Figure 3. Poly2 and Poly3 exhibit greater association with siRNA in self-assembled polyplexes than Poly0 or Poly2O.

A) In the fluorophore exclusion titration assay, binding of polymer to siRNA displaces a pre-bound fluorophore, decreasing the fluorescent signal. With this assay, the effective equilibrium binding coefficient (K_eff) can be assessed between the polymers, where a lower value indicates stronger binding of polymer to RNA. Relative binding is shown as both a w/w ratio (PBAE:siRNA) and a nitrogen (from the polymer) to phosphate (from the siRNA) ratio. The data was fit using a variable slope sigmoidal relationship in Graphpad Prism. K_eff,w/w was estimated from the curve fit. Representative of 2 biological replicates. n=3 technical replicates. Mean±S.D. B) Relationship between hydrophobicity (as ClogP of the polymer repeat unit) and the binding equilibrium between polymer and siRNA (as K_eff,w/w). A least squares linear regression showing relationship was fit. R²=0.99. Color indicates the corresponding polymer. C) z-average particle hydrodynamic size and polydispersity index (PDI) measured using dynamic light scattering (DLS) for PBAE and siRNA complexed into polyplexes at four w/w ratios in 50 mM sodium acetate buffer. Representative of 2 biological replicates. n=3 technical replicates. Mean±S.D. D) Zeta potential surface charge of assembled polyplexes containing PBAE and siRNA at four w/w ratios after dilution 14-fold in water to reduce charge shielding. Representative of 2 biological replicates. n=3 technical replicates. Mean±S.D.