Introduction: Gene therapy holds immense therapeutic potential, but its efficacy relies on safe and efficient gene delivery systems. Non-viral vectors, such as polyethylenimine (PEI), offer advantages over viral methods, including lower immunogenicity and easier fabrication. The primary challenge of non-viral gene delivery vectors lies in their lower transfection efficiency compared to viral systems. This study investigates novel PEI-based ternary complexes utilizing anionic peptides to enhance plasmid DNA delivery by mitigating the significant cytotoxicity and non-specific interactions associated with cationic vectors.

Methods: We developed ternary polyelectrolyte complexes comprising plasmid DNA, PEI, and anionic, glutamate-rich peptides (E6p, cRGD-E6p, E6Hp, E6p0), which varied in their histidine content and the presence of an αvβ3 integrin-targeting ligand (cRGD). Their transfection efficiency was evaluated in vitro using PANC-1 cells and in vivo via intramuscular injection in m. quadriceps f. in mice. Reporter genes (lacZ and GFP) were used to quantify efficiency of delivery through β-galactosidase assays and fluorescence microscopy.

Results: Optimal charge (Phosphorus/Nitrogen/Carboxyl) ratios for high transfection efficiency were identified. For the E6p0 peptide, the most effective DNA/PEI/peptide ratio was 1/16/2, while for E6p, cRGD-E6p, and E6Hp, it was 1/16/4. Reducing the injection volume to two-fold did not compromise transfection efficiency but minimized tissue damage in vivo. All formulations achieved statistically significant transfection levels compared to the control group. Peptides E6p0 and E6p showed the highest performance in vivo, evidenced by a greater number of GFP-positive muscle fibers.

Conclusion: The designed ternary complexes exhibit high efficacy for plasmid DNA delivery both in vitro and in vivo, highlighting their strong potential as versatile vectors for therapeutic gene delivery applications.