Nanoparticle formation and characterization
PBAE and plasmid DNA were each dissolved in sterile 25 mM magnesium acetate buffer, pH 5.2, then combined to yield a weight ratio of 60:1 PBAE:DNA and incubated at room temperature for 15 minutes to allow for nanoparticle self-assembly. Linear PEI of average molecular weight 25 kDa (Polysciences) was prepared according to manufacturer’s instructions and stored at -80°C. To prepare particles, PEI was thawed, diluted in OptiPRO SFM, and incubated for 15 minutes at room temperature before being combined with plasmid DNA, also diluted in OptiPRO SFM, for resulting weight ratios of 2:1 PEI:DNA for HEK cell transfections or 3:1 PEI:DNA for CHO cells. The PEI:DNA mixtures were then incubated at room temperature for 15 minutes to allow for nanoparticle self-assembly. To characterize the resultant particles, 1:20 dilutions were prepared in FreeStyle 293 Expression Medium (HEK cells) or FreeStyle CHO Expression Medium (CHO cells). Size, as measured by hydrodynamic radius determined via dynamic light scattering, and surface charge, as measured by zeta potential determined via electrophoretic light scattering, were measured using a Malvern Zetasizer Pro (Malvern Panalytical). Measurements were reported as mean values from n =3 independently prepared replicates.