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.