Gene therapy has emerged as a promising strategy for the treatment of diverse diseases, ranging from genetic disorders to cancer and neurodegenerative conditions. Initially focused on repairing defective genes through DNA delivery, the field has expanded to include small nucleic acids such as siRNA and antisense oligonucleotides. Although viral vectors have been widely used, safer alternatives have been researched due to safety issues and production challenges. Among nonviral approaches, polyplexes, i.e. complexes formed between cationic polymers and nucleic acids, have emerged as promising delivery platforms. Polyethyleneimine (PEI) is one of the most widely studied polymers in this category, due to its high positive charge density and ability to form stable polyelectrolyte complexes. PEI is available across a broad range of molecular weights (Mw) and branching degrees, offering versatility in design. While many efforts have been made to study the delivery mechanisms of nucleic acids from complexes into cells, the formulation process, specifically how nucleic acids and polymers are combined, as well as the kinetics, has often received less attention. Furthermore, the structure of the resulting complexes and their stability have been less studied. In this work, we investigated how the order of component addition, the volumes used, and the kinetics of addition influence the physicochemical properties, stability and biological activity of PEI/plasmid peGFP-C3 complexes (plasmid encoding green fluorescent protein). Two strategies were compared: (i) rapid addition of either PEI to peGFP-C3, or peGFP-C3 to PEI with vortexing, and (ii) slow, progressive addition of either PEI to peGFP-C3, or peGFP-C3 to PEI under gentle stirring. Four PEI samples of different molecular weights (0.8, 20, 25, and 60 kDa) were tested, forming complexes at a charge ratio of 10 (R= [N⁺]/[P^-]). The hydrodynamic diameter of the complexes was calculated using Dynamic Light Scattering (DLS), and their surface charge was determined through zeta potential measurements. To assess the impact of mixing conditions on biological activity, GFP expression was evaluated by flow cytometry (FACS), and cell viability was measured using ATP (adenosine triphosphate) assays. Our results indicate that the method of component addition and the kinetics of addition influence the properties and biological activity of PEI/peGFP-C3 complexes. Rapid addition of peGFP-C3 to PEI with vortexing generally produced smaller hydrodynamic diameters, whereas slow, progressive addition of either PEI to peGFP-C3 led to the formation of larger complexes. Among the PEI samples tested, PEI 25 kDa formed smaller complexes and showed higher transfection efficiency in HEK293T cells compared to other molecular weights. Transfection efficiency appeared to be influenced by the mixing conditions; this effect, together with ATP-based measurements of cell viability, is currently under investigation in ongoing experiments.

Ana Sofia Rojas Noriega acknowledges financial support the Institute of Chemical Sciences of Rennes. Lourdes Mónica Bravo-Anaya acknowledges funding from the ANR DIMECO ANR-21-CE06-0027-01