Abstract
PURPOSE: Lipopolyplexes (LPP), i.e. hybrid ternary complexes of cationic polymers, nucleic acids and liposomes, represent a second-generation non-viral vector aiming to overcome the limitations of the first-generation polyplexes and lipoplexes like in vivo toxicity and ineffective transfection efficiency. Although their potential has already been proven in vitro and in vivo, lipopolyplexes are still poorly explored as gene delivery systems. Here, we provid evidence of the effect of lipopolyplexes composition on their physicochemical features, cytotoxicity, and biological activity (i.e. cell uptake, endosomal escape, and transfection efficiency). METHODS: Lipopolyplexes were prepared by either bulk mixing or a two-step microfluidic process consisting of i) the formation of polyplexes by complexing a plasmid DNA encoding the green fluorescence protein with a panel of cationic polymers (either chitosan, poly-L-lysine (PLL) or polyethyleneimine (PEI)) followed by ii) the formation of the ternary complex by mixing polyplexes with neutral liposomes. The optimal polymer/DNA/lipid Nitrogen/Phosphate ratios and microfluidic operating parameters (volume ratio and total flow rate (TFR) were preliminarily defined to obtain lipopolyplexes with desired properties. RESULTS: The optimized conditions led to obtain lipopolyplexes with a mean diameter of ~180 nm, a PDI < 0.2 and a slightly positive or neutral z-potential. FRET, SAXS and Cryo-EM analyses demonstrated the formation of a ternary complex in which the type of polymer dictated particles' structure. Lipopolyplexes displayed negligible toxicity in vitro, while promoting higher protein expression compared to the corresponding polyplexes and control 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) lipoplexes. Moreover, despite the three lipopolyplexes displaying similar uptake kinetics, those made of PEI showed the highest endosomolytic activity and promoted the most effective DNA transfection. CONCLUSION: Overall, this study demonstrates that lipopolyplexes are a valid platform for pDNA delivery, with PEI lipopolyplexes being the best performing formulation, and that the type of cationic polymer plays a major role in the nanoparticles intercellular trafficking.