Structure-function relationships for polymeric gene carriers are derived from crude, heterogeneous mixtures of free polymers and polymer-pDNA complexes (polyplexes). Recognizing that our current understanding of polyplex solution properties may be erroneous, we separated free polymers from crude polyplexes to gain important, previously inaccessible perspectives into polymer-mediated pDNA delivery. Free polymers, the numerically dominant species in crude polyplexes, impose characterization and quality control challenges similar to empty viral capsids and lipid nanoparticles. Polyplexes are frequently formed at large stoichiometric excesses of cationic groups in polymers relative to phosphates in pDNA resulting in nitrogen-to-phosphorus ratios between 5-100. Most polymers exist as "free polymers" in dynamic equilibrium with pDNA-bound polymers. For the first time, we quantified the contributions of free polymers to polyplex solution properties and biological performance. We separated free polymers from polyplexes by applying asymmetrical flow field-flow fractionation (AF4), which removes free polymers under gentle laminar flow, minimizing shear stress and keeping polyplexes intact. While batch light-scattering analysis of unfractionated crude polyplexes severely underestimates polyplex size and pDNA loading by 2-4×, AF4-aided removal of free polymers sharpened measurements by eliminating background light-scattering contributions. Polyplexes with 78% free polymers aggravated inflammatory responses in macrophages by 2-20× compared to polyplexes devoid of free polymers. 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 using AF4, we corrected systematic biases in structure-function trends gleaned from crude polyplexes. For instance, microstructure-dependent trends in pDNA loading reported in our earlier work originated from differences in free polymer content rather than the polymer microstructure itself. Our study establishes that free polymers skew measurements of polyplex properties and biological performance and offers design guidance to navigate trade-offs between transfection efficiency, immunogenicity, and toxicity.
