Figure 4. Poly2 and Poly3 exhibit slower polyplex disassembly of siRNA in pH 7.4 biologically-relevant conditions.

A) Polyplexes containing siRNA with Poly0, Poly2, Poly2O, or Poly3 were assembled in pH 5.2 sodium acetate buffer at a 10:1 w/w ratio (PBAE:siRNA) to ensure complete charge reversal. Disassembly of these polyplexes was carried out in PBS at pH 7.4 and 37°C, and samples were rapidly frozen in liquid nitrogen after various time points to assess the remaining degree of binding between polymer and siRNA. B) Gel mobility shift assay in 4% agarose gels was used to assess the degree of disassembly between PBAE and siRNA. Comparison to siRNA (si) as reference. Representative of two biological replicates. Images brightened and contrast-enhanced to better visualize bands. Polyplexes were also released in PBS at pH 7.4 and 37°C over 8 hours for DLS measurements. DLS was used to assess z-average size (C) and derived count rate (D), which is the counts of particles concentration normalized to linear attenuator setting. Horizontal dashed lines indicate z-avg sizes for the polyplexes in pH 5.2 sodium acetate buffer before disassembly in PBS. One biological replicate. n=3 technical replicates. Mean±S.D.