Innovative Design of Raft-like Nanostructured Lipid Carrier for Nucleic Acid Delivery

Rationale: RNA-based therapies delivered via lipid nanoparticles (LNPs) represent a promising therapeutic approach, yet high cationic lipid content limits biocompatibility. This study aimed to develop novel nanostructured lipid carriers (NLCs) incorporating cationic lipid raft microdomains to enhance nucleic acid delivery efficiency, reducing cytotoxicity. Methods: Raft-like NLCs were synthesized from POPC, cholesterol, sphingomyelin, dimethyldioctadecylammonium bromide (DDAB), and ALC-0315 at a 50:25:10:10:5 molar ratio, based on the principle that clustering electrostatic binding points enhances RNA binding [1,2]. Physicochemical characterisation (hydrodynamic diameter, PDI, ζ-potential, colloidal stability) was performed, and ordered-phase microdomains were analysed by Laurdan GP fluorescence spectroscopy. siRNA and mRNA loading efficiency, transfection assays, cellular internalisation by confocal microscopy (BODIPY-labelled NLCs; AF647-labelled siRNA) in hCMEC/D3 cells, and cytotoxicity (MTT assay, 48-hour exposure, 2 mM) were assessed and compared against Covid-like and conventional cationic LNPs. Results: The NLCs showed a diameter < 120 nm, PDI < 0.2, and ζ-potential of 40–50 mV, with colloidal stability retained for up to 100 days post-synthesis. Laurdan GP fluorescence spectroscopy [3] confirmed ordered-phase microdomains (emission peak 438 nm vs 480 nm for POPC liposomes), preserved up to 40 days, with gel-to-liquid crystalline phase transition at 38–39 °C. Charge clustering within raft domains enabled effective siRNA and mRNA complexation with reduced cationic lipid content, while preserving endosomal escape and improving transfection rates relative to conventional LNPs. In hCMEC/D3 cells, confocal microscopy confirmed efficient cellular internalisation and intracellular AF647-labelled siRNA co-localisation; MTT assay demonstrated improved biocompatibility and reduced cytotoxicity compared to Covid-like and conventional cationic LNPs, without compromising delivery efficiency. Conclusions: Raft-like NLCs achieve effective siRNA and mRNA complexation and intracellular delivery with a reduced cationic lipid content, while displaying superior biocompatibility compared to conventional LNPs in vitro. These findings support the further preclinical development of raft-based NLC platforms as safer and more efficient RNA delivery systems. References: 1. Manning AM et al. (2023) Small 19, e2302917. 2. Staneva G et al. (2021) Methods Mol Biol 2187, chap. 4. 3. Harris FM et al. (2002) Biochim Biophys Acta 1565, 123–128.