Tuning DLVO Interactions Alters Polymer-Mediated pDNA Delivery in a Cell Type-Dependent Manner

Polycations bind, protect, and deliver nucleic acid payloads, such as plasmids (pDNA), mRNA, and genome editor proteins, overcoming steep intracellular gene delivery barriers. The size of polycation-pDNA complexes (termed polyplexes) governs transgene expression, cell viability, and cellular internalization pathways. However, researchers struggle to decouple the effect of polyplex size from polycation composition. Here, we map the polyplex size preferences of diverse cell types by applying colloidal science principles to realize differentially sized, yet compositionally equivalent polyplexes. We generated polyplexes ranging from nanometric to micrometric sizes (hydrodynamic radii of 40, 85, 136, 213, 349, 719, and 827 nm) merely by tuning interpolyplex interaction potentials. Derjaguin-Landau-Verwey-Overbeek (DLVO) analysis guided the selection of pH and ionic strength to orchestrate kinetically controlled polyplex aggregation and tune the polyplex size distributions. We first formed polyplexes at a low pH and low ionic strength and induced aggregation via polycation deprotonation and Debye screening. Acidification at different time points arrested polyplex aggregation and thus controlled polyplex size. Static light scattering quantified the pDNA loading per polyplex and revealed that polyplexes pack and condense pDNA more loosely with increasing hydrodynamic volume. Next, we compared the transgene expression mediated by differentially sized polyplexes in kidney cells, retinal cells, and macrophages. Polyplex size preferences diverged between cell types. For example, 85 and 136 nm polyplexes performed best in kidney cells, while retinal cells preferred a wider range of polyplex sizes (85 to 349 nm). We observed that size-dependent cellular internalization limited pDNA delivery efficiency and that we could traverse trade-offs between toxicity and transfection efficiency merely by tuning the polyplex size. Overall, our work exploits colloidal science principles to discover important correlations between polyplex size and the biological outcomes of polymer-mediated pDNA delivery.