Tailoring Zein-based lipid–polymer hybrid vectors for efficient pulmonary siRNA delivery by nebulization

For the local treatment of lung diseases, siRNA inhalation could represent a promising approach, but it requires suitable formulations for efficient RNAi therapy. In this work, we developed a cationic and amphiphilic copolymer, obtained from the grafting on α, β-poly(N-2 hydroxyethyl)-DL-aspartamide (PHEA) of 1,2-Bis(3-aminopropylamino) ethane (bAPAE) and zein, with a derivatization degree (DD%), respectively, equal to 35±0.3 mol% and 2.0±0.2 mol%. The obtained graft PHEA-bAPAE-g-zein copolymer self-assembled in water and efficiently complexed siRNA. From this, nanoparticles (NPs) were produced by complexation with siRNA at N/P ratios of 10 and 20; by surface coating with phospholipid shell, polymer-lipid hybrid systems (LPHNPs) were also produced. These carriers showed nanometric size which was confirmed by TEM analysis. Nebulization with two commercial VMNs preserved the size and stability of most formulations, producing droplets suitable for inhalation (3.5􀀀 4.5 μm). Specifically, the Aerogen Solo device achieved higher product recovery than the Beurer IH 55. All samples, except for LPHNPs 10, demonstrated a high cellular uptake and efficient eGFP knockdown pre and post nebulization and mediated a sustained endosomal escape, validated through Pearson’s correlation coefficient analysis. NPs 20 and LPHNPs 20 samples showed the highest eGFP knockdown compared to the scrambled control sequence. Finally, the muco-diffusive properties, evaluated using two air–liquid interface (ALI) cellular models with different muco-secretory activity, showed that the LPHNPs 20 sample was the most internalized vector, as its diffusion and cellular uptake were less influenced by the amount of mucus on the cells.