essential for nucleic acid loading (Mendes et al., 2022); however, the liquid-crystalline structure is highly sensitive to these molecules due to charge, packing, and responsiveness to intracellular pH, particularly in the case of cationic and ionizable lipids (Rajesh et al., 2021; Sarkar et al., 2020; Vicentini et al., 2013; Yu et al., 2024; Yu et al., 2023) Furthermore, subtle variations in poly(ethylene oxide) (PEO)/poly(propylene oxide) (PPO) block composition can alter surface corona organization, membrane insertion dynamics, and intracellular targeting, ultimately affecting biological performance..

Here, we systematically investigated how poloxamer architecture and polycation incorporation modulate the colloidal behavior, internal mesostructure, membrane insertion, and siRNA delivery efficiency of reverse hexagonal LCNs. To establish a well-defined and biologically relevant model for interfacial studies, Langmuir monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), a major phosphatidylcholine component of mammalian cell membranes, were employed to probe nanoparticle–membrane interactions under controlled conditions. These biophysical insights were complemented by cellular uptake, functional gene silencing, and in vitro skin distribution analyses to provide a multiscale evaluation of delivery performance. In this context, siTNFα was used as a proof-of-concept anti-inflammatory payload to validate functional intracellular delivery. Although not restricted to a specific disease model, this strategy is particularly relevant for TNFα-driven inflammatory skin disorders (Andretto et al., 2023). Overall, our results demonstrate that stabilizer-driven interfacial organization, rather than surface charge alone, plays a central role in dictating biological performance across molecular, cellular, and tissue levels.