In recent years, natural and synthetic polycations have been investigated as vectors for nucleic acid-based therapies such as gene therapy, which relies on the delivery of genetic material into specific cells for the treatment of various diseases. These polycations form complexes with DNA or RNA through electrostatic interactions, generating nanoparticles commonly known as polyelectrolyte complexes (PECs) or polyplexes. Among them, synthetic polypeptides such as poly-L-lysine (PLL) have attracted considerable interest due to their biocompatibility and chemical versatility. Despite an extensive use of complexes in gene delivery, the physicochemical mechanisms governing their stability, their disassembly after endosomal escape, and their interactions with other biological molecules remain poorly understood. 

In this work, two synthetically polypeptides, typical poly-L-lysine (PLL) and a novel analogue with new properties called poly(N-aminobutyl)glycine (PNL), were synthetized with different degrees of polymerization (DP= 30, 60 and 100 for PLL; and DP= 20, 50 and 100 for PNL) and were used to study the characteristics, stability and biological performance of polycation/ plasmid DNA (peGFP-C3) complexes. The stoichiometry of PLL/DNA and PNL/DNA complexes was determined by gel electrophoresis, conductivity and ζ-potential measurements. Then, we investigated how the polycation structure, degree of polymerization (DP), and charge ratio (R = [N+]/[P−] = 3, 5, 10, 15) influence complex size, polydispersity index (PDI), and surface charge. Colloidal stability was then evaluated in biological media and in presence of competitive biomacromolecules such as anionic polysaccharides. Finally, cell viability and transfection efficiency were studied in HEK293 cells.  

Overall, the stoichiometric analysis showed that PLL/DNA complexes require less volume of PLL to reach the isoelectric point (IP) compared to PNL/DNA complexes, which require larger volumes of PNL, with no significant influence of molecular weight. The characterization of complexes revealed that particle size increases with higher molecular weight, while ζ-potential increases with both molecular weight and charge ratio. We found that some morphological differences were mainly driven by the chemical structure of the polycation. Furthermore, we found that temperature variations induced reversible and reproducible changes in complexes size. Regarding stability, complexes prepared with classical structures and high molecular weight were more resistant in biological media, while stability against the exposition of heparin was favored by higher molecular weight and charge ratio, independent of chemical structure. Notably, both PLL/DNA and PNL/DNA complexes maintained their physicochemical properties for at least seven days, highlighting their suitability for potential gene delivery applications. 

Lizeth Montserrat Bravo Lozano acknowledges financial support from CONAHCYT, and the CNRS through the Institut de Physique de Rennes, as well as technical facilities provided by the University of Guadalajara, the Institut des Sciences Chimiques de Rennes and the École Nationale Supérieure de Chimie de Rennes. Lourdes Mónica Bravo-Anaya acknowledges funding from the IEA P2NanoBio program (CNRS).