Poly(l-glutamic acid) augments the transfection performance of lipophilic polycations by overcoming tradeoffs among cytotoxicity, pDNA delivery efficiency, and serum stability.

Polycations are scalable and affordable nanocarriers for delivering therapeutic nucleic acids. Yet, cationicity-dependent tradeoffs between nucleic acid delivery efficiency, cytotoxicity, and serum stability hinder clinical translation. Typically, the most efficient polycationic vehicles also tend to be the most toxic. For lipophilic polycations-which recruit hydrophobic interactions in addition to electrostatic interactions to bind and deliver nucleic acids-extensive chemical or architectural modifications sometimes fail to resolve intractable toxicity-efficiency tradeoffs. Here, we employ a facile post-synthetic polyplex surface modification strategy wherein poly(l-glutamic acid) (PGA) rescues toxicity, inhibits hemolysis, and prevents serum inhibition of lipophilic polycation-mediated plasmid (pDNA) delivery. Importantly, the sequence in which polycations, pDNA, and PGA are combined dictates pDNA conformations and spatial distribution. Circular dichroism spectroscopy reveals that PGA must be added last to polyplexes assembled from lipophilic polycations and pDNA; else, PGA will disrupt polycation-mediated pDNA condensation. Although PGA did not mitigate toxicity caused by hydrophilic PEI-based polycations, PGA tripled the population of transfected viable cells for lipophilic polycations. Non-specific adsorption of serum proteins abrogated pDNA delivery mediated by lipophilic polycations; however, PGA-coated polyplexes proved more serum-tolerant than uncoated polyplexes. Despite lower cellular uptake than uncoated polyplexes, PGA-coated polyplexes were imported into nuclei at higher rates. PGA also silenced the hemolytic activity of lipophilic polycations. Our work provides fundamental insights into how polyanionic coatings such as PGA transform intermolecular interactions between lipophilic polycations, nucleic acids, and serum proteins, and facilitate gentle yet efficient transgene delivery.