Fresh polyplexes (1.2 μL, 0.1 μg pGFP) were mixed with 6X loading buffer (2 μL) and loaded into a 1% agarose gel containing SYBR Safe Stain (10,000X, Water). Electrophoresis was set up in 1XTAE buffer at 120 V and kept for 30 min. Chemidoc Imaging System was used to assess DNA location. The hydrodynamic diameter (nm), polydispersity index (PDI), and surface charge were measured by Dynamic Light Scattering (DLS) on Zetasizer Nano ZS (Malvern Instruments Ltd, United Kingdom, 4-mW laser) at 25 ºC using 633 nm of laser wavelength and 173 º signal detector. Three measurements of each polyplex batch were performed with 30 runs per measurement. The Z potential was also measured by DLS. 1H-NMR spectra were recorded in a Varian 400 MHz spectrometer using chloroform-d as a solvent. For 1H NMR, the peak of the TMS (0.0 ppm) was used as a reference.