Design and characterization of a SLN-based DNA delivery platform encoding highly conserved regions of the SARS-CoV-2 Spike S2 subunit

The continuous emergence of SARS-CoV-2 variants underscores the need for immunization strategies targeting highly conserved viral regions, such as the Membrane Proximal External Region (MPER) within the Spike S2 subunit. While lipid nanoparticles are widely used for mRNA delivery, their application in DNA delivery remains underexplored, despite advantages in stability and cost-effectiveness of DNA. This study describes the design, optimization, and characterization of a multicomponent solid lipid nanoparticle (SLN)-based vector for the delivery of DNA encoding the MPER sequence. SLNs were prepared via solvent emulsification-evaporation (EE) or hot-melt emulsification methods. The nanoplatform consisted of an SLN core surrounded by DNA complexed with the cationic peptide protamine and polysaccharides (hyaluronic acid [HA] or dextran). The cationic lipid DOTAP was present in all formulations, and the incorporation of ionizable lipids (DODAP or MC3) and gold nanoparticles (AuNPs) was evaluated. MC3-containing formulations achieved the highest transfection efficiencies, while AuNPs were essential for stabilizing DOT/MC3 EE-based systems and maintaining particle sizes around 200 nm. The selected vector (AuNP-SLN HA DOT/MC3 EE) exhibited robust DNA binding (>88%), protection against DNase, and positive zeta potential (+27 to +49 mV). Transfection efficiencies reached 35% in HEK-293 cells. Importantly, expression of MPER at the cell surface was inferred from antibody recognition of a surrogate sequence, supporting its potential accessibility for immune recognition. Furthermore, the nanoplatform efficiently delivered a larger plasmid containing the full-length Spike sequence. These findings establish this multicomponent SLN-system as an adaptable nucleic acid delivery technology supporting its potential as a platform for future immunization strategies.