Penetratin Decoration Increases RNAi Silencing Effects of Polymeric Carriers

This study reports the development of a polymeric carrier for pulmonary siRNA delivery via inhalation. Poly-(α,β-N-(2-hydroxyethyl)-d,l-asparamide) (PHEA) was first functionalized with divinyl sulfone (DV) to form PHEA-VS, enabling controlled grafting of 1,2-bis-(3-aminopropylamino)-ethane (bAPAE, of about ∼25 mol %) and poly-(2-methyl-2-oxazoline) (PMeOx, of about ∼5 mol %). The resulting PHEA-VS-g-(PMeOx;bAPAE) copolymer contained protonable amines for efficient siRNA complexation. Potentiometric titration confirmed strong buffering capacity, while fluorescence studies indicated pH-responsive membrane interaction, suggesting improved endosomal escape. Polyplexes formed starting from a polymer/siRNA ratio of 5, with diameters below 40 nm, showing stability in mucins and pulmonary surfactant and protection against RNase degradation. Surface decoration with the cell-penetrating peptide penetratin (Pen) via a terminal alkyne on PMeOx enhanced cellular uptake and siRNA release. Biocompatibility tests on 16-HBE cells showed viability over 80% at high polymer concentrations. Functional assays in MDA-MB-231-LUC cells demonstrated effective gene silencing, particularly at a polymer/siRNA ratio of 5. Combined with the favorable aerosolization properties of aqueous dispersions, these results highlight PHEA-VS-g-(PMeOx;bAPAE) as a versatile platform for pulmonary siRNA delivery, offering stability, biocompatibility, and targeted intracellular release for potential treatment of respiratory diseases.