Impact of the physico-chemical properties of surface-initiated polymer brushes on siRNA binding and transfection efficiency

Abstract

Rational design of polycationic gene delivery vectors requires understanding how polymer chemistry governs RNA complexation, cellular uptake, and intracellular release kinetics. Poly(dimethylaminoethyl methacrylate) brushes were found to display striking potential for the delivery of siRNA, with high knockdown efficiency and low cytotoxicity. However, the impact of the physico-chemistry of a broader range of polymer brushes on RNA delivery has not been explored. Using a library of 10 cationic polymer brushes spanning tertiary amine and quaternary ammonium chemistries, we reveal how hydrophobicity, pKa, rigidity, and quaternisation modulate siRNA delivery and gene silencing. Combining ellipsometry and surface plasmon resonance, we find that RNA binding capacity correlates directly with pH-responsiveness and the chain rigidity of brushes. Quaternisation with short methyl groups fundamentally altered binding mechanisms, eliminating pH sensitivity while improving colloidal stability and reducing cytotoxicity. To investigate the impact of the physico-chemical properties on transfection efficiency, the uptake of brush-RNA complexes was examined by confocal microscopy and flow cytometry and we quantified cytosolic release using a competitive binding microscopic assay. We observe excellent correlation between the impact of the brush physico-chemistry on release kinetics and long term silencing efficiency. Remarkably, we report that the binding capacity of RNA within brushes does not always correlate with cellular internalisation, competitive desorption and transfection efficiency: rigid imidazole brushes achieved robust gene silencing despite binding substantially less RNA than poly(alkylamine methacrylate) brushes. These findings expand the delivery vector design space, enabling the rational engineering of next-generation polymeric carriers for controlled RNA delivery kinetics.