Smart Inhalation Therapy: Boosting siRNA Efficacy with Inulin-Based Multifunctional Polymers

Therapeutic delivery of siRNA via inhalation holds significant promise for managing severe pulmonary diseases. However, an effective delivery platform capable of overcoming the lung's physical and biological barriers is essential to achieve efficient gene silencing in the airway epithelium. Here, we describe the synthesis of an inulin (INU)-based copolymer, INU-VS-g-(PMeOx; bAPAE), designed for siRNA inhalation. A stepwise synthesis was employed: first, INU was functionalized with divinyl sulfone to form INU-VS, allowing controlled conjugation of 1,2-bis(3-aminopropylamino)ethane (bAPAE) and poly(2-methyl-2-oxazoline) (PMeOx) at, respectively, 25 and 5 mol % on the total INU repeat units. The resulting copolymer exhibited protonatable amine groups essential for nucleic acid complexation. Stable formation of siRNA polyplexes was found at low polymer/siRNA weight ratios (R = 5), with a mean size below 30 nm. Potentiometric titrations confirmed efficient buffering capacity while fluorescence microscopy demonstrated pH-dependent membrane destabilization, indicating an enhanced endosomal escape potential at low pH values. Stability studies in mucus and pulmonary surfactant revealed that polyplexes remained intact even at high mucin concentrations (5 mg mL^-1) and exhibited high muco-diffusivity. Biocompatibility assessments on 16-HBE showed excellent cytocompatibility with over 80% cell viability even at high polymer concentrations. Uptake studies confirmed polyplex internalization and siRNA release. Experiments on MDA-MB-231-eGFP cells demonstrated siRNA-mediated silencing. Overall, together with the excellent aerosol performance of the polyplex aqueous dispersions, these findings highlight the potential of INU-VS-g-(PMeOx; bAPAE) as a versatile and effective siRNA carrier for pulmonary administration, paving the way for future therapeutic applications in respiratory diseases.