Small-angle X-ray scattering (SAXS) experiments were performed using a graded-multilayerfocused Cu Kα (λ = 1.54 Å) radiation from a Nonius FR-591 rotating anode fine-focus X-ray generator operating at 45 kV and 20 mA. Samples were sealed with a bath of buffer solution at room temperature in a custom-built sample holder before measurements. The flight path from the sample to the detector was filled with helium gas to minimize air scatter and sample-to-detector distance was determined using silver behenate standard. Collimation of the primary beam used a fine rear aperture beam tunnel. Diffraction patterns were recorded with a Pt-135 CCD detector with phosphor optimized for Cu Kα radiation. Images were analyzed with Fit2D and OriginPro software. Bragg scattering peaks were used to determine interaxial DNA-DNA spacings. For a hexagonal lattice, the relationship between the Bragg spacing and the actual interaxial distance between DNA helices (Dint) is calculated as Dint = (2/√3) DBragg. For different samples equilibrated under the same buffer conditions, Dint values were reproducible to less than ± 0.1 Å. Typical exposure times were 2 minutes. No significant sample degradation was observed due to either exposure time or pH buffer conditions. PEI Quantification by Ninhydrin Assay. The concentration of bPEI released from polyplexes in the supernatant was quantified using a ninhydrin colorimetric assay. Fibrous bPEI-DNA condensates were prepared in the same manner as those used for X-ray experiments using 10 mM HEPES (pH 7.5) buffer. HEPES was chosen here to avoid reaction with ninhydrin. Fibrous condensate was centrifuged, removed from solution, washed thoroughly with double distilled water to remove unbound PEI and patted dry. After washing, the condensates were then immersed in fresh 20 µL of either 10 mM acetate buffer (pH 4) or 10 mM HEPES buffer (pH 7.5) and equilibrated for 24 hr. After equilibration, supernatant from these samples was transferred to a new tube and diluted to a total volume of 250 µL with additional buffer. In parallel, an 8% (w/v) ninhydrin solution was prepared in acetone and combined with 0.2 M sodium acetate buffer (pH 5.2) in equimolar volumes. Subsequently, 125 µL of the equimolar ninhydrin-acetate mixture was added to 250 µL of the sample solution and the mixture heated at 100 °C for 15 min followed by rapid cooling using ice. After cooling, 62.5 µL of ethanol was added to each sample. The absorbance of supernatants was measured at 570 nm and the PEI concentration quantified using a bPEI standard curve (10-200 µg/mL) prepared in HEPES.