Asymmetrical flow field-flow fractionation reveals the true biophysical properties of polymer–pDNA complexes by gently separating free polymers

Due to their low manufacturing cost and versatility in delivering diverse nucleic acid payloads such as plasmids (pDNA) and genome editor proteins, polymers have emerged as promising vectors in gene therapy. To encapsulate and deliver anionic nucleic acids, researchers add cationic polymers to payloads (e.g., pDNA), forming highly fragile, complex, and heterogeneous polyplex assemblies. Large stoichiometric excesses of protonotable amine groups (in the polymers) over the phosphate groups (in the pDNA) are typical of polyplexes, which are frequently formed at nitrogen-to phosphorous ratios between 5–100 . Rather than binding irreversibly to pDNA payloads, polymers predominantly exist as “free polymers” which dynamically exchange with pDNA-bound polymers. Analogous to empty lipid nanoparticles and empty viral capsids, free polymers pose complex characterization, quality control, and regulatory challenges that may hinder clinical translation. We urgently need to quantify how free polymers modify critical polyplex properties (e.g., pDNA loading per polyplex) as well as biological outcomes (e.g., immunogenicity, pDNA delivery efficiency, and toxicity). We developed a unique analytical approach that unveils hitherto unsuspected roles played by free polymers during the complexation and intracellular delivery of pDNA. Applying asymmetrical flow field-flow fractionation (AF4), we gently separated free polymers from crude polyplexes while conserving polyplex integrity. Unlike batch light scattering analysis of fractionated crude polyplexes, which severely underestimate polyplex size and pDNA loading per polyplex, AF4 sharpened measurements of polyplex molecular weight, pDNA loading, and radius of gyration by removing free polymers and their background scattering contributions. Comparing solution properties via light scattering, we discovered that AF4-purified polyplexes appeared over twice as large, loading twice as much pDNA as crude polyplexes. AF4 uncovered contrasts in the biological performance of crude and purified polyplexes. Free polymers aggravated inflammatory responses in macrophages by up to 22%. Removing free polymers depressed transfection efficiency by 50% while improving cell viability by 30%; reintroducing free polymers abrogated these contrasts. By separating and quantifying free polymers in polyplexes, AF4 illuminated a far more complex portrait of polyplex solution properties that has hitherto remained elusive to conventional characterization methods. Our work has revealed pathways to ameliorate polymer-associated toxicity and inflammation during pDNA delivery.