Poly(2-Propylacrylic Acid) Increases In Vitro Bioactivity of Chitosan/mRNA Nanoparticles

Chitosan-based nanoparticles have been extensively studied for the delivery of nucleic acids. Previous results suggest that these nanoparticles have limited ability to escape the endosome, one of the main cellular barriers hindering nucleic acid delivery. Escape can be improved by the addition of endosomolytic agents during the formulation process or by developing delivery systems with intrinsic properties to disrupt endosomal membranes. In this study, Poly(2-Propylacrylic Acid) (PPAA), an anionic synthetic polymer with known membrane lytic activity was added to the binary chitosan/mRNA nanoparticles to improve bioactivity. The ionization behavior of PPAA was characterized to identify conditions in which PPAA is sufficiently charged to interact electrostatically with chitosan and thus form nanoparticles. The physicochemical characteristics (hydrodynamic diameter, polydispersity index, ζ-potential) and the in vitro transfection efficiency (bioactivity) of this new family of CS/mRNA/PPAA ternary nanoparticles were evaluated. The addition of PPAA to CS/mRNA nanoparticles was shown to be an efficient strategy to augment in vitro bioactivity. The optimal formulation reached an expression level  ~86% of the commercial lipid control at pH 6.5 without any signs of metabolic toxicity. In this paper, we report the effect of salt and pH on the ionization behavior of PPAA and demonstrate 1) successful incorporation of PPAA into/onto nanoparticles, 2) improved bioactivity with PPAA, and 3) that the kosmotropic effects of trehalose play a minimal role in the apparent increase in bioactivity in presence of trehalose.