Building on growing evidence that ionizable lipids improve RNA delivery, in this work, we developed ionizable lipid/poly(lactic-co-glycolic acid) hybrid nanoparticles (iLipid@PLGA hNPs), consisting in a PLGA core modified at surface with either 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA), 1,2-dioleoyl-3-trimethylammonium-propane (DODAP), or the branched-tail proprietary amino lipid ALC0315. iLipid@PLGA hNPs were engineered to meet key requirements for inhalation. Thorough physicochemical characterization revealed how the choice of ionizable lipid influences pH responsiveness, surface composition, and architecture of iLipid@PLGA hNPs. In vitro studies demonstrated effective siRNA encapsulation, adjustable release kinetics, and poor interactions with mucus components, as assessed by combined UV-Vis, Dynamic Light Scattering, and Small Angle X-ray Scattering analyses. Confocal microscopy analysis of A549 cells transfected with iLipid@PLGA hNPs showed reduced colocalization of AlexaFluor647-labeled siRNA with lysosomes over time, suggesting enhanced endosomal escape in the case of DODMA@PLGA hNPs. Functional validation using GAPDH-targeting siRNA (siGAPDH) confirmed cellular uptake and gene silencing in normal human bronchial epithelial (NHBEs) cells, confirming the superior performance of DODMA@PLGA hNPs. Finally, representative fluorescently labeled DODMA@PLGA hNPs successfully diffused across a 3D air-liquid interface (ALI) cell model, simulating the human bronchial epithelial barrier. These findings highlight the successful integration of ionizable lipids into polymeric nanoparticles, establishing iLipid@PLGA hNPs as versatile and efficient carriers for siRNA therapeutics. This breakthrough supports their continued development in respiratory nanomedicine and in the local treatment of lung diseases.
