Influence of Biological Medium pH on the Stability and Gene Delivery Efficiency of Chitosan/peGFP‐C3 Polyelectrolyte Complexes

ABSTRACT Natural and synthetic polycations are commonly used in DNA‐ and RNA‐based transfection for their ability to condense and protect nucleic acids, and the subsequent cellular uptake. While the formation of these complexes is well‐documented, fewer studies have examined their stability under both extracellular and intracellular conditions. Chitosan, a biodegradable natural polysaccharide, has shown promise for gene delivery. However, its pKa of ∼6.5 makes its ionization state highly sensitive to pH fluctuations around this value. In this study, we examined how pH‐induced changes influence the stability of chitosan/peGFP‐C3 complexes and their effects on cell viability, proliferation, and transfection efficiency in HEK293T cells. The highest transfection efficiency was achieved by transfecting the complexes at pH 6.5, followed by a medium replacement at pH 7.1, particularly with high molecular weight chitosans. Cell viability was found to be higher at pH 7.1, whereas acidic conditions (pH 6.5) affected proliferation, leading to S‐phase accumulation, suggesting potential interference with cell cycle progression. These findings underscore the critical role of pH in optimizing gene delivery and maintaining favorable cell growth conditions.