Hybrid core-shell lipid-polycation-nucleic acid nanoparticles (LPNPs) provide unique delivery strategies for nonviral gene therapeutics. Since LPNPs consist of multiple components, involving different pairwise interactions between them, they are challenging to characterize and understand. Here, we propose a method based on fluorescence cross-correlation spectroscopy to elucidate the association between the three LPNP components. Through this lens, we demonstrate that cationic lipid shells (liposomes) do not displace polycations or DNA from the polycation-DNA cores (polyplexes). Hence, polyplexes and liposomes must be oppositely charged to associate into LPNPs. Furthermore, we identify the liposome:polyplex number ratio (ρN), which was hitherto an intangible quantity, as the primary parameter predicting stable LPNPs. We establish that ρN ≥ 1 ensures that every polyplex is enveloped by a liposome, thus avoiding coexisting oppositely charged species prone to aggregation.
Keywords: LPNP; fluorescence cross-correlation spectroscopy (FCCS); gene delivery; lipid−polycation−DNA (LPD); lipopolyplex; membrane-coated nanoparticles.