Pullulan (PULL) has emerged as a promising natural material to produce carriers for pulmonary siRNA delivery. In this study, controlled acidic hydrolysis was employed to tailor PULL molecular weight, reducing it from ~405 kDa to ~45 kDa. The resulting polymer was functionalized with 1,2-bis(3-aminopropylamino)ethane (bAPAE) to obtain a cationic copolymer (DD ≈ 33 mol%) and further modified with all-trans-retinoic acid (Ret) (DD ≈ 4.3 mol%) to introduce hydrophobic component. Potentiometric titration of PULL-bAPAE revealed pKa values of 4.73, 7.00, and 9.31, indicating suitable buffering capacity for endosomal escape, while the amphiphilic PULL-bAPAE-Ret copolymer exhibited self-assembly behavior. Complete siRNA complexation occurred at low polymer/siRNA weight ratios: 6 for PULL-bAPAE and 5 for PULL-bAPAE-Ret, corresponding to N/P ratios of 1.45 and 1.17, respectively. Polyplexes maintained integrity in mucin dispersions at lower N/P ratios, exhibited controlled siRNA release and effectively protected siRNA from RNase degradation. Nebulization using a vibrating mesh device preserved polyplex integrity, with siRNA and polymer recovery >90%. Aerodynamic assessment revealed a mass median aerodynamic diameter of ~2 μm and a fine particle fraction >80%, indicating suitability for deep lung deposition. Biological evaluation showed high cytocompatibility and demonstrated enhanced cellular uptake and significantly improved endosomal escape for PULL-bAPAE-Ret polyplexes in comparison with PULL-bAPAE, resulting in superior gene silencing efficiency. At a polymer/siRNA weight ratio of 6, luciferase expression was reduced to ~38% after nebulization, confirming retention of functional activity. These results demonstrate that rationally engineered PULL-amphiphilic copolymers provide an inhalable formulation for siRNA delivery, combining biological efficacy with tunable physicochemical properties.
